Bacterial Toxins & Secretion Systems

Bacterial Toxins

Ø Sepsis is a process involves an uncontrolled inflammatory response by the host cells that may result in multi organ
failure and death
Ø Toxins induce the dysregulation of the immune system leading to septic shock

AB organization of toxins: The A and B domains may be linked by a disulfide bond or associated by non-covalent
interactions.
1. The A domain/ effector: is usually an enzyme
2. The B domain: the receptor-binding function, providing tropism to specific cell types. The B domain also
includes a domain that translocates the A domain
Ø Bacterial toxins often act distanced from the site of infection
Ø Bacterial pathogens damage the host via invasion and toxin secretion:
1. Invasion involves the ability production of: Extracellular enzymes that damage host tissue such as lipases and the
modulation of the host response system such as the up regulation of cytokine expression and immune evasion.
2. Bacterial damage the host through the action of toxins are often non-invasive and have limited capacity to
disseminate in the host

A. Diphtheria toxin:
Ø A domain: enzyme: ADP-ribosyl trasferase activity
Ø B domain: binding to GFR and allowing entry to cells through receptor mediated endocytosis
Ø The toxin undergoes conformational changes under certain pH

B. Cholera toxin:
Ø V. cholera releases cholera toxin (A1, A2, and B subunits).
Ø B domain: attachment to a ganglioside receptor, which undergoes conformational changes to allow the entry
of A1 & A2.
Ø A2 dissociates leaving A1 (which has the enzymatic activity) activating adenylyl cyclase, which leads to
increased cAMP causing loss of Cl and H2O (persistent activation of adenylate cyclase)

C. Heat stable enterotoxins: (ST) are a family of conserved peptides expressed by pathogenic strains of Escherichia coli.
Ø ST elicits fluid accumulation in the intestines, which is often responsible for diarrhea in travelers and young
children in developing countries.
Ø ST includes two subfamilies, STa and STb.

D. Pore forming toxins: (PFT) are a large group of protein toxins which forms pores in the membranes of bacteria,
plants, and mammals, causing membrane permeability and ion imbalance.
Ø Inactive PFT monomers (soluble) bind to the membrane (specific to a receptor) thus becoming active.
Ø The monomers come together and form a pre-pore intermediate phase, which inserts itself into the membrane.
Ø Once it inserts itself, a PFT is formed
Ø The PFT punches pores into the membrane causing the exchange of large molecules / ATP / ions.
Ø PFTs are classified into two groups based on the multimeric structure involved in membrane insertion:
1. α‐ PFTs: insert into membranes as an α-helix: E. coli and Cholera toxin
2. β‐ PFTs: insert into the membrane as β-sheets, GP pathogenic bacteria such as Lysteria,
Streptococcus pneumoniae, and Bacillus anthracis

E. Endotoxins: glycolipid, LPS

macromolecules that make up about 75% of
the outer membrane of gram‐negative
bacteria that are capable of causing lethal
shock.
The structure of LPS generally consists of:
1. A lipid A domain (effector/enzymatic
activity),
2. A polysaccharide core (transmembrane;
translocation),
3. Outermost O-antigen polysaccharide
(binding)

Vaccines developed against LPS (ex:

pneumococcal) target the O-antigen thus
preventing entry in the first place.
 Superantigens:
Ø SAgs are non-specific; bind MHC II and stimulate peptide-independent MHC II/T cell receptor (TCR)
interaction and immune activation.
Ø Responsible for Toxic Shock Syndrome (TSS) and food poisoning
Ø Superantigen - like toxins (SSL) do not bind MHC II or activate T cells, but do target the innate immune system
Ø Cause an exaggerated immune response
Ø Staphylococcus aureus superantigen SEC3 binds to the β chain of TCR acting as a wedge to encourage TCR and
MHCII interaction and activation lacking foreign peptide specificity along with cytokine release

Secretion Systems

Ø Bacterial pathogens utilize many methods to invade hosts, damage tissue sites, and evade the immune system.
Ø One essential component of these strategies for many bacterial pathogens is the secretion of proteins, small
molecules, and DNA across phospholipid membranes.
Ø One essential prokaryotic cell function is the transport of proteins from the cytoplasm into the environment (other
bacteria or eukaryotic cells) a process known as protein secretion
Ø Secretion systems are found in almost all bacteria (more commonly in GN)
Ø The secreted substrates have three possible fates:
1. They remain associated with the bacterial outer membrane (OM),
2. They are released into the extracellular space, or
3. They are injected into a target cell (either a eukaryotic or bacterial cell).
Ø These substrates have key roles in the response of a bacterium to its environment and also in several physiological
processes such as adhesion, pathogenicity, adaptation and survival. Due to their bacterial specificity, they can be used
for antimicrobial development
Ø These secretion systems are not continuously expressed (only when the microbe finds the right environment with good
nutrition)

