Introduction to viruses, viroids and prions

A. General Characteristics:
Particles that are dependent on the host for replication
Much smaller than bacteria: Too small to be seen by light microscopy. They all need an
electron microscope except for pox virus which can be seen by light microscopy.
Generally made up of nucleic acid and a protein that will form the capsid.
Found in 2 shapes:
* Helical: the majority are helical and made up of a nucleic acid surrounded by protein
capsid
* Spherical: the capsid is called a coat and each protein component of the coat is called
capsomere.
In simple viruses, each capsomere can be made of 1 protein. But, usually this is
not the case for most viruses.
* The larger the capsomere the larger the virus you get (can accommodate
more nucleic acid in the genome).
Viruses can be either :
* Naked viruses: only capsid protein surrounding the nucleic acid.
* Enveloped viruses: has lipid bilayer acquired from host. This bilayer is similar to the eukaryotic
membrane except for the presence of glycoproteins which are of viral origin. Additionally, any protein on
the membrane is encoded by the viral gene.
Most enveloped viruses infect animals (much less bacteria and plants)
The specificity and some aspects of the viruses penetration are controlled in part by the virus
membrane.
Note: * Viruses cant down regulate these viral proteins because of their importance in tissue tropism. In
fact, envelope proteins are very important to attack and initiate the invasion of E. coli cells in the urinary
tract.
* It is worth noting that many of these viral proteins are vaccine candidates.
If the virus has no envelope then the attachment proteins will be part of the capsid which will anchor them
to allow binding to host receptors.
RECALL: Glycosyation is done by the host cell because the virus cant glycosylate its own proteins.
B. Shapes of viruses:
In both helical and spherical viruses, the capsid is a very nice geometrically structured array of proteins.
Rod: helical viruses they have helical symmetry
The longer the genome the longer the helix. Thus the virus needs more capsid to encapsulate the genome.
The width of virus has to do with subunits that make up the virus.
Spherical
Most look spherical however they have an icosahedral symmetry.
It is a geometrical structure with many equilateral triangles that is highly energetic and efficient way to
build a very stable capsule (capsid).
These proteins can interact with same protein around it by putting each protein on the vertices.
This is because one protein can interact with many different regions to form a stable structure (one type of
protein can be enough).
Watson and Crick said that the best way to build a viral capsule is to use 1 single protein over and over to
build this structure. This can be due to the fact that viral genomes are small (5-10 genes). So if the virus

uses one gene that encodes the protein capsid then the rest can be used for other stuff. Viruses thus prefer
not to dedicate most of the genome to build the capsid.
3 subunits in every triangle x 20 (faces or triangle number) = 60 subunits.
The number of circulating viruses having only 60 subunits in their capsid is very small. It is because a 60
subunits capsid builds a small sphere that cant accommodate the viral genome.
How do you increase viral genome without replication?
* By increasing the S.A of the equilateral triangles which will also increase the size of sphere without
changing the protein type. So this can be done by dividing triangle into more triangles (triangulation of
the sphere).
* By building larger S.A triangles that can be divided into 4 triangles and then proteins are added in these
places.
The total number of subunits (N) in a virus capsid = 60T (where T is triangulation #)
Exception: Complex viruses: Bacteriophage T4 of E-coli
Made of several parts with different shapes and symmetries
T4 phage has an icosahedral head and helical tails
Have additional tails, pins and tail fibers to infect bacteria.
Have contractile tail made of several proteins: similar to T6SS and uses ATP
to inject the genome.
Have tail fibers: important to bind PS on bacteria and pins.
Tail pins: important to interact with the outer membrane of bacteria.
Around 160 bp- 168kb. Other viruses have larger genomes.
Steps in forming this complex structure.
- 1st nucleocapsid is assembled
- 2nd the tail is added
- 3rd the tail fibers formed from another protein are added to make mature virion
The complexity is dictated by the cell wall of bacteria.
C. Viral genomes:
Viral genomes are small (5-10 kbp), and encode the functions that they can NOT adapt from the host. Inside
the cell the virus depends on the host structural and metabolic components
Different type of genomes: ssRNA, dsRNA, ssDNA, dsDNA, segmented or non-segmented, + or - sense.
In segmented viruses, each segment carries a gene.
For all of these viruses, in order to replicate, they must produce mRNA.
* Replication depends on protein synthesis which uses the ribosome that can only recognize mRNA.
Enzymes: Some viruses carry additional enzymes to package nucleic acids and other extra enzymes.
* Some enzymes are involved in early infectious processes: the bacteriophages have lysozyme.
* Mammalian RNA viruses that interact as DNA intermediate thus are well packaged with polymerase: RNA
dependent DNA polymerase (reverse transcriptase) which is a specific polymerase that allows transcribing
RNA to DNA. These enzymes allow reverse-transcription.
* RNA viruses in sense genome must produce mRNA to get + sense. For this reason, they use RNA
dependent RNA polymerase which is found in mammalian viruses.
D. Quantifying viruses:
Like in the case of bacteria but here, we count plate forming units (PFUs).
We can perform this procedure for bacterial and eukaryotic viruses: say
have tube containing a certain virus and you want to quantify the number

of viruses (phage/ bacterial virus). Take an agar plate and mix the phage with bacteria and soft agar. Pour
the mixture on agar plate. Get full coverage of bacteria on the plate (confluent growth of bacteria).
Results: Each plate corresponds to a lysed bacterium by the virus.
This experiment gives fairly a good idea of how many viruses there are but might give an underestimation
if counting PFU (more than one virus might give a single lysed area if found in same place).
Why use soft agar? To prevent virus from spreading. If a virus lyses a cell and produces virions, the virus
can diffuse if we dont have agar or any other solid medium. In this case, we will no longer see 1 or 2 lysed
cells. We dont want the virus to spread very far since this would defeat the whole aim of the experiment.
A more accurate way is through electron microscopy.
For Eukaryotic Viruses:
* Grow a monolayer of eukaryotic cell, and then add the virus and soft agar medium.
* Plaque assay can also be done with animal cell lines growing as a monolayer in a petridish.
* Confluent growth on plate. Afterwards, add the virus and soft agar medium (the soft agar causes
jellification of medium and prevent viral spreading).
* Problem: The efficiency of infection might be poor since bacterial viruses are more infectious than
mammalian viruses.
Disadvantages of Plaque Assay
Low platting efficiency: not all viruses in cell mixture will infect a bacteria or animal cell (thus plaqueforming units is always lower than counts made of the viral suspension with an electron microscope.)
* Several reasons for this case
- Some virions might be inactive.
- Infectious conditions not optimal for phages to bind and interact with bacteria.
- The culture is not optimal for infection. Whenever we have a certain virus frozen to be used later, not all
of the particles will infect.
* Animal viruses efficiency can only reach 0.1-1% whereas bacteriophages are >50% efficient.
Some viruses dont infect any cell line (dont have cell-line to that permissive for the virus)they grow
endlessly since we don`t know to what type of tissue they bind or what is their specific tissue properties.
Lethal dose 50 (LD):
* Know the type of the virus by infecting animals and checking the LD (lethal dose) 50.
* What dilution that you inject causes the death of 50% of X animals.
E. Viral infectious cycle:
Difference if bacterial or mammalian virus.
Bacterial virus
Virus remains outside the cell (capsid stays outside) and only the genome
is injected into the infected cell.
Injected genome takes over the machinery of the host. Promoters (of viral
genes) are directly recognized by bacterial polymerases, and compete well
with bacterial promoters.
Once transcribed, the virus produces its own proteins. Then, it replicates its
genome. Afterwards, it assembles new viroids; package the copies of the
genome. Finally, lysis occurs and many viroids are released.
LPS or surface molecules are important ligands for interaction. Penetration
requires injection of genome then the synthesis of nucleic acid and proteins.
Mammalian virus
Attachment to host cell involves many different ligands that range from proteins to sugars and lipids
(minority).

The whole virus enters the cell.

The mechanism changes depending on whether it is an enveloped or non-enveloped virus
Mammalian viruses are taken in by endocytosis or fusion.

Growth curve simpler than that of bacteria

Latent period: unfolding of virus then synthesis of early enzymes
(polymerases for replicaiton). Replication and synthesis of protein
capsid.
Divided into two stages: Eclipse and maturation
Eclipse: when virus nucleic acid separates from the protein coat
Maturation: when newly synthesized nucleic acids become
packaged into protein coats
Assembly and release: at the end of the latent period virions are
released by lysis or budding or secretion depending on the virus.
Bacterial virus only gets out by lysing the host cell.
Mammalian viruses can either lyse cell, bud out of membrane (glycoproteins embedded in membrane),
or translocate to Golgi through secretoy pathway and gets secreted from there.
Note: Most virus attachment is mediated by 1 or 2 proteins. Usually we dont see a lot of proteins on
surface of virus compared to bacteria. In general most ligands are protein ligands; in some cases like
influenza they bind to sugars or cyenic acid. So they might have 1 or more receptors with redundancy.
If one ligand is mutated, others will take over and fulfill its function, since some viruses use more than one
receptor.
NOTE: a cell that allows the replication of a virus inside it is called a permissive cell.
The problem with designing vaccines for viruses arises from the mutations that occur and changes in their
profile of surface proteins.
o RSV (Respiratory Syncytial Virus):
Virus that infects infants by age of 5 and causes severe respiratory infections
Has 2 proteins ligands but we still havent found a vaccine that works well due to high mutation rates.
Some animal viruses invade through 1 single step by interacting with 1 receptor. For others, they need
interaction with more than one receptor: it is a stepwise interaction (>1 receptor)
E.g in HIV: 2 important glycoproteins (receptors) on surface of
HIV
gp 41: anchored in the membrane and gp-120 bound to gp 41
gp 120 which binds to protein ligand CD4 on T-cells and
then triggers conformational change in the second gp120
This will release the binding site for another ligand: gp120
binds to ligand that is a chemokine receptor.
These is a change in conformation of gp41 which reveals
hydrophobic helices that can insert in the membrane
gp41 digs in and bring membrane of virus close to membrane of cell leading to fusion
Viral fusion mediated by hydrophobic proteins that have to insert in one membrane to get membranes
close to each other.
This doesnt mean that all enveloped virus fuse at level of plasma membrane
Some bind to receptor on plasma membrane and are endocytosed whole virus now in an endosome.
Fusion occurs with the endosomal membrane (in early endosome pH of 6).
Fusion triggered by conformational change of gp41 mediated by interaction with the ligand

Fusion is mediated by low pHpH of 6 or 5.5 then surface protein undergoes conformational change
revealing hydrophobic domain that insert into the endosomal membrane; viral and endosomal membrane
fuse and virus escapes.
* In late endosomal period, the virus is destroyed because of the low pH (therefore fusion and escape of the
virus only occurs in early endosmal period).
Conformational change occurs in endosome and is triggered by drop in pH
Naked Virus
Binds to receptor then endocytosed or taken in by endocytosis.
In early endosomes (pH 5-6), when pH drops some proteins in capsid undergo conformation change, insert
in membrane and form a pore.
Pore formation through which the virus exit the endodome into the cell.

F. Outcomes of viral infections:

Some viruses cause transformation of cell into tumorous cells.
Many viruses produce regulators that might activate genes involved in cell division or promoters which
lead to cell division and signaling pathways.
E.g HTLV (human T-cell leukemia virus)
o Can cause tumors; leukemia in T-cells once it infects the T-cells
Some viruses cause persistent infection: remain in the host and eventually replicate very slowly virus
remains and produces very few virulence over a period of time(productive persistent infection since can
detect virulence)
o Might also remain latent: remain inside cell put wont divide.
o In latent infection: the virus is there but we cant detect virulence or virus at some point the virus
activates itself and might lyse the cell and produce a lytic infection.
E.g HIV: cause a very long latent infection. The virus is not producing virions but it is hiding. It integrates its
genome in the chromosome and becomes integrated in all cells. When these cells are activated, their
promoters get active and virus starts replicating.

G. Replication:
dsRNA or ssRNA replicate in the cytoplasm while most DNA viruses replicate in nucleus only.
Most DNA viruses replicate in nucleus because they use DNA polymerase of host and the host has protein
sensors in cytoplasm of eukaryotic cells that can sense DNA activate immune response (viruses evolved
to replicate in the nucleus in order not be detected).
* Their Proteins are produced in ribosome, transported back to nucleus where encapsulation and
packaging occurs. Afterwards, the virus escapes from the cell.

EXCEPTION to process above: Small Pox (caused by variola virus)

Exception since replicates in cytoplasm.
o Uses its own polymerase encodes its own RNA & DNA polymerase
Not surprising because it has a large genome (one of largest viruses).
Since it replicates in cytoplasm, it needs to escape the host. And it does this by :
o Escaping host detection by replicating in specific domains in cytoplasm called VIROSOMES
Organelle like structure that are surrounded by a membrane where viral synthesis and packaging occur
Specific for small pox
Virosomes are assembled by viral proteins and are derived from ER membrane.

o
o
o

o
o

RNA viruses
All replicate in cytoplasm very close to ER
+ sense RNA viruses are in sense of mRNA
Genome already infectious since mRNA is present.
Once injected in eukaryotic cell, the virus is produced without replication and it can directly bind to
ribosome
Once genome released in cytoplasm it can directly bind to ER and start translating its mRNA.
Translating the genome means virus starts producing its RNA polymerases (RNA viruses must use their
polymerases since not found in host)
Process
First translation occurs to produce enzymes needed for replication as well as the capsid proteins.
When enough proteins produced, there is a switch from translation to replication: + sense RNA converted
to sense RNA (1or 2 copies)
Convert these sense RNA to several/many + sense RNA.
Ratio of + to is like 50:1
- to + replication much more efficient since it gives many templates. This has to do with differences in
promoters: -ve templates can have stronger promoters
Viruses can enhance these strategies by producing primers which enhance replication of - cell.

Strategy is always translation followed by replication

Same template need to replicate and translate: both cant occur at same time so translation always first.
They cant occur on same template since they would overlap in this case.
Note: the protein coat is synthesized before the genome because RNA genomes are highly sensed by
receptors in cytoplasm of eukaryotic cells
Virus cant tolerate to keep its genome for a long period in cytoplasm packaged to protect from
recognition by cytoplasmic protein of eukaryotic cells.
o Mechanism to escape immune response: genome therefore produced once there are enough proteins.

Negative sense RNA viruses: doesnt have an infectious genome

Virus must enter with its own polymerase
Virus enters with sense genome + an RDRp (RNA dependent RNA polymerase) prepackaged in virus
can`t get translated by ribosome so the virus genome has to be converted to + sense.
In cytoplasm must convert sense directly to + sense
When enough proteins produced have switching to replication.
The + is no longer used and are used to produce - genome to be packaged.
dsRNA viruses have both + and - is replicated to form the + sense
+ sense is translated
When have enough proteins, we will have replication
Packaging of + sense occurs when we have enough proteins present and then we will have replication.
First layer of proteins have + sense genome
Second genome (dsRNA) is produced within the virus. This is done because dsRNA in cytoplasm is a potent
trigger of immune system. They package the single strand in + sense and convert to sense in the capsid
Capsids have pores
If look at virus, the dsRNA produce + sense in capsid and mRNA comes out from pores in the capsid; try to
hide dsRNA genome as much as possible

o
o
o

First production of + sense dsRNA done in capsid

Presence of dsRNA in cytoplasm will trigger immune response
Release + sense dsRNA from pores in capsid
Goes to ribosome to produce proteins
Recall some RNA viruses have segmented genomes;
E.g influenza :
Can have up to 8 segmented segments
- sense RNA genome
Because of many pieces in cytoplasm, the virus need to choose one a, one b, one c one d to package
Problem if virus for example uses two b.
This will make the virus genetically different
Problem with segmented viruses is that they can shuffle their genome if have 2 viruses infecting the same
cell because signals for picking up are similar in both genomes. This is why influenzae viruses tend to
diversify quickly
Not very common though, happen once every 20 yrs. Mutations can occur but no that severe.

Retroviruses (class VI, ssRNA +)

Much more complex because switch between RNA and DNA
Initially ssRNA (HIV + sense RNA) but doesnt use it as mRNA to produce proteins convert to cDNA
(sDNA)
This sDNA is converted to dsDNA
ssRNAssDNAdsDNA
step 1 and 2 occur by reverse transcriptase
dsDNA integrate into genome of the host and gets transcribed
o Transcription uses RNA polymerase of the host
o Virus will use host RNA polymerase and some host transcription factors of host can recognize viral
promoters
Class VII (dsDNA)
Small group
Include Hepatitis A
dsDNA but replicate via an RNA intermediate (opposite of retro-virus or HIV)
DNA genome that Replicate through an RNA intermediate
By transcription produce +sense RNA which is converted to a ssDNA and the ssDNA to double stranded
DNA
o Also use reverse transcriptase
Dont integrate in genome
Not clear why they do all this

o
o
o
o

H. Viral proteins
Early Proteins
Mainly enzymes
Enzyme needed for replication
Enzymes that Interfere with host physiology
Synthesized in low amounts (needed in small concentration)

 Late Proteins
o Synthesized later
o Include Protein of the virus coat& capsid
o Produced in large amounts (needed in large concentration)

o
o
o

I. Characteristic of bacterial viruses

Bacterial viruses also have same diversity dsRNA, ssRNA... however most know about T4 phage others
not well understood
Huge variety - Can have tail/ no tail, neck/ no neck region
T4 Phage
Model phage
Huge virus (250 proteins): can guess this from its elaborate structure
Infect because of the tail that interacts with LPS on the surface of bacteria
Once fibers interact with LPS, they change conformation so that fibers bend bringing pins
closer to the surface of wall
Contraction of sheath similar to T6SS
Uses ATP to contract the sheath proteins sending a tube across the outer membrane across
PGNalso have lysozymes packaged in virus which help cleave part of PGN
Eventually inject genome into cytoplasm
Genome released to periplasm and then transported to cytoplasm
Not directly to cytoplasm
Gets to cytoplasm by a transportation process
Phage assembles and packages ATP from previous infections
Whenever it has infected a cell it packages ATP (around 150
molecuels of ATP) together with lysozyme

Tail and head contractile

Head contraction mediate injection of genome into host
Eukaryotic cells protect themselves from viruses by :
Sensors in cytoplasm that detect viral genome
Bacteria defense mechanism to viruses
By use of restriction enzyme / endonuclease
If virus has not methylated its genome then it will be broken down
o Viruses can escape by glucosylating and methylating genome
o Defensive mechanism of virus against bacteria
Ignore slides not covered.

Viroids
Infectious RNAs
Do not have any protiens associated with them
Specific for plant kingdom (don`t find them in animals)
Circular dsRNA
Very stable
Can be carried by insects; usually enters wounds of bee
Doesnt encode any protiens
o Use polymerase of plant cell to replicate

o Symptoms in plants usually growth related (slower development)

Work by interfering in growth;
o Don`t encode any proteins so must work by interfering have complementary domains to mRNA or rRNA
that might inhibittranscriptionwhen they bind to them.

o
o

o
o
o

o
o
o
o
o
o

o
o

Prions
Protein particles; not associated with nucleic acid
Originated from normal host proteins that undergo mutations to become
prion proteins
Responsible for disease known as encephalopathy spongiform (brain
becomes like sponge, has many holes and lots of degradation); bovine
spongiform
Mad cow disease is an example
Encephalopoathy- symptoms include anything related to brain
degradation , loss of vision and motor activities
Scrapie in sheep
Kuru and a form of Creutzfeld-Jakob disease in humans (long time ago, in
the 50s) found only in Montenegro because practicing cannibalism.
Diseases caused by protein usually found in the brain
PrPc protein and as such it is involved in cell-cell interaction
If some mutations occur in gene of protein, then get protein with conformational change that is
significantly different from main protein
Mutation seen in folding ; protein folding disease
The normal protein is expressed on the cell, anchored in the membrane by a GPI linker.
Diseased protein is rich in B-helices and is an aggregated protein (structure very different)
Highly resistant to enzymes,pH& detergents
Cant break them down even in lysosomes (lysosomal compartment)in fact they can lyse the lysosomes
Scientists are trying to solubilize them however are failing
PrpSc the mutated form; PrPs normal
Conformation change is transmitted if an abnormal protein binds to normal one changing it to abnormal.
PrPSc can convert normal PrPs to abnormal conformation
Have some abnormal prion proteins which form aggregates on the surface of the cell, &can recruit normal
proteins and convert them to abnormal conformation
Eventually cell try to get rid of them by endocytosing ; fuse with lysosomes however some lysosomes lyse
and aggregates become found in cytoplasm
They interfere at some point with proteasome function by forming like protein magnets and starting
toadsorb hydrophobic domain from many unfolded proteins intoxicating the cell
If they are released to extracellular domain, they will build up to form breaks
Cause Amyleoid break any form of abnormal protein aggregation
Seen in Alzheimers and Parkinsons
Macrophages interact with these breaks as defense mechanism and activate immune response leading to
inflammation in brain since macrophages try to get rid of them (nothing else working)
End up with destruction of brain due to aggregation and the immune response.Only way to get rid of
them is by burning everything.
Not clear how prion is transported from stomach to brain.

Microbial Interactions with Humans

The human body is constantly exposed to microorganisms (MOs) and many harmless MOs live on
and inside certain regions of our body. Most of the interactions are beneficial, only a small
percentage is harmful.

A typical human body contains 1 x 1013 body cells, yet harbours an estimated 1 x 1014 bacterial cells
so bacteria have a larger genome which has an implication on the physiology such as competing
with pathogenic bacteria, colonize surfaces to trigger production of AMPs, aids digestion, vitamins,
immune system maturation

What types of bacteria grow normally on different parts of the body?

A science paper in 2006-7 tried to identify the types of bacteria that grow normally on different
types of the body. Samples were taken from the mouth, skin, epithelium, stomach, esophagus and
colon. They did meta-genomic analysis and large profile sequencing to see what species were living
in every place. What did they find?
Mouth:
-

Most bacteria are gram positive, like firmicutes & actinobacteria, and are referred to as lowGC bacteria because the percentage of GC in their genome is low (20-30%).
Very few gram negative bacteria like bacteroidetes & proteobacteria

Skin:
-

Majority is gram positive because they are more resistant to dryness (thick PGN)
An unexpectedly substantial number of gram negatives like proetobacteria & e. coli

Vaginal endothelium: mostly gram positive, which has an effect on pH

Gut: Most are gram positive
Colon: Half are gram positive, half gram negative
Esophagus:
-

Mostly gram positive firmicutes

Very few bacteroidetes (they increase towards the colon)
Proteobacteria (e.coli, salmonella, shigella, klebsiella) are present at very low concentrations.
These are the ones that cause disease. If they dominate, it is called dysbiosis, which is
abnormal and can cause disease or cancer (eg. Colon cancer).

This composition is dynamic with time, age (infants, children, adults and elderly) and habitat. What
influences this dynamicity?

