Helicobacter pylori Multitalented

Adaptation of Binding Properties

Sara Henriksson

Department of Medical Biochemistry and Biophysics

Ume 2012

Responsible publisher under Swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-487-4
ISSN: 0346-6612 New series nr: 1524
Cover: Helicobacter pylori adhering to epithelial cells of gastric mucosa, made by
Stefan Lindstrm
Elektronisk version tillgnglig p http://umu.diva-portal.org/
Tryck/Printed by: VMC-KBC, Ume University
Ume, Sweden 2012

The years of anxious searching in the dark, with their intense longing, their
intense alternations of confidence and exhaustion and the final emergence
into the lightonly those who have experienced it can understand it.
Albert Einstein

To My Family!

TABLE OF CONTENTS

ABSTRACT ................................................................................................................ III
LIST OF PAPERS ........................................................................................................ V
ABBREVIATIONS .................................................................................................... VII
ENKEL SAMMANFATTNING .............................................................................. VIII
INTRODUCTION ........................................................................................................ 1
HISTORY ..................................................................................................................................... 1
GENERAL FACTS ON H. PYLORI ................................................................................................ 1
Infection and colonization of the stomach .............................................................. 2
Motility and chemotaxis ................................................................................................... 3
Urease ...................................................................................................................................... 3
TRANSMISSION, EPIDEMIOLOGY, AND DISEASE OUTCOME ................................................. 4
Transmission and epidemiology ................................................................................... 4
Disease progression and outcome ............................................................................... 5
GENETIC AND GEOGRAPHICAL DIVERSITY ............................................................................ 7
VIRULENCE FACTORS ................................................................................................................ 9
cagPAI .................................................................................................................................... 10
VacA ........................................................................................................................................ 10
OUTER MEMBRANE PROTEINS ............................................................................................. 11
BACTERIAL ADHERENCE ....................................................................................................... 12
Receptor specificity .......................................................................................................... 13
Detachment ......................................................................................................................... 13
H. pylori adherence and detachment ....................................................................... 14
BabA ....................................................................................................................................... 16
SabA ........................................................................................................................................ 19
Mucin interactions ............................................................................................................ 20
THE RECEPTORS FOR H. PYLORI .......................................................................................... 21
The ABH antigens ............................................................................................................. 21
Lewis antigens .................................................................................................................... 23
AIMS OF THE THESIS ............................................................................................................. 24
RESULTS ................................................................................................................... 25
PAPER I .................................................................................................................................... 25
PAPER II .................................................................................................................................. 27
PAPER III ................................................................................................................................. 30
PAPER IV ................................................................................................................................. 31
DISCUSSION AND CONCLUDING REMARKS ................................................... 33
ACKNOWLEDGEMENTS ....................................................................................... 36
REFERENCES ........................................................................................................... 38

ii

Abstract
Helicobacter pylori infects and persists within the stomach of most of the
worlds population causing chronic inflammation of the stomach that can
result in gastritis, peptic ulcer disease and cancer. Adherence to gastric
epithelial cells is important for the establishment of infection and is mainly
mediated by the two adhesins BabA (Blood group Antigen Binding Adhesin)
and SabA (Sialic Acid Binding Adhesin). BabA binds the ABO/Leb blood
group antigens (Leb), and SabA binds to sialylated antigens. Both adhesins
demonstrate high-affinity to their receptors. This very strong protein-glycan
type of interaction is both complex and also seemingly contradictive within
the dynamic environment of the stomach. This is so because the high
turnover rate and exfoliation of the gastric epithelium would otherwise have
managed to shed and discard of attached bacteria into the acidic and
bactericidal stomach lumen.
We have found that BabA mediated binding is regulated by acid and binding
to Leb is inactivated at low pH, but recovers when pH is neutralized. The
SabA adhesin behaves similarly, which suggests that this reciprocity in
binding properties is a unique and important feature of H. pylori. The acid
lability of the binding interaction was traced to specific amino acid positions
in the BabA protein. These mutations were also found to correspond to
differences in local acidity of different parts of the stomach and to human
populations with different patterns of gastric disease. Mutations in similar
amino acid positions were identified in BabA also from a 5-year animal
infection study. Our hypothesis is that when BabA is exposed to a pHgradient the binding is gradually decreased and lost at lower pH, i.e., when
the epithelial cells are discarded and shed into the acidic lumen. Such acid
regulation of binding propertied allows the bacteria (and hence the infection)
to detach from the dying cells and by their flagella bacteria can swim back to
the intact stomach wall and hence, recycle the infection. Thus, H. pylori can
take advantage of pH-regulated binding affinity of its adhesins for functional
recycling and continuous adaptation to the stomach environment in order to
maintain persistent life-long infection.
H. pylori strains from southern Europe have a lower prevalence of Lebbinding ability. Seventy-five percent of strains in Sweden but only 40% of
strains in Spain bind Leb. Among the Spanish isolates, we identified one
strain, 812, that binds poorly to Leb but adheres strongly to histo-tissue
sections of gastric mucosa. This binding was not perturbed by removal of
sialylated carbohydrate, and, therefore, SabA binding sites. These novel
results suggest alternative BabA-mediated binding properties and receptors

iii

for H. pylori. We first identified this new receptor as a glycolipid that was
independent of secretor phenotype, i.e., Leb, in secretions such as saliva and
milk. However, glycan arrays demonstrated stronger binding of 812 to blood
group A and B antigens compared to blood group O, the Leb/ALeb ratio on
glycan array was 0.65 compared to 1.18 for the reference strain 17875/Leb.
These results suggest that some H. pylori strains have adapted their binding
properties to individuals of blood groups A and B.
To investigate the impact of secretor status in BabA-positive H. pylori
infections, we studied a large group of volunteers from Porto, Portugal, for
factors that could affect adherence. We found a significant correlation
between BabA expression and other virulence factors such as CagA and
VacA, pre-dysplastic appearance, and positive secretor status. BabAmediated in vitro adherence of H. pylori to the gastric tissue correlated with
positive secretor status.
Detailed investigations of H. pylori BabA-mediated binding to cells in
culture have been difficult because H. pylori is a human-specific pathogen
and most cell lineages are derived from immortalized cells with dysplastic
glycosylation
expression
patterns.
We
transfected
cells
with
glycosyltransferases necessary for expression of the Leb or Lea blood group
antigens; Leb-expression levels were correlated to binding of H. pylori.
Thus, these cell lineages could be used for detailed studies of BabA-mediated
binding and what effect this interaction has on the host cell.

iv

List of papers
This thesis is based on the following papers:
I. J. Bugaytsova*, O. Bjrnham*, S. Henriksson*, P. Johansson*,
M. Mendez*, R. Sjstrm, K. Brnnstrm, C. Aisenbrey, A. Shevtsova,
G.Bylund, J. Mahdavi, J. gren, D. Ilver, R. H. Gilman, A. Chowdhury,
A. K. Mukhopadhyay, L. Engstrand, S. Oscarson, C. G. Kelly, J. S. Younson,
S. Odenbreit, J. Solnick, G. Grbner, R. Haas, A. Dubois, S. Schedin,
D. E. Berg, A. Arnqvist, and T. Born
pH Regulated H. pylori Adherence: Implications for Persistent Infection and
Disease.
Manuscript in final progress
*These authors contributed equally to this work
II. S. Henriksson, M. Mendez, J. Bugaytsova, K. Brnnstrm, M.
Westermark, J. Nordn, Douglas E. Berg, O. Blixt, S. Teneberg, and T. Born
Clinical isolates of Helicobacter pylori demonstrate alternative BabAmediated binding properties for adherence to the gastric mucosa.
Manuscript in progress
III. M. Azevedo, S. Eriksson, N. Mendes, J. Serpa, C. Figueiredo,
L. P. Resende, N. Ruvon-Clouet, R. Haas, T. Born, J. Le Pendu, and
L. David (2008) Infection by Helicobacter pylori expressing the BabA
adhesin is influenced by the secretor phenotype.
J Pathol, vol. 215, no. 3, pp. 308316
IV. J. Lfling, M. Diswall, S. Eriksson, T. Born, M. E. Breimer, and
J. Holgersson (2008) Studies of Lewis antigens and H. pylori adhesion in
CHO cell lines engineered to express Lewis b determinants.
Glycobiology, vol. 18, no. 7, pp. 494501

Please note the change in name from S. Eriksson to S. Henriksson

Papers not included in this thesis

Y. Y. Fei, A. Schmidt, G. Bylund, D. X. Johansson, S. Henriksson,
C. Lebrilla, J. V. Solnick, T. Born, and X. D. Zhu (2011) Use of Real-Time,
Label-Free Analysis in Revealing Low-Affinity Binding to Blood Group
Antigens by Helicobacter pylori.
Analytical Chemistry, 83(16), pp.63366341.

vi

Abbreviations
BabA
CagA
CBD
FITC
FucT
Gal
GalNAc
GlcNAc
HC
Hop
Hor
HU
Le
Lea
Leb
MALT
OMP
RIA
SabA
Se
SPR
T4SS
VacA