Protein secretion by Gram Negative Bacteria

I. Type 1 secretion system (T1SS): transport their substrates in a one step process across both the inner and outer
bacterial membranes
Ø T1SSs have three essential structural components:
1. ABC transporter: protein in the inner membrane:
Ø Catalyzes ATP to provide the energy to transport the substrate
Ø Interacts with the MFP
Ø Participates in substrate recognition
2. Membrane fusion protein (MFP): that crosses the inner membrane and bridges it to the
OMF:
Ø Associates with the ABC transporter and spans the periplasm to associate with the OMF
Ø It plays a role in substrate selection
3. Outer membrane factor (OMF): Generates a pore in the outer membrane, through which the substrate passes
in an unfolded state (inactive). Interestingly, T1SSs often use the multipurpose protein TolC as their
OMF. TolC & MFP do not come in contact & bind unless IMC binds to a substrate.
Ø Pathogens utilizing T1SS:
1. Vibrio cholerae, which uses its T1SS to secrete the MARTX toxin linked to evasion of host immune system
and facilitating colonization of small intestine.
2. Serratia marcescens, which secretes the hemophore HasA.
3. Uropathogenic E. coli secretes HlyA hemolysin protein. This toxin inserts into the membranes of both
erythrocytes and nucleated eukaryotic cells, causing them to rupture (helps pathogen in attaining iron). Rupture
of host cells can help the bacteria to cross-mucosal barriers, and additionally, can damage effector immune
cells, which prevents clearance of the infection.
 II. Type 2 secretion system (T2SS): Transport folded proteins from the periplasm into the extracellular
environment. Only found on the OM and hence needs Sec/Tat secretion pathways (helpers in the periplasmic space
which transfer protein substrates across the inner membrane)
Ø Proteins transported across the cytoplasmic membrane by the Sec pathway must be folded in the periplasm prior to
export through the T2SS.
Ø They’re usually enzymes: Lipases/proteases
Ø T2SSs are complex and consist of as many as 15 different proteins, which can be broken into four
subassemblies:
1. Outer membrane complex: channel through which folded periplasmic T2SS substrates are
translocated. Channel is composed of a multimeric protein called the secretin. The secretin
has a long N terminus (to interact with the IM platform)
2. Inner membrane platform: composed of multiple copies of at least 4 proteins, is embedded
in the inner membrane and extends into the periplasm, contacting the secretin, pseudopilus, and the ATPase to
coordinate export of substrates
3. Secretion ATPase: ATPase is located in the cytoplasm and provides the energy to power the system
4. Pseudopilus: pseudopili retract to push the folded T2SS substrate through the outer membrane channel: piston
model.
Ø Utilized by GN bacteria that secrete toxins (V.cholerae and P.aeruginoa). Also used by other pathogens to secrete
their enzymes (to help them adapt to their environment): Legionella pneumophila, E. coli (ETEC and EHEC), and
K. pneumoniae.

III. Type 3 secretion system (T3SS):

Ø Long structure ensuring secretion across the 3 membranes (inner, outer, host)
Ø T3SSs have been described as injectisomes and needle and syringe like apparatuses.
Ø Their structure can be broken down into three main components:
1. Base: spans the inner and outer membranes, and is socket like
2. Needle: extends through the secretin complex (OMP) to the extracellular
space. Has a hollow core allowing the unfolded effector to be secreted.
The tip complex on the outer end of the needle is essential for:
Ø Sensing of contact with host cells
Ø Regulation of secretion
Ø Insertion of translocon into host
3. Translocon: Translocon is essential for passage of effectors through host
cell membranes, but not for secretion of effectors outside of the bacterium.
Translocons are assembled upon contact with host cells and form a pore
that is essential for effector delivery.
Ø Yersinia, Salmonella and Shigella

IV. Type 4 secretion system (T4SS):

Ø Related to bacterial DNA conjugation
Ø Secrete substrates into a wide range of target cells, including other bacteria and eukaryotic cells.
Ø Transport substrates (DNA/proteins) directly across both the inner, outer and host membranes.
Ø N. gonorrhea, H.pylori

V. Type 5 secretion system (T5SS): Autotransporter: secrete themselves

Ø The substrate with its autotransporter enters the periplasmic area via the Sec system.
Ø The autotransporter folds forming a B-barrel, which inserts into the OM to form a channel to allow the substrate to
leave.
Ø Because protein secretion by T5SSs only occurs in the outer membrane, these proteins must first be translocated
across the inner membrane and into the periplasm in an unfolded state by the Sec apparatus Therefore, T5SS
proteins carry an N‐terminal Sec signal sequence that is cleaved off as they pass into the periplasm.
Ø Important for pathogens secreting toxins that are very vital for their virulence, receptor-binding proteins that
participate in cell-to-cell adhesion and biofilm formation hence cannot wait for a secretion system to be assembled.