Genetics or environment? Is the composition of the flora inheritable? If we take two monozygotic
twins, do they have same flora if they live in different places?

 The environment (diet and habits) controls the floral composition. Genetics plays only a very
minor role. The composition does not seem to be heritable as much as it seems to be affected by
the environment.

According to the way MO establish an interaction with the human host we can classify them into 3
broad groups:
a. Normal microbiota (aka commensals): MO that establish a more or less permanent
residence in the host but do not cause disease under normal conditions; they can differ in %
between different people.
We cannot call them symbiotic (although often called symbionts); if a bacteria is symbiotic it
has to be found in every human individual because the interaction would be essential for
both, and there are no such bacteria reported. Plus they do not have to be beneficial.
b. Transient microbiota: MO that may be present for several days, weeks or months and then
disappear. These also do not cause disease (staph or strep)
c. Pathogens: MO that are able to inflict damage on the host although they dont establish a
permanent interaction.
Opportunistic pathogens: These cause disease only in the absence of normal host resistance
(Immuno-compromised) (eg. fungus).

I.

Normal Microbiota

A) Normal microbial flora of the skin

- An average adult human has 2 m2 of skin that varies greatly in chemical composition and moisture
content
Areas of the skin are favourable for growth of MO:
i.

Epidermis:
-

ii.

It is not a favourable place for abundant growth of MO as it is

subject to periodic drying.
Apocrine sweat glands:

Most skin MO are associated directly or indirectly with these

glands

These are secretory glands located in the dermis and found mainly in the external genitalia,
nipple of the mammary gland and the underarm or axilla (Underarm odor develops as a result of
bacterial activity in the apocrine secretions)

The apocrine glands are activated during puberty and secretions are released to the hair canal
so the flora is subject to changes according to age

The apocrine secretions collected aseptically are odourless, but develop odour on inoculation of
bacteria. The secretions include sialomucins, which are rich in sialic acid. Bacteria ferment these
sugars producing ammonia that gives the smell.

Note: Apocrine gland secretions contain protein, carbohydrate, ammonia, lipid and other organic
compounds. Apocrine glands become functional at puberty as with axillary and pubic hair.
iii.

Sebaceous glands
-

Sebaceous glands are activated during puberty develop as an outgrowth of the external root
sheath of the hair follicle found in the face, the skull of the head, the upper chest and the back.

They produce sebum secreted directly to the canal, oily substance rich in nutrients such as
amino acids, salts, lactic acid and triglycerides (triglycerides are broken to fatty acids by bacteria
on the skin surface, and the free fatty acids may be an irritant in the formation of acne lesions)
due to propionobacterium acnus associated with pimple.

Propionibacteria ferment lactic acid which acidifies the medium and if pores are clogged, the
sebaceous glands become necrotic to release the sebum, but cell debris cant be excreted to the
outside and over-proliferation of bacteria in this small area causes inflammation.

In addition there are the Ecrine Glands (normal sweat glands):

Not associated with hair follicles. They have ducts and they secrete directly to the surface. They cover
the whole surface area of the skin providing it with the necessary nutrients (water).

Characteristics of the normal microbiota of the skin

1. It consists of resident and transient populations of MO. (The skin is continually being inoculated with
transient MO from the environment, virtually all of which are unable to multiply and usually die)
2. To know the species, we take from dry areas, sweat glands, and sebaceous glands and perform
meta-genomic analysis, which is scraping then purifying DNA then sequencing 16S rRNA and
compare sequences. Three Gram-positive genera are stable residents of the skin and predominate
of other species: these are Staphylococcus (Firmicutes), Propionobacterium and Corynebacterium
(both belong to Actinobacteria) forming about 75% and this is due to their resistance to dryness
since they have a thick PGN
3. Beta-proteobacteria and bacteroidetes (Gram-ve) are abundant on dry areas of the skin
4. Few yeast species may be found on the skin.

In sebaceous Propiobacteria reside due to fermenting lactic acid so pH drops so inhibits other
bacteria from survival
In armpit, nostrils there are corynebacteria (thick cell wall) and staph
In dry skin, proteobacterea were dominant and it was a surprise.

5. The composition of the microbiota is affected by several factors:

a. Weather: an increase in temperature and moisture increases the density of the skin microbial
flora
b. Age: Young children have a more varied microflora and usually carriers of pathogenic bacteria
than adults
c. Personal hygiene: Unclean individuals have higher microbial population densities on their skin
Most of the MO in direct contact with the skin do not become residents because:
Secretions from sweat and oil glands have some antimicrobial activities
Keratin is a resistant barrier to the invasion of deeper softer tissues of the skin
The low pH of the skin secretions (ranges from 4 to 6) inhibits growth of many MO
Low moisture content of the skin
The skin is not a mucosal tissue. Mucosal tissues include mouth, intestinal tract, respiratory tract and
urogenital tract.

B) Normal microbial flora of the mouth

-

Saliva contains a variety of microbial nutrients, but it is not an especially good medium for microbial
growth because nutrients are present at low concentrations

Saliva also contains a number of antimicrobial agents. Examples include:

a) Lysozyme: an enzyme that cleaves glycosidic bonds in the peptidoglycan of the bacterial cell
wall)
b) Histatins: small histidine-rich salivary gland proteins that have moderate antifungal activities

However, despite the presence of antimicrobial activities, the abundant moisture, warmth and
constant presence of food make the mouth and ideal environment that supports a very large and
diverse microbial populations (600 genera) on the tongue, cheeks, teeth and gums

The teeth and dental plaque

-

In the mouth bacteria are especially adapted for growth on teeth

surfaces and gingival crevices (hard to clean). They start at the
crevices and spread to over the teeth.

Bacterial colonization of tooth surfaces begins with attachment of

single bacteria to a thick film of acidic glycoproteins produced by
salivary gland that attach to the tooth from the saliva

This film is a firm attachment site for bacterial cells

Streptococci are mainly found on tooth

Experiment:
Microbial attachment to the tooth surface is quick
(A) Colonies growing on a model tooth inserted into the mouth for 6h
(B) Note the diverse morphology of MO present, and the slime layer they produce to recruit other
bacteria

Major Bacteria Colonizing Tooth

-

Colonization of the glycoprotein film involves only a few species of Streptococcus (S. sanguis, S.
sobrinos, S. mutans and S. mitis), aerotolerant anaerobes (strict anaerobes)

It starts with streptococcus and then extensive growth of these bacteria results in a thick bacterial
layer called dental plaque. Then bacteria start moving deeper until they reach the bones, at which
point and immune response is induced.

Note: The plaque builds preferentially near the gum line, first occurring directly adjacent to the mucous
membranes of the gingiva

If the plaque continues to form, other bacteria will associate with it including:
a) the filamentous anaerobic Fusobacterium species (Gram ve bacteria causing periodontal
disease which results in bone loss around the teeth),
b) In heavy dental plaques, filamentous Actinomyces (Gram +ve obligate or facultative anaerobes)
may predominate. In rare cases they may cause disease
c) other gram +ve and ve cocci.

The anaerobic nature of the oral flora may seem surprising considering the intake of oxygen
through the mouth

However, anoxia develops due to facultative bacteria growing aerobically on organic material at the
tooth surface where they deplete O2.
Furthermore, as the plaque builds up it produces a dense matrix that decreases O2 diffusion to the
tooth surface forming an anoxic microenvironment.

Electron micrograph of a thin section of a plaque

-

Bottom is the base of the plaque (anaerobes), and top is the portion
exposed to oral cavity (facultative anaerobes)

Note:
-

Streptococci are the predominant MO (dark cells are S. sobrinus which

appear as 2 distinct chains)

An extensive slime layer surrounds the S. sobrinus cells forms the matrix on
which other cells will stick.

We get rid of them by scraping (mechanically) the upper layer. Listerine and
other mouth washes will not help.

Dental Caries
-

As dental plaques accumulate, the resident microflora produces locally high concentrations of
organic acids (e.g. Lactic acid), which cause decalcification of the tooth enamel and dental caries
(tooth decay) result

Diets high in sucrose are especially cariogenic because lactic acid bacteria ferment sugars to lactic
acid

Two MO implicated in dental caries are Streptococcus sobrinus and S. mutans, both homofermentative (produce only lactic acid from glucose fermentation)

a. S. sobrinus has specific affinity for salivary

glycoproteins found on smooth tooth surfaces (first
bacteria associating)
b. S. mutans, leading cause of dental caries, has an
enhanced ability to attach to tooth surfaces due to
production of Dextran polysaccharide that is strongly
adhesive
o S. mutans produces dextran only when sucrose
is present, using the enzyme dextransucrase to
give chains of glucose (and use fructose for
energy)
The figure shows the sticky dextran material holding the cells together as filaments
Susceptibility to tooth decay varies and is affected by inherited traits in the individual as well as by
diet and other factors

C) Normal microbial flora of the gastrointestinal tract

The human gastrointestinal tract, the site of food digestion, consists of the stomach, small intestine
and large intestine.
Microorganisms colonize all these compartments to varying degrees as the physiological conditions
vary among these 3 compartments

i. The stomach
- The stomach fluids are highly acidic (~ pH 2) hence the stomach is a chemical barrier to entry of MO
to small intestine
As a result very few bacteria are able to colonize the stomach: MOs such as Helicobacter pylori can
colonize the stomach wall (mucous) and causes ulcers in susceptible individuals.
- Helicobacter infects 40-45 % of the population and can remain for decades, they synthesize
enzymes which produce NH3 to buffer acidity.
- Actinobacteria, Proteobacteria and Bacteroidetes are also present. There are also firmicutes,
streptococcus and staphylococcus.
Nonetheless, we do not know how long they will stay there. Are they transient or do they stay there?

ii. The small intestine

-

The small intestine is composed of the duodenum, jejunum and ileum

The duodenum, adjacent to the stomach, is fairly acidic and resembles the stomach in its lack of a
microbial flora. It also contains bile made of bile acids that kill gram +ve bacteria

From the duodenum to the ileum, the pH gradually becomes less acidic and bacterial numbers
increase. In the lower ileum, bacteria numbers of the order of 105-107 / gram are common

iii. The large intestine

- In the colon, bacteria are present in enormous numbers. The pH of the colon is neutral and favours
colonization by several genera of bacteria (both facultative anaerobes and anaerobes)
- However, facultative anaerobes like E. coli are present in small numbers (total count of facultative
anaerobes is 107/gram of intestinal contents) since they are inflammatory.
- These consume the remaining oxygen making the environment strictly anoxic.
- Many strict anaerobes colonize the colon. The Bacteroides and gram-positive bacteria (firmicutes)
constitute the majority, others include Clostridium, Enterococci etc
- The total count of obligate anaerobes in colon is 1010-1011 / gram of contents
- The normal microbial flora of the colon protect the host from colonization by pathogenic bacteria
(e.g. E. coli produces bacteriocins that inhibit growth of pathogenic Salmonella and Shigella)
- People with chronic inflammation have 3 different enterotypes:
o Bacteroidites (gram -ve protein and fats)
o Prevotella (gram ve vegetarians)
o Ruminococcus (gram +ve vegetarians)
- Every individual has a has a unique origin flora
Purpose of Study:
o The enterotypes are related to the disease (fingerprint)
o Possibility of changing the flora by diet change
- Importance of the Flora:

Compete with pathogens. How?

- Metabolic competition (for nutrition)
- Cover space
- Produce bacteriocins that kill virulent bacteria
Produce vitamins
Development/maturation of the immune tissues in the colon (gnotobiotic/germ-free mice that
are raised in sterile environments lack many immune tissues compared to mice that have
normal gut microbial flora)
The initial colonization of the babys tissues starts from the birth canal and then from peoples
skin, from the milk
They reduce inflammation (example, bacteroidetes that make 50% of the bacteria, their LPS
doesnt activate the immune cells)
Important for differentiation and regeneration of colonocytes, which are the epithelial cells of
the colon. Bacteria break down of fibers and ferment the products to produce short-chain fatty
acids like propionate, which are a signal for the differentiation of colonocytes and are also a
source of nutrients to them (through Beta oxidation).
o Differentiation of the colon (prevents metastasis), maintenance of the colon (anti-cancer
components)
Flora of the gastrointestinal tract

These are local effects. Are there any non-local roles for the gut microbial flora?
-

The composition of microbial flora may have an effect obesity

Microbial flora in the gut has an effect on mucosal tissues elsewhere. It was found that mice that
have a normal microflora in the gut are better able to fight viral infections in the respiratory tract
than gnotobacteria. The connection is still unclear though.

D) Normal microbial flora of the respiratory tract

a) In the upper respiratory tract MO
- Live in areas bathed with secretions of mucous membranes (nasopharynx, pharynx, larynx)
- Most of the bacteria that enter the upper respiratory tract from the air are trapped in the nasal passages
and expelled in nasal secretions

Resident MO most commonly found are staphylococci,

streptococci, gram-negative cocci and others
Pathogens that may exist:
Staphylococcus aureas (found in 20% of the population; it is
the most important pathogen causing pneumonia)
Streptococcus pneumonie (found in 40% of the population;
if it goes to the lower RT, it causes pneumonia).
Also gram negative Neisseria, which causes meningitis.
Also there is Haemophilus influenzie.
These are called carriers but do not normally acquire disease because growth of pathogenic
bacteria is limited by resident MO. Plus, these bacteria can be eliminated by immune system.
Components of the immune system (antimicrobial peptides, IgA antibodies) are particularly active at
mucosal surfaces and may also inhibit growth of pathogens.

b) The lower respiratory tract (trachea, bronchi)

-

It has no resident microflora

As air passes into the lower respiratory tract, the flow rate decreases and MO settle on the walls of the
passages. However, The ciliated epithelium pushes bacteria and other particulate matter toward the
upper respiratory tract where they are expelled in saliva and nasal secretions. Moreover, the epithelia
of the lower respiratory tract produce the highest amount of lysozymes. Additionally, there are
phagocytes in the lungs that eat bacteria if they reach it.

E) Normal microbial flora of the urogenital tracts

- In males and females the bladder is sterile but the epithelial cells lining the distal urethra (last 2 cm)
and the female vagina are colonized by facultative anaerobic Gram-negative rods and cocci as well
as by facultative and aerotolerant anaerobic gram-positive rods and cocci
The Vaginal flora
-

The female vagina is weakly acidic because Lactobacillus acidophilus, a resident organism in the
vagina, ferments glycogen to produce lactic acid (lactobacillus species are the predominant MO in a
healthy female genital tract; 107 to 108 CFU/gram of vaginal fluid in healthy premenopausal women)
However, before puberty and after menopause, glycogen production is absent and pH rises and
hence microbial flora is different. So this happens during the reproductive phase of a females life
during which estrogen level is high (related to glycogen storage).

Lactobacilli are believed to interfere with pathogenic bacteria by different mechanisms

a) competitive exclusion of genitourinary pathogens from receptors present on the surface of the
genitourinary epithelium (lactobacilli adhere well to uroepithelial cells)
b) inducing a low pH (around 4) environment by producing lactic acid so kill pathogenic bacteria
c) production of antimicrobial compounds, such as hydrogen peroxide, and bacteriocin-like substances
Why not use lactobacillus to inhibit HIV infection?
We can design bacteria that produce peptides that can block HIV receptors. This is called
Paratransgensis = Modifying the microbes living in the host to fight pathogens. This is actually done in
mosquitos gut flora to allow it to fight the dengue virus and malaria parasites, and hence these
pathogens will no more be transmitted to humans by mosquito bites. Scientists modify endosymbiont
bacteria that are transmitted vertically (from mother to progeny) to make sure all progeny has it. Eg.
Wolbachia bacteria.

Transgenesis is when the host itself is modified to become resistant to pathogens

Biological outcomes of the presence of resident microbial flora
1.
2.
3.
4.

Help competing with pathogens such as Salmonella in the GI

Provide us with vitamins (several intestinal bacteria)
Harm us by promoting diseases (dental caries)
Stimulate the immune system maintaining a basal immune activity and maturation of colon
(especially in GI tract)
5. Compete for space and food
6. Cause neither help nor harm or dont know their function yet
What is the importance of studying the human microbiota
1. Determine whether healthy individuals share a core microbiome
2. Determine whether changes in the microbiome can correlate with changes in human health
(obesity, allergy, inflammatory)
The ability to study native microbial communities represents a fundamental shift in microbiology and
is one whose implications can only be imagined

II. Harmful microbial interactions with humans

A- Entry of pathogens into the host
For a pathogen to cause disease it should be able to adhere to and
penetrate the skin, and mucosal surfaces that normally act as microbial
barriers. It can remain on the surface of the cell to grow in number then
invade and colonize deeper tissues. It can also invade and replicate inside
the cell.
Some pathogens are specific to the tissue they invade, others have a
broader range of targets and be transported though the blood and
lymphatic systems to reach distant sites.
Note: invasiveness is not characteristic of all microbes.
Tissue damage is not only cause by bacterial invasion but also from
toxins (which can act locally or diffuse further to other tissues).

Adherence
Pathogenic MO are able to bind to epithelial cells. A pathogen may not adhere to all epithelial cells
equally but selectively adhere to cells in a particular region of the body
Adhesion of pathogens to host cells is mediated by several mechanisms:
i- Bacterial surface proteins that recognize receptors on target cells
ii- Slime layers (loose network of polysaccharide polymers extending outward from the cell which are
bound by sugar receptors on the eukaryotic cell)
iii- Capsules: a polysaccharide coat consisting of a
dense, well-defined layer surrounding the cell
Note: slime layers and capsules also allow bacteria
to adhere to each other and escape certain immune
reactions like phagocytosis
iv- Fimbriae /pili: both function by binding host
cell glycoproteins initiating attachment event

Note: V. Cholera is not an invasive bacterium, while

EPEC is.

Invasion

Vibrio cholerae adhering

to the rabbit brush
border of rabbit villi

Enteropathogenic E. coli
attached to brush border of calf
villi via a well-defined capsule

- Organisms can be pathogenic in 2 ways:

i) by producing toxins (few)
ii) by penetrating the epithelium (invasion). This characterizes most pathogens
- Growth of pathogens is established
i) At point of entry or
ii) On intact mucosal surfaces (especially if the normal flora is altered or eliminated by
antimicrobial therapy) eventually invading the tissue
iii) At a distant site from the original point of entry (access to distant organs is through the blood or
lymphatic circulatory systems)
- If a pathogen gains access to a tissue it is said to colonize the tissue
- Successful colonization requires:
i) Appropriate environmental conditions (e.g. pH)
ii) Appropriate oxygen levels
iii) Most importantly, the availability of nutrients (sugars, amino acids, vitamins, trace elements
such as Iron)
iv) Receptors on tissues
- Although the host seem to be a nutritional paradise for MO, not all nutrients are plentiful
- For instance the host proteins transferrin and lactoferrin bind iron decreasing the accessibility of MO to
iron pool
- However, certain bacteria produce siderophores like the aerobactins of E. coli which can strip iron from
transferrin
- Bacteremia: It occurs when bacteria growing in tissues are shed in large numbers into the blood
- Septicemia: stage after bacteremia where bacteria has already infected many organs

Virulence
Virulence is the ability of parasite to cause disease through toxicity and invasiveness
How is virulence measured?

Virulence can be estimated by measuring the lethal dose 50 (LD50):

the dose of an agent that kills 50% of the animals in a test group
Highly virulent pathogens frequently show little difference in the
number of cells required to kill 50 and 100% of the population

Note: virulence is often lost when pathogens are kept in laboratory cultures.
Such pathogens are said to be attenuated.
The MO doesnt want to kill the host quickly, otherwise transmission will be
aborted. Influenza for example requires a long incubation period before
showing symptoms and its not lethal.
Exceptions exist, like the Ebola virus which kills within 2/3 days, hence epidemics disappear quickly.
Virulence Factors

i- Invasiveness
It is the ability of an organism to grow in host tissue in such large numbers that the pathogen inhibits host
function
a- Capsules and slime layers are virulence factors since they facilitate invasiveness.
e.g. Streptococcus pneumoniae can cause extensive host damage because their capsulated forms
are highly invasive and grow in lung tissues in enormous numbers.
Non-encapsulated forms are quickly and efficiently destroyed by phagocytic cells.
b- Enzymes that will change the physiology of the host
- These also promote spreading of pathogens by breaking down components of the extracellular
matrix (hyaluronidase and collagenase).
- Some fibrinolytic enzymes dissolve clots at the sites of infection (these clots are often formed at
the site of microbial invasion to isolate pathogens to a local region). Some strains can hijack
enzymes of host used to break fibrin clots.
- Coagulases produced by virulent strains of Staphylococcus aureus produce fibrin and deposit it
on the surface of the bacteria. The fibrin coat protects the bacteria from immune response of the
host.
c- Fimbria and short pilli
ii- Toxins

The structure of alpha-hemolysin in membrane bilayers

a- Exotoxins
These are toxic proteins that may travel from a local area of
infection and cause damage at distant sites. These toxins fall into
one of three categories:
1- Cytolytic toxins: they work by enzymatically attacking
cell constituents or inserting into membranes causing lysis.
Examples include hemolysins. Can either be Phospholipases
or have a pore forming activity (seen in E. Coli).
2- A-B toxins: Called the two component toxins. They consist of 2 covalently bonded subunits
- The B subunit generally binds to a cell surface receptor, allowing the transfer of the A subunit
across the membrane into the cell where it functions to damage the cell.
- Examples of A-B toxins include Diphtheria toxin, tetanus and botulinum toxins.
- Showed a new way of delivering proteins to host cells: the Endocytic pathway (similar to
mechanism of viruses). We have already seen the direct injection method in T3SS, T4SS and
T6SS.
- A-B toxins are endocytosed after binding to the receptor. Then they escape from early
endosomes. A drop in the pH (to 5-6) triggers the escape of A to the cytoplasm through B which
is now a pore-like structure in the endosomal membrane. It is thought that the low pH also
allows A to unfold and translocate through B. Also, A fold in the cytoplasm with the help of host
chaperones.
- Some A-B toxins take the Retrograde pathway: got from endosome to the golgi, ER ad get
out of the ER to the cytoplasm through the Sec61 pore (translocon) mimicking proteins of the
host.