Blood group antigen binding adhesin

Cytotoxin-associated gene A
Carbohydrate binding domain
Fluorescein isothiocyanate
Fucosyltransferase
Galactose
N-AcetylGalactosamine
N-AcetylGlucosamine
High cancer
Helicobacter outer membrane protein
Hop-related
High ulcer
Lewis antigen
Lewis a
Lewis b
Mucosa-associated lymphoid tissue
Outer membrane protein
RadioImmunoAssay
Sialic acid binding adhesin
Secretor
Surface plasmon resonance
Type 4 secretion system
Vacuolating cytotoxin A

vii

Enkel sammanfattning
Redan som barn blir ungefr hlften av jordens befolkning infekterad av
Helicobacter pylori, en kronisk infektion som kan botas med
antibiotikabehandling. De flesta br p infektionen utan tydliga besvr, men
ungefr 10-20% kommer f magsr och 1-2% drabbas av magcancer. I
utvecklingslnder r frekomsten av H. pylori infektion nnu hgre dr
80-90% kan vara infekterade.
Magsyran r toxisk fr H. pylori som drfr koloniserar det mer neutrala
mukusslemlagret som skyddar vr underliggande vvnad. I mukuslagret
simmar de flesta bakterierna omkring men ngra vidhftar till epitelcellerna.
Vidhftning r en viktig del av infektionsprocessen d den mjliggr fr
bakterierna att komma nra inp vra celler och p s stt ta del av dess
nringsinnehll, men ocks fr att kunna utsndra faktorer som kan
pverka, dmpa alt. frndra eller frsjunka vrdens immunfrsvar.
H. pylori anvnder sig frmst av tv proteiner, adhesiner, som uttrycks p
bakteriens membran och vidhftar till epitelcellerna i magen, dessa
adhesiner heter BabA och SabA. Adhesinerna vidhftar till s.k. receptorer p
vrdcellens yta. Receptorerna fr bde BabA och SabA r
kolhydratstrukturer p proteiner och lipider belgna p cellens yta. BabA
binder AB0-blodgruppsantigener, de kolhydrater som bestmmer vilken
blodgrupp vi tillhr, Leb/H1(bg0), ALeb (bgA) och BLeb (bgB). Dessa
blodgruppsantigener associerar vi med rda blodkroppar men de finns ocks
i
magslemhinnan.
SabA
binder
sLex,
speciella
sialylerade
kolhydratstrukturer som upptrder i magen nr den blir inflammerad ssom
vid H. pylori infektion. Vidhftningen av H. pylori till epitelcellerna r
mycket stark vilket blir komplicerat d epitelceller bara lever ngra f dagar.
Bakterierna riskerar drmed att flja med cellerna nr de lossnar och stts
av och p s stt skulle H. pylori infektionen med tiden frsvinna. H. pylori
mste drfr kunna balansera sin frmga att vidhfta och slppa taget fr
att inte skljas bort.
Syftet med denna avhandling var att studera hur H. pylori anpassar sitt
vidhftningsprotein BabA till att binda till AB0-antigen trots de
pH- frndringar som H. pylori utstts fr under infektion. Jag ville ven
studera stammar frn olika befolkningar och underska deras
bindningsegenskaper d vr grupp tidigare visat att mnga isolat inte tycks
binda till blodgruppsantigenerna.

viii

Nr vi studerade bindningen av H. pylori till vvnadssnitt av magslemhinna

vid lgt pH fann vi att vidhftningen minskade kraftigt d bindningsstyrkan
av BabA till blodgruppsantigenerna minskade. Vi testade ven SabA och
fann att ven den bindningsfrmgan var pH-knslig. Lgt pH denaturerar
de flesta proteiner, vi kan dock visa att H. pylori har en unik egenskap med
att den kan terskapa bindningen nr pH igen neutraliseras, H. pylori kan
drigenom teranvnda sina adhesiner. Denna minskning av
bindningsstyrkan var olika fr varje isolat, dr ngra var mycket knsliga och
frlorade nstan all sin bindningsfrmga redan vid pH4 medan andra
kunde binda nda ner till pH2. Vi kunde genom olika frsk spra denna
egenskap till adhesinets proteinsekvens. Vi jmfrde H. pylori som isolerats
frn vre och nedre delen av magscken eftersom dessa har olika pH. Vi
kunde visa att proteinsekvensen hos dessa BabA r ngot olika och r
anledningen till dess frmga att behlla bindningsfrmgan ven vid lgre
pH. Vi kunde se mnster av dessa aminosyrafrndringar i BabA frn
befolkningar som uppvisar olika typer av H. pylori relaterade sjukdomar, en
som ger upphov till lgt pH i magen och en som hjer pH. Vi kunde ven
pvisa med H. pylori infekterade Rhesus apor att dessa specifika aminosyror
kan bytas ut genom sekvensutbyte med ett BabA-liknande protein dvs.
genom s.k. rekombination. Vr hypotes r att H. pylori vidhftar till cellerna
i magslemhinnan och drigenom sitter fast nr cellerna dr och sakta stts
ut och glider mot den sura magsyran i magen. Nr pH sjunker kan bakterien
slppa taget om sina receptorer och drefter simma tillbaka ner till cellerna
fr att terigen vidhfta.
H. pylori som isolerats i sdra Europa har tidigare visat sig ha en lgre
frekomst av bindning till AB0-antigenerna. Vi ville underska hur dessa
H. pylori isolat binder till magslemhinnan. Anvnder de fortfarande BabA,
men har frndrat det fr att binda en ny receptor eller binder de med ett
nytt, tidigare oknt, adhesin? Vi kunde visa att mnga av dessa stammar,
trots att de inte kunde binda, nd uttrycker mycket BabA. Det r ovanligt
att bakterier uttrycker proteiner som de inte har anvndning fr. Vi
underskte drfr om dessa stammar anpassat sig till att binda en helt ny
receptor i magslemhinnan. De flesta av dessa stammar kunde inte binda till
vvnadssnitt av magslemhinna men vi fann en stam som hade en unik
egenskap dr Leb-bindningen var mycket svag men den kunde nd uppvisa
stark bindning till vvnadssnitt av magslemhinna. Denna bindning beror p
BabA men den binder annorlunda jmfrt med vr typstam 17875. Genom
att testa med flera kolhydrater kunde vi visa p att 812 verkar ha frflyttat
sin bindning p blodgruppsantigenerna, dvs. den binder till en annan del p
kolhydratkedjan. Vi kunde ven visa p att det inom denna stam finns en
variant med tv aminosyror frndrade i BabA vilket resulterade i att detta
BabA kade sin bindningsstyrka till blodgruppsantigenerna. Intressant i

ix

sammanhanget r att ven dessa frndringar uppkommit genom

sekvensutbyte med ett annat BabA-liknande protein.
Ungefr 20 % av Europas befolkning r s.k. icke-sekretor och saknar drfr
blodgruppsantigener i trvtska, saliv och brstmjlk, de har inte heller
blodgruppsantigener i sin magslemhinna. Istllet har de en kortare
kolhydrat som H. pylori inte kan binda till och som kallas Lewis a (Lea). De
vriga 80 %, vilka har AB0 blodgruppsantigener i magen, kallas sekretor
positiva. Vi underskte H. pylori infektion hos sekretor positiva fr att se
om de oftare blev infekterade av H. pylori som har BabA. Nr vi studerade
vidhftning H. pylori med BabA till vvnadssnitt frn magslemhinna kunde
vi se att sekretorindivider uppvisade hgre bindning. Vi kunde ocks se att
individer som har blodgruppsantigenerna i magen r i strre utstrckning
infekterade med H. pylori som har BabA. Infektion med BabA-uttryckande
stammar pvisade fler bakterier i nra kontakt med vra celler i epitellagret,
medan de H. pylori stammar som saknar BabA var p strre avstnd frn
epitelcellerna. Vi fann ett samband mellan frekomst av BabA och tv andra
sjukdomsassocierade faktorer hos H. pylori, CagA och VacA-protein
Frekomst av BabA var ocks vanligare hos individer med mer avancerad
sjukdom ssom intestinal metaplasi, ett frstadium till magcancer.
Om man vill studera hur vidhftningen av H. pylori pverkar vra celler kan
man inte anvnda vvnadssnitt d dessa r fixerade och inte lngre levande.
Det finns levande cellkulturer fr att studera H. pylori interaktioner men
dessa har inte ett intakt kolhydratsmnster och r drfr svra att anvnda
till studier av BabA-medierad vidhftning. I mitt fjrde arbete konstruerade
vi celler som uttrycker blodgruppsantigener. Vi kunde ven visa att
H. pylori kan binda dessa celler i olika grad.
Genom att bttre frst hur bakterien vidhftar och drigenom hur man
skulle kunna frhindra att de kommer i kontakt med vra celler s kar
mjligheten att hitta nya principer fr lkemedelsbehandling mot magsr
och cancer.

xi

Introduction

History
Helicobacter pylori infects the stomachs of more than 50% of the worlds
population and has lived in such close association with modern humans
since before they migrated from East Africa more than 58,000 years ago
(Linz et al. 2007). Before the discovery of H. pylori in the early 1980s,
stomach disorders such as gastritis and peptic ulcers were ascribed to bad
diet, too much coffee, or a stressful lifestyle. The disorders were treated
accordingly with drugs such as antacids and proton pump inhibitors to
reduce the acidity of the stomach and thus eliminate the symptoms. Bacteria
had been seen in the stomach as early as 1874, but these findings were
ignored because nothing was believed to survive the acidic environment in
the stomach. In the early 1980s, H. pylori was isolated from the antrum of
patients with gastritis and ulcer disease, and later experiments fulfilled
Koshs postulates and, importantly, antibiotic treatments got rid of the
infection and the inflammation dissapeared (Marshall & Warren 1984). For
this finding, Marshall & Warren were awarded with the Nobel Prize in
Medicine and physiology in 2005. Hence, H. pylori was confirmed as the
cause of gastritis and the more serious peptic ulcer diseases. In
epidemiological studies, H. pylori infection was also found to correlate with
gastric cancer, i.e. H. pylori is considered as an onco-pathogen (Parsonnet et
al. 1991). In 1994, the World Health Organization listed H. pylori infection
as a carcinogen (IARC 1994). The understanding that H. pylori infection is
the causative agent of overt gastric disease opened a paradigm shift that has
completely changed the treatment of stomach disorders, which are now
considered as infectious diseases.

General facts on H. pylori

H. pylori is Gram negative, helical shaped bacteria, and has a tuft of 57
polar flagella located at its distal end. H. pylori is microaerophilic and has its
optimal growth conditions at 519% O2, 510% CO2, 37 C, and high
humidity; similar to conditions that are found in the gastric mucosa.
Although being a gastric pathogen, the high acidity of the stomach juice is
lethal for the bacteria. H. pylori, therefore, resides in the slimy mucus layer
that lines and protects the epithelial cells from the acidic gastric juice.

Infection and colonization of the stomach

H. pylori colonizes the stomach and, in particular, the less acidic antrum
(Figure 1). During disease progression, pH initially decreases due to
hypersecretion and H. pylori might move into the first parts of the intestine,
the duodenum. This region is less resistant to infection and peptic ulcer
could develop. Long-term hypersecretion can cause atrophic gastritis to the
mucosa and even loss of the acid producing parietal cells and higher stomach
pH. Atrophy can also result in a gastric ulcer formation, which is sometimes
a precursor to gastric cancer (described in the section on Disease progression
and outcome). Within the mucus layer, H. pylori is mainly confined to the
100 m of mucus closest to the epithelial cells where pH is more neutral.
Thirty percent are found within the first 5 m and around 20% are found
tightly attached to the cells (Schreiber et al. 2004; Hessey et al. 1990). The
colonization of H. pylori is restricted to the superficial epithelial cells,
colocalizing with the expression of the mucin MUC5AC (Van den Brink et al.
2000; Hidaka et al. 2001). The strict colonization of the superficial zone
might relate to the glandular mucin MUC6 that possesses terminal
1,4-GlcNAc. This structure inhibits cell wall synthesis in H. pylori thereby
making these glandular regions toxic to H. pylori (Kawakubo et al. 2004). In
addition to the extracellular habitat, H. pylori has also been found between
cells, deeper in the tissue, and in intracellular vesicles of both cultured
gastric epithelial cells and in gastric biopsies (Amieva et al. 2002; Necchi et
al. 2007; Aspholm et al. 2006). These invasive bacterial cells can repopulate
the extracellular environment suggesting that the intracellular lifestyle might
be a way for H. pylori to escape the immune system as well as antibiotic
treatment (Amieva et al. 2002 and (reviewed in Dubois & Born 2007).