Note: A-B toxins have very few lysine residues, in order to avoid ubiquitilation in the host and
targeting to the proteasome.
3- Superantigens: these function by stimulating large numbers of immune response cells (5-25%
of lymphocytes, particularly the T-cells) resulting in extensive, non-specific inflammatory reactions
which eventually cause tissue damage (damage not resulting from MO itself).

b- Enterotoxins
These are exotoxins whose activity affects the small intestine, generally causing massive secretion of
fluid into the intestinal lumen leading to vomiting and diarrhea (e.g. Cholera toxin also an A-B toxin)

c- Endotoxins
Many gram-negative bacteria produce potentially toxic lipopolysaccharide (LPS) as part of the outer
layer of their cell envelope. These are called endotoxins. The lipid A portion of LPS is responsible for
toxicity, while the polysaccharide fraction makes the complex water soluble and immunogenic (both
fractions are required in vivo to induce toxicity). Endotoxins are cell-bound and are released in large
amounts when cells lyse (in contrast to exotoxins which are secreted products of living cells).
Once it reaches the blood, LPS triggers a strong inflammatory response in the spleen and the liver. It can
also cause damage to endothelial cells (triggers apoptosis). Endotoxins are pyrogens (cause fever). Thus,
pharmaceuticals such as antibiotics and intravenous solutions must be endotoxin-free.
Endotoxin is detected by the Limulus Amebocyte Lysate (LAL) assay:
The coagulation cascade of the horseshoe crab (Limulus polyphemus) amebocyte lysate is very sensitive
to small amounts of LPS (10 pg/ml). In the presence of LPS the cascade is activated causing gelling and
precipitation of the extracts resulting in change in turbidity.
The LAL assay uses enzyme components of the cascade and lead to the breakdown of a chromogenic
substrate yellow color = positive test.
It is used to detect endotoxin in serum, cerebrospinal fluid, drinking water, and fluids used for injection
and drug formulation

Host risk factors for infection

i- Age
Infectious diseases are more common in the very young (immature immune system) and in very old
(have senescence of immune cells).
Infants, especially in the days following birth, are susceptible to pathogenic strains of E. coli and
Pseudomonas aeruginosa because their normal intestinal flora is not established as that in adults.
Clostridium botulinum is found almost exclusively in infants under 1 year of age, because intestinal flora
in older children and adults prevent C. botulinum colonization. One source of Clostridium is honey.
ii- Stress

Stress can predispose a normally healthy individual to disease. In studies with rats and mice,
physiological stressors such as fatigue, strenuous exercise, poor diet, dehydration, or drastic climate
changes increase the incidence and severity of infectious diseases.
Hormones produced in stress conditions may influence the immune system (e.g. Cortisone which is
produced at much higher levels than during normal periods, is an effective anti-inflammatory agent).
iii- Diet
Diet plays a role in host susceptibility to infection. Examples:
- A poor diet can weaken mucosal immunity (need for carbohydrates and vitamins).
- Malnutrition alters the composition of the normal flora, allowing opportunistic pathogens a better
chance to multiply and increasing susceptibility of host to pathogens (e.g. The number of Vibrio
cholerae that can cause cholera is reduced in malnourished individuals).
- In some cases, absence of a particular dietary substance may prevent disease by depriving a pathogen of
critical nutrients (e.g. Effect of sucrose on dental caries).

iv- The compromised host

Cancer patients may have altered immunity because of aggressive therapies they receive.
The stress of surgery and anti-inflammatory drugs given to reduce pain and swelling may also reduce host
resistance. Organ transplant patients are treated with immunosuppressive drugs which can reduce the
patients ability to resist infections.
Smoking and excess consumption of alcohol. Infections with HIV causes an immunocompromised state
Note: hospital-acquired infections are called nosocomial infections

Physical, chemical and anatomical barriers to infection

Person to person microbial diseases

Airborne Transmission of diseases
Aerosols such as those generated by sneezing are important for person-to-person transmission of many
infectious diseases. Most respiratory diseases are spread exclusively in this fashion..
Airborne pathogens:
- Most organisms found in air originate from soil, water, plants, animals, people etc...
- most organisms survive poorly in air and so can be effectively transmitted only over short distances;
however certain human pathogens survive under dry conditions and remain alive in dust or inanimate
objects for long periods of time.
-Most airborne pathogens are Gram+ because of their thick cell wall which resists dehydration (see
example below)
Examples:
- Staphylococcus and Streptococcus (Gram+ve are in general more resistant to drying than Gram-ve
because of their thick, rigid cell wall). Capsules also play an important role in avoiding dehydration.
- Mycobacteria have a waxy layer of cell walls which resist drying. They can stay in sputum for days.
- Endospores of certain Gram+ve bacteria are extremely resistant to drying but are not generally passed
from human to human in the endospore form
The air flow slows down as it moves through the respiratory tract, and as the air flow slows down,
particles stop moving and settle down. The large particles settle down first and only particles smaller than
3m actually reach the bronchioles. Sine all airborne pathogens are smaller than 3m, size does not play a
role in tissue colonization. The reason why different MOs colonize different parts of the respiratory tract
depends on unique metabolic requirements and virulence factors associated with each pathogen.

Bacterial respiratory pathogens

Most bacterial pathogens affecting the respiratory tract inhabit only humans and are transmitted from
person to person (hence humans are the only reservoirs).
Many person to person respiratory pathogens are Gram +ve bacteria. Because these MOs are mainly G+,
they respond rapidly to antibiotics.

I- Streptococcal diseases
Characteristics of Streptococci:
- Most common respiratory disease
- Streptococci are non-sporulating Gram +ve bacteria arranged in pairs or chains.
- They belong to the lactic acid bacteria group in the phylum Firmicutes(low GC
group).
- Are aerotolerant anaerobes (peroxidase positive).
- Are homofermentative (ferment carbohydrates into Lactic acid only).
- They are catalase-negative (if bacteria produce catalase, then after adding a
drop of 30% hydrogen peroxide to a colony of cells, bubbles are produced
immediately as a result of oxygen release). 2H O 2H O + O
2

Note: not all the cocci are catalase-negative (Staphylococcus and Micrococcus are catalase positive can
differentiate them)

Organisms in the genus Streptococcus have been divided into 2 subgroups of related species mainly
based on the pattern of hemolysis on blood agar
a- Pyogenes (-hemolytic):
These strains produce Streptolysin O or S that cause complete red
blood cell hemolysis.
Most virulent streptococci. E.g.: Streptococcus pyogenes.

Zones of hemolysis
around a -hemolytic
Streptococcus

b- Viridans subgroup (-hemolytic, but not hemolytic):

Many Streptococci, Lactococci and Enterococci do not produce
hemolysins but instead cause the formation of greenish or brownish zones
around colonies on agar (the bacteria produces an enzyme that converts the
Fe2+ in hemoglobin to Fe3+. Hemoglobin bound to Fe3+ is called methemoglobin and results in the discoloration of blood to a green-brownish
color that can be seen as brownish zones around the colonies.). E.g.:
bacteria that cause caries.
c- -hemolytic
Dont lyse RBCs and dont produce met-hemoglobin

Note: Another classification (Lancefield classification) is based on the type of serum (A, B, C, D, F, G,
etc.) that recognizes a sugar in the bacterial cell wall.
E.g. serotype A includes S. pyogenes.
A third classification is based on the biochemistry of the bacteria.

A. Streptococcus pyogenes
It is transmitted by respiratory route through aerosols
I- Virulence Factors
1. Streptolysins S and O
- Cell bound hemolysins
- Lyse erythrocytes, leukocytes and platelets. Are pore forming toxins, responsible for the -hemolysis.
- Can stimulate the release of lysosomal contents after engulfment with the subsequent death of the
phagocytic cell.
- Note that the difference between the S and O streptolysin is that O is immunogenic while S is not.
2- Streptokinases (not a kinase)
- Form 1:1 complexes with plasminogen. The complex starts cleaving other plasminogens, ultimately
plasmin replaces plasminogen in the complex with streptokinase (higher affinity).
- Plasmin cleaves fibrin and fibrinogen, hence lysing blood clots, facilitating the spread of S.
pyogenesin infected tissues.
- Streptokinase used in the form of Streptase in pharmaceutics to treat ischemic heart disease.
3- C5a Peptidase:
Surface bound endopeptidase (break peptide bonds of non-terminal amino acids) that cleaves the
complement component C5a (chemo-attractant + inflammatory protein) which attracts phagocytic cells
to the bacteria.
4- M Protein:
Main adhesive protein in Streptococci + prevents lysis by complement.
A dimeric protein anchored in the cytoplasmic membrane, extends through the cell wall and protrudes
above the cell surface.
Functions in attachment to host cells and prevents bacterial opsonization by complement proteins.
M protein can recruit (hijack/bind to) Factor H in the serum which is one of the host proteins that cleave
complement proteins.
The principle function of Factor H is to regulate the complement pathway to ensure that it is directed
towards pathogens and not self-cells. (It protects them from the complement system). When bacteria
bind/recruit Factor H, they develop resistance to the complement system, and thus increased virulence.
Now trying to block colonization by targeting M protein with vaccine.
5- Protein G:
A bacterial surface protein that binds antibodies with high affinity from their constant region, thus
inhibiting their opsonic potential.
Commercially used to purify antibodies.
6- Exotoxins:
- Produced by lysogenic strains of streptococci (previously infected by a lysogenic phage).
- Act as superantigens that activate large numbers of T-cells leading to an exaggerated inflammatory
response with symptoms including shock and organ failure seen in patients with streptococcal shock
syndrome.

A-Streptococcus Pyogenes:
I-Virulence Factors:
- An additional virulence factor produced by some streptococcus are capsules; not found in all strains
- Capsules are a dynamic structure; strains that are expressing the capsule are not always expressing it
- Example: A streptococcus strain isolated from a skin lesion of the mouse shows to have a very little capsule
with small sugars. If this strain is grown in the lab, it again produces very little sugars in the capsule. Once
this strain is isolated from the blood stream, it has a very elaborate capsule. Why? The capsule plays a role in
protecting the bacteria from phagocytosis. In addition, the blood compartment is the most dangerous place in
a sense that all the antibodies and proteins that might interfere with the bacterial phagocytosis are present
there. Therefore, the bacteria will not have this capsule during invasion but will develop a more complex
capsule in the blood stream to protect itself.

Streptococcus from
mouse skin lesion

Streptococcus grown in
the lab

Streptococcus from
the blood stream

II-Diseases Caused by Streptococcus:

1- Tonsillopharyngitis: - The most common disease caused by streptococcus
- Affects both the tonsils and the pharynx
- This disease causes the strep throat where both tonsils and the pharynx are swollen,
with white structures on the sides.
- These white structures are composed of bacteria, dead cells and immune cells
- Most common in children and infants but can infect others
2- Otitis Media: - It is the infection of the inner ear (most common in children)
- How does it reach the ear? There is a structure that connects the pharynx to the ear named
the eustachean tube across which the bacteria can reach the ear
3- Infections of the superficial layers of the skin (impetigo): rare infections; it causes the pealing of the skin.
Streptococcus cannot invade the skin. However, in case there is a wound, the virulent strain can grow in the
skin and cause this rare but severe skin infection.
4-Scarlet fever: - Also a rare infection
- Caused by the strains that has Exotoxin A
- These strains have a lysogenic phage that encodes for this pyrogenic Exotoxin A
- Exotoxin A is a superantigen that can activate a very strong and powerful immune response
- Usually this fever starts as a pharyngitis which then develops into a rash on the chest, that
will then go over the skin

- This rash is due to the systemic activation (over-activation) immune system

- It can lead to a toxic shock which means that patients with this infection can have a very
high fever, they might faint out or lose blood volumes which will affect the kidneys.
REMEBER: this happens only due to strains with Exotoxin A
III-Delayed Complications:
-These complications occur due to untreated or insufficient treatment of the S. pyogenes infections mainly in
the poor countries
a- Rheumatic Fever: - It is a type of auto-immune disease
- The antibodies start reacting against self-antigens on the heart, joints, kidney resulting
in damage of these tissues
- This fever is due to M protein
- There are 120 serotypes of the M protein
- The problem with the M protein is that some of its domains are similar to the domains
found in the proteins of many other organs like the heart tissues
- So if these domains of the M protein are recognized by the immune system, the
antibodies will be cross-reacting with the tissues of the heart
NOTE: There's a genetic pre-disposition for developing auto-immune diseases
b- Glomerulonephritis: - It is the inflammation of the kidneys
- It is caused by the antigen-antibody mega-complexes that will accumulate in the
blood and from the blood they move to the kidneys
- It is a painful case but will eventually be resolved by itself once the antigen-antibody
complexes are removed from the nephritis
- Can last for 2-3 days
NOTE: Most of the bacterial infections need no treatment; the body will remove them
spontaneously. However, when the bacteria is present in the body for a long period of
time, it will cause more complications.

B-Streptococcus pneumoniae:
- It is also a major pathogenic streptococcus species causing lung infections (elderly and young children) and
recurrent ear infections in young children. It can also cause meningitis.
- The most common virulence factor in S. pneumoniae is the capsule
- This capsule helps in bacterial dissemination and protects it from phagocytosis
- There are 91 different types of capsules
- They differ in the composition of the sugar
- When diagnosing patients for infections with S. pneumoniae, the sugars of the capsules are recognized
- The body will produce antibodies against the polysaccharides of the capsule
- Also, the vaccines will target the sugars of the capsules. However, the vaccines can't target all 91 different
capsules; they will target the most prevalent capsules of the most commonly isolated strains. This is figured
out from the epidemiological data
- Streptococcus pneumoniae infect the respiratory tract and then spread to the blood stream
- It is one of the three bacterial causes of meningitis

- S. pyogenes have capsules with hyaluronic acid which is similar to some components of the human tissues.
Thus, there are no vaccines for pyogenes yet, but there are vaccines against S. pneumoniae that can protect
against 2/3 of the circulating strains (the capsules of S. pneumoniae are more diverse). These vaccines
(multivaccines) are a mixture of capsule polysaccharides.
-These vaccines are usually given to the elderly, health-care providers, individuals with compromised
immunity and to the patients that are genetically predisposed to bacterial infections; rarely given to children

Corynebacterium diphteriae:
-Also a respiratory tract bacteria
-Belongs to the phylum Actinobacteria (coryne + myco)
-It is Gram +ve, non-motile, rod-shaped, aerobic bacterium that forms irregular shaped, club-shaped or Vshaped cell arrangements
-Belongs to the high GC gram-positive bacteria group (the genome has more than 50% GCs in terms of
composition)
NOTES: a) Streptococcus is the low GC group
b) Mycobacterium and Streptomycin also belong to the Actinobacteria group
-Actinobacteria are rod to filamentous, primarily aerobic and are common inhabitants of soil and plant
materials.
-Under the microscope, diphteriae show arrangement of cells in short chains ("V" or "Y" configurations) or in
clumps resembling "Chinese letters"; it has a smaller size than usual
-It can be identified by these shapes.
-Infections and diseases by this bacteria is not very common now due to vaccination
I-Clinical Diseases:
-Diphteriae can cause 2 diseases:
1- Cutaneous diphteria:
-In this case, the bacteria invades the skin through cuts and injuries upon contact with an infected patient
(example: if an infected person sneezes and some droplets land on an injury)
-It causes an ulcer in the skin=the skin tissues die=causes necrosis of the skin tissue
-It is a rare infection
2-Respiratory diphteria:
-Most common disease
-Transmitted by contaminated aerosols
-The bacterium enters the body via respiratory tract and attaches to throat and tonsils.
-Symptoms usually start as those of a viral infection: fatigue, malaise, sore and painful throat,
and low grade fever, but then there is development of a thick membrane in the pharynx
-It is called a pseudomembrane which is composed of bacteria, lymphocytes, fibrin, and dead tissues that can
block respiration and is hard to remove (well-attached)
-This membrane is unique, which allows the distinction between diphteria and other infections
-This infection which used to be fatal doesn't occur anymore because of the vaccines

II-Virulence Factors:
-The virulence of diphtheria is due to its A-B exotoxin (diphteria Exotoxin)
-Certain strains of C. diphteriae are lysogenized by bacteriophage that contain a tox gene encoding an A-B
exotoxin
-Exotoxin is secreted at the site of infection
-This exotoxin is also an enzyme that is made of 2 components: A and B which are covalently bonded
-A is an enzymatic component= ADP-ribosylase
-B is a peptide which has a receptor in the tonsil
-B binds to receptor of the epithelial cells in the respiratory tract which will trigger the internalization of A
into the cytosol of the cell following proteolytic cleavage
- A using nicotinamide adenine dinucleotide (NAD), removes an ATPribose from NAD+ and covalently links it to the elongation factor-2
-Elongation factor-2 is important for translation. Hence its
adenylation aborts translation.
-Therefore, A-B Exotoxin blocks translation (no protein synthesis=cell
death). This will cause a severe damage in the epithelial cells, which
will activate the immune response
-This toxin is very effective; a single molecule can kill one cell

III-Prevention:
-If the toxin is blocked then the bacteria becomes indirectly harmful
-The vaccine component is the diphteria toxin itself; it is part of the DTP vaccine (diphteria, tetanus,
pertussis)
-The DTP vaccine is now called the DTaP vaccine (diphteria, tetanus, acellular-pertussis)
-This vaccine is acellular which means that the components or the bacterial toxins of this vaccine have been
deactivated
-Example: the diphteria toxin is formaldehyde-deactivated: the toxin is isolated and then treated with
formaldehyde to prevent it from damaging the cells. But still, the immune system will be able to produce
antibodies against this toxin
NOTE: the vaccine for pertussis used to be bacterial=attenuated vaccine, but now it is acellular

Mycobacterium
-Belongs also to the Actinobacteria group
-Found in many organisms
-Non-spore forming, aerobic rods that can grow as filaments
-The name myco, meaning fungus-like, was derived from their occasional
exhibition of filamentous growth
-These filaments are due to a glycolipid called the cord factor which allows
the rods to interact with each other
-These filaments can be easily broken by shaking the culture of the
Mycobacteria leading to the formation of rods => not true filaments

-There are several groups of the Mycobacteria: human pathogen, animal pathogen, fish pathogen
-The outermost layer of the mycobacterial cell wall is made up of mycolic acids which form a waxy, waterresistant layer (this layer is stained with the red basic fuchsin dye, aka acid fast stain)
-Mycobacteria are very resistant to drying because of their waxy cell wall (they can survive for weeks in
dried sputum)
-Mycobacteria are also resistant to antiseptics and disinfectants because they cannot penetrate the waxy
layer and they are scavenged
-That's why it is a highly infectious bacteria: if a patient sneezes and the droplets contain mycobacterium, the
bacteria can survive harsh conditions for a long period of time
-Initially, the patients infected with Mycobacterium were isolated (in quarantines and sanitariums). Need at
least 5 months antibiotic treatment.
- No vaccine yet
-Mycobacterium is characterized by their slow growth, especially the virulent strains
-it sometimes takes weeks for visible colonies to appear (generation time is 20hrs or longer)
-This is a problem for diagnosis: culturing cannot be used to detect if a patient is infected with
Mycobacterium since the growth rate is really slow
Notes:
- Mycolic acids are branched chain hydroxy lipids, attached to the PGN layer in the cell wall
- Mycobacteria are not readily stained with Gram stain because of the high surface lipid content
-Mycobacterium cell wall:

B: PGN
C: arabinogalactan
D: mannose capped lipoarabinomannan (ManLAM)
E: Cell wall associated proteins
F: mycolic acids
G: glycolipids associated with mycolic acids

Why is the infection with Tuberculosis still present?

i- The economy in the poor countries: treatment for tuberculosis needs a long time. This treatment includes
placing the patients in sanitariums where they would receive rest, good food rich in vitamins and sun light.
Some patients took 1, 2 or 3 yrs to be treated. Light is important to enhance immunity
ii- Treatment with Tuberculosis antibiotics must be sustained for a long period of time (some multidrug
resistant strains take up to 20 months to be cleared form the body)
iii- There's still no strong vaccine

I-Clinical Diseases:
Mycobacterium can cause 2 diseases for the humans due to 2 strains:
M. tuberculosis:
- Still present and highly prevalent in the poor world (South-East Asia and Africa)
- It is an acid-fast bacillus= it needs a special stain that can break the cell wall
- Aerobic gram +ve
- Still causing 1.2-1.5 million deaths/year worldwide; it is the number-1 cause of deaths
-There is no toxin released by the Mycobacterium; its resilience leads to its virulence=>it is very hard to be
killed by macrophages
-M. tuberculosis is transmitted by the respiratory route, and even the simple act of talking can spread the
organism from person to person
-most infections in immunocompetent patients are restricted to the lungs and are cleared by the immune
system. Some infections might spread to other parts of the body (ex: joints)
-If a healthy individual who is on a normal diet is infected with the Tuberculosis mycobacterium, the immune
system can eliminate the infection, or if not, it can constrain the infection in the lungs for 10-20 years
-If after these 10 yrs the body experience a stress the tuberculosis will be reactivated
-It is a major problem in HIV patients; most of these patients die because of the Mycobacterium tuberculosis
bacteria since the HIV overtakes the immune system (tuberculosis and HIV have a high co-incidence)
-Usually, pneumonitis (active disease) develops in individuals with compromised immunity. In most cases of
tuberculosis obvious acute infection does not occur (only in 5% of infections)
-Mycobacterium tuberculosis infects the alveolar macrophages
-Once the mycobacterium enters the respiratory tract, it is surrounded by macrophages, but these bacteria
are not killed by the macrophages
-they are resistant to the acidic conditions of the lysozome and to the ROS (oxygen or nitrogen radicals) since
mycobacterium can neutralize the radicals
-More macrophages will migrate to the site of infection and this structure grows with the Tubercle bacilli in
the middle
-This can be seen in patients by X-ray

-These structures are called tubercles. They are controlled as long as the immune system is healthy
-If something happens, the macrophages break out and the bacteria will spread
-Culturing is not used for the diagnosis of mycobacterium
-It can be diagnosed by PCR: extract DNA from the sputum or tough material from the patient and do PCR for
the genes
-Also can be diagnosed by a skin test= a tuberculin test where cell wall components are injected under the
skin. 24 hrs later, we check for redness which is a delayed response that indicates the presence of T-cells
against the bacteria. This will be followed by X-ray
II-Prevention:
-There's a vaccine that consists of mycobacterium bovis, a strain that infects cattle. It is very similar to the
mycobacterium tuberculosis strain
-It is an attenuated bacteria that has the cell wall component that will activate the immune response
-It is good for children but it doesn't provide a good protection for adults
-Usually this vaccine is not given unless a child is threatened to be in direct contact with the bacteria. This is
because in case of endemics, the skin test will show positive for all population in case they were vaccinated
which makes it hard to detect who is infected and who is not

Bordetella pertussis: the whooping cough

-It is a component of the DTP vaccine
-Gram ve bacteria
-obligate aerobic coccobacillus
-Belongs to the proteobacteria phylum that is subdivided in 5 different subfamilies (, , Gamma, Delta,
epsilon). This division depends on the 16S ribosomal RNA
- Bordetella is a Beta proteobacteria
I- Clinical disease:
- Infection is initiated when infectious aerosols are inhaled and bacteria attach to and
proliferate on ciliated epithelial cells
- The first symptoms resemble a common cold (sneezing, malaise, low-grade fever)
then develop into a whooping cough
- Whooping cough develops because ciliary action is compromised which leads to the
accumulation of mucus, and the patient desperately tries to cough out these mucus
accumulations
- It infects the trachea of the lower respiratory tract, killing the cilia of the trachea epithelial cells
II-Virulence Factors:
a- Pertactin:
-an autotransporter that has an adhesive domain expressed on the surface of the bacteria (T5SS)
-mediates the binding of the bacteria to the epithelial cells
b- Hemagglutinin:
-Binds to glycoproteins on the surface of epithelial cells, mostly glycosylated integrins on glycoproteins