Figure 1. Simplified anatomical illustration of the stomach

Motility and chemotaxis

For chemotactic orientation, H. pylori senses the pH and bicarbonate
gradient in the mucus and swims away from the gastric juice and towards the
neutral environment close to the epithelial cells (Schreiber et al. 2004).
The spiral shape of the bacteria, caused by specific crosslinking of the
peptidoglycan and coiled-coil-rich proteins, together with flailing
movements of the flagella, enables H. pylori to penetrate the slimy mucus
layer in a cork screw-like motion, a function that is essential for survival
(Eaton et al. 1992; Eaton et al. 1996; Sycuro et al. 2010; Specht et al. 2011;
Ottemann & Lowenthal 2002). The flagella are composed of three structures:
the basal body, the hook, and the filament that ends with a bulb (Chevance &
Hughes 2008). The hook and filament protrude from the basal body and are
covered in a membranous sheath that is contiguous with the outer
membrane, a sheath that probably functions to protect the filament from low
pH or against the host immune response (C. S. Goodwin et al. 1985) and
(reviewed in Lertsethtakarn et al. 2011). The flagella filaments are mainly
composed of the two proteins, FlaA and FlaB (Kostrzynska et al. 1991;
Suerbaum et al. 1993). During assembly of the filament, both FlaA and FlaB
are heavily glycosylated with pseudaminic acid, a carbohydrate that
resembles human sialic acid (Josenhans et al. 2002; Schirm et al. 2003).

Urease
In addition to the chemotactic and swimming capabilities that H. pylori
utilizes to escape the acidic gastric juice, it also possesses the cytoplasmic
enzyme urease. Urease is a large 1.1-MDa complex that neutralizes the local
environment by converting urea to NH3 and HCO3-. NH3 neutralizes protons
in the periplasm and HCO3- acts as a buffer to maintain a pH of 6.1 (Scott et
al. 2007; Boyanova et al. 2011). Lysis of bacterial cells in the surrounding
releases urease and buffers into the microenvironment of the live bacteria,
thus the bacterial cells are buffered both internally and externally (Dunn et
al. 1997; Marcus & Scott 2001). This neutralization of the mucin also changes
its rheological properties and makes it easier for H. pylori to penetrate (Celli
et al. 2009).

Transmission, epidemiology, and disease outcome

Transmission and epidemiology
H. pylori infects around 50% of the population, is acquired during
childhood, and typically persists in the stomach throughout life unless
treated with antibiotics. Overcrowding, poor sanitation, and low economic
standards are the risk factors for acquiring H. pylori infection, which is
inversely correlated with socioeconomic status (reviewed in Khalifa et al.
2010). Because of the improved living standards in developed compared to
developing countries, the infection rate is still much higher in developing
countries where it can sometimes reach 80-90%. Related to this is the birth
cohort effect that is seen in developed countries where the H. pylori
prevalence is much higher in the older generation because of a lower
acquisition rate in the younger generation and wide spread use of antibiotics.
In a study of Peruvian children, 71% were infected by the age of 6 months
(determined by urea-breath test) compared to 1.2% of 6-month-old Swedish
children (determined by serological test) (reviewed in Delport & van der
Merwe 2007). The increased growth rate of developing countries proposes
that the total number of infected individuals in the world will increase even
further (reviewed in Khalifa et al. 2010). Consequently, gastric disease
related to H. pylori infection will also increase.
No natural reservoir has been found to be responsible for the spread of
H. pylori, and it is probably transmitted from person to person through
oral-oral transmission because bile in the fecal environment is toxic for
H. pylori. The most plausible route of transmission is during events of
gastroenteritis where H. pylori can be isolated particularly from vomitus but
also from the air. During such disease, H. pylori can also survive and spread
through diarrhea (Perry et al. 2006); and (reviewed in Delport & van der
Merwe 2007). In developed countries, H. pylori is mainly transmitted in an
intra-familial manner from parents to children but also between siblings. In
the developing world with poor sanitation and other family arrangements,
extra-familial spread is more common (Schwarz et al. 2008); and (reviewed
in Delport & van der Merwe 2007).
In 1020% of infected individuals, the infection progresses towards peptic
ulcer disease, 12% acquire gastric cancer, and <1% acquire mucosaassociated lymphoid tissue (MALT) (Figure 2). Because of the high
prevalence of H. pylori infection, gastric cancer has become the second most
common form of cancer in the world, second only to lung cancer. In Sweden
there were 9323 deaths due to gastric cancer between 1991-2000
(Sonnenberg 2011). However, during the same period, 4373 individuals died

due to bleeding peptic and gastric ulcers that can also be associated with
H. pylori infection. Considering that these numbers represent Sweden, a
country with a relatively low prevalence of H. pylori, they are expected to be
even higher worldwide. However, infection does not necessarily lead to these
disorders and many infected carriers have no symptoms. Because of the long
relationship with humans, some scientists almost consider this a commensal
relationship, and H. pylori infection has suggested to protect against
diseases such as gastroesophageal reflux disease (reviewed in de Vries &
Kuipers 2010).

Disease progression and outcome

During the primary, acute infection, the stomach responds by releasing
mucins and sheds the cells of the superficial cellular layer in an attempt to
remove the bacteria. This is accompanied by hypochlorhydria (low acid
secretion) that can continue for weeks until the acid secretion returns to
normal (Schubert & Peura 2008). For very few individuals the infection is
spontaneously cleared, and it is more common that the bacteria will persist
in the stomach for the individuals lifetime unless treated with antibiotics.

Figure 2. Disease progression of H. pylori infection. Adapted from (Suerbaum & Josenhans
2007)

Hyperchlorhydria-related diseases
All H. pylori infected individuals develop gastritis, primarily of the antrum
where the bacteria mainly colonizes. Gastritis is characterized by an
increased infiltration of mononuclear leucocytes and PMN cells that causes
an inflammation of the mucosa (reviewed in Correa & Piazuelo 2012).
During this stage of disease, the acid secretion from the parietal cells is

increased because of an increase in gastrin production by the G-cells in the

antrum. For most H. pylori infected individuals, the disease progression will
stop here and they might carry the infection unnoticed for the rest of their
lives. Some infected individuals, however, are more sensitive to the high
acidity (low pH) and will develop duodenal ulcers (reviewed in El-Zimaity
2007). Eradication of H. pylori at this step will allow the ulcer to heal
(Graham et al. 1992).
Hypochlorhydria-related diseases
If the infection of the antrum progresses, it will eventually degrade the
G-cells and gastrin secretion will decrease. This causes a reduction in the
flow of gastric juice and the corpus becomes less acidic. H. pylori can then
migrate towards the corpus and cause a corpus-predominant gastritis or
pangastritis, further degrading the parietal cells and eventually causing
corpus atrophy and loss of normal glandular tissue (reviewed in Correa &
Piazuelo 2012). During this step, some individuals develop gastric ulcers,
that, unlike duodenal ulcers, occur in an environment of low acidity (high
pH) (reviewed in El-Zimaity 2007). Intestinal metaplasia is the primary step
towards cancer development and is characterized by a progressive spread
and replacement of the stomach cells by intestinal cells accompanied by a
loss of the normal mucins of the stomach, MUC5ac and MUC6. These are
then replaced by more intestinal-like sialylated and sulphated mucins, such
as MUC2 (reviewed in Correa & Piazuelo 2012). These mucins are secreted
by large vacuoles of the intestinal Goblet cells, and are easily detected with
Alcian blue staining, a typical hallmark of intestinal metaplasia. Intestinal
metaplasia advances into dysplasia that almost irreversibly progresses
towards gastric cancer. During the cancer stage, very few H. pylori have
remained in the stomach due to unfavorable growth conditions. The
prognosis for gastric cancer is poor, usually because it is detected at a late
stage. However, better prognosis is seen for patients receiving anti-H. pylori
therapy simultaneously with surgery (Fukase et al. 2008).
The rate at which this entire process of disease progression occurs is slow,
and if it progresses all the way to gastric cancer this will occur later in life,
usually >60 years of age. The exact mechanism that triggers the progression
towards corpus-predominant gastritis and gastric cancer is not known. Some
host factors such as polymorphisms of IL-1, TNF-, and IL-10 might be
involved. IL-1 is particularly important for gastric cancer development
because of its inhibitory effects on acid secretion. Additional host factors
involve high salt consumption and cigarette smoking (reviewed in
Wroblewski et al. 2010). Bacterial virulence factors such as a prevalence of

CagA and the VacAs1 allele are also correlated with gastric cancer (described
in the section on virulence factors).

Genetic and geographical diversity

To persist in the gastric niche, H. pylori must quickly and constantly adapt
to new environments. H. pylori demonstrates a substantial heterogeneity
and allelic diversity making it one of the most diverse bacterial species
known. No two isolates are alike and, despite being classified as the same
strain, differences are found even in isolates from the same individual at
different time points (Kuipers et al. 2000; Kersulyte et al. 1999; Israel et al.
2001; Falush et al. 2001; Gressmann et al. 2005; Salama et al. 2007). This
diversity was noticed early using simple techniques, and modern
developments of more elaborate genome sequencing have enabled detailed
studies on the mechanisms that confer this variability. Sequence comparison
of seven different genomes identified 4.9 to 12.4% of the total gene pool to be
strain specific, and microarray analyses have found this number to be as high
as 27% (Fischer et al. 2010; Gressmann et al. 2005). Diversification of
H. pylori is conferred by two main processes. First, during replication the
genome is subjected to changes such as point mutations, rearrangements,
and slipped-strand mispairing. Second, these changes are further distributed
between genetic variants within the strain, or between strains in a mixed
infection, by natural transformation and recombination (Figure 3). Because
of inversions, many strains also demonstrate different syntenies (gene
orders) (Lara-Ramrez et al. 2011). The outcome of this diversity is a quasipanmictic population structure where, in a changing environment, one
genetic variant is superior and could surpass the other clones when they are
challenged by changes in the environment (Suerbaum et al. 1998); and
(reviewed in Dorer et al. 2009)
The microenvironment in which H. pylori lives is a constant source of
mutagens such as free radicals and low pH. In addition, H. pylori also uses a
polymerase that does not possess proof reading activity and results in a
hypermutator phenotype when it is overexpressed (Garca-Ortz et al. 2011).
In line with having a small genome, the mechanisms to cope with such high
mutation rate are limited and no homolog for the mismatch repair system
has been identified (reviewed in Dorer et al. 2011). Because of the lack of
many conventional repair mechanisms, homologous recombination is
important for DNA-repair. This is supported by the findings of Dorer et al.
(2010) that upon DNA damage, H. pylori upregulates a lysozyme and genes
involved in natural competence such as the unique comB Type IV secretion
system to acquire new genes from lysed cells in the microenvironment

(Hofreuter et al. 2001; Dorer et al. 2010). The average length of imported
gene fragments is estimated to be 417 base pairs and is much shorter than
that seen in other bacteria (Falush et al. 2001). Interestingly, it appears that
the uptake of external DNA consists of larger pieces that are then digested to
smaller fragments before being imported into the chromosome to further
increase the genetic diversity of the organism (Kennemann et al. 2011; Lin et
al. 2009; Kulick et al. 2008).

Figure 3. Mechanisms of genetic variability in H. pylori. Allelic change can be the result of a
point mutation in an existing gene (1) or acquire of a new gene from other strains by natural
transformation, which could either replace a gene (2) or add a new gene to the genome (3).
H. pylori can turn off expression of genes by phase variation and slipped strand mispairing (4).
Intra-genomic recombination can create either mosaic genes (5) or complete gene conversion
creating two identical genes (6)

In a genome-wide analysis, the average mutation rate was 2.5 105 per site
and year and this can be compared to the recombination rate, 5.5 10-5 per
initiation site and year, measured on the same set of samples (Kennemann et
al. 2011). This mutation frequency is even higher than that seen in the
Escherichia coli mutator phenotype (Bjrkholm et al. 2001). In a previous
analysis of related samples, it has been demonstrated that only 1 out of 650
recombination events gives rise to a loss or gain of a gene, and that
recombination is responsible for three times as many substitution mutations
making it the most important factor in generating diversity (Morelli et al.
2010; Kennemann et al. 2011). Deletion mutants of RecA, the H. pylori
recombinase, are sensitive to both UV and DNA-damaging agents and are
also defective in successful colonization demonstrating the necessity for

recombination in generating the genetic diversity required for a persistent

infection (Amundsen et al. 2008; Thompson & Blaser 1995).
Related to recombination are the numerous restriction enzymes (R-M
system) that H. pylori utilizes to digest and restrict the import of DNA from
other species or strains. Humbert and colleagues (2011) indicated in their
discussion that the recognition sites for the R-M system are not evenly
distributed in the genome indicating a selection for recombination of specific
loci (Humbert et al. 2011). In line with this, it has recently been shown that
the outer membrane proteins are among the highest frequency genes
involved in recombination (Kennemann et al. 2011; Yahara et al. 2012).
The quasi-panmictic population of H. pylori and its long association with
humans since before they migrated out of Africa, has resulted in a diversity
that expands globally (Linz et al. 2007; Falush 2003). Human migrations
have limited the exchange of alleles between strains and isolated groups of
H. pylori ancestral strains have evolved into seven population types that
reflect the migrations of humans: hpEurope, hpEastAsia, hpAfrica1,
hpAfrica2, hpAsia2, hpNEAfrica and hpSahul (Falush 2003; Linz et al. 2007;
Moodley et al. 2009). Interestingly, the cag pathogenicity island (cagPAI)
was acquired after the populations split in Africa as it is completely missing
in the hpAfrica2 strain (Gressmann et al. 2005). These different population
classes thus reflect different genotypes that, to some extent, are responsible
for the dissimilar prevalence of H. pylori-related diseases worldwide. An
example of this is gastric cancer being more common in Japan but duodenal
ulcers being more common in India (reviewed in R. Suzuki et al. 2012).

Virulence factors
Virulence is defined as the ability of a bacterial species to induce disease.
Consequently, the outcome of a bacterial infection is highly dependent on
the prevalence and status of its virulence factors. The genetic diversity and
variability of H. pylori is mirrored in the wide range of virulence factors that
vary by disease, age, country, and ethnicity (Boyanova et al. 2011). To be
defined as an H. pylori virulence factor, the protein must be correlated with
disease both in vitro and in vivo and with epidemiological disease patterns
(reviewed in Lu et al. 2005). Three main virulence factors of H. pylori are
the cytotoxin-associated gene pathogenicity island (cagPAI), the vacuolating
cytotoxin (VacA), and the outer membrane proteins (OMPs). Many of the
OMPs are proposed to be involved in disease-associated mechanisms such as
adherence and manipulation of the immune response. VacA and CagA are,

together with BabA, associated with the more severe cases of gastric disease
(described in the section babA and pathogenesis).

cagPAI
The cagPAI is a pathogenicity island in the H. pylori genome and encodes
numerous genes that, upon cell contact, are expressed and assembled into
the needle-like type 4 secretion system (T4SS) (Rohde et al. 2003). The T4SS
is evolutionarily conserved among many Gram-negatives such as
Agrobacterium, Bordetella, and Legionella, but differs in different
organisms in terms of what substrates are transferred. H. pylori T4SS binds
the integrin 1 receptor that is located on the basal membrane and transfers
the cytotoxin associated gene A (CagA) which is also encoded by the cagPAI
(Odenbreit et al. 2000; Backert et al. 2000; Stein et al. 2000; Kwok et al.
2007; Jimnez-Soto et al. 2009). Once inside the cell, CagA is
phosphorylated on specific EPIYA motifs by host kinases, and
phosphorylated CagA goes on to manipulate the cell by interacting with
numerous host cell proteins. In addition, injected non-phosphorylated CagA
manipulates proliferation and immune response of host cells(M. Suzuki et al.
2009). Cultured epithelial cells respond by forming the characteristic
hummingbird phenotype that is the effect of both cell scattering and
elongation (reviewed in Tegtmeyer et al. 2011). CagA is not considered as a
virulence factor (although the cagPAI is), but it is considered an oncoprotein
and is associated with development of gastric adenocarcinoma (Parsonnet et
al. 1991). H. pylori infections of Mongolian gerbils resulted in more gastric
adenocarcinomas in a CagA-dependent manner and so did mice that were
transgenic for CagA expression (Ohnishi et al. 2008; Franco et al. 2005).

VacA
Vacuolating cytotoxin A (VacA) is a multifunctional secreted cytotoxin. The
vacA gene is found in all H. pylori isolates though there are differences
among the alleles. The s1 allele, especially in combination with the m1 allele,
is highly associated with the risk of developing peptic ulcers and gastric
cancer (reviewed in Palframan et al. 2012). The VacA toxin forms large
vacuoles in gastric cells; however such vacuoles are not seen in biopsies.
VacA localizes to, and exerts effects on, the mitochondria where it triggers
the apoptotic cascade and induces cell death by mitochondrial fission. The
detailed molecular mechanisms for this, however, are not known (Jain et al.
2011) and (reviewed in Palframan et al. 2012). In addition, VacA has been

10

found to bind the integrin subunit CD18 on T-cells and suppressing their
activities (Gebert et al. 2003; Sewald et al. 2008).

Outer membrane proteins

The OMPs of H. pylori comprise about 4% of the genome, more than in any
other bacterial species. They are divided into five families with the largest
group being the Hop family. The Hops are comprised of 21 genes that all
encode a conserved N-terminal motif (AEX[D,N]G) (Alm et al. 2000).
Related to the Hop family are the 11 Hor genes, which are homologous in
sequence to the Hop family but lack the N-terminal motif. Together the Hop
and Hor families form the major OMP family (Alm et al. 2000). The Cterminal domains of the OMPs are homologous and have alternating
hydrophobic and hydrophilic domains that are proposed to form antiparallel amphiphatic -sheets that assemble into a -barrel in the outer
membrane (Bina et al. 2000; Tomb et al. 1997). Phylogenetic analyses based
on the homologous C-terminal domains reveal clustering of certain genes.
High nucleotide similarities in the 5' and 3' regions of the genes indicate a
preference for recombination between these alleles and this has indeed been
show for babA, babB, and babC, and more recently for sabA, sabB, and
hopQ as well (Bckstrm et al. 2004; Solnick et al. 2004; Talarico et al.
2012). Located between the N- and C-terminal domains, and exposed to the
extracellular environment, is the hypervariable region that is unique for each
OMP and is proposed to determine their functions. This variable region not
only varies between the OMPs, but also varies for the same OMP between
different strains (Alm et al. 1999). Because this region is exposed to the
outside of the cell, it is part of the face towards the immune response and is,
therefore, under constant selective pressure to avoid recognition.
In relation to the diversity seen in the variable region, some genes (sabA,
sabB, hopZ, oipA, babB, and babC) are also proposed to have CT repeats that
can be regulated by slipped-strand mispairing to undergo phase variation
(Alm et al. 1999; Tomb et al. 1997; Oh et al. 2006). By readily turning
proteins on and off, the bacterial community is more dynamic and able to
prepare for sudden changes that can occur during the course of the infection.
Although functions are still not assigned to all OMPs, their cellular
localization hints that these proteins are involved in adherence or transport
of nutrients. The first five characterized OMPs were classified as porins, and
two of the OMPs, BabA (HopS) and SabA (HopP), are defined and
characterized as adhesins (Ilver et al. 1998; Mahdavi et al. 2002). Many
others are proposed to be involved in adhesion such as HopZ, HopQ and

11

AlpAB (HopB and HopC) but no receptors for these proteins have yet been
identified (Peck et al. 1999; Loh et al. 2008; Senkovich et al. 2011).
The OMPs are thought to have been acquired as a single gene event that
underwent duplication and divergence before the speciation of H. pylori
because they are also present in their closest relative, Helicobacter
acinonychis, though the gene repertoire is somewhat dissimilar (Gressmann
et al. 2005). The evolution of the OMPs has probably been a continuous
process because some genes are still found as duplicates (hopJ/K and
hopM/N) indicating a recent duplication event (Alm et al. 2000). The
geographic variation that can be seen for other virulence factors of H. pylori
such as CagA and VacA is also seen for the OMPs. Kawai et al. has recently
published a comparison between 20 complete genomes from East Asia,
Europe, West Africa, and among Amerindian populations and found distinct
differences in the prevalence of babC (HopU), which only seems to be
present in the European strains (Kawai et al. 2011).