Pertactin and hemagglutinin are adhesive proteins; they are not the only ones but they are the major.
Bacteria that have many adhesive molecules usually have a redundancy between them=if one is inhibited, the
bacteria can still bind
Because Pertactin and hemagglutinin are important adhesive molecules, they are part of the vaccine. They
will inhibit the first contact of the bacteria with the epithelial cells thus inhibiting colonization and infection
c- Pertussis Toxin (PT):
-Enzymatic A-B toxin
-Consists of 1 A subunit and a pentameric B subunit
-B subunit binds to glycosylated molecules on the surface of many types of mammalian cells. Although the
bacteria infects only the tracheal epithelial cells, the toxin can bind to a wide variety of sugars that are found
on cells (WBCs, fibroblasts, etc.)
-After binding, the toxin is taken in by endocytosis
-A is the enzyme that translocates to the cytosol
-A is an ADP-Ribosylase that acts on the G-proteins, specifically the G-inhibitory subunit which has a
regulatory function (inhibitory)
-G-proteins are molecules that are associated with G-protein coupled receptors that activate important
signalling cascades
-One of these G-proteins is Adenylate Cyclase that converts ATP to cyclic AMP
-Cyclic AMP is an important secondary messenger that can activate many signalling pathways inside the cell
-Usually, the enzyme Adenylate Cyclase is activated by the subunit of the G-protein. Part of the subunit is
inhibitory that will control Adenylate Cyclase; otherwise the ATP will depleted from the cell
-The toxin will ribosylase the Gi subunit (the inhibitory subunit) inhibiting its activity=>no more control on
Adenylate Cyclase and there will be abnormally high levels of cyclic AMP that will lead to increased secretion
of mucus if this occurs in the cells of the respiratory tract
-This is dangerous since at the same time there are no cilia and it will be very hard to get rid of the excess
mucus=> much more coughing
-The effect of the toxin differs from one cell to another depending on the relevance of the cyclic AMP in the
pathways of each cell
-The toxin can have a broad activity on the different subunits of the G-coupled proteins (some might bind to
the activating subunit)
-If this occurs in the immune cells that are infiltrating the trachea and the level of cAMP increases,
phagocytosis will be inhibited and the migration of immune cells is also inhibited by modifying the
cytoskeleton of these cells
-This increase in cAMP in the epithelial cells of the trachea can inhibit them from attracting the immune cells
(disturbs proper signalling pathways leading to cytokine production)
-Because of its toxicity, PT is another component of the pertussis vaccine (in addition to Hemagglutinin and
Pertactin)
d- Tracheal Cytotoxin (TCT):
-Responsible for the elimination of the cilia
-It is a piece of the PGN of the bacteria
-When the bacteria divide and renew their PGN, they release a piece of the PGN
-The pieces released are usually called TCT and they are made of the 2 sugars and the small peptide (4 amino
acids)=disaccharide-tetrapeptide monomer
-In general, bacteria recycle TCT and don't shed it

-Bordetella is unique since is sheds a large amount of TCT

-Initially the TCT will bind to the epithelial cells and cause ciliostasis where the cilia are still present but not
functional anymore. More build up in the toxin will cause the lyses of the cells and shedding of the cilia from
the respiratory tracts
-It also interferes in the regeneration of the cilia
d- Adenylate Cyclase/Hemolysis toxin (bifunctional enzyme):
-The bacteria itself produces this enzyme
-It is a cyclase that converts ATP to cyclic AMP in the cytoplasm
-The second part is a hemolysin that is a perforate toxin; it can lyse cells since it forms oligomers on the cells
-It is functional only in the Eukaryotic cells since it needs to bind to calmodulin which is only found in the
cytoplasm of the eukaryotic cells

III-Prevention:
-Done by a whole cell inactivated vaccine that contains pertactin, hemagglutin and the Pertussis toxin
-Very protective vaccine that all children receive
-It is part of the DTP vaccine
-How is infection with Bordetella detected? Check for the toxin genes by PCR or we can culture a sputum on
an agar for Bordetella

II- Direct contact transmission of diseases

-These can be transmitted across the respiratory tract but don't necessarily cause infection in it
Staphylococcus
-member of the microbial flora of the skin
-In general, Staph. Is found in 2 places: the microbial flora of the pharynx and the microbial flora of the skin
-Facultative anaerobe, catalase +ve
-Gram +ve cocci forming clusters of grape-like shape
-Causes the common infections of the respiratory tract, skin and wounds
-Can't invade the skin unless there is an opening in it
-They are non-sporulating but resistant to drying and heat (60C for half an hour)
-most infections result from the transfer of staphylococci in normal flora from an infected but asymptomatic
individual to a susceptible individual
-There is a large variety of staphylococcus depending on the components of their sugar capsules=several
serotypes that can interact with the different antibodies by their different sugar components
- 2 Staph. species cause human diseases:
1. S. epidermidis: causes mainly skin infection
2. S. aureus (more common pathogen): causes both skin and respiratory tract diseases
Note: both species frequently occur in the normal microbial flora of the upper respiratory tract or on the skin
-There are no reservoirs for Staph.; Humans are their reservoirs
-When there is no reservoir, we can't perform in vivo assays

I-Clinical Diseases:
-It can cause a variety of diseases like pneumonia in the upper respiratory tract, skin infections, as well as
arthritis=infection of the joints
-serious staphylococcus infections often occur when the immune system of the host is compromised because
of illness or treatment with drugs that suppress immunity
II-Virulence Factors:
1- Capsule: - A very important virulence factor
- Not all are capsulated
- Those that are capsulated can have both a capsule and a slime layer (consisting of monosaccharides, proteins and small peptides)
- There are 11 different types of capsules with different types of sugars that are targeted by
different antibodies
- Protects the bacteria from phagocytosis
- The slime layer around the capsule allows the bacteria to stick to layers
- Staph can stick and survive for several years on many inanimate objects and on the skin
2- Protein A: - Imbedded in the PGN of S. aureus and interacting with it
- It is similar to the protein G of Strepto
- Has a unique affinity for the Fc region of IgG isotype antibodies, blocking the domain of
antibody needed for the binding to the bacteria
- It can also secrete protein A to the extracellular environment which will help block the
antibodies away from the surface of the cell, protecting it more
- Protein A is commercially available and used for purification of antibodies
3- Hyaluronidase: -Enzymes that break down Hyaluronic acid by hydrolysis, the acidic polysaccharides found
in extracellular matrix of connective tissue
- It facilitates the spread of the bacteria in tissues
- produced by > 90% of S. aureus strains
4- Lipases: -these enzymes hydrolyze lipids
-they may help the bacteria to invade cutaneous and subcutaneous tissues
5-S. aureus toxins:
a) cytolytic or membrane-damaging toxins
-These will cause the lysis of the cell
-These include the , , , and Panton-valentine (leukocidin) toxins
-Similar to the Hemolysins of the E.coli
-these are cytolytic to many types of cells including RBC, leukocytes, hepatocytes, fibroblasts and
platelets.
-Different strains have different combinations of these toxins
b) Enterotoxins:
-Related to food poisoning
-Enters the body if we consume a food with Staph. where the bacteria will release the enterotoxin=>
the food that we consume will already have the enterotoxin

-They are highly resistant to heat; even if food is boiled at 100C, they will survive
-Also very resistant to acids; resistant to hydrolysis by gastric and jejunal enzymes (these are sources
of food product contamination)
-They act as superantigens and cause inflammation in the intestine (not severe; the disease can
subside by itself)
-they are produced by 30-50% of S. aureus strains
-Ex: enterotoxin A causes a form of food poisoning. After ingestion of food, the toxins stimulate Tcells localized along the intestine resulting in massive T-cell response, inflammation and increased
permeability of the intestine.
-Symptoms: severe diahhrea (short-lived) and vomiting
c) Toxic shock syndrome toxin-1 (TST):
-A superantigen but not acid-resistant
-So it will not be expressed in Staph. if it is going through the gut
-Expressed when Staph is in the skin or is in the blood stream
-Activates a very strong inflammatory response that will affect all the body
-toxic shock syndrome is characterized by high fever, rash, vomiting, low blood pressure diarrhea and
occasionally death
-The difference between TST and the enterotoxin is that the effect of enterotoxin is local (in the gut
for example) and it cannot translocate to the blood. TST is unique is that it can translocate to the
blood compartment
-If you consume food that is contaminated with TST, it will reach the blood stream before reaching
the gut, and it can cause a systemic response
-They are found on mobile pathogenicity islands on the chromosome which are mobile in the genome.
Thus these have landed on the chromosomes by conjugation events

III-Prevention:
-No vaccines are available
-20-30% of the population are carriers of Staph (Asymptomatic carriers)
-They carry different strains in the nasal cavity, as well as in the skin

Helicobacter pylori
-Not transmitted by respiratory tract but by food
-It colonizes in the stomach
-Gram ve flagellated
-Proteobacteria
-It can contaminate water and food in non-hygiene conditions
-Have a high ability to survive in the stomach but not all are virulent

-Inflammation in the stomach starts due to a virulent strains. It colonizes the non-acid secreting mucosa of
the stomach and the duodenum causing gastritis, gastroduodenal ulcers. Chronic infections due to absence of
treatment may lead to gastric carcinomas.
-two factors lead to this gastric carcinoma: the protein secreted by the bacteria that will trigger cell migration
and the continuous damage to the cells of the stomach
-How does the bacteria survive in the acidity? It does not live in the lumen of the stomach; it colonizes the
surfaces of the gastric mucosa (Hence protected from stomach acids by the gastric mucus layer) and
associates itself with cells that do not produce acid
I-Virulence factors:
1- Urease and the urea transporter (UreI):
-Not found in other bacteria
-Urease is found in 2 places in the bacteria: cytoplasm and on the surface of the cell
-When we consume food contaminated with Helicobacter, as the food goes down, acidity starts to drop down.
Once it reaches low acidity, the urea transporter becomes active because it is proton gated transporter. Urea
that is found in the stomach secretions starts being translocated into the cytoplasm of the bacteria. Urea is
eventually converted by Urease into bicarbonate and Ammonia
-Ammonia will keep the pH of the cytoplasm buffered
-The urease which is on the surface of the bacteria will act on the Urea which is extracellular, producing
ammonia and again buffering the medium around the cell
-10% of the total proteins in the bacteria are urease
-Urease helps in 2 ways: keeping the pH of the medium buffered and controlling the pH of the cytoplasm

Helicobacter pylori: Virulence factors:

a. Urease and the urea transporter (UreI)
b. Vacuolating cytotoxin A (VacA):
Characteristics:
i. It is a multifunctional toxin secreted through the T5SS autotransporter pathway.
ii. the secreted toxin can assemble into water-soluble oligomeric structures that can insert into
lipid bilayers to form anion-selective membrane channels + can reach the cytoplasm and insert
into the mitochondrial membrane
iii. called Vacuolating because when it enters the cells it forms vacuole- like structures
iv. Binds to receptors on the surface of the cell its endocyted (it interferes with the endocytic
trafficking in the cells), But eventually there is no acidification of the vacuoles
Effects/phenotype:
i. Swelling of vacuoles: Forms pores in the endosomes though these pores only chloride ions
will enter (selective for anions) Chloride ions accumulate as well as ammonium eventually
water will follow vacuoles will get swollen (+ proton pump in the endosomal membrane
which tries to acidify the medium)
ii. Vacuolation in eukaryotic cells
iii. Apoptosis: translocates from OM to membrane of mitochondria forms pores and releases
cytochrome c Damages mitochondria reduce ATP synthesis triggers apoptosis
iv. Inhibit immune cells: Once the barrier between epithelial cells is disrupted VacA goes in
between the tight junctions of the cells and binds immune cells sitting beneath the epithelium
and inhibit their activity (T cells or B cells)
v. Activates signalling pathways: It can
activate intracellular signalling pathways
in epithelial cells to trigger inflammation
vi. Increases transepithelial flux of certain
molecules, including urea (most probably
through Vac A channels)
All strains of H. pylori that have been
isolated from humans contain the VacA
gene, which suggests that production of
VacA is important for colonization or
persistence of H. pylori in the human
stomach.
c. Adhesions:
There are several adhesions that allow the bacteria to stick to the epithelial cells (specifically bind
to gastric epithelial cells)
Virulence factors that allow the bacteria to colonize
d. CagA
Unlike Vac A, only in some strains: not all people colonized with helicobacter have cagA otherwise
50% of people would have gastric ulcers
It is an effector protein injected by Helicobacter pylori T4SS
Once injected, it triggers actin polymerizations and thus migration of epithelial cells

Cells injected with/expressing cagA extend pseudopodial processes and also move between and
under neighboring cells in a manner consistent with the loss of cellcell adhesion
Stains that have cagA are more prone to cause cancer (A direct causal link between CagA and
oncogenesis in vivo was achieved by the generation of transgenic mice expressing CagA)

II.
-

Sexually transmitted diseases:

Several important human pathogens are transmitted exclusively by sexual contact. These include a
wide variety of bacteria, viruses, protozoa and even fungi
3 main genera that cause SDS: Treponema, Chlamydia, and Neisseria
Sexually transmitted pathogens are found only in body fluids from the genitourinary tract.
Most STD (except HIV) are curable and virtually all are controllable with appropriate medical
intervention (however delayed treatment can lead to long term problems like infertility, cancer,
heart disease, genitalia damage, etc...)

1.
a.

Treponema pallidum:
Characteristics:
Causative agent of syphilis
T. pallidum is a gram-negative, micro-aerophile spirochete
Extracellular microbe: divides in extracellular medium + obligate parasite
Within spirochetes(order): There are 3 genera that cause disease in humans, which are
Treponema, Borelia (transmitted by arthropods), leptospira
These spirochetes are long but slender (less than 0.2 micron wide): cant see them with light
microscope you need dark field or florescent microscope
Have special endoflagella (axial filaments):
i. 2 or more axial filaments axial filaments that wrap around the body inserted at each end,
which is typical of spirochetes (In Treponema: 3 from each side)
ii. The axial filaments enclosed in the periplasmic space = not exposed to extracellular
environment.
iii. The outer membrane is called a sheath.
iv. rotation of the axial filaments allow the cell to rotate like a corkscrew which is very efficient
in moving the organism through liquids
v. The hide the flagella in the sheath which is important because Bacterial flagellin subunits
are detected by the immune system, so by hiding them they eliminate the risk of losing
flagellin subunits that can trigger immune system

vi. Thats why its one of the bacteria that are poorly detected by immune system called
stealth pathogen (can survive for many years in the host)
vii. Extracellular microbe that preferentially divides in the Extracellular medium.
There are many peculiarities about Treponema:
i. We know little about its biology: no animals model can replicate what happens in humans
(best model is rabbit, which you cannot practice genetic knock out on)
ii. Slow growth: generation time = 20 hrs Cannot grow in tissue culture/agar plates
iii. Very efficient in escaping immune system and escaping the host:
(*) Very little antigenic surface proteins + no LPS though gram ve
(*) The few known surface proteins have antigenic variation: change their profile from time
to time (change surface part of the protein by translating different exons/alternative
splicing) thus escape the immune system.

Facts from the genome sequence of T. Pallidum:

o Absence of many metabolic pathways , including the tricarboxylic acid cycle, components
of oxidative phosphorylation, lipopolysaccharide and lipid biosynthesis mechanisms, and
most biosynthetic pathways means its highly dependent on the host (obligate parasites)
Import all building molecules from host (FAs + polar heads of lipids+ phosphatidyl
choline) (no known environmental or zoonotic reservoir human only reservoir)
o No surface proteins associate with virulence or antigenicity have been identified so far
(one or two that are continuously varying by exon splicing)
o Very small genome (1/4 of E.coli genome; around 1.1 Mbps)
Disease caused by Treponema:
Syphilis: extremely slowly-progressing disease which in the same individual may evolve through
three phases.
i. T. pallidum gains entry to dermis of genitalia through intact mucosal surfaces or microscopic
skin abrasions
ii. primary syphilis:
- Is characterized by 1 or more lesions (called chancres) at the dermis of genitalia. (appear in
couple of weeks)
- In most cases the chancre heals spontaneously and the organism disappears from the site
within 2 months giving the patient a false sense of relief= because this only means bacteria
have spread from skin dermis to bloodstream or lymphatic vessels.
iii. secondary syphilis:
- Spread of bacteria in bloodstream or lymphatic vessels and are disseminated to various
parts of the body.

- Spread from initial point of infection (i.e. skin dermis of the genitalia) to the mucosal
surface/skin of the whole body skin rash appearing all over the body (50% of cases)
- flu like symptoms develop at that stage followed by the rash (in most cases the rash and
symptoms gradually resolve at that stage)
- After secondary, person may go through a Latent phase: clinically no symptoms for several
years (bacteria still found on mucosal surfaces or dermis) Most individuals stop at this
stage and get healed (bacteria eliminated)
iv. late syphilis
- Only 30% of the cases progress to late syphilis. But this occurs after several years. Reasons
for progression unknown.
- Many tissues become infected like eyes, joints, bones and central nervous system, causing
lesions. (death may occur if the disease is untreated)
- This however requires several years.
Notes:
- T. pallidum can be transmitted across the placenta to the unborn fetus (congenital syphilis) where
it causes severe disease that may be fatal
- In both males and females initial infection is usually on the genital organs. in 10% of the cases
infection is extragenital (usually in the oral region)
- No vaccine for these bacteria BUT efficient antibiotics.
- Cases of syphilis mostly in underdeveloped countries.

2. Chlamydia trachomatis:
a.Characteristics:
Gram-negative, coccoid bacteria
Intracellular microbe = divides intracellularly (e.g. live inside epithelial cells) + Obligate parasite
Dont have PGN But have IM & OM
Advantages: (1) No E spent on PGN synthesis (2) Escape host immune system (escape
recognition in host cytoplasm since they are intracellular)
Several serotypes exist based on antigenic differences in outer membrane protein
Cell cycle: Chlamydia have a unique developmental cycle (~ 48 hrs) that is perhaps their most
distinguishing characteristic (alternate between spore and dividing cell)
i. When a cell is infected and lyses it releases elementary bodies and reticulate bodies
- Elementary bodies
The EB is the only infectious stage of the chlamydial developmental cycle.
It functions as a tough "spore-like" electron dense body whose purpose is to permit chlamydial
survival in the non-supportive environment outside the host cell
the EBs lack the peptidoglycan layer and their outer membrane proteins are extensively crosslinked by disulfide bonds between cysteine residues (surface resembles coat of spores)
Oxidized cysteine residues disulfide bridges btw proteins proteins non-functional
metabolically inactive
- Reticulate bodies:
Reticulate bodies die outside cell they are non-infectious.
Stage of the chlamydial developmental cycle responsible for intracellular replication

ii.

iii.

iv.

v.

Reticulate bodies are metabolically active, so their cytoplasm is rich in ribosomes, which are
required for protein synthesis.
Peptidoglycan has never been detected in RB although the Chlamydia genome encodes all the
putative enzymes required for PGN synthesis: This means that the bacteria are in the process of
eliminating these genes from the genome (genetic erosion).
Reduced cysteine residues no disulfide bridges btw proteins proteins are functional
metabolically active
EB invade either through endosomes or phagosomes (inclusion body): they bind to receptors on the
host cell and they are taken either by endocytosis (if epithelial cell) or phagocytosis (if phagocytic
cell)
However whether its phagosome or endosome: it
is not allowed to mature i.e. bacteria inhibits
the phago-lysozomal fusion preventing
acidification of inclusion body & degradation of
bacteria
Bacteria will differentiate from EB to RB and
divide in the inclusion body: There is an
intermediate body btw EB and RB and occurs
inside membrane bound organelles.
Eventually, the cell will lyse and most of the RB
will differentiate back to EB (because they are
exposed to extracellular env.)
Facts from the genome sequence of Chlamydia trachomatis:
i. Chlamydia possess an essentially complete set of genes for the synthesis of PGN, despite the
fact that PGN are not detectable in the bacteria, either by electron microscopy or by
biochemical methods .The bacteria is most likely in the phase whereby they are losing the
PGN genes due to genomic erosion.
ii. Chlamydiae are energy parasites, depending totally on the host for generating ATP pump
ATP from host to cell. Though Chlamydia has functional glucose-catabolizing (glycolysis)
enzymes, hence it has its own capacity to produce ATP BUT they prefer pumping host ATP
(common to intracellular parasites). In the future, they will most likely lose all pathways for E
production.
Tissue tropism = Target cells = include non-ciliated epithelial cells found on the mucous membrane
of the urethra, fallopian tubes, respiratory tract and conjunctivae (one serotype called a serovar
infect phagocytes in the lymphatic system), anal and rectal region + Only one serotype can infect
macrophage
Clinical manifestations of Chlamydia infections are due to direct destruction of cells during
replication and host inflammatory response --Chlamydia dont cause disease by releasing toxin but
by the damage they cause to cells
Chlamydia trachomatis causes several clinical diseases depending on the serotype:
i. Trachoma (chronic conjunctivitis): characterized by vascularization and infection of the cornea
repeated infections scarring of the cornea eventually can cause blindness (more
common in children).

- In newborns eye infections can develop (during passage of an infant through infected birth
canal) which if untreated can disseminate leading to infant trachoma.
- Infection of the eyes is actually cross-contamination from genitalia to eye.
- If child has trachoma + have dripping from eye exudates contain the bacteria flies can
transmit the bacteria btw individuals from exudates.
ii. Infections of the genitourinary tract (it is one of the leading sexually transmitted diseases)
Infections epithelia of urethra, cervix, fallopian tubes if not treated can lead to problems witt
fertility.
iii. Infections of phagocytes in lymph nodes: swelling of lymph nodes
Disease is treatable + no vaccine + hygiene and safe-sex measures are most important prevention
method

Arthropod transmitted microbial diseases

A.

Pathogens can be transmitted to hosts from the bite of a pathogen-infected arthropod vector
(mosquito, flies, lyse, ticks, bed bugs or fleas)
In many cases such as rickettsial illness, humans are accidental hosts for the pathogen.
Only few cases (malaria and dingi), humans primary host of mosquito infected humans can
also play a critical role in the life cycle of the pathogen and its vector such mosquitoes are called
anthropohilic have olfactory receptors that detect human smell.