Adherence
Bacterial cells that are attached to the host cells have a benefit over freeswimming, planktonic bacteria because of the higher supply of essential
nutrients, especially iron, close to the host cells. A tight adherence to the host
cells also enables the bacteria to manipulate and control the immune
response through delivery of their effector molecules and to promote
invasion for an intracellular lifestyle. Adhesins are proteins expressed by the
bacteria that mediate attachment to specific receptors on the host cell
surface. FimH and PapG are the two most described bacterial adhesins are
so called fimbrial adhesins that are found at the tip of fimbriae. Afimbrial
adhesins, such as in H. pylori, are found integrated in the membrane. Such
adhesins are proposed to promote more cellular damage because of their
tighter association with the host cell, and have been demonstrated to be
important for the delivery of effector molecules (reviewed in Kline et al.
2009). The most common receptors for bacteria and viruses are
carbohydrates on glycoproteins and glycolipids. Such interactions are
typically weak, but by combining adhesins and adhering to glycosylated
domains in the host membrane, such as lipid rafts, multivalency increases
the affinity and is commonly known as the Velcro effect (reviewed in
Moran et al. 2009).

12

Receptor specificity
The interactions between adhesins and their cognate receptors determine, to
some extent, the host specificity and tissue tropism (Klemm & Schembri
2000). Some bacteria, like H. pylori, colonize a narrow niche whereas other
bacteria can colonize and spread through multiple niches. A shift in niche is
sometimes preceded by a switch in receptor phenotype that benefits an
adherent clone over the non-adherent clones, and consequently leads to
outcompetition by the adherent clone. A rapid way of acquiring new receptor
specificity is to express multiple adhesins that can easily be switched on and
off to fit the local receptor repertoire. Such multiple adhesins are present in
many bacterial species e.g., E. coli, where different pathogenic strains
possess distinct adhesin-receptor repertoires (reviewed in Kline et al. 2009).
Additionally, allele changes could arise resulting in a change of the protein
structure, either directly in the binding site or at a distant site causing
secondary effects to the conformation or orientation of the binding site. Such
small changes can be acquired by point mutation, recombination, allelic
exchange, or gene rearrangement (Ofek & Doyle 1994). PapG is the adhesin
of the P-fimbriae in uropathogenic E. coli and has been demonstrated to
regulate its tissue tropism by allelic exchange. Initial receptor specificity
studies by Strmberg et al. identified three allelic variants of the adhesin
based on differential recognition of isoreceptors for the globoseries
glycosphingolipids (GbO) (Strmberg et al. 1991). All alleles are specific for
the GbO determinant Gal(1-4)Gal epitope, but different specificities for the
extended GbO structures discriminate among the alleles. The PapGI adhesin
binds GbO3, expressed on uroepithelial cells, though this allele is
uncommon. PapGII, which is correlated with pyelonephritis and bacteremia,
prefers GbO4 that is also found on uroepithelial cells. PapGIII is correlated
with human cystitis and genitourinary infections in cats and dogs and
prefers GbO5, Forssman, or GloboA, the latter of which is found in the
human urinary tract (Strmberg et al. 1991; Lindstedt et al. 1989) (reviewed
in Lane & Mobley 2007). Such drifting in receptor specificity is not restricted
to bacteria as viruses are also known to possess such capability. Recently,
point mutations in the hemagglutinin receptor binding site of avian
influenza virus (H5N1) were shown to promote airborne transmission
between ferrets (Herfst et al. 2012; Morens et al. 2012).

13

Detachment
Despite the importance of attachment for colonization, it is also an obstacle
that needs to be overcome under certain circumstances. A tight adherence
could trigger a tremendous immune response that could be risky for the
attached bacteria. A strong permanent attachment would also limit the
possibility to inhabit other surfaces. An obvious way for a bacteria to cope
with this is to simply turn off expression of the adhesin, but not all situations
support this type of regulation. Such an environment is in the urinary tract,
where a loss of adherence during conditions of low urinary flow would
present a risk for the bacteria of being flushed out during urination. Both
commensal and uropathogenic E. coli express the FimH adhesin, the tip
protein of Type I fimbriae. The most common receptor for FimH is trimannose and enhanced binding is accomplished by multivalency.
Uropathogenic strains, especially those causing pyelonephritis, are found
with a different allele that, in addition to the binding of tri-mannose, also
can bind mono-mannose that is common in the urinary tract, binding to
mono-mannose had 10 times higher affinity (Sokurenko et al. 1997). Such
FimH adhesins increase the affinity for mono-mannose under conditions of
high shear stress, such as during urination, and enable the bacteria to
withstand 10-fold higher flow rates. The bacteria can then revert back to the
lower affinity state when the flow has ceased (Thomas et al. 2002).
Consequently, nearly permanent high affinity binding is limited to just those
situations in which it is required. However, this type of regulation requires
changes in flow conditions that not all colonized sites experience. Neisseria
meningitidis uses Type IV pili for colonization of the nasopharynx
epithelium where it multiplies to microcolonies and then detaches and
spreads to a new host or causes an invasive disease. It has recently been
shown that detachment of N. meningitidis from epithelial surfaces is
phosphorylation-dependent and caused by increased expression of the pptB
gene during the multiplication phase. Phosphorylation of a serine on the pili
causes destabilization of the pilus fiber and subsequent detachment, thus
enabling the bacteria to restart the infectious cycle (Chamot-Rooke et al.
2011).

H. pylori adherence and detachment

During infection, around 20% of the H. pylori infectious load bacterial cells
are found adherent to human gastric mucosa (Hessey et al. 1990). The
adhesins of H. pylori are membrane-bound, and this enables a more
intimate contact with the host cells compared to fimbrial adhesins.

14

Adherence of H. pylori is beneficial for the colonization, establishment, and

propagation of infection due to three factors (reviewed in Amieva & El-Omar
2008): 1) adhesion enables an intimate contact with host cells that can
induce cellular damage and inflammation that will lead to an increased
release of nutrients by the host cell; 2) adhesion helps the bacteria to avoid
mechanical clearance and supports invasion and persistence; and 3) the host
cell surface could be used as a site for H. pylori replication as recently
demonstrated by Tan et al. 2009. Nonetheless, the majority of H. pylori cells
are found free-swimming, which can be rather challenging for the bacteria.
In the outer most part of the mucin layer, the immune response is less active
but the bacteria are at increased risk of swimming towards the acidic lumen.
Therefore, attachment would appear to be a solution to this problem.
However, close to the epithelial cells peristaltic movements and immune
responses are more active and the bacteria are at risk of being cleared. Also,
epithelial cells have a short lifespan of around 3 days, and they carry any
attached bacteria with them as they shed (Hidaka et al. 2001 and reviewed in
Moore et al. 2011). Thus, for H. pylori, the attachment dilemma is
particularly evident, and tight regulation of attachment and detachment is
necessary for the bacteria to persist in the proper environment. In addition
to phase variation, that disassembles the bacterial adherence machinery, we
have recently identified a functional regulation of H. pylori adhesins by
affinity reduction in an acidic environment. This helps the bacteria to detach
from the epithelial cells before they reach the acidic lumen enabling them to
use chemotaxis for proper orientation back towards the epithelium
(Paper I). Adherence by H. pylori must also be flexible enough to adapt to
changes in the receptor repertoire that occurs during infection and is
triggered by both the bacteria and the host cells. To date only two H. pylori
adhesins are characterized; BabA and SabA. BabA binds almost
constitutively to ABO/Leb blood group antigens whereas the more phasevariable SabA binds inflammation-associated sialylated structures (Born et
al. 1993; Ilver et al. 1998; Mahdavi et al. 2002; Aspholm-Hurtig et al. 2004).
In addition to adherence to the epithelial cells, BabA and SabA are known to
interact with their carbohydrate receptors when they are expressed on the
mucins that serves to protect the gastric mucosa from acidity (described in
the section Mucin interactions) (Van de Bovenkamp et al. 2003). Numerous
additional OMPs have been demonstrated to be adherence-related, such as
HopZ, AlpAB, HorB, and HopQ, though no clear mechanism or receptor has
been described (Snelling et al. 2007; Loh et al. 2008; Peck et al. 1999;
Odenbreit et al. 2002; Senkovich et al. 2011).

15

BabA
The Blood group Antigen Binding Adhesin, BabA, mediates binding to the
ABO/Leb blood group antigens. The first hint of the existence of an adhesin
was provided by application of FITC-labeled H. pylori to paraffin-embedded
tissue sections of human gastric mucosa and observed adherence to the
foveolar epithelial cells (Falk et al. 1993). Inhibition with various substrates
such as human colostrum from secretors and non-secretors, antibodies, and
glycoconjugates identified the receptor as the H1 and Lewis b (Leb) blood
group antigens (Born et al. 1993). H1/Leb are terminal carbohydrate
structure that defines blood group O. They are found on red blood cells and
on gastro-intestinal (GI) epithelial linings such as in the stomach (described
in the section The receptors for H. pylori). Related to H1 and Leb structures
are the A and B blood group antigens, but neither of these structures were
identified as receptors, nor did H. pylori bind the related Lea structure,
demonstrating specificity for a fucose moiety (Born et al. 1993). The
cognate adhesin, Blood group Antigen Binding Adhesin (BabA), was
identified by use of the re-tagging technique (Ilver et al. 1998). This
technique utilizes a cross-linker attached to the receptor glycoconjugate.
Binding of this receptor exposes the adhesin for cross-linking and enables
detection by streptavidin-biotin via SDS-PAGE and mass spectrometry to
identifies the cognate adhesin. The strain used in these studies, CCUG17875,
was found to contain two babA alleles of which one is silent due to defects in
the translational sequence and signal peptide (Bckstrm et al. 2004; Ilver et
al. 1998). This allele was called babA1 and the allele encoding a functional
BabA protein was called babA2. Deletion of babA2 confirmed that the BabA
protein is the functional adhesin for binding to H1/Leb. During these
studies, another gene was identified and called babB. It was homologous to
babA at the 3' and 5' ends but divergent in the middle (Ilver et al. 1998).
Both of these proteins were later classified as belonging to the Hop family of
OMPs (Alm et al. 2000).
H1 and Leb were identified as receptor structures for BabA, whereas ALeb
and BLeb did not demonstrate any binding to the Peruvian isolate that was
used in the original studies (Born et al. 1993). When Aspholm-Hurtig et al.
investigated the worldwide receptor specificity for BabA, they identified
strains that, in addition to Leb/H1, could also bind the ALeb and BLeb
structures. These strains were termed generalists while those that were
restricted to Leb/H1 were termed specialists (Aspholm-Hurtig et al. 2004).
Interestingly, the specialist strains originated from parts of the world, such
as South America, where O is the predominant blood group. The affinity of
BabA for its receptors is high, with values in the M to nM range, though

16

they vary substantially between clinical isolates (Aspholm-Hurtig et al.