Rickettsial diseases:
One of the common arthropod infections.
Rickettsia are small, gram-negative, cocci or rod-shaped bacteria
They are obligate intracellular parasites
Have not yet been cultivated in the absence of host cells.
Rickettsia do not survive long outside the host and this may explain why they must be transmitted
from animal to animal by arthropod vectors
But because they are highly infective, they can survive outside the host/extracellularly for a small
period of time and infect. Proof: they were used as weapons by the Russians (in the form of
powder Rickettsia is lyophilized or aerosols water droplets). Bacteria can easily kill a guinea
pig.
The rickettsial cell wall is very similar to that of Gram negative bacteria (contains 3 layers). Have a
very minimal/ thin PGN layer. Rickettsia also have a crystalline protein layer (S-layer) surrounding
their cell wall
Metabolic dependency of Rickettsia on host cells:
i. They are energy parasites depending totally on the host for generating ATP pump ATP from
host to cell.
ii. No glycolysis genes are found in Rickettsia (since the ancestor of Rickettsia acquired
transporters for ATP and ADP, the glycolytic pathway became non-essential)
iii. But they contain:
- Pyruvate transporter
- Enzyme pyruvate dehydrogenase that converts pyruvate to acetyl-coA, the first substrate in
the TCA cycle
- TCA and electron transport chain (Rickettsia possess a complete respiratory chain and are
able to transport electrons using NADH as electron donor)
- They can transport pyruvate from host (since no glycolysis) and synthesize ATP from it BUT
prefer pumping ATP from host.
ii. The pathways for amino acid biosynthesis are absent or imperfect (missing enzyme) in most
Rickettsia, implying the use of host-derived amino acids
iii. The genes for the biosynthesis of many vitamins (riboflavin, vitamin B6 and nicotinamide) are
absent from Rickettsia
iv. Rickettsia are not capable of producing nucleoside monophosphates
NOTE: Usually all bacterial DNA is coding; however, around 24% of Rickettsia DNA is non-coding
mainly genes involved in metabolic functions (genome is very small). Probably these will be lost by the
process of genetic erosion. Eliminated by evolution. 1 Mbp genome.

 Life cycle of Rickettsia:

i. When the arthropod takes a blood meal from an infected person, and acquires the Rickettsia
ii. The Rickettsia penetrates the epithelial cells of the gastrointestinal tract of the arthropod ,
multiply and appears later in the feces or in saliva.
iii. When the arthropod feeds on an uninfected individual, it then transmits the bacteria by
contaminating the bite with its feces or by injection from saliva (saliva is injected because
contain anticoagulant)
If saliva injected epidermis dermis
If feces defecated scratch skin (infected people always sratch) damage skin epidermis
dermis
iv. Either by injection or scratching introduce bacteria to dermis (which is highly vascularized)
v. Rickettsia infects mostly the endothelial cells of the vascular system (layer lining the interior
surface of blood and lymphatic vessels) and multiplies within them. The resulting inflammation
causes local blockage and rupture of the small (venules, capillaries, and arterioles) and
medium-sized blood vessels. NOT by releasing toxin
vi. Rickettsia are divided into 2 groups based on clinical diseases they produce:
1- Typhus group (Rickettsia prowazekii)
2- The spotted fever group (Rickettsia rickettsii)
1. Rickettsia prowazekii:
R. prowazekii is the etiologic agent of epidemic typhus, also called louse-borne typhus. 1 Mbp
genome.
The principal vector is the human body louse (pl. lice) (Pediculus humanus corporis). This is the
only one among other louse that infects humans.
Hence, humans are the primary reservoir of typhus (squirrels are reservoirs also however their
vector: fleas because human lice dont feed on squirrels)
Lice die from their infection within 2-3 weeks (Note: if bacteria kills the arthropod
transmission rate is low. The longer the half life of the arthropod, the more it lives and bites,
hence the more it can transmit diseases)
epidemic typhus occurs among people living in crowded, unsanitary conditions that favour
spread of body lice
Clinical disease
o Caused mainly by destruction of the endothelial cells (non-endocytic) of blood vessels.
o The bacteria enter endothelial cells by stimulating phagocytosis (since non-endocytic). After
engulfment, they degrade the phagosome/endosome membrane by producing a
phospholipase and hence are released in the cytoplasm where they divide.
o Bacteria induce lysis of the cells and are consequently released to the extracellular medium
and infect other cells.
o Symptoms
Edema: loss of intravascular fluid/blood leaking into tissue spaces
Rash: spreads over the body (except the face, palms of hands and soles of the feet).
Swelling due to the serum leaking from blood vessels.
Complications from untreated typhus involve damage to the CNS, lungs, kidneys and heart.
In most cases, infections cleared by the individuals.
Geographical distribution
The disease is found in cool mountainous regions of Africa, Asia and Central and South America

 Prevention:
An inactivated typhus vaccine is available, and its use is recommended in high risk population
Control:
Louse-control and hygiene measures help control the disease during an epidemic
Sequencing of the Rickettsia prowazekii genome revealed that they can produce ATP by means of
the tricarboxylic acid cycle.
However, these bacteria use the host ATP as long as it is available. They also depend on many if
the hosts biosynthetic pathways (E parasites)
2. Rickettsia rickettsii:
The agent responsible for Rocky mountain spotted fever
Reservoirs are wild rodents
It is transmitted to humans by various ticks, most commonly the dog and wood tick (most imp:
The wood tick /Dermacentor andersoni) - other types for example are heart ticks or Ixodes ticks
in Europe
NOTE: Rickettsia rickettsii in female ticks are usually passed from one generation to anothervertical transmission- through their eggs (transovarian passage).
Tick, unlike other arthropods, feeds for a long period of time: up to 6 days.
To become infected a person must be exposed to the tick for a lengthy period = 24-48 hrs (takes
its time to attach to host very well. Grabs the skin by introducing its appendages into the
epidermis of the skin).
The tick swells as it feeds and can reach 2-3 mm in size.
NOTE: If you eliminate the tick in the first 24 hours, you are probably not infected yet because it
takes time for attachment. Removal must be done by a dermatologist because if mouth with
salivary glands remains stuck infection follows.
The dormant avirulent Rickettsia are activated by the warm blood meal then must be released
from the tick salivary glands to penetrate the human blood
Clinical disease
o Caused mainly by destruction of the endothelial cells (non-endocytic) of blood vessels.
o The bacteria enter endothelial cells by stimulating phagocytosis (since non-endocytic).
o After engulfment, they degrade the phagosome/endosome membrane by producing a
phospholipase and hence are released in the cytoplasm.
o Cells of Rickettsia can divide now within the cytoplasm as well as nucleus (1st difference) in the
of endothelial cells
o 2nd difference: Mode of transmission: They spread from cell to cell without being exposed to
extracellular space using actin-based motility (NO cell lysis)
Process: Actin polymerizes at one pole of the HOST cell. The longer the actin filaments, the more it
will push the bacteria. Filaments will grow until the bacteria reach the cell membrane. Then it will
shoot the bacteria from the cell membrane into the membrane of another cell. Then it will be
taken by in an endocytic vesicle in the other cell.
o When faced with such microbe, antibodies are not helpful because antibodies are found in the
serum of the extracellular fluid
o Primary manifestations of the disease appear to result from the replication of bacteria in
endothelial cells, with subsequent damage to the cells and leakage of the blood vessels.

o Symptoms include initially fever and later rash appears on the whole body including palms and
soles.
o Mortality can reach 20% if not treated. Most people can heal spontaneously.
o Vertical transmission from female infected tick to eggs.
Geographical distribution
Rocky mountain spotted fever is prevalent in Mexico, South and Central and North America
Prevention:
o There is no vaccine for Rocky mountain spotted fever
o Use of protective clothing and insect repellents and the prompt removal of attached ticks are the
best preventive measures
R. rickettsii has a lethal effect on the ticks themselves which may
explain the low prevalence of
infected ticks in nature
50000 cases of infection in the States every year.

B. Lyme disease:
It is caused by the spirochete Borrelia Burgdorferi (a Gram-negative microaerophile). They are
extracellular bacteria
Borrelia has between 7 and 20 endoflagella (depending on the species) responsible for its twisting
motility (in periplasmic space). Flagella emerge from each pole
At least 10 Borrelia species are responsible for Lyme disease in man
It is a tick-borne disease that affects humans and animals.
Lyme disease is spread primarily by the deer tick Ixodes scapularis
Note: other Ixodes species can transmit the disease in different geographical areas
Reservoirs
o field mice are primary reservoirs of B. burgdorferi
o the ticks that carry the bacteria feed on the blood of birds, domesticated animals, various
wild animals (deers) and occasionally humans (man s an accidental host)
Life Cycle: Usually the female is infected. Follow the picture:
* Larvae: small six-legged arthropods seek blood meal field mice and act as reservoirs for hours
to days and transmit B. Burgdorferi. The mice are reservoirs of B. Burgdorferi. Larvae feed until
completely full. They try to concentrate red blood cells as much as possible as source of proteins.
Once full, they fall from mice to ground and become dormant. Feed in summer remain dormant
through fall and winter develop into a nymph
* Nymph: slightly larger eight legged arthropod seeks blood meal animal/human and transmit
B. Burgdorferi. They infect humans/animals (prefers deer) during the summer and develop into an
adult by the winter.
* Adult male and female now choose a host on which they will feed and MATE deer. Female lays
the eggs and die.

Clinical disease
o The bacteria is transmitted to human blood during a tick blood meal, and then spread to many
organs
o The first symptom of Lyme disease is usually a rash that appears at the bite site after a few
days. It is a red area that clears at the center and is darker red at the edges. It is large are with a
diameter up to ~ 15 cm)
o Eventually rash will resolves But this means that the bacteria is spreading into the blood
circulation.
o Flu-like symptoms appear in a couple of weeks as rash fades.
o If untreated the disease can cause complications like arthritis (due to immune response to the
bacteria at the joints level). It can resolve without treatment.
Prevention:
o NO vaccines
o Vaccines are available for veterinary use
o Best prevention is to avoid tick attachment (protective clothing); insect repellents
o Remove attached ticks (after attaching the tick does not feed for about 24h)
The ticks feed for several days but blood does not coagulate throughout feeding period. The ticks
inject saliva containing anticoagulant to keep the blood in a fluid state. Thats why they were
studied for their anticoagulant activities for possible use in medicine. It has alteration between
feeding and injection of anticoagulant. They also have anticoagulant in their stomach so that the
blood it feeds on doesnt coagulate in the stomach.

C. Plague (zoonosis):
Pandemics of plague have been responsible for more human deaths than any other infectious
disease (except malaria and tuberculosis) - Almost always fatal if not treated
Plague killed 25-33% of Europes population in epidemics in the Middle Age
Caused by a Gram-negative, facultative aerobic rod called Yersinia pestis
Rats (also rabbits and squirrels) are the primary disease reservoirs and humans are only accidental
hosts. Most infected rats die soon while in other wild-rodents (e.g. rabbits) the disease is not fatal
Yersinia is transmitted to humans by the rat flea (Xenopsylla cheopsis)
Note: A plague-infected flea is always hungry for a meal, because the growth of the bacteria blocks
its digestive tract inducing the quick regurgitation of ingested blood
Referred to as black death because it results in a black skin in patients
Clinical disease
o the 2 clinical manifestations of Y. pestis are bubonic plague and pneumonic plague
o Bubonic plague:
- Symptoms start 7 days after a flea bite
- Bacteria are injected directly into the dermis and spreads there into the lymph nodes through
blood or lymph vessels
- Symptoms include fever, painful buboes (inflammatory swelling of lymph nodes in groin or axilla
tender and hot then rupture)
- Y. Pestis have unique ability to escape phagocytosis. Dont produce toxins.
- Y. pestis cells from buboes eventually enter the blood stream causing generalized septicaemia
and multiple hemorrhages produce dark splotches on the skin
- Bacteraemia develops rapidly in patients and if untreated as many as 75% die
o Pneumonic plague
- occurs when cells of Y. pestis are inhaled or reach the lungs during bubonic plague (i.e. reaches
lungs from blood circulation)
- It gives the symptoms of pneumonia
- Once this happens infected person coughs the Y. pestis are transmitted now by aerosols
eliminating the need of flea vector
- Within 1-2 days pulmonary signs develop (ex: bloody sputum). It causes 90% mortality
o Virulence factors: all targeted towards phagocytosis
1- Adhesions: mediate attachment to epithelial cells, macrophages and ECM proteins (laminins).
2- Some have and additional protease activity (cleave complement and inhibit coagulation)
3- Yop (Yersinia outer protein) virulon:
- It is an extra-chromosomal 70 kb plasmid encoding the Yop effector proteins, as well as a type
III secretion system (TSS)
- T3SS: is a needle-like translocation apparatus highly conserved among Gram-negative
pathogens (Salmonella, Shigella, and pathogenic E. coli) and is used to inject effector proteins
from the bacterial cytoplasm into the host cytosol into macrophages
- Through the use of its T3SS, Y. pestis delivers six different Yop effector proteins into
macrophages: Yop H, Yop A Yop T, Yop J, Yop E and Yop M. Their job is to paralyze the
macrophages. They interfere with their migratory capacity as well as bactericidal capacity.
Even if they were taken up:
4- Inhibits killing by macrophages even if taken up: inhibits phagosome-Lysosomes fusion.
5- It can inhibit complement system and kill macrophages.

6- Y. pestis has 2 additional plasmids that encode virulence genes:

a. F1 gene: codes for an anti-phagocytic protein capsule surround the cell inhibit phagocytosis
b. Plasminogen activator protease gene:
* It degrades complement components (that trigger phagocytosis) preventing phagocytosis
and phagocytic migration.
* It also degrades fibrin clots (prevent dissemination/spread of bacteria) permitting Y.
pestis spread rapidly favouring the dissemination of bacteria
All virulence factors are targeted against phagocytosis.

Should be treated rapidly and certain death if not treated.

Infected rats and other rodents should be destroyed.
Sporadic infections still occur but not a major problem anymore.
No vaccine is present.

(Continued) Arthropod transmitted microbial diseases

D) Plasmodium AND Malaria (parasite not bacteria):
The reason we talk about malaria here is that it is an arthropod born disease. This decease had caused so
many deaths in the past and is still causing many deaths.
It is a disease caused by the eukaryotic protozoan of the genus Plasmodium which is a unicellular parasite,
belongs to the group of parasites we call: Apicomplexa
It is transmitted by several types of mosquitoes that belong to the same genus that is anopheles; ONLY
Anophelinis can transmit malaria. And among anopheles only around 20 species can transmit malaria.
Plasmodium is one of the most important human pathogens: Around 500 million infections of malaria/year
and 1 million deaths mostly children below the age of 5 in Sub-saharan Africa (malaria regions). This result in
an economic burden: hospitalization Death used to be 1-2 million (used to be the first killer), now its rank
decreased to third, its after tuberculosis and HIV now its around 800-900 death per year mostly in Africa
(90%), and majority are children (before the age of 5). The reason why mostly children are the victims is that
they are more prone since their immune system has not meet malaria often, once they pass the age of five
and they have been subjected to malaria often they develop a partial immunity (not complete, so the
disease in not that severe and is less fatal).
Malaria is a public health problem today in >100 countries (mostly in the tropics and the sub-tropics and
sometimes the temperate areas) inhabited by 2.4 billion people (~ 40% of the worlds population!)
4 Plasmodium species cause disease in man {a, and b are the major problem}:
a. Plasmodium falciparum (the most serious/ fatal disease) [found in south and central America, Africa,
south east Asia]
b. Plasmodium vivax [more spread since we find it in more temperate regions,]
c. Plasmodium ovale [much less]
d. Plasmodium malariae [much less]

Distribution of Malaria:

Malaria used to be everywhere before the

60s (Europe, UK, northern America), spraying
of the DDT insecticide is the cause of the
removal of malaria from the northern
hemisphere, kills mosquitoes. Because the
developed countries had very good mosquito
surveying programs and very good usage of
DDT, they eventually eradicated the
mosquitoes that carry malaria from their

countries {in lebanon it was eradicated

in 1960}. As a result malaria remained
in poorer counties (central and south
America {mostly brazil}, sub-Saharan
Africa until south Africa and
Madagascar, south western part of the
Arab peninsula, turkey, Iraq, Iran, Syria,
south-eastern part of Asia). The
problem in poor counties, and especially Africa the infection continued because of the rice agriculture (these
countries depend on the rice agriculture), rice fields need a high dose of H 2O, (swamps) mosquito breeding
sites were huge! So eradicating the mosquitoes using the same method as developed countries was
impossible! So what happened people used massive amount of insecticides on these mosquito breeding sites,
but as a result of this massive use; the insects became resistant to insecticide so populations also started to
grow quickly. Problem: Insecticide resistance is growing. Insecticide used for 2-3 years, the insect develop
resistance to the insecticide then you change to another insecticide 2-3 another population develops
resistance again people are always looking for insecticides that are also safe, (DDT are not used anymore they
are not safe, only allowed in Africa because there is no other way to kill the insects). Insecticide is the major tool
used to eradicate arthropod born deceases because this is what worked in history. In Africa there is an
additional problem to the rice fields (large surface areas where mosquitoes can breed) and poverty, which is the
presence of anthropophilic mosquitoes (mosquitoes that love biting men): Anopheles gambiae is one of them,
and the second is A. funestus and many others but these are the most popular man oriented, therefore in these
mosquitoes endemic places the decease can spread very quickly, and they peak in rain seasons almost 2% of
mosquitoes are infected (huge number); in these endemic places a person get around 100 bites per night (these
bites at night after sunsets) 2% infected then at least 2 are infected and one bite is enough to deliver enough
parasites to cause the disease.

The Plasmodium life cycle

So initially the human are bitten by an
infected mosquito which has the parasite
in the salivary gland. Then where the
mosquito bites it releases saliva (the
saliva has an anti-coagulant property); by
releasing saliva the sporozoites are
released and injected with the saliva to
the dermis of the skin (a single bite can
release tens of these sporozoites). The
sporozoites eventually will access the
blood circulation in the dermis in the
small blood vessels and they flow with
the blood till they reach the liver (usually

the liver is the place where microbes in the blood are filtered {liver sinusoids/macrophages}, we have
macrophages so if any microbe reaches the liver the macrophages will capture it and wont allow it to remain in
the circulation). The problem is that sporozoites are highly invasive so they meet the macrophages and invade
them and cross them and cross the liver sinusoids which are small blood vessels of the liver that are covered by
endothelial cells, but in between the endothelial cells we have Kupffer cells (macrophages = phagocytes) that try
to trap all microbes in blood. Now here once the sporozoites reaches the Kupffer cells it can invade and doesnt
stop so it crosses the macrophage and endothelial cells and goes to the hepatocytes and then they move from
one hepatocyte to another. Then at some point we dont know what happens, it decides to stop invading the
cells and eventually form a mature cell we call the schizont. The schizont is a mature cell that will undergo a lot
of mitosis (so initially one sporozoite has invaded the liver the sporozoite has developed into a schizont
schizont undergoes many mitotic divisions to form thousands of cells). Eventually this hepatocyte (where mitosis
is taking place) will rupture and what is found inside we call them the merozoites are released. So one
sporozoite can give many merozoites these are now released into the blood circulation and here is where they
start infecting the blood cells. The first stage of the disease is where the liver is involved we call it hepatic
schizogony (hepatic because its the liver, which is infected, schizogony: schizont is this multi cellular parasite
that is developing in the liver and releases the merozoites into the blood circulation. Now the merozoites infect
red blood cells (cannot infect hepatic cells again). Now one merozoite infects a RBC can invade the cell and
becomes a small ring stage (the cell at this stage has a light cytoplasm with a dense nucleus at the periphery);
now this ring stage matures and becomes larger and becomes what we call a trophozoite and here it matures
and starts covering most of the cytoplasm of the RBC, and eventually the trophozoite fills the whole RBC and
becomes a schizont and eventually the schizont undergoes several mitotic divisions giving rise to 16-32
merozoites (depending on the specie of plasmodium), rupturing the RBC and producing the merozoites. So from
what we said we can see that this parasite doesnt produce any toxin! The decease is caused because of the
rupturing of the RBC. Then outcome of the decease is the loss of RBC and becoming anemic. So this is the
Erythrocytic cycle that keeps going on and on hepatic schizogony occurs at the beginning only during the
sporozoite migration. Now as this cycle is going on: ring stage trophozoite schizont ring stage. Some
of the rings they dont go through this cycle they differentiate into sexual stages the gametocytes. Some
develop into a male and some to female gametocytes. These opposite sexes gametocyte cant survive for long,
they remain in the blood for several hours and then they disintegrate. Now these have to be picked up by
mosquitoes. So if this infected person is now bitten by a
mosquito that is not infected, the mosquito will ingest blood,
the blood will contain all of these stages but only these 2 (male
and female gametocyte) will develop in the gut of the mosquito
and the other stages (ring/trophozoite/schizont) will die. Now
when the gametocytes are in the mid-gut of the mosquito, they
first mature to male and female gametes then fuse and form a
zygote (fertilization). This fusion occurs very quickly (30 mins
usaully). Now the zygote develops and matures to a motile
body called ookinete; this happens within 12 hours after blood
feeding. Now this ookinete is a motile cell it will invade the gut
of the mosquito by going through the layer of epithelium. And
will stay in the basal lamina where it will mature, and round up
to form an oocyst. The oocyst will grow for the next 12-14 days to form a larger cell in which several mitotic
divisions will occur to produce sporozoites. So one ookinete can generate thousands of sporozoites, and at

some point the oocyst will rupture and the sporozoite will be released to the blood of the mosquito, then they
migrate to the salivary gland and invade them and wait till they are injected to another person. Now its a long
cycle, from the first bite of a mosquito from an infected host, till the sporozoite reaches the salivary gland we
need like 17 days. So only after 17-21 days the mosquito becomes infections.
Question: if a mother carries malaria, will it deliver it to its fetus? No they dont cross to the blood of the fetus
but they can cause an inflammatory response to the placenta and affect the uptake/exchange of nutrients.
NB the mosquito should take both gametocytes to become infections. If 0.5 % red blood cells is infected then
the human is critically sick (parasitemia -presence of parasites in the blood- cant be high in humans)! Rodents
are much more resistant 10-20% of their blood infected and they still act normally! But there is a big difference
between rodent malaria and human malaria.
Now there are there stages as we have noticed in our explanation that are invasive:
1. Merozoites: these infect RBC
2. Sporozoites: these infect the hepatocytes
3. Ookinete: these infect the epithelial cells in the mosquitos gut
So these 3 stages are equipped with invasive proteins which can probably invade any type of cells (hepatocyte,
RBC, epithelial cell).
Sporozoite and ookinete cell traversal involves common molecular mechanisms:
Sporozoites require cell traversal to breach skin cells and other
barriers on the way to successful hepatocyte entry, whereas ookinetes
use it to breach the mid-gut epithelia before forming an oocyst
So the parasite uses the same strategy whether in the mammal or in the mosquito and this is shown in the
following figure:
a) Is a sporozoite that goes through several hepatic cells and then becomes a schizont
b) Is an ookinete in the midgut of mosquito it invades the epithelial cells and goes from one cell to another until it
decides to exit from the basal part and stops at the basal lamina to form the cyst.
NB: Of course, every cell invaded becomes apoptotic.
Now what allows the parasite to go from cell to cell is a complex of proteins that you find at the apex of these
invasive stages as shown in these figures:

The long slender cell is the sporozoite, and the one above it is the ookinete, and the one above is the
merozoite. And as we can see ate one apex they have lots of granules, which they secrete and help them
invade and eventually wander around in tissues. And that is why we call them of the group Apicomplexa
they have an apical complex of organelles that help them during the infection process. Now there are 4
types of organelles and we find different combinations of these depending on the function of each cell.
The 4 types of organelles (colored in the figure) are the rhoptries, exonemes (at least in merozoites),
micronemes, and dense granules.
The first group is the orange ones that we see in the figure in all 3 stages are the micronemes, and are
used by all of them (the different stages). They contain enzymes and adhesive molecules that are
needed for all invasive processes. The micronemes are secretory membrane bound organelles, they are
rich in adhesive proteins as well as proteases enzymes, which are released on the surface of the
parasite they equip the parasite with adhesive receptors that allow it to bind to cells, and with enzymes
that allow it to destroy the membranes and eventually move into cells. So micronemes are found in all
these stages because all of them involve invasion of cells.
The second group is the rhoptries; they are these rabbit ear shapes that are colored blue which are
found only in sporozoites and merozoites. The ookinete does not have rhoptries so this means the
rhoptries are involved in a certain function that is not important in ookinete. The importance of
rhoptries is to do 2 things: the first is for invasion it has molecules in its organelles that help in invasion,
and the second is that the molecules in the organelle contribute to the formation of a vacuole which is
known as a Parasitophorous Vacuole. What is this Parasitophorous Vacuole? If we go back to the
hepatocyte, you see that the sporozoite at some point it stops and grows within this membrane bound
organelle, it does not grow in the cytoplasm, it grows in a vacuole and all of the cell division grows in the
vacuoles. And again the same thing for the merozoite in the RBC during the Erythrocytic cycle, the
merozoite invades and then forms a vacuole around it and a ring occurs within this vacuole and a
trophozoite forms within this vacuole and the schizont also. So there is a membrane vacuole like an
endosome (endocytic vesicle) in which the parasite develops. Now the ookinete stage does not produce
vacuoles because it matures extracellularly (crosses the epithelial cells in a mosquito and matures to a
cyst extracellularly). What is this vacuole? This vacuole largely originates from the membrane of the cell,
it is very rapidly produced (10-20 sec after the parasites invade the cells the vacuole is produced), most
of the vacuole lipids come from the membrane of the cell that is infected, but also the rhoptries and the

micronemes (mostly) during invasion the membranes of the micronemes become imbedded in this
granule, eventually they enrich the vacuole, but its mostly lipids that originate from the host. What do
you see on the vacuole? We dont see any protein that comes from the host (these proteins will be
excluded), we see only parasite proteins which means that this vacuole cant fuse with lysosomes which
can degrade the parasite, so the vacuole remains as an intact membrane bound organelle inside the cell,
and the parasite transports and exports material through this vacuole, it has transport proteins that can
be recruited whenever in contact with whatever is found in the cytoplasm. So remember the vacuole is
found in the hepatocytes and RBC but not in mosquito cells. This vacuole will grow and grow till it
ruptures.
The third group is the dense granules are the ones in purple in the figure are important for the
formation of Parasitophorous Vacuole. So these dense granules are released after the parasite has
invaded the cell and these seem to contribute to the establishment of this vacuole.
Forth and most recently described group are the exonemes, and from its name it is needed for the exit
of the parasite from the cell. These we see them in green in the figure and all the sporozoites and
merozoites have it. Again it is needed to exit from the vacuole (the vacuole rupturing and the release),
so exonemes have lots of hydrolytic enzymes that break the vacuole.
These stages have different quantities of these granules. If you look at the micronemes, the ookinete
has many of these micronemes (orange), sporozoites also have many, while merozoites have few of
these micronemes. The explanation for this distribution is that merozoite is a one-time invader to the
RBC and doesnt go from one cell to another; while sporozoite and ookinete go from one cell to another
because, the ookinete moves between the epithelial cells and the sporozoite moves between the
hepatocytes, so need several granules because they are multi cell invaders.

Distribution of plasmodium:
P. vivax: (second major parasite)widest geographical distribution (tropical, subtropical and temperate
regions) {this is the one seen in Syria and turkey} the problem with vivax is that it is the only parasite
that can go through a dormant stage, it forms hypnozoites.
P. malariae: subtropical and temperate but less prevalent
P. ovale: tropical zone in Africa, Asia and South America, also much less prevalent
P. falciparum: (major parasite) exclusively in tropical and sub-tropical regions.

What makes falciparum a major parasite?

It causes the most serious often deadly disease by invading any RBC at any stage in its existence in the
host. And another characteristic of falciparum is that several merozoites can invade a single RBC, usually
3 to 4, rodent parasites can also do this. In addition ring stage parasites that develop within a single RBC
(this is specific for falciparum) are not central but seen at the very edge of the cell membrane (this is
called the accol position and is distinctive for this species)

In figure 1 shows 2 ring stages developing but this is not falciparum because
these asexual stages gametocytes are not specific for falciparum. (more than 1
ring stage but no crescentric gametocytes so this can be a rodent parasite for
instance)
In Figure 2: is a falciparum infection because the asexual stages of
falciparum forms crescentic gametocytes (banana shaped gametocytes)
that are diagnostic of this species and look like ookinetes in the
mosquito. The blood schizonts can have up to 36 merozoites. In
addition, they do not produce hypnozoites (dormant stages).
Figure 3: Schizont in a RBC with many nuclei: each black dot is a
merozoite. Can be counted.
Another important characteristic of falciparum is that you rarely see
mature stages in the blood smear, growing trophozoites and schizonts are
rarely seen in blood films but we mostly see the ring stages and the
reason for that is that mature stages are sequestered in blood vessels and
capillaries, meaning they adhere to the epithelial cells and dont tend to
circulate in the blood and they do that because these mature stages
produce receptors which recognize sugars on epithelial cells, so
eventually they are sequestered in the small blood vessels and only in
very heavy infections they can be found in the circulation; but mainly in regular stages you see the ring
stages and the sexual stages (gametocytes) in a blood smear.

Virulence of P. falciparum
Now whats unique about these mature stages are the Cytoadherence
and this is the problem with falciparum. Cytoadherence is the
adherence to the endothelial cells of the blood capillaries. What
happens if we have a lot of these cells adhering to the walls of cell capillaries and adhering to each other
is that they clot the capillaries. So trophozoites and schizonts they adhere to the capillaries and to noninfected red blood cells and form like rosettes (large clumps of cells) which are marginalized on the wall
of the capillaries blocking them, so if this occurs in the capillaries going to the brain you get cerebral
malaria if in kidney capillaries then kidney failure so the damage that results from falciparum is 2
things: first is anemia because the RBC are being ruptured, and the second is the blocking of the
capillaries and it depends where the blocking will occur we will get different effects. Now this adherence
is due to an erythrocyte protein known as P. falciparum erythrocyte membrane protein 1 (PfEMP1)
which is encoded by 60 var genes. This protein is expressed by the parasite but it is delivered to the
membrane of the cell, so eventually will localize on the membrane surface of the RBC, so the RBC will
carry the PfEMP1 and this infected RBC will be in fact adhering to the capillaries. PfEMP1 is encoded by
60 var (variable) genes and this is why immunity is not very protective after first infection. 60 var genes
means at some point when the parasite has infected the host is expressing gene nb.1, after a certain
replication in the cell it stops expressing gene nb.1 and start expressing a different variable; so
eventually if your body has produced anti-bodies against the first variable then they will not work on the
second variable. So antigenic variation is very quick inside the host and this is one of the major

problems against vaccine development in plasmodium parasite. Now PfEMP1 binds to CD36 receptor
found on endothelial cells, chondroitin sulfate A which is a proteoglycan that we find in many different
places in (endothelia and in placenta and this is why infected mothers large number of parasites adhere
to the circulation of the placenta because of the high expression of chondroitin sulphate A, this will
result in a stong inflammation in the placenta and this will affect the exchange of nutrients between the
mother and the fetus.

Immune evasion by P. falciparum:

Immune evasion is a problem with falciparum and thats why there have been no vaccines so far,
although there has been many attempts to produce one, the produced ones give protection only for the
first few month. This is largely attributed to switching in the expression of PfEMP1 genes since only a
single PfEMP1 gene is expressed by a mature parasite and switching to the expression of another
PfEMP1 changes the antigenic properties of the parasite surface. (therefore an accepted vaccine can be
a vaccine that gives 50% protection)

Clinical disease
early symptoms are flu-like, the parasite then produces chills and fever (up to 40C) which first occurs on
daily basis accompanied with nausea vomiting and diarrhea then becomes tertian.
Because of RBC lysis the person becomes anemic and with time malaria causes enlargement of the
spleen because this is where RBC are cleared out of the blood and in this case someone can have
Splenomegaly. Splenomegaly has been shown in mummies which point out to that they have been
infected with malaria. Although any malaria infection may be fatal, P. falciparum is the most likely to
cause death if left untreated (mainly because of cytoadherence) and one of the severe problems is the
cerebral malaria. There are treatments and drugs that can act very well but are very expensive. Another
problem with these drugs is that you cant take them for long periods of time because they have strong
side effects on the liver. So if someone is going to Africa for a week or 2 they can take anti malarias, but
if you spend 2-3 month you should not take drugs.

Fatal complications
Infected RBC that adhere to walls of capillaries in tissues and to adjacent RBC can plug these capillaries.
Plugging of capillaries in the brain can cause cerebral malaria (coma and death)
Kidney failure (blackwater fever): black water means the urine becomes dark, due to the release of
large amounts of hemoglobin into the blood from massive numbers of lysed RBC. Now it is not due to
the parasite so what is causing this symptom is still not clear but it looks like an auto-immune decease,
something is happening and lysing the RBC (massive not due to the parasite), this lyses results in a huge
amount of hemoglobin released which cant be cleared and turn the color of urine dark.

Prevention
There are no vaccines for malaria yet but we have some under trials now. Anti-malarial drug treatment
is expensive and for short-term use and malaria can develop resistance to it, hence the best preventive
measure is to avoid/eliminate the mosquitoes:

a) Elimination of breeding habitats (drainage of swamps), it is the elimination of huge water

areas but we cant do this in places where rice is grown or else there will be a hug economic
problem.
b) Insecticide sprays (although DDT was efficient in eradicating the mosquitoes from several
areas of the world in early 60s in Mediterranean and the Middle-East i, however it is toxic
and it is not allowed to use it now so there are other, many mosquitoes are resistant to it)
c) Use of insecticide-impregnated bed-nets, and this came by an initiative 7 years ago where
they decided to go to these endemic villages in Africa and provide them all with bed-nets
that are impregnated with insecticides especially for children under the age of 5. So if they
sleep under a bed net (anopheles bite only at night) in this case the net will protect them
and kill the mosquito. These bed-nets where given for free and changed every 6 month. It
helped to reduce the incidence of malaria
d) Improve the socio-economical conditions in endemic countries, very important because
malarias are mostly due to poverty. They depend on agriculture of rice, they cant afford to
treat themselves, their houses have no windows, no nets
e) Eliminate mosquito population using transgenic mosquitoes or sterile insect technique.
The sterile insect technique is the growing of male mosquitos in the lab in large numbers
and irradiate them so they have non fertile sperms so they are sterile then you release these
males into the population. Now these males will compete with the wild type males to mate
with the females, and when they mate with the females there is no progeny since they are
sterile, so we can reduce the population, but for this to work we have to release every
month or 2 month. They did it in Brazil to eliminate a screw worm that effect cattle, but this
had economical impact, so they were able to eliminate the population. Mosquitoes are
more difficult, the problem in this technique is that the female apparently in the field knows
a sterile male from a non-sterile male, and it will mate with the one that is not sterile. So the
mosquito irradiation causes a fitness cost and these release males are not really competitive
with the wild type males in terms of mating, so this makes it a big barrier for the use of this
technique. Now in order to come with new ideas why cant we release transgenic mosquitos
that will suppress the population? This sounds difficult to do but eventually in oxford
university they came out with a nice strategy that seems to work out in the lab at least. They
produced mosquitoes that will generate flightless females so in this case they release males
that are fertile and these males are transgenic so they contain an actin promoter which is
only expressed in adult females. So these males carry an actin promoter that drives the
expression of a gene which TTA (tetracycline trans-activator), TTA bind to a TTA responsive
promoter to drive the expression of another gene (conditional expression: promoter +
substance Y will drive the expression of TTA, TTA is a transcription factor that will bind to the
promoter and drive the expression of gene X. So only when you put the substance Y we
activate this promoter). By mistake they found here that part of TTA domain inhibits a
protein in the flight muscle of the female, so the males that contain the actin promoter
released into a cage where there are females, they will mate with the females and the
female progeny that will have the actin promoter for TTA, this specific actin promoter will
be active only in the female progeny and not the male progeny. Now the females as they
mature actin promoter starts to be expressed TTA will be expressed actin promoter

f)

this specific actin promoter is active only in the flight muscle of the females once the
female develop they become pupae and then hatch to become adults and they want to fly
from water they cant fly and we see them walking. So the actin promoter works specifically
in the females and the females we call it a flightless phenotype and they cant escape from
the water. It worked perfectly in the lab and they started doing trials in specific endemic
area. The only problem is that the actin TTA you have to introduce should enter somewhere
in the genome without causing a cost effect, and this we cant know since these flies are
transgenic so they may interfere in the fitness of the males and wont be competitive as wild
type strains, so it is very important that transgenic males are as fit as wild type, and if it
works we will be eliminating the population, because the hatched males can carry this gene
and mate while female become flightless and cant mate anymore.
The use of para-transgenic approach to cure mosquitoes and kill the parasite in the
mosquito and this is by using the microbial flora. So they are looking at bacteria that are
found in all this specific strain and which are transmitted vertically (mother to child) and
with these bacteria If we can produce a transgenic bacteria that can produce a toxin against
the malaria parasite for example, then if we release 1000s of these mosquitoes carrying the
flora, when they mate their progeny will carry this transgenic bacteria because it is vertically
transmitted and the toxin will kill the malaria parasite. Now this worked so far against the
Dengue virus, not done on anopheles yet. For this virus we use the bacterial symbiont called
wolbachia which is a bacteria that is found in many mosquitoes in the wild, but there are
certain strains that they removed from drosophila and that they put into Aedes mosquitos
that transmit dengue virus and this bacteria eventually spreads to the progeny vertically and
the presence of this bacteria inhibits the development of the virus in the mosquito. Now
they started releasing some of these in Australia and they found that this bacteria is
spreading very strongly. So here we are introducing a bacteria that spread itself into the
population and interfere with the replication of the virus and this is what is known as a paratransgenic process.

We think we will use a combination of these techniques to eliminate malaria in the future.

Waterborne microbial diseases

We will discuss here only two major bacteria: Cholera and Legionellosis. Several microbes which can be
transmitted by water which are variety of bacteria, viruses, fungi (the less prominent within this group)
and protozoans. Here the dosage is important, so it doesnt mean that each time you drink water
containing bacteria it might get decease, even if the bacteria has Cholera, it depends on how much you
CFUs are found in a certain ml of water, and this means the immune statues of the host is also
important. So, the exact numbers of waterborne pathogens necessary to cause disease depend on the
virulence of the pathogen and the general ability of the host to resist infection.

The most important common source of infectious diseases is contaminated water (a failure in a single
step in drinking water treatment may result in exposure of millions of individuals to an infectious agent)
There are 2 sources where you can catch waterborne diseases (1 and 2 are portable water and 3 is
recreational water):
1) Drinking water, and here infection occurs because of poor quantity, meaning there is no proper
filtration of water and sewers mixing with drinkable water.
2) Consumption of contaminated water from non-regular and non-tested sources such as private
wells, streams or lakes may also cause isolated local outbreaks, which might be contaminated by
fecal material
3) Recreational water and this is water were we swim and it includes fresh water recreational
areas such as ponds, lakes streams and pools. Again a person can be contaminated by bacteria.
The 2 major water born deceases are Vibrio Cholera and Salmonella Typhimurium. Salmonella
typhimurium is a food born disease also so we will discuss it with food born disease. Now cholera we all
know it that a very strong diarrheal disease and mainly restricted to developing counties and it occurs as
outbreaks (large populations are infected). It is caused by Vibrio cholerea, a Gram-negative, curved
(comma-shaped) rod, proteobacteria. Vibrio is a
facultative an anaerobe, can ferment a large number
of sugars as well as it can undergo respiration and its
one of the most important waterborne microbe.
Vibrio species can grow on a variety of simple media
within a broad temperature range (14 40C), and
they are aquatic Bactria meaning that you find them
in fresh water and in salt water. They are highly
susceptible to acids. Vibrios tolerate a wide range of
pH (6.5 9.0) but are susceptible to stomach acids.
Hence a large inoculum (108 109 vibrios) is needed
to initiate a Cholera infection. If the person uses pills
to decreases the acidity of the stomach then a
person might be infected by a lower doze, so if gastric acid production is reduced or neutralized by
bicarbonate, the infectious dose of cholera decreases to as few as 104 cells. Most vibrios have a polar

flagellum cells and have a cytochrome C oxidase so they are oxidase positive. All strains of vibrios have
LPS in their outer membrane. Vibros are aquatic bacteria yet only colerae among the sevela vebreos that
humans can grow in the absence of salt. Other species like Vibrio parahaemolyticus and Vibrio
vulnificus, they need salt so you need to add up to 6-7% salt in the medium for them. Vibrio
parahaemolyticus cause diarrehtic decease but milderand is resolved within 3 days usually, Vibrio
vulnificus causes wound infection (if you are swimming and wounded it can get to the wound and enter
the circulating and infect other organs so it is more serious, can consentrate in shell fish). There are
several serotypes of vibreo ~ 140, and they differ in the O-polysaccharide of the LPS, so each one will
correspond to a different anti-body (O1-O140). Not all serotypes cause cholera, V. cholerea O1 and
O139 serotypes mainly because these serotypes carry a bacteriophage in the chromosome, and this
phage is what carries the cholera toxin, they produce cholera toxin and cause epidemics of cholera while
the other groups do not produce cholera toxin and are not associated with disease.

Virulence factors
Vibrio is not invasive so wont invade the epithelia nor the blood, so the bacteria will remain at the
epithelium surface and will release toxin. So following ingestion of a substantial inoculum of V. cholerea,
the bacteria attaches to intestinal epithelial cells, where it grows and releases cholera enterotoxin and
other virulence factors including:
1) Colonization factor: important for adhering to intestinal epithelia, because if the vibrio cant
attach it does not cause the decease. So adhesion proteins which bind to the intestinal epithelia
in the small intestine and this is where the toxins work.
2) A type 4 pillus, and we call it a toxin correlated pillus (encoded by tcp gene complex). Its a pillus
that has several roads so this pillus is important for bacterial-bacterial interaction, adhesion to
the epithelial wall, and for pillus-pillus interaction for the colonization of bacteria and their
growth as a micro-colony. The importance of growing as a micro-colony at the surface of the
epithelium is that the concentration of the toxin is higher compared to individual growing cells.
This pillus is also a receptor for the cholera toxin carrying lysogenic bacteriophage CTX [this
bacteriophage encodes the genes for the 2 subunits of the cholera toxin (ctxA and ctxB). After
infecting the bacterial cell the CTX genome becomes integrated into the bacterial genome]
Note: non-adherent strains are unable to establish infection of the intestinal mucosa
3) Zonnula occludens toxin: It disrupts the tight junctions (zonnula occludens) of the small
intestine mucosa, increases the paracellular permeability, and contributes to diarrhea or
loss of water and increase of water secretion into intestinal tract.
4) Accessory cholera enterotoxin: induces increased fluid secretion from the intestinal
mucosa
It activates Ca2+ influx into epithelial cells, rise in Ca2+ opens chloride channels,
chloride Cl- flows out of cell sodium Na+ follows water follows loss of water
from intestine (chloride important ion in intestine that control water movement and
secretion)

a.

Cholera toxin:
Cholera toxin is major one responsible for disease
also act on chloride channels but in a different mechanism
Is an A-B toxin consisting of an A subunit and 5 pentameric B subunits.
the B subunit binds to the receptor on intestinal epithelium cells (GM1 ganglioside)
(GM1 ganglioside): a complex glycolipid in the cytoplasmic membrane of epithelial
cells
itself the B subunit does not alter the membrane permeability, but binding to GM1
ganglioside is followed by the internalization of the active portion, the A subunit,
into the cell where it activates the G-protein Gs subunit by ADP-ribosylation which
in turn activates the enzyme adenylyl cyclase that converts ATP to cAMP (an
important mediator of ion balance)
Increased cAMP levels inside the cells induce the secretion of potassium, chlorine
and bicarbonate ions from the cells into the intestine lumen. This is followed by
massive water movement into the lumen

How does this happen?

1. AB toxin binds to epithelial cells it is endocytosed into cell .To enter to the
cytoplasm, it goes through retrograde pathway where it goes to golgi, then to ER. In
ER, A and B separate. One way by which AB toxin go into cytoplasm is accessing ER
degradation pathway. Usually, proteins destined for degradation are released from
ER to be ubiquinated in cytoplasm. AB uses same pathway but wont be ubiquinated
because it has very few lysine. All AB toxins that go through retrograde pathway to
ER then are released to cytoplasm dont have lysine so they escape ubiquination and
make it to cytoplasm.
2. A eventually splits into 2 components A1 (contains enzymatic activity) and A2. A is
an ADP ribosylase, adds an ADP ribosyl group to the alpha subunit of G-protein
bound to GTP and locks G protein in GTP bound state (always activated), this alpha
subunit in GTP bound state activates enzyme adenylyl cyclase which increases
amount of CAMP in cell by converting ATP to CAMP.
3. Because GTP is locked, it is an Irreversible process there is huge amount of CAMP
produced. CAMP will disrupt ionic balance within epithelium and activate specific
type of chloride channels called CFTR (cystic fibrosis transmembrane
conductance regulation channel) different than channels activated by ACE toxin.
Cystic fibrosis is disease where the channels is inhibited
CFTR:
Regulate water movement across cell
it is a chloride channel

 broken ABC transporter: similar to ABC transporter it uses ATP to function but unlike all
ABC transporters it allows ions to move down their concentration gradient not up.(ABC
transporters usually concentrate ions against their gradients)
the rise in cAMP activates a Protein Kinase A, PKA phosphorylates CFTR(because in order to
open CFTR, CFTR needs to be phosphorylated) CFTR opens, since chloride in cell higher
chloride ion released out of the cell into lumen of intestine, sodium follows to keep
electric balance across membrane.
Also, Activated CFTR inhibits Na+ channels(thru which Na gets into cell)
NET Result: We have an increase in Na and Cl outside of cell. Water will follow by osmosis
Note: In cholera, the rate of water loss is greater than the rate of its reabsorption by the large
intestine. CAMP produced for long period of time at high concentrations, the person will lose a huge
amount of water (up to 20 litres a day) if person becomes dehydrated death will occur.
Mortality rate may be ~50 % if untreated but can be much greater under conditions of malnutrition
Main problem with cholera: dehydration + loss of electrolytes
Treatment: give water, electrolytes and antibiotic to eliminate bacteria and effect of toxin.