2004). Interestingly, the affinity for H1/Leb is stronger than for ALeb or
BLeb, and this could explain the higher risk of peptic ulcer disease in blood
group O individuals (Aspholm-Hurtig et al. 2004 and reviewed in Anstee
2010). In addition to the BabA-mediated attachment to the epithelial cells,
BabA also interacts with the mucins MUC5AC and MUC1 (described in the
section Mucin interactions).
Clinical isolates have exceedingly diverse babA sequences, with the highest
variability in the middle domain. In addition, not all strains express BabA,
and
not
all
expressed
BabA
proteins
are
functional
(Paper II) (Aspholm-Hurtig et al. 2004). Thus, the presence of a babA gene
is not necessarily evidence of a functional BabA protein. The mechanism by
which this diversity is obtained is not known, but phylogenetic analyses of
the BabA variable region in different populations reveals heterogeneous
selective pressures, such as escape from host immune response, receptor
specificity, and affinity, that act on the protein (Aspholm-Hurtig et al. 2004).
During H. pylori infection studies in Rhesus macaques, mice, and gerbils,
expression of BabA was frequently lost (Solnick et al. 2004; Ohno et al. 2011;
Styer et al. 2010). The loss of BabA in Rhesus macaques might be because of
a higher inflammatory response to BabA-expressing adherent bacterial cells.
However, it could also be because of the lower prevalence of the ABO/Leb
blood group antigens in gastric mucosa during infection and hence selection
against BabA-expression (Lindn et al. 2008). Interestingly, BabA is not
immunogenic in humans indicating an evolved function to resist human
immune responses (Kimmel et al. 2000; Haas et al. 2002).
babA, babB, and babC recombination
Phylogenetic analysis of the OMP C-terminal regions reveals a close genetic
relationship between BabA, BabB, and BabC where BabA and BabC are most
similar. Despite this similarity, only BabA has been assigned a function (Alm
et al. 2000). BabA and BabB are most prevalent, and BabC is less so and was
recently associated with strains of European origin (Kawai et al. 2011). The
three alleles are commonly found at three different loci, A, B, or C, that are in
each third of the genome (Tomb et al. 1997). The existence of three similar
OMPs suggested that H. pylori utilizes them to switch antigenic appearance
to avoid the immune response (Alm et al. 2000). Indeed, after the first
genome comparison between strains 26695 and J99, it was clear that babA
and babB were found at inverted loci. In addition, 26695 has the babC gene
at the C locus, which J99 is missing (Alm et al. 1999). Comparisons of
clinical isolates demonstrated that these alleles frequently recombine and
switch loci to form duplicates, deletions, and chimeric genes at a frequency

17

of about 3 10-6 gene conversions per cell division (Colbeck et al. 2006;
Pride & Blaser 2002; Hennig et al. 2006; Amundsen et al. 2008).
Interestingly, the C-terminal regions of BabA and BabB are more
homologous within a genome/strain than between genomes/strains, and this
suggests a concerted evolution between BabA and BabB (Pride & Blaser
2002). No such analysis has been done for BabC, but the close relationship
between these genes suggests similar results. The Leb-binding function of
BabA is not affected by the loci from which it is expressed (Hennig et al.
2006). However, expression of BabA from the B or C loci could result in a
gain of function because BabA could then make use of their CT-repeats for
faster switching of expression (Bckstrm et al. 2004; Colbeck et al. 2006).
The recombination frequency is very high, and a seemingly homogeneous
population from a single clone can retain a subpopulation of single clones
that demonstrate an altered arrangement of the bab genes caused by
recombination (Colbeck et al. 2006). Such rearrangements can also revive
lost BabA expression demonstrating that loss of a functional BabA is
reversible (Bckstrm et al. 2004). Recombination is also seen during
experimental infections, such as in the Rhesus macaque model, where the
expression of BabA is sometimes switched off by babA-babB recombination.
BabA and pathogenesis
The pathogenic importance of an ABO/Leb-mediated attachment by BabA
was demonstrated in H. pylori-infected, Leb-transgenic mice that had higher
inflammatory scores compared to their non-transgenic littermates (Falk et
al. 1995; Guruge et al. 1998). This study was later confirmed in Mongolian
gerbils where the animals infected with a babA mutant had lower infiltration
of inflammatory cells and a reduced cytokine response (Sugimoto et al.
2011). In 1999, Gerhard et al. updated the old Type 1 (CagA+, VacA+) and
Type 2 (CagA-, VacA-) classifications of H. pylori by adding a third
denominator, babA2 (Gerhard et al. 1999). Genotypic studies demonstrated
that babA2 was correlated with cagA and the more pathogenic vacAs1 allele
and was significantly associated with adenocarcinoma and better
discriminated against gastritis compared to the conventional Type 1 vs. 2
definition. These results indicated that BabA expression has an important
role in the disease process, and these strains were termed triple-positive
strains (Gerhard et al. 1999). Many groups tried to repeat the task of
correlating babA2 with gastric disease but obtained inconclusive results
(reviewed in Yamaoka 2008). Because the prevalence of babA2 is not
equivalent to expression of BabA, additional studies have investigated the
correlation with BabA expression. Such studies demonstrated a correlation
between BabA, CagA, and VacA, and also to more severe gastric disease such
as intestinal metaplasia (Odenbreit et al. 2009; Yamaoka et al. 2002;

18

Yamaoka et al. 2006; Azevedo et al. 2008) (Paper III). Interestingly,

Fujimoto et al. demonstrated a stronger correlation with duodenal ulcer and
gastric cancer for strains with low levels of BabA expression than for those
with high or no expression (Fujimoto et al. 2007).
Evidence for the functional correlation between BabA, CagA, and VacA is
now being elucidated. Adherent H. pylori are frequently found associated
with the intercellular junctions where they would have immediate access for
penetration of the mucosa (Amieva et al. 2003). Such tight adherence,
mediated by BabA (or other adhesins), simplifies the secretion and delivery
of VacA to host cells that could trigger separation of the cellular junctions
and facilitate penetration of H. pylori through the intercellular space. On the
basal side, these bacteria have access to the T4SS integrin 1 receptor that is
located where the bacteria can deliver CagA (reviewed in Wessler & Backert
2008). Indeed, CagA-positive bacteria are found more tightly associated with
epithelial cells during infection (Camorlinga-Ponce et al. 2004). This
hypothesis has recently been supported by Ishijima et al. who demonstrated
that the BabA-Leb interaction increases T4SS-mediated induction of mRNAs
for pro-inflammatory cytokines and precancerous factors in cell cultures,
and stimulates intracellular levels of phosphorylated CagA (Ishijima et al.
2011). BabA, SabA, and CagA have also recently been demonstrated to share
regulatory mechanism upon interaction with mucins (Skoog et al. 2012).
Although not all studies were able to demonstrate a correlation between
BabA, CagA, and VacA in disease progression, there is a strong indication
that BabA itself can cause damage to the epithelial cells. A recent finding has
suggested that BabA is involved in induction of double-strand breaks and
such grave DNA damage could tilt the disease state towards more aggressive
pathogenesis (Toller et al. 2011). However, it is difficult to determine if these
effects are mediated by direct binding to the receptor, or are secondary and
caused by the tighter association with host cells.

SabA
SabA, the Sialic-acid binding adhesin, was discovered some years after BabA.
Mahdavi et al. observed adherence of the CCUG17875 babA2 mutant to
human gastric mucosa from a gastritis patient (Mahdavi et al. 2002). The
receptor was characterized to be sialyl-dimeric-Lewis x antigen (sdiLex), but
more detailed analysis of the binding specificity has identified the minimal
binding epitope to be NeuAc2-3Gal with a polymorphism for the core chain
(Mahdavi et al. 2002; Aspholm et al. 2006). Such sialylated antigens are
expressed by inflamed tissues to recruit neutrophils and are, therefore,
triggered by H. pylori infection. SabA expression is highly variable with both

19

a PolyT tract in the promoter and CT-repeats in the coding region, and it has
also recently been shown to be controlled by the acid-responsive ArsRS twocomponent system that also regulates urease and carbonic anhydrase (A. C.
Goodwin et al. 2008). Similarly to the recombination between babA and its
related alleles, sabA can also recombine with its related allele sabB, and to
some extent with hopQ (Talarico et al. 2012). This occurs at a frequency of
1.4 10-9 gene conversions per cell generation, which is lower than that seen
for babA and babB recombination.

Mucin interactions
Mucins are large, heavily O-glycosylated proteins and represent the major
component of the mucus layer. This layer covers and protects mucosal
surfaces from the external environment such as in the stomach where it also
serves to maintain the pH gradient (Bhaskar et al. 1992). Healthy gastric
mucosa contains the two secreted gel-forming mucins, MUC5AC and MUC6,
and the cell surface-bound MUC1. MUC5ACac is the major constituent of the
mucus and is secreted from the surface foveolar cells and pit cells and is
reflective of their glycosylation pattern. MUC6 is secreted from the glands in
lower amounts (Phillipson et al. 2008). H. pylori specifically interacts with
MUC5AC via the BabA adhesin, and this interaction triggers proliferation
and faster division of the bacterial cells (Van de Bovenkamp et al. 2003;
Skoog et al. 2012). Contradictory reports have been written about changes in
distribution of these mucins during H. pylori infection. Byrd et al. reported a
downregulation of MUC5AC and upregulation of MUC6, but later studies by
Teixeira et al. and Van de Bovenkamp et al. saw no such changes (Byrd et al.
1997; Teixeira et al. 2002; Van de Bovenkamp et al. 2003). Though the
results are inconsistent, similar effects are seen in acute infections in Rhesus
macaques (Cooke et al. 2009; Marcos et al. 2008). A downregulation of
MUC5AC could be triggered by the bacteria, as it would thin the mucin layer
and diminish the Leb structures in the mucus that otherwise might hinder
the bacterial cells from reaching the cell surface. The increase in MUC6 could
reflect a host response because, as described previously, H. pylori is unable
to colonize MUC6-producing regions due to the toxic effects of the 1,4GlcNAc moieties (Kawakubo et al. 2004) and (reviewed in Moran et al.
2011).
MUC1 is a membrane-bound mucin found on the surface epithelium and
glandular tissues. Both BabA and SabA can mediate binding to Muc1 and
Muc1 could potentially mediate cell signaling through its cytoplasmic tail
(Lindn et al. 2009) and (reviewed in Moran et al. 2011). However, the
interaction of H. pylori with MUC1 triggers shedding of its highly

20

glycosylated extracellular domain, thus acting as a releasable decoy. MUC1

also blocks bacterial cells that are unable to bind the mucin and acts as a
steric hindrance at the cell surface (Lindn et al. 2009; McGuckin et al.
2007). This is supported by findings that alleles coding for shorter versions
of MUC1 can enable a closer contact between H. pylori and host cells and are
associated with more severe gastritis and gastric cancer (Carvalho et al. 1997;
Vinall et al. 2002).