II. Sources of cholera infections: Vibrio cholera is spread by

i- Contaminated water
ii- consumption of raw shellfish (contaminated by untreated sewage) Shell fish have high
ability to concentrate bacteria in their tissues. If farmed in untreated sewage water, shell fish will
have vibrio. However, Dosage is important, you need high doses to cause disease. If someone
consumes 10000 bacteria, no disease since most of them will be degraded by the acidity. It should
be high colony forming unit per ml of water
iii- consumption of raw vegetables: washed by contaminated water
Note: - Vibrios in general are found in aquatic habitats including fresh and salty water
Note: There is No vaccine for cholera.
III. Clinical Disease:
symptoms start 2-3 days from ingesting the bacteria
onset of watery diarrhea and vomiting
As more fluid is lost the stools become odourless and colourless and speckled with
mucus (rice-water diarrhea)
No vaccine.
IV. Prevention:
A variety of cholera vaccines have been developed but none providing long-term
protection.

adequate sewage treatment and a reliable source of safe drinking water are
the most important measure for preventing cholera(avoid sewers getting in contact
with drinking water)
avoid untreated water and raw foods in endemic areas
Control safety of wells, ponds, lakes

B. 2nd water Bourne Disease: Legionellosis:

Second water disease also common
Not as severe as vibrio
The genus Legionella contains 48 species, with L. pneumophila responsible for almost
85% of infections
Caused by the bacterium Legionella pneumophila:
thin, gram-negative
obligate aerobic rod
has complex nutritional requirements including an unusually high iron requirement
and presence of L-cysteine in the growth medium
Was first discovered as the pathogen that caused an outbreak of pneumonia in
American Legion members in Philadelphia in 1976.
grows in fresh water ( unlike Vibrio: both salt +freshwater )
Disseminated in humidified aerosols, water droplets. You inhale droplets, bacteria
comes in contact with epithelia in respiratory tract
Human infection is via airborne droplets but infection is not spread person to
person, i.e. it cant be caught from human to human contact only from aerosols (Air
cooling systems)
L. pneumophila is commonly present in lakes and streams as well as in the water of
air-conditioning cooling towers (release warm particulate aerosols) and in water
systems (ex: showers, hot tubs etc.)
Can survive in moist environments for long time at relatively high temperatures (3545C) and in the presence of low levels of disinfectants such as chlorine.
More resistant to chlorine than other bacteria since it hides in an amoeba. In
water, it is eaten by amoeba (bacteria eaters). Amoeba usually degrades whatever it
eats (food vacuole will fuse with lysosome and be degraded). However, legionella
will inhibit this process. Although, it is taken in the food vacuole, it inhibits the
fusion of the lysosome it will survive in amoeba which will protect it from chlorine
and detergents found in water so it uses the amoeba as a vehicle to survive.
I.

Pathogenisis:
L. pneumophila is a facultative intracellular bacteria (Spend most of life in cell of
amoeba or other eukaryotes, although it can spend some time out of a cell )
can multiply in alveolar macrophages, monocytes and in nature in free-living amoeba
(amoeba provide a protective environment for bacterial growth)
It Kills amoeba , gets eaten up by other amoeba grow in them as well

Upon inhaling aerosols containing L.pneumophila, first cell to pick them up are macrophages in
mouth sitting on alveoli and are in contact with air we breathe alveolar macrophages will
phagocytoze legionella legionella taken into phagosome inhibit acidification and fusion of
phagosome with lysosome can survive in macrophage and divide producing proteolytic enzymes,
phosphatase, lipase and nuclease which eventually kill the host when the vacuole is
lysedeventually it can kill macrophage infect others

How do macrophage pick up ameoba? By complement system

The infection is initiated by binding of complement to a bacterial outer membrane porin protein
and the deposit of C3b (opsonin) protein on the bacterial surface. This permits the bacteria to bind
to C3 receptor on alveolar macrophage which phagocytoze the bacteria. However, the bacteria
remain alive inside the phagocyte.
Once amoeba reaches tissue in lung, complement system (immune proteins) catch
bacteria macrophage will detect these proteins and eliminate bacteria.
Macrophage will initially struggle against bacteria but later when T cells come and
help them, they eventually clear the infection. The bacteria are not killed until
sensitized T-cells activate the parasitized macrophages.
Healthy individual can resolve infection without treatment i.e. system can clear
bacteria. Thus, It is Self-subsiding infection unless patient very young or very old, or
has compromised immune system. In these cases it can develop into pneumonia.
II.

III.

Main virulence of L.pneumophila:

Inhibit phagolysosome fusion (most imp). allow it to survive in both amoeba and
macrophages.
Enzymes allow it to lyse cells: proteolytic enzymes, phosphatase, lipase and
nuclease
Clinical disease
Asymptomatic infections with Legionella are relatively common(looks like flu and then
subsides)
Symptomatic infections may be: mild (Pontiac fever) or severe (legionnaires
disease)
Pontiac fever: like common cold, characterized by fever, chills, myalgia, malaise and
headache, no symptoms of pneumonia, no inflammation of lower respiratory tract.
symptoms will resolve in 2-5 days spontaneously with no antibiotic treatment
Legionella disease: a more severe disease which may cause 15-20% mortality if
untreated occurs in susceptible individuals (elderly, people with compromised
immunity, weak respiratory system), can develop pneumonia (inflammation in the
lower respiratory tract). Multi organ disease: from lungs it can spread to blood
circulation and can affect the gastrointestinal tract, kidney, liver, CNS...

IV.

Prevention

Reduction of the bacterial load in contaminated water sources: water treatment (usually filtering,
hyper chlorination and heating water at > 63% are efficient in killing the bacteria)
We have seen bacteria but Viruses and parasites are also an important cause of water borne
diseases. Major problem ARE parasites.
What distinguishes Parasites from bacteria is their high resistance to chlorination and UV
treatment. Usually one way to disinfect water is by UV machines. But here the only option is to
filter water.
Public health and water quality
Testing drinking-water quality:
Specific indicator microorganisms are used to signal that a water source might be
contaminated with pathogens.
The most widely used indicator for microbial water contamination is the coliform
group of microorganisms
Coliforms:
1- Many of them inhabit the enteric system: intestinal tract of humans and animals in large
numbers, but can also be found in aquatic environments and soil. Found in many organisms: coldwarm blooded
2- They are facultative anaerobic, gram-negative, non-spore forming, rod- shaped bacteria that
ferment lactose with gas formation within 48 hours at 35C
3- Coliform group includes a variety of organisms, most of which are members of the enteric
bacterial group (ex: E. coli and Klebsiella pneumoniae)
4- Look in coliform for their ability to ferment lactose and generate gas within 48 hours of plating
them.
5- When you do coliform, interested in fecal coliforms not total coliform
Total coliform: all coliform generated from warm + cold blooded organisms
Fecal: Generated from warm blooded
What distinguishes fecal from total is temperature (fecal are more resistant to higher
temp)
Ecoli, Klebsiella can survive up to 41C, by putting plates in high temp you are selecting for
fecal coliforms coming from warm blooded individuals not those coming from lizards,
reptiles...
The coliform test:
The most common procedure used to test for coliforms is the membrane-filter (MF)
procedure:

1- 100 ml of water sample is passed through a sterile membrane filter which removes the
bacteria
2- The filter is then placed on a plate of eosin-methylene blue (EMB) culture medium, which
is selective/differential medium. (Bacteria ferment lactose acid the acid precipitate
methylene blue get dark colonies)
3- Following incubation the number of coliform colonies is counted, compare number
colonies to standards, know if water safe or not.
If you do many collections, get Average fecal coliform/ml water. According to US standards,
drinking water is not considered safe if the number of faecal coliforms is higher than:
a- 1 per 100 ml (of tested water) as the arithmetic mean of all samples examined per
month
b- 4 per 100 ml in more than one sample when fewer than 20 samples are examined per
month(more stringent)
Disinfection of drinking water (chlorination and UV irradiation)
Chlorination(most common).A residual chlorine level of 0.2-0.6 ug/ml is suitable for most
water supplies and kills microorganisms except for certain pathogenic protozoa like cysts of
Cryptosporidium and Giardia which are resistant to levels of chlorination used to disinfect
water)
In Europe UV irradiation (253 nm) is commonly used to disinfect water from bacteria
,viruses, parasites except cysts of Cryptosporidium and Giardia are also resistant to UV
However, the main advantage of using UV over chlorine is that UV irradiation is a physical
process (does not introduce chemicals to the water)
UV radiation preferred more than chlorination.

Food Preservation and foodborne microbial diseases

Some microbes can be found in both categories water and food since microbes found in water can
contaminate food.
example:salmonella(food +water borne microbe)
Foodborne diseases can be separated into 2 categories:
1. Food poisoning or food intoxication:
Disease results from ingestion of foods containing preformed microbial toxin(bacteria has grown in food
and released toxin) .you consume food and toxin act immediately,symptoms quick(1 hr or 2).The
microorganisms that produced the toxins do not have to grow in the host and are often not alive at the
time the contaminated food is consumed.When you heat cook food ,bacteria dies ,but toxin heat stable.
2. Food infection:
Disease results from an infection resulting from ingestion of pathogen-contaminated food.food contains
bacteria,bacteria grows in intestine ,cause disease,symptoms delayed(12 hrs later)
i.
Food poisoning:
A. Staphylococcal food Poisoning

A very common food poisoning is caused by Staphylococcus aureus(Gram-positive)

why? S. aureusis frequently associated with food poisoning because:

itcan survive at 6% salt(halotolerant).we usually salt food to inhibit water activity and
kill microbes.But S. aureus can grow
Many people are carriers of it (found in skin and naso-pharynx) ,so contamination of
food is easy since we are the reservoirs doesnt come from environment.
can grow in many common foods and some strains produce several heat-stable
protein superantigens called enterotoxins (Enterotoxin A is the most commonly
associated with disease)
food poisoning is due to super antigens called enterotoxins
Not all strains carry enterotoxin
Estimate :30-50% have enterotoxins
S. aureus enterotoxins:
1- At least 7 different but related enterotoxins(A,B,C,D,E,F,G) are produced by S.aureusstrains (Most
strain produce only one or two of these toxins and some produce non)
2- The most common enterotoxin produced is Enterotoxin A

3- they are stable to heating at 100C for 30 minutes.bacteria dies but toxin remains.stapp lands
on food ,devide,produceenterotoxin,If you consume food(even if bacteria has perished),the toxin is
highly stable and causes disease.
4- they are resistant to hydrolysis by gastric and jejunal enzymes.it resists degradation through intestinal
tract
5- when ingested through contaminated foods they cause a gastroenteritis characterized by
nausea, vomiting and diarrhea (Symptoms quick usually within 1-6 hours of ingestion)
Enterotoxin A:
Super antigen,trigger strong inflammatory response in intestine which is not very severe and subside
by itself.
Prevention : S. aureusfood poisoning can be prevented by
hygiene measures both in food production and food preparation steps
storage of food at low temperatures to inhibit bacterial growth.refrigerate food or heat
it then consume it .never keep food at room temp.
How to detect food poisoning?
Check food for presence of bacteria+toxin since bacteria might not be living.
Measure toxin by ELISA(detect toxin using proteins)quantify how much toxin is there per mg of
food.fix antibody specific to toxin in a plate,grind food place over antibody ,if toxin is there
,toxin will bind to antibody ,then you have another antibody with a fluorescent probe .
Usually in diarrheic disease, culturing of stool is helpful in diagnosis.in such disease
most important indicator is food and not stool cultures since bacteria might not be there.
B. Clostridium perfringesfood poisoning
second most important source of food poisoning is clostridium

Clostridium perfringes:
obligate anaerobe + aero tolerant (can resist exposure to O2 )
gram-positive rod
spore-forming
the organism grows rapidly in tissues and in culture
imp component in microbial flora of gut of organisms :humans,animals
it is hemolytic (it produces toxins that lyse red blood cells in blood-agar plates),similar to
streptococcus pylori),produce halo around colony
ubiquitous , mainly found in soil
strictlyfermentative,grow very quickly.ferment many diff organic
molecules:sugar,aminoacids,purines..

Pathogenesis
The disease results from the ingestion of a large dose of vegetative cells of C. perfringes(>108
cells) in foods, especially meat, poultry and fish
After consumption of contaminated food, many bacteria perish in the stomach acidity(very
susceptible to acidity of stomach). many bacteria will die as they go through stomach and upper
part of small intestine.The few survivors multiply and eventually undergo sporulation in the
intestine.
Main source: proteinacious food (meat,poultry,and fish)since likes to ferment amino acids
How they contaminate food?
Spores land on food,germinate on food,become vegetative cell,devide,produce toxin as they
aredeviding ,we consume food,food poisoning.
After consuming food, many die in stomach ,others multiply in intestine and eventually sporulate in
distal part of small intestine(ileum, colon).what triggers sporulation is alkaline PH=7.
Organisms will then shed spores through stools to the environment.
The pathogenicity of C. perfringesis attributed primarily to its arsenal of at least 12 toxins and
enzymes produced by this organism. (however each individual C. perfringesisolate expresses a
diff combination of these toxins).we Use this diff cobinations to classify Strains as type A,B,C,D,E.
C. Perfringes toxins include:
a- Alpha-toxin:
the most important toxin and the one produced by all isolates(Most common)
Highly lytic to cells
it is a lecithinase (phospholipase C) that lyses erythrocytes, leukocytes, platelets, and
endothelial cells.Targets phosphatidyl choline which is absent in bacterial membrane.
causes massive hemolysisof RBC s, increased vascular permeability(vessels will leak
,especially small vessels which dont have a lot of smooth muscles around
them,endothlial cells will lyse lots of serum goes to tissue causes swelling)and
bleeding (augmented by the destruction of platelets), tissue destruction (myonecrosis)
Clostiridium perfengin: cause gangrene disease
b- Beta toxin:
Causes necrosis(death) of the mucosa of the small intestine
it assembles into complexes that form pores in the membrane of intestinal epithelial cells
leading to cell lysis and allowing bacteria to go to abdominal wall,to cross intestinal
epithelia.
c- Epsilon-toxin
it is a pore-forming prototoxin that is activated by trypsin in small intestine
it crosses the intestinal barrier and is uptaken by the vasculature (blood vessels) of the gut
where it interacts with vascular endothelial cells and increases the vessel wall permeability by

direct damage/lysis of the endotheliumcausing edema in many tissues(swelling due to leakage

of blood vessels)
Not local toxin :Activated extracellulary ,cross epithilium to blood vesselsand spread through
the circulation causing edema in many tissues
d- iota-toxin:
It is an A-B toxin that ADP-ribosylates actin leading to cell death.
A(ADP ribosylase),ribosylate actin monomers,A gets into cell ,rybosylate actin and
interferes with actin remodelling.ifepithilial cellwere elongated,they become
circular.toxin ,lead to cell roundingand death (remodeling of cell leading to apoptosis)
Note: the production of 1 or more of these toxins is used to subdivide C. perfringens isolates into 5 types
(A-E)
e- Enterotoxins:
it is produced primarily by type A strains
it is a heat-labile toxin (degraded if food cooked)whose activity is enhanced 3 times
followingexposure to trypsin
the enterotoxin forms a complex on the apical membrane of intestinal epithelial cells
that alters the membrane permeability of the cells (The toxin bind to certain claudins
which are members of tight junctions that allow epithelial cells to bind tightly to each
other)trigger internalization of ecludindisrupt tight juction between epithelial cells
more permeablitymore leakage of water, diarrheaspores shed into environment.
it also acts as a superantigen:activating large number of T cells ,triggering an
exaggerated inflammatory response
it is produced during the phase transition from vegetative cells to spores (the toxin first
accumulates in the cytoplasm of the sporulating cell and is released into the lumen
when the mother cell lyses to release its spore).Toxin is Produced during sporylation in
intestine of mother cell,as bacteria reach colon and ileum,they start sporylating.mother
cell(Vegetative) produce enterotoxin,still in cell,once mother cell
degarded,sporereleased,toxin released to extracellular medium.
due to presence of these we classified it as food poisoning
Toxin only produced through phase transition from vegetative cells to spores
Benefecial for bacteria .enterotoxin is the one that cause the diarrheic disease. once
diarrhea is caused ,this helps in spreading bacteria(spore from host into environment )
Multifunctional toxin :Trigger diarrhea by forming pores in epithilialcells,disrupt ionic
balance channels ,allow anions like chloride to leak from cells.

Clinical diseases:many type of disease depending on how bacteria enters

I.

Soft tissue infection:

Virulent strains of the bacteria can access wounds infecting the soft- tissues causing disease
that can range from mild to severe-life threatening.if you have wound, contaminated by soil

II.

III.

containingclostridiumbacteria bacteria start dividing in wound since it is highly

fermentativelyse cells ,go deeply to muscles.
Gas is produced as a result of the metabolic activity of the rapidly dividing bacteria in tissues
(hence the name gas gangrene)
Muscles metabolically favored place for them because they produce lots of proteases and
enzymes that degrade extracellular matrix.so they degrade protein in tissues and ferment
the amino acids
Due to Fermentative activity(gas is produced in tissues )
Gas production in tissues help bacteria spreading: it open spaces for bacteria to spread and
go deeper and more quickly
Gas gangrene: generation time is fast ,you cant stop spreading by medication ,only way by
amputation. Cant stop bacteria
Gas gangrene Due to 2 imp characteristics:
Highly fermentative of sugar +amino acids
Enzymes they produce(proteases) that allow to degrade tissues
Anaerobes so if they go deaper in tissue they are ok away from O2
Food poisoning:
it is due to ingestion of large numbers of enterotoxin-containing C.perfringes type A (it is widely
distributed in nature mainly in soil and water contaminated with fecal material). As bacteria
reaches ileum ,bacteriasporulate ,enterotoxin released,cause diarrheic disease.foodpoisining
occur due the enterotoxin produced during transition from vegetative cell into spore.
it initiates 8-24 hrs after ingestion of the bacteria
symptoms include abdominal cramps, watery diarrhea ,symptoms much less severe, canresolve
by itself ,unlike soft tissues infection.
Nectorizing enteritis:
Necrosis: killing cell
Enteritis: occurs in enteric system .
Killing the epithelial cells of small intestine
characterized by abdominal pain, vomiting, bloody diarrhea and perforation of the intestinal
wall leading to peritonitis
the Beta-toxin is responsible for this disease.Occurs with strains containing Beta-toxin,lyse
epithelial cells of intestinall wall,Causes bloody diarhhea and vomiting
presence of blood in stool is an indication of Necrotizing enteritis
more severe than diarhhea where there is no blood.blood means Sthg invading or causing
severe killing of cells.
Bacteria can cross from intestine into abdominal wall due to pores created in intestinall walls,

Prevention and control:

Proper wound care to avoid contamination by the bacteria
proper cooking of food following by consumption or refrigeration(bacteria and toxin killed since
toxin here not heat labile ).spores wont die.But Spores not problem, problem is sporulation of
vegetative cells.
it is difficult to control the bacteria as it is ubiquitous

Type A most common in nature( soil)

B,C,D not in soil,in guts of organism
No vaccines
Common source of clostridium infection is canned food:
No O2
Can ferment whatever found in cancan grow on sugars ,proteincan swells(gas)
C. Clostridium botulinum and foodborne botulism
More severe disease ,Life threatening
Botulinum
It is a spore-forming, anaerobic rod
the bacterium normally inhabits soil(Ubiquitous) or water, but its endospores may contaminate
raw food before harvest or slaughter (If foods are not properly processed so that endospores
are washed away, these endospores may initiate growth and toxin production
I.
Pathogenesis:
disease occurs following the ingestion of food contaminated by the botulinum toxin. spores land
on food germinateproduce toxin.so you are consuming the bacteria and the toxin.
7 antigenically distinct botulinum toxins (A-G) have been described; human disease is
associated with types A, B, E and F
Note: botulinum toxins are the most potent biological toxins known (1 milligram is enough to kill more
than 1 million guinea pigs !)+ stable neurotoxin(can survive very long time in the body)

Problem for adults is not consuming the bacteria,because vegetative cells dont survive in gut
because of microbial flora.theycant establish a niche there.what will remain is the stable toxin
that will cause the disease.

botulinum toxin :

A-B toxin.
But A notribosylase!A is Zinc endopeptidase which cleaves proteins. while the B chain is nontoxic.
the botulinum toxin is complexed with non-toxic proteins that protect it during passage through
the digestive tract.
Unique in its journey
Translocation of the toxin through the intestinal epithelium

- The botulinum toxin is an unusually potent oral poison, which means that the toxin must have an
efficient mechanism for escaping the lumen of the gut to reach the circulation. This occurs through two
processes:

1- Interaction with auxiliary proteins that protect the toxin from enzymatic activity in the stomach(If
you consume food containing neurotoxin,toxins act at neuromuscular junction . it goes from gut to
bloodstream,to neuromuscular junction. the toxin doesnt perform this long journey by itself. it is held
by auxiliary protein,protect it from PH and enzymatic activity of stomach.untill delivered to blood
circulation)
2- Transcytosis of botulinum toxin through intestinal epithelial cells
When Toxin reaches stomach, it is translocated across epithelial cells by transcytocis .
toxin bind to apical part of epithelial cell,taken in an endocytic vesicle,vesicle fuses with the basal
part,releasing the toxin to the other side(rather than taking it to normal endocytic pathway to lysosome
).so it gets released through basal part where it can access blood circulation then it goes to
neuromuscular junction .once in neuromuscular junction, AB toxin is translocated into neuron by
endocytosis

Mechanism of action of the botulinum toxin

o The B chain of the toxin binds to specific glycoproteins on the surface of motor neurons
and stimulates endocytosis of the toxin molecule
o acidification of the endosome stimulates the release of the A chain from the B subunit .
o The A polypeptide translocates to the cytosol where (through its zinc-dependent
endoprotease activity) it cleaves proteins involved in the exocytosis of acetylcholine
containing vesicles at the neuromuscular junctions.
o acetylcholine is required for muscle contraction((what causes muscle contraction usually
is release of vesicles containing acetylcholine to the neuromuscular juction,then AC
binds to the receptors on muscle ,lead to contraction)
o The proteins needed for trafficking of these vessels are the target so no more vesicles
released at neuromuscular junctionparalysis
o After consuming botulinum toxin ,usually paralysis is gradual .starts by blurred vision
,then inability to move muscles of face, then peripheries...