The receptors for H. pylori

Surrounding the surface of all cells is the glycocalyx, a halo-like structure
that is a result of substantial post-translational glycosylation that takes place
in the cell. Glycosylation is the most diverse post-translational modification
because it is not encoded in the genome but is accomplished by the action of
several glycosyl transferases that polymerize monosaccharides into long
chains of polysaccharides. These chains can be found as glycoconjugates,
connected to proteins or lipids, but also as free glycans. The polysaccharide
chains vary in linkages, are linear or branched, and can have different
endings such as acetylation or sulfation. It is estimated that 12% of the
human genome is involved in the synthesis of glycans, and that more than
70% of human proteins are glycosylated (Apweiler et al. 1999). In addition,
glycosylation is tissue and cell specific, and glycosylation levels change in
response to intrinsic or extrinsic factors and during disease progression. One
such disease is cancer where several specific glycans are involved in the
pathophysiological steps that are characteristic of malignancy (reviewed in
Fuster & Esko 2005). Recent advances in new technologies for studying
glycans has resulted in high sensitivity mass spectrometry, high throughput
HPLC, and glycan arrays (Adamczyk et al. 2012; Rillahan & Paulson 2011;
Blixt et al. 2004). This has made glycobiology an emerging field in not only
the diagnosis and treatment of cancer, but also, with the use of glycan arrays,
in the study of pathogens and their receptor specificities.

The ABH antigens

The ABH-blood group antigens are the terminal carbohydrate structures that
define the human blood groups. Despite the name, most of the ABH antigens
are not found on erythrocytes but instead in other tissues such as gastric
mucosal cells and their secretions, saliva, milk, and tear fluid where they are
designated histo-blood group antigens. These terminal structures are found
on four different poly-N-acetyllactosamine core chains: Type 1 (Gal13GlcNAc1-R) and Type 2 (Gal1-4GlcNAc1-R), which are N-linked, Type 3

21

(Gal1-3GalNAc1-R), which is O-linked, and Type 4 (Gal1-3GalNAc1-R),

which is found on glycolipids. The majority of the antigens are found on
Type 1 and 2 core chains. Differential expression of these core chains
together with the substrate specificity of the glycosyl transferase determines
what terminal structure will be formed. In the stomach, the ABH antigens
are found on the Type 1 chain in epithelium and foveolar cells and on the
Type 2 chain in the deeper glands, though the border between the two is not
always obvious. Type 3 is found on stomach mucins. BabA-mediated binding
of H. pylori is restricted to the terminal structures of the Type 1 and Type 3
chains, and there is no binding to the Type 2 chain or its derivatives.
There are three antigens defining the blood groups A, B, and H (blood group
O) in which H is a disaccharide and the precursor of the A and B
trisaccharides. These antigens are synthesized by enzymes encoded in three
loci (H, Secretor (Se), and ABO). The H-determinant is formed by an 1-2
fucosyl transferase (FucT) encoded by the H or Se locus. The H 1-2FucT is
almost 100% prevalent in humans and forms the H-antigen on Type 2 or
Type 4 core chains on erythrocytes. However, in epithelial cells and in
secretions the Se 1-2FucT is the active enzyme and forms the H-antigen on
Type 1, 2, and 3 core chains.

The ABO locus is the blood group-determining locus because it contains the
A and B alleles as well as the O allele that encodes a non-functional enzyme.
The A and B alleles each encode the A or B transferase that strictly modifies
the fucosylated galactose of the H-antigen with a GalNAc (A) or Gal (B). The
A and B transferases are not 100% efficient, resulting in residual levels of A
and B structures in the tissues.
Secretors and non-secretors
Around 20% of Caucasians are homozygotes for a non-functional Se 12FucT, mainly caused by a G428A point mutation in the gene that results in
an early stop-codon and a non-functional protein (Kelly et al. 1995). Because
the A and B transferases are specific for the H-determinant, this results in an
almost complete lack of all histo-blood group antigens on epithelial cells and
in secretions. Such individuals are termed non-secretors. Another point
mutation in the Se 1-2FucT, A385T, renders the enzyme weaker and fewer
histo-blood group antigens are formed. This phenotype is more common in
Asia and these individuals are termed weak secretors (Henry et al. 1996;
Lindn et al. 2008 and reviewed in Anstee 2010). Neither of these
phenotypes shows any obvious negative side effects. On the contrary, nonsecretors are protected from infection by some Noroviruses that use the ABO

22

antigens as receptors (Thorven et al. 2005; Carlsson et al. 2009). In very rare
cases, there is a complete lack of both the H and Se transferases, termed the
Bombay phenotype, and such individuals are completely devoid of ABO
antigens but they do not demonstrate any obvious side effects.

Figure 4 Schematic illustration of the synthesis of the ABO/Leb-antigens

Lewis antigens
Related to the ABH blood group antigens are the Lewis antigens (Figure 4).
These are the result of concerted action between the Se transferase and the
Lewis (Le) transferase, which is encoded by the Le locus. The Le transferase
is a fucosyl transferase that modifies the ABO antigens (H1, A1, and B1) by
adding a fucose in an 1-3 or 1-4 linkage to the sub-terminal GlcNAc

23

thereby forming Leb, ALeb, and BLeb, respectively. The concurrent

expression of the Le transferase and Se transferase converts almost all ABO
antigens expressed on the stomach epithelium into Le antigens. In nonsecretors, Lea is formed from the unmodified Type 1 core chain and is,
therefore, the main antigen found in the stomach mucosa of non-secretors.
Lea is not a substrate for the Se-transferase, thus minute levels of Lea will,
therefore, be found in secretors. The Le transferase also modifies Type 2
chains generating Lex (corresponding to Lea) and Ley (corresponding to
Leb). Because of the location of these core chains, such antigens are
predominantly found in the glandular region. Le antigens can be detected on
erythrocytes but, despite the specificity of the Le transferase for Type 2
chains, it does not modify ABH blood group antigens on erythrocytes. These
Le antigens are instead glycolipids that have passively adsorbed onto the
membrane.
Ten percent of the European population is Lewis-negative in that they lack a
functional Le transferase. Together with the phenotypes of the secretor
transferase (secretor, weak secretor, or non-secretor), this generates four
different phenotypes designated by the antigens that are expressed: Lea-b-,
Lea-b+, Lea+b-, and Lea+b+.
The lack of terminal structures on the Gal of Lea and Lex makes them targets
for modifications such as sialylation and sulfation. Such antigens are
common in healthy gastric mucosa of non-secretors, but not of secretors.
However, their expression is triggered during inflammation to recruit
neutrophils and is also triggered by H. pylori during infection (Sakamoto et
al. 1989; Mahdavi et al. 2002; Lee et al. 2006; Lindn et al. 2008; Marcos et
al. 2008).

Aims of the Thesis

1.

To investigate how H. pylori adherence to human gastric mucosa is

affected by low pH.
2. To investigate adherence mediated by the European non-Leb
binding strains.
3. To investigate how secretor status affects the H. pylori infection.
4. To create transgenic cell-lineages for detailed investigation into
BabA-Leb interactions.

24

Results

Paper I
pH Regulated H. pylori Adherence: Implications for Persistent
Infection and Disease
This study focused on the attachment dilemma of H. pylori, where a tight
adhesion to the gastric cells during infection could be detrimental because
permanently attached bacteria would be lost when dead cells shed into the
acidic lumen.
We initially observed that in vitro adhesion of FITC-labeled H. pylori strain
17875 was lowered if the adhesion was carried out at a pH below 7. The loss
of adherence was attributed to a reduction in affinity for the Leb receptor,
and, interestingly, the ability to bind Leb was fully restored when pH was
reconditioned and returned to neutral. We compared this property of BabA
with other lectins, and despite these other lectins also losing binding at low
pH, none of them had the ability to recover their binding properties when
the pH was neutralized. The stability of complexes of bacterial cells, Leb
conjugates, and BabA at low pH indicates that the acid-lability is attributed
to a functional property of the BabA adhesin. We detected a similar property
for the SabA adhesin, although SabA was more sensitive to low pH compared
to BabA.
The polymorphism of BabA in terms of both affinity and specificity for the
receptor is also reflected in its acid-labile property. In a large-scale screen of
clinical isolates, we found that while some strains still bound Leb at pH 2,
other strains had lost most of the binding already at pH 4. We concluded that
the acid lability of the BabA-Leb interaction is reflective of their diversity.
Purified BabA from H. pylori demonstrated a specific binding to the human
gastric mucosa at neutral pH, and low pH caused detachment of BabA.
However, pretreatment of BabA by low pH followed by neutralization
restored binding. Pure BabA from two bacterial isolates with different acidlability were compared in a Leb-binding ELISA. The acid-lability that each
protein demonstrated correlated well with the acid-labile binding that the
bacterial cells of each strain demonstrated. These results confirmed the
finding that the BabA protein has a unique ability to restore its binding
ability after exposure to low pH.

25

To determine how the structure of BabA is affected by low pH, we analyzed

BabA protein stability at lower pH by circular dichroism. We only detected
subtle changes, probably because of rearrangements in the -helices, and no
further changes to the global structure occurred below pH 2.5. Crossover
experiments of BabA from an acid-tolerant isolate to an acid-sensitive isolate
showed that the recipient improved in Leb binding and became almost as
acid-tolerant as the donor. This demonstrates that most of the unique acidsensitivity in BabA-mediated binding is encoded within the babA gene.
We further tested pH tolerance/sensitivity of isolates from the same
individual, but collected from different regions of the stomach that are
characterized by low vs. high acidity (antrum and corpus). The aim was to
test if such putative differences could relate to the distinct acidity of their
gastric niche. One patient, rebro 2, demonstrated differences in acidlability between isolates from the antrum vs. corpus tissue. Sequencing
revealed close to identical babA sequences with the exception of two amino
acid positions. In the antrum strain, we detected Leu in the putative
carbohydrate binding domain (CBD), i.e. the Leb-binding site, and Glu in an
area distant from the binding site, the C-terminal domain. In the corpus
strain, these amino acids were, instead, Pro and Gly. We cloned these babA
genes and expressed them as recombinant BabA. In addition, we also
constructed recombinant BabA where the two amino acids were swapped
giving a mosaic BabA genotype. These recombinant proteins were tested for
Leb binding at lower pH by surface plasmon resonance (SPR). The
recombinant proteins reflected the acid-sensitivity of the antrum isolate and
the more tolerant corpus isolate. In the mosaic BabA, we detected no
differences in acid-lability when exchanging Glu for Gly, but exchanging of
Leu for Pro revealed a shift towards higher acid tolerance suggesting that the
Pro residue is important for acid tolerance.
H. pylori-related diseases are reflective of the different acid profiles of the
stomach: ulcers are typically related to high acid secretion and cancer to low
acid secretion. A comparison of acid-lability from high peptic ulcer (HU) vs.
high cancer (HC) prevalent regions revealed that strains from HU regions
were more acid sensitive in binding compared to HC regions. These findings
were somewhat contradictory but might reflect the different niches that
these strains colonize. Duodenal ulcers are associated with high acidsecretion that typically arises from an antrum-predominant colonization
where bacteria in the antrum are adapted to the acidic antrum conditions.
Sequencing of babA from these regions showed that almost all BabA from
HC regions have the Pro-position in the potential CBD. In addition,
sequences from HU regions had several amino acids missing in this region as

26

they lacked the Pro and contextual amino acids. Instead HU strains had
required the antrum-associated Glu position in the C-terminal. These results
further emphasize the importance of these amino acids for adapting to local
pH and regional gastric patterns of disease.
To investigate evolution in BabA acid-lability during chronic infection, we
analyzed H. pylori from Rhesus macaques that had been experimentally
infected for five years. From one monkey, we found clones from the antrum
and corpus that differed in acid-lability. Antrum isolates and the original
infecting strain were similar in acid-lability whereas the corpus clone was
more acid-sensitive. We sequenced babA from these clones and found
identical sequences in input and output bacteria from the antrum except for
some mutations in the -barrel domain. However, in the corpus clone we
identified several amino acid differences in the same region as the Glu and
Gly in the rebro 2 antrum and corpus. These mutations were confirmed to
affect acid-lability by SPR experiments, which also confirmed that this
corpus strain was similar to the corpus strain from rebro 2. We could trace
these mutations in the macaque corpus BabA as coming from a
recombination event between babA and a babA homolog.
In conclusion, we propose that the unique method by which H. pylori
regulates attachment and detachment is vital during infection. By keeping
the binding activity of the adhesins acid-regulated, H. pylori is able to detach
from a dead cell that moves towards the toxic acidic lumen, orient itself by
chemotaxis towards the epithelial cells, and start a new cycle i.e. biopanning
for the most fitting phenotypes and then recycle the infection.

Paper II
Clinical isolates of Helicobacter pylori demonstrate alternative
BabA-mediated binding properties for adherence to the gastric
mucosa
This project was initiated during the large screen for ABO/Leb binding
among different populations (Aspholm-Hurtig et al. 2004). The phenotype
of Leb binding in European populations decreased towards southern Europe.
We were interested to test if these strains utilize alternative mechanisms for
adherence to gastric mucosa.
We initially screened strains from Spain for Leb binding properties and in
vitro adhesion to human gastric mucosa. Most of the strains that were low in
Leb binding were similarly low in adherence to gastric mucosa with some

27

exceptions. One isolate, 812, was low in Leb-binding as measured by RIA

(RadioImmunoAssay) but demonstrated strong adherence to the human
gastric tissue sections. We expanded the screen to include strains from
northern Scandinavia, Sweden, Spain, and Portugal. We correlated Leb
binding activity with expression of BabA and identified a set of strains that
were low in Leb binding but high in BabA expression, indicating additional
functions of BabA. We tested these strains in the in vitro adherence assay,
i.e. binding to the human gastric mucosa, and found a group of strains with
binding patterns similar to the previously identified 812, although only one
of those strains demonstrated adherence as strong as 812. We concluded that
although many strains from Europe are high in expression of BabA they still
dont adhere to gastric mucosa. The functional reason for the high BabA
expression but low Leb binding in these strains needs further investigation.
We then focused our study on the adherence properties of the strain 812.
Periodate treatment and trypsinization of sections from human mucosa
suggests the receptor for 812 to be glycolipids. Strain 812 also showed a
different adherence phenotype when we tested adherence to porcine gastric
mucosa and Leb transgenic mice; the reference strain 17875/Leb (possessing
ABO/Leb-mediated binding) adhered strongly to all tissues but 812 adhered
weakly if at all. Interestingly, also adherence to Rhesus macaque gastric
mucosa seemed to be complex; 17875/Leb could adhere to all macaque
mucosal samples, 812 only bound to every second Rhesus macaque gastric
mucosa.
We attempted to characterize the receptor of 812 by inhibition tests and
found no inhibition by use of free Leb glycan, which argues that Leb is not
the main receptor for 812. In support, both human secretor and non-secretor
milk inhibit adherence of 812, which taken together suggests that 812 binds
to carbohydrate that lack 1,2-fucose alt. to fucosylated antigens that are not
dependent and expressed due to individual secretor phenotypes.
We found that adherence of 812 to human gastric mucosa was mediated by
BabA because an isogenic BabA deletion failed to bind and because crossover
experiments transferred binding phenotype of 812. However, recombinant
812 BabA demonstrated a specific but much lower binding to human gastric
mucosa (Figure 10b) than was expected comparing with the strong
adherence of the 812 bacterial cells (Figure 10b).
To expand the search for the cognate 812BabA adhesin binding site, i.e. the
best receptor, we applied 812 to a glycan array with 350 different
carbohydrates but detected no adherence to any of these carbohydrates. We
expanded the array to also include the albumin-glycoconjugates used for

28

RIA-assay that might present the ABO/Leb-antigens further away from the
array surface. By this array, 812 binds to the series of ABO/Leb blood group
antigens, and a stronger binding to ALeb and BLeb than to Leb and H1. We
also analyzed binding of 812 to glycosphingolipids by TLC found affinity for
A-hexa structures indicating that 812 might have shifted its receptor binding
epitope towards GalNAc.
We further analyzed single colonies of 812 for Leb binding properties and
found a marked heterogeneity with a mixture of three Leb-binding
phenotypes, which we named red, circle, and darker according to their
appearance on the membrane after the Leb-binding colony screening test.
The dominant red colonies were very low in Leb binding, circle colonies
bind similar to Leb as red but appeared red in the middle with a darker
ring, and the darker colonies were stained because they bound stronger to
Leb, although the frequency of darker colonies were as low as 1/500. In
addition to Leb binding, the darker clone also showed stronger binding to
ALeb and A-hexa, as confirmed by RIA. Such an increase in Leb binding
could come from higher BabA expression, but immunoblots actually revealed
decreased BabA expression in the darker clone. Sequencing demonstrated
identical BabA sequences in red and circle clones, but the darker one
had two amino acids replaced in the C-terminal region: Asn to Ala and Arg to
Lys. We could trace these amino acid replacements to a recombination event
with a babA homolog. Interestingly, the deletion of this homolog had no
effect on adherence.
Glycosylation is a common post-translational modification and often
involves Asn, such as the Asn we found replaced to Ala in the darker clone.
By staining carbohydrates we identified a different glycosylation pattern in
the red clone. These experiments revealed that the red and darker
phenotypes exhibit additional differences accompanying previously observed
amino acid substitutions in BabA. We proceeded to study the clones that
adhered to human gastric mucosa to determine the phenotype they belonged
to. We made use of tissue sections from gastric mucosa as a bio-panning tool
to select and to isolate a population of adherent clones. Colony screening of
these clones by printing membranes and incubating with Leb as bait and
further babA sequencing showed that they were of the red, low Leb binding
genotype.
In conclusion, we have found that many of the European strains express
BabA but dont bind Leb, and that the majority of such strains cannot bind
sections of human gastric mucosa. Further investigations are needed to
determine if these BabA proteins have modified binding properties. Our
studies focused on strain 812 that demonstrates low-affinity binding to

29

ABO/Leb but fully maintained strong binding to gastric mucosa. Glycan

array analysis suggested that 812 has shifted its binding epitope towards the
Gal/GalNAc epitope and displays increased preference for the ALeb and
BLeb structures. We also detected single clones with higher Leb binding due
to amino acid replacements acquired by recombination event with a BabA
homolog.

Paper III
Infection by Helicobacter pylori expressing the BabA adhesin is
influenced by the secretor phenotype
This study focused on investigating how the secretor phenotype, i.e.
expression of ABO-antigens in secretions and the GI-tract epithelium,
influences H. pylori infection and adherence. We hypothesized that secretors
are more likely to be infected with a BabA-expressing strain.
For these types of studies, it is crucial to select non-biased subjects. We
screened volunteers from a gastric pathology survey at a shipyard in
northern Portugal, which could be considered a cross section of the
population. BabA status was determined from biopsies by both PCR and
immunofluorescence. In accordance with previous findings, the prevalence
of the babA-gene and the BabA-protein did not correlate. Therefore, to
assure that we investigated expressed BabA, we assessed BabA by histotissue
immunodetection.
First, we analyzed BabA for correlation with other virulence factors. Positive
BabA status was correlated with both CagA and VacAs1. This result is in
accordance with the definition of triple-positive strains. We next analyzed
how BabA status affects the pathological state of the stomach. We found
BabA to be correlated with a higher H. pylori density. BabA was also found
to be correlated with intestinal metaplasia and degenerative alterations. The
histopathology found in BabA-positive cases is possibly related to the
correlation between BabA, CagA, and VacAs1 (Gerhard et al. 1999). Of
particular relevance, BabA was recently demonstrated to