Most problematic part of disease: muscles of diaphragm which can affect respiration
No major toxin other than botulinum toxin
Before Disease fatality high 60,70%,now due to supportive care and ventilation it drops to 10%.
Here is an Example where you dont need antibiotic because bacteria by itself cant grow .if
someone is suspected to be infected with botulinum by consuming food .best way to fight

disease ,inject serum (antibodies that block toxin in blood circulation,so it wont reach
neuromuscular junction).
II.

Clinical disease:
Disease mostly due to food borne botulism where toxin is consumed and cause all the
symptoms.
1. Foodborne botulism:
- After ingesting the toxin, the peripheral muscles eventually weaken and death may occur due
to respiratory paralysis
Note: The neurotoxin persists in the neuromuscular junction for long time hence complete
recovery often requires many months to years (mortality has been reduced to 10% through
supportive care mainly in managing respiratory complication)
2. Infant botulism
- In contrast to foodborne botulism, this disease is caused by toxin produced in vivo by C.
botulinum colonizing the gastrointestinal tracts of infants (sometimes from raw honey but more
often from no identifiable source). So ,In infants less than 1 year old ,it is not food intoxication
but C. botulinum causes food borne infection .reason: C. botulinum can colonize intestinal
tract,can proliferae and produce toxin in vivo leading to symptoms similar to those in adults .
- most common source of botulism in infants is honey contaminated by spores of bacteria.

Note: although adults are exposed to the organism in their diet, C. botulinum cannot survive and
proliferate in their intestines because of competition with microbial flora.After age of 1 year
,microbial flora becomes very similar to that of adults and doesnt allow growth of bacteria.
3. Wound botulism:
- a rare condition which develops from toxin production by the bacterium in contaminated
wounds. C. botulinum proliferates in wound since In wound ,no microbial flora bacterium can
multiply,produce toxin ,spread to neuromuscular junction.
- symptoms are identical to those of foodborne disease.
III.
Prevention
heating of food (all botulinum toxins are destroyed by heating at 60C to 100C for 10 minutes
- Toxins Heat labile,quickly degraded
infant botulism has been associated with consumption of honey contaminated with spores.
(children < 1 year should not eat honey)
note : Clostridium perfringes more common source of food borne infection than foodborne botulism
ii.

Food Infection:
consume vegetative cells,Vegetative cells grow,cause infection
salmonella v. Common source of infection

I-

Salmonellosis (caused by bacteria of the genus Salmonella)

Salmonella:

they are gram-negative, facultative anaerobic rods (catalase positive and oxidase negative)

they are enteric bacteria that normally inhabit the gut of animals and are thus found in sewage

virtually all Salmonella are pathogenic for humans. S. typhi causes the serious human disease
typhoid fever

Salmonella can colonize virtually all animals including poultry, surface of eggs, livestock,
rodents, reptiles, domestic animals birds and humans

Ubiquitous,also found in soil

Most infections result from the ingestion of contaminated food products or from direct fecaloral route in children.
Normal to find salmonella in chicken farms,but what you need to avoid is contact of intestinal
material with fresh meat when you cut chicken.

most common sources of human infections are poultry, eggs, dairy products and foods
prepared on contaminated surfaces

species like S. typhi are highly adapted to humans and do not cause disease in non-human host.
S. typhi infections occur when food or water are contaminated by infected food handlers is
ingested

There is no animal reservoir, ,human are carriers

Hard classification:

Genus Salmonella initially split into 2 groups:

Salmonella enterecia:this is subdevided into more than 2000 subspecies

Shiggela bongoli

We refer to Salmonella typhi: imp human pathogen

But ,Proper name: Salmonella enterecia(group) Serovar typhi
Serovar:serotype of bacteria(Could be typhi,paratyphi)
-

Almost all salmonella cause disease in humans. Although all salmonella can cause disease in
human, it is usually mild.only problem is with typhi and paratyphi
2 most major causes of severe infection are salmonella typhi (typhoid fever)and salmonella
enteric serovac paratyphi
Rodent salmonella ,typhimurium(used in research)cause typhoid like disease in mice.
person with salmonella shed a lot of salmonella in stool so diagnose by testing stool sample for
salmonella.

Pathogenesis
-

Salmonella cause disease due to several virulence factors:

1- Fimbriae :attachment
- Salmonella species-specific fimbriae allow the bacteria to bind to the M (microfold cells) cells that
are located in Peyers patches in the small intestine
-uses Fimbrae to bind to epithelial cells of intestine (which are not phagocytic)
-but salmonella need to go in cell and replicate.thus,they need to trigger their own uptake by
epithelial cells (vibrio binds to cells doesnt invade it,but produces toxins and toxins go inside cell
unlike salmonella).

There are Microviliated epithelial cells ,rich in microvilli+ no microvilliated epithelial cells ( microfold
cells .hiding under these are immune tissues.
Importance of microfold:cells active in macropinocytosis.they take material from lumen of gut,by
transcytosis ,they release it to immune cells underneath,so immune cells can always sense what is found
in the lumen.they are not phagocytic,they can not phagocytoze salmonella
Salmonella uses microfold cells to cross epithelium.but microfold are not phagocytic.salmonella must
trigger these microfold to become phagocytic using T3SS which inject protein inside triggering actin
polymerization.

2- Type III secretory system (T3SS):

Two Salmonella type III secretion system exist: with diff roles,activated sequentially
a- SPI-1 (for Salmonella pathogenicity island-1) introduces salmonella-secreted invasion proteins into
the M cells which causes rearrangement of the host cell actin cytoskeleton with subsequent membrane
ruffling formation

First machine help bacteria get into epithelial cells

What salmonella does it injects thru the medium molecules that can trigger actin
polymerization (flowering of cell)Trigger ruffling of surface of
cell(invaginations)salmonella goes deep into rufflesonce ruffles fuse salmonella is
inside cell(it is in endocytic vesicle in cytoplasm)
Salmonella triggers ruffling to infect cell then repair damage to bring cell back to normal
because it wants to survive(if Continous rufflingapoptosis)
causes transient damage to get into the cell then repairs everything

Once in cell , salmonella starts deviding and uses diff type 3 machine .it injects effector proteins into
cytoplasm of cells to avoid being targeted to lysosomes and interfere with many signaling pathways.thus
the phagosome in which salmonella is deviding will not fuse with lysosome.
b- SPI-2 which is a multifunctional virulence system which facilitate replication of intracellular bacteria
within membrane-bound Salmonella containing vacuoles (SCVs)
- ruffled membranes surround and engulf the Salmonella bacteria
- the bacteria replicate intracellularly in phagosomes with subsequent cell death and spread to
adjacent epithelial cells and lymphoid tissue. eventually salmonella typhi will escape from microfold
cells it will lyse them and infect other cells .or it will captured by immune cells(macrophages) which
engulf salmonella .here it doesnt need needle since they are phagocytic cells so dont need first
pathogenicity island(doesnt need first T3ss).Second needle:interferes with phagocytic cell ,so
bacteria remains intact in phagosome
note :in case of salmonella typhi the infection can spread .Some of these phagosytic cells drain to lymph
nodes and also spread to the blood stream and infect liver, spleen..

The red arrow points to an M cell

unaffected by Salmonella infection.
The white arrow points to an M cell
exhibiting extensive membrane
ruffling in response to Salmonella
infection

Note: virulence factors cluster in pathogenicity islands and are known from GC content.

3- Tolerance to acids in the stomach and in phagocytic vesicles (the Acid tolerance response
requires a set of at least 50 genes that allow bacteria to survive in stomach and small intestine).this
is also common to enteric bacteria like ecoli.
Called acid tolerance genetically regulated program
Clinical disease
1- Gastroenteritis: (infection of the gastro intestinal tract)
- is the most common form of salmonellosis
- symptoms appear 6 to 48 hrs after consumption of contaminated food or water. These include nausea,
vomiting and non-bloody diarrhea (no invasion of gut)

- symptoms can persist 2 days to 1 week before spontaneous resolution, can subside by themselves
2- Septicemia
- serious disease resulting from the presence of the bacteria in the blood (bacteremia).
(allow bacterial spreading to blood )
- The risk of Salmonella bacteremia is higher in young children, elderly and immunocompromised people
Problem with salmonella typhi:bacteria can spread to blood and infect spleen +infect tissue bone
marrow
You can see bacteria in lymph nodes draining from intestine ,liver,spleen,bone marrow
The place where it will remain for long periods of time is gall bladder.after the symptoms of someone
infected with salmonella typhi dissapear ,they can still carry bacteria for a year in gall bladder ,since gall
bladder empties into intestine .person will be shedding bacteria although no symptoms
Gall bladder in 1-5% of patients that recover from disease is storage site for bacteria

3- Enteric fever
-

S. typhi produces an illness called typhoid fever characterized by gradually increasing fever,
following bacterial invasiveness of the intestinal epithelium

The fact that bacteria is in blood triggers strong inflammatory response (high fever)

fever high 41,42C due to presence of bacteria

Prevention and control

- cooked foods heated to 70C for at least 10 minutes are considered safe if consumed immediately or if
held at 50C or stored at 10C or lower. dont eat raw meat and poultry because they are a main
source.
- No vaccines for salmonella.

II- Escherichia coli

-

The genus Escherichia consists of 5 species, of which E. coli is the most common and clinically most
important
They are flagellated, gram-negative, facultative anaerobic rods
They are catalaze positive, oxidase negative (common characteristics to all Enterobacteriaceae,
which E. Coli, Klebsiella & Shigella belong to)
Most strains of E. coli are not pathogenic and are common commensals found in the intestines of
humans
However, around 200 strains are known to be pathogenic and can cause life-threatening diarrheal
disease and urinary tract infections (these strains become pathogenic by acquiring virulence factor
genes on plasmids, bacteriophages or pathogenicity islands)

Pathogenesis
-

Pathogenic strains cause disease because they possess a broad range of virulence factors:

1- Adhesins
-

E coli is able to remain in the urinary tract and gastrointestinal tract because it can adhere to the
epithelial cells at these sites and avoid being eliminated by the flushing action of voided urine or
intestinal mobility
Adherence is mediated by several factors including the colonization factor antigens, fimbriae/short
pili. In general when E. coli attach to epithelial cells, they stay local and produce toxins. The toxins
can either remain local or translocate.

2- Exotoxins
a. Shiga toxins (Stx-1 and Stx-2) (first found in patients diagnosed with Shigella)

both are acquired by lysogenic bacteriophages

both have 1 A subunit and 5 B subunits (A-B toxins)
E. coli have both Stx-1 and Stx-2.
o Stx-1 is conserved. It is similar to the one found in Shigella.
o Stx-2 is more varied. (Around 60% sequence homology with Stx-1)

Mechanism of action:
-

The B subunit binds to specific glycolipid receptor (GB3) which is found at high concentrations in
the intestinal villus, renal endothelial cells and smooth muscles of the intestines
This is followed by the internalization of the A subunit which is cleaved inside the Golgi of the
host cell into 2 fragments A1 (larger) and A2. A1 and A2 were initially linked by disulfide bonds.
The fragments are subsequently partially unfolded by chaperones in the ER. The ER mistakes
them to be misfolded proteins and releases them to the cytoplasm for degradation. However,
the A1 fragment has no Lysine residues and hence cannot be ubiquitinated for degradation.

A1 acts is an enzyme, called RNA N-glycosidase; it depurinates (removes specific adenine

residues) from the 28S ribosomal RNA, which stops the t-RNA binding to the ribosome. This
disrupts protein synthesis.
These toxins lead to the destruction of the intestinal villus resulting in decreased absorption
with a relative increase in fluid secretion.

A1 also activates signalling pathways leading to apoptosis. It also induces the release of
inflammatory cytokines that enhance inflammation. Inflammation is important because the
inflammatory cytokines induce an increase in the expression of the GB3 receptors on the cells.
Hence more toxins can enter the cell!
Note: Most of the disease symptoms are associated with the Stx-2, which is variable among different
strains. Stx producing E. coli do not necessarily produce both Stx-1 & Stx-2; some express one of
them and some both.

b. Heat-stable toxins (ex: STa)(There is also STd, but not important)

-

STa is associated with human disease

Sta is a short peptide that binds to the extracellular domain of guanylyl cyclase C (located in the
brush border of intestinal epithelial cells) activating the enzyme leading to increased levels of
cGMP production and subsequent hypersecretion of fluids.
- STa can do that because it molecularly mimics the ligand of the receptor.
Note: Activation of guanylyl cyclase C in small intestine results in a dual action:
1- stimulation of Cl- and HCO3- secretion into the lumen through a Cl- channel
2- Inhibition of Na+ absorption by blocking an apical Na+/H+ exchanger
How? cGMP activates a cGMP-dependent kinases called GK2. GK2 phosphorylates chloride channels
making them open, and it phosphorylates Na+/H+ exchanger blocking it and keeping sodium outside the
cells. This increases the electrolyte concentration outside the cells, moving water out (reducing
absorption) and causing diarrheal disease.

Sta structurally resemble physiological

ligands of guanylyl cyclase C
The role of guanylyl cyclase C in ion transport

c. Heat-labile toxins (LT-I)

LT-1 toxin mode of action

It is an A-B toxin consisting of 1 A subunit and 5 B subunits (very similar to cholera toxin)
The B subunit binds to the same receptor as cholera toxin (GM1 ganglioside) as well as other surface
glycoproteins on epithelial cells in the small intestine
Following endocytosis the A subunit translocates across the membrane of the vacuole.
The A subunit adds an ADP ribosyl group on Gs protein that regulates adenylate cyclase. Activated
adenylate cyclase leads to increased cAMP levels with enhanced secretion of chloride ions and
decreased absorption of Na, leading to a watery diarrhea.

3- Microvilli attachment & effacement (virulence of enteropathogenic E coli (EPEC))

Mechanism
1. In the first stage, EPEC cells express on their surface 3 things: bundle-forming pili (Bfp), the intimate
adhesin intimin, and short surface-associated filaments (EspA filaments that function as unique long
T3SS needle).
2. In the second stage, EPEC cells adhere to the epithelial cell via Bfp and EspA filaments, and a type III
secretion system injects the translocated intimin receptor (Tir) and a number of effector molecules
directly into the host cell

Effector molecules activate cell-signalling pathways, causing alterations in the host cell cytoskeleton
and resulting in the depolymerization of actin and the loss of microvilli (this is physiologically
indicated by the reduction of nutrient absorption disease)
- The initmin receptor gets phosphorylated by host kinases and insert itself in the membrane of the
host cell.
3. The bacterial adhesin intimin binds to the modified Tir, resulting in a strong intimate attachment; and
accumulation of actin and other cytoskeletal elements occurs beneath the site of bacterial adherence.
This strong and close attachment is important to stay away from the microbial flora above the mucosal
layer and to avoid competition.
4. Massive accumulation of cytoskeletal elements at the site of bacterial attachment results in the
formation of the characteristic EPEC pedestal structure.
- The injected effector molecules disrupt host cell processes, resulting in loss of tight-junction integrity
end eventually to both electrolyte loss and eventual cell death.

Note: EPEC cells form AE (attachment/effacement) lesions, which are characterized by the loss microvilli
and intimate adherence of the bacteria to the host cell membrane.

Clinical diseases
1- Gastroenteritis
The strains of E. coli that cause gastroenteritis are subdivided into the following major groups:
A- Enteropathogenic E. coli (EPEC):

the EPEC strains attach firmly to the epithelial cells of the small intestine forming the cup-like
pedestals (more than 40 bacterial genes mediate attachment and destruction of the host mucosa)
they cause watery diarrhea which result mainly from malabsorption caused by microvilli destruction
they are a major cause of infant diarrhea in impoverished countries (disease is rare in older children
and adults probably because they acquired protective immunity)

B- Enterotoxigenic bacteria (ETEC):

They are also mostly common in developing countries, affecting mainly young children
ETEC bacteria produce heat-labile (LT) and heat-stable (ST) toxins
Symptoms include mainly watery diarrhea and abdominal cramps

C- Enterohemoragic E. coli (EHEC):

One of the most severe infections: EHEC strains express a shiga toxin and induce A/E lesions on
epithelial cells. They carry a plasmid, which encode as well other virulence factors.
Disease caused by EHEC ranges from mild uncomplicated diarrhea to hemorrhagic colitis with severe
abdominal pain, bloody diarrhea with little or no fever. Bloody diarrhea occurs because Stx-2 can
translocate from the lumen to the cells abdominal wall and disrupts them causing bleeding, and
some of the blood reaches the intestines.
The shiga toxin (Stx-2) produced by certain strains can also translocate from the blood to the
kidneys and destroy the glomerular endothelial cells resulting in decreased glomerular filtration and
renal failure. (but this rarely happens, only about 5% of E. coli infections)
The most common enterohemoragic strain is O157:H7 (epidemic outbreaks). It is found in cattle and
domestic animals, in which it doesnt cause symptoms. The symptoms only occur in humans if
transferred through meat contamination.

D- Enteroinvasive E. coli (EIEC):

These strains are rare. They can cause septicaemia/sepsis because they can reach the blood.
EIEC cells encode virulence factors that mediate invasion of the epithelium of the colon. Bacteria are
first taken up in phagocytic vacuole, which they eventually lyse and replicate in the cytoplasm. EIEC
can move form the cytoplasm of one cell into another using actin tails. Actin tails are when the cell

produces actin at one pole pushing the bacteria out of it and into another cell. (It is physically
mediated)(explained more elaborately at the end)
Symptoms include initially watery diarrhea. A minority of patients can progress to a more severe
disease consisting of fever, abdominal cramps, and bloody diarrhea.

2- Urinary tract infections (UTIs)

Most E. coli that cause UTIs originate in the colon, contaminate the urethra, ascend into the bladder
and may migrate to the kidney or prostate
Almost most strains of E. coli can produce UTIs
90% of the UTIs are due to E. coli

III- Listeria monocytogenes

- Listeriosis is a gastrointestinal food infection that may lead to bacteremia and meningitis. It is caused
by Listeria monocytogenes.
Listeria monocytogenes

it is a gram-positive, non spore-forming rod that is acid tolerant, psychrotolerant (cold-tolerant) and
salt-tolerant.
It is a facultative anaerobe. It is found widely in soil and water and virtually no fresh food source is
safe from contamination with this bacteria
Refrigeration, which limits bacterial growth, is ineffective against L. monocytogenes.
The bacteria can be isolated from the feces of mammals, birds, fish, insects and other animals
(hence the organism is ubiquitous).
Because the organism is ubiquitous, exposure and transient colonization are likely to occur in most
individuals (hence healthy people can be asymptomatic carriers of the bacteria)

Clinical disease
1- Asymptomatic disease (or mild flu-like symptoms):

In most healthy adults listeria infections are asymptomatic or occur in the form of a mild flu-like
disease. Gastrointestinal symptoms develop in some patients.

2- Meningitis: is one of the major causes of listeria infection in adults where immunity (in particular cellmediated immunity) is weak. Ex: cancer, transplantation and HIV patients
In these people (+ children and elderly), Listeria can cross the GI tract to the blood and pass the bloodbrain barrier infecting the nervous system and causing meningitis.

3- Neonatal disease:

Can be acquired trans-placentally in utero or acquired at or soon after birth. The disease in the fetus
is of high mortality (very weak immune system) unless treated promptly

Virulence factors of L. monocytogenes

-

Three tight barriers (the intestinal, blood-brain, and placental barriers) can be crossed by this
pathogen. These events are central for human listeriosis pathogenesis.
In the cytosol, Listeria uses an actin-based motility process to propel itself in the cytosol; it invades
neighbouring cells via a double-membrane protrusion, resulting in the formation of a so-called
secondary vacuole.

1- Internalins:

Bacterial surface proteins (receptors) that mediate

attachment and invasion of target cells.
The N-terminal end (outer part extending from the
bacteria) contains leucine-rich repeats (LRRs) that
interact with adhesion glycoprotein receptors on target
cells.
LRRs are variable and can interact with many different
ligands on different target cells.
Listeria internalins with established role in virulence
include:
a- Internalin A
o Its C-terminal domain is bound covalently to PGN in bacterial cell wall
o Internalin A binds to E-cadherin glycoprotein receptor on the surface of epithelial cells
o Required for invasion of intestinal epithelial cells and epithelial cells of the placenta

b- Internalin B
o
o
o

Its C-terminal domain is bound non-covalently to lipoteichoic acids in the cell wall
Receptors for Inl B include a receptor tyrosine kinase on hepatocytes and gC1q receptor. gC1q is
a ubiquitously expressed receptor for complement protein C1q.
Is involved in entry of Listeria into a broad range of cell lines including hepatocytes and nonepithelial cells (fibroblast, endothelial cells)

2- Listeriolysin O (LLO)

It is a secreted pore-forming toxin whose receptor is cholesterol in host bilayers

It is involved in Listeria escape from primary and secondary vacuoles
Optimal pore formation by LLO (oligomerization of LLO) occurs between pH 5.5 & 6.0 (pH of the
early phagosome)
Intracellular vacuole lysis is also aided by phospholipase C enzymes secreted by the bacteria and
which hydrolyse a wide variety of phospholipids

3- Act A protein:
-

Localized on the cell surface at one end of the bacteria

After lysis of the vacuole, cytosolic Listeria induces polymerization of actin filaments that promote
bacterial intracellular movement and cell-to-cell spread. This is achieved uniquely by Act A protein.
The amino-terminal region of Act A binds and activates Arp2/3, a seven-protein host complex that
induces actin polymerization and the generation of a dendritic array of actin filaments (Actin tail).
The actin tail is fixed to the cytoskeleton from one side. Actin monomers are added to the tail
pushing the bacterium to towards the plasma membrane (#4) until it extends through it (#5) and
reaches another cell (#7) where it detaches from the first cell. The bacterium ends up in a double
membrane (one from each cell). Then it escapes to the cytoplasm through LLO and PLC.
Arp2/3 forms a platform on which actin polymerization occurs.

Other bacteria as well as viruses and parasites may cause foodborne infectious diseases

Foodborne infections may be caused by other bacteria such as Yersinia enterocolitica (gram ve rod) and
Bacillus cereurs (Gram +ve, endospore forming)
-

The largest number of annual food borne infections is thought to be caused by viruses. Viral foodborne illness consists of gastroenteritis characterized by diarrhea, often accompanied by nausea and
vomiting. Recovery is spontaneous and rapid usually within 24-48 hrs (known as the 24-hour bug).
Important foodborne protozoans include Giardia lamblia, Cryptosporidium parvum and Toxoplasma
gondii. (all these parasites can be spread via food, presumably contaminated by fecal matter in
untreated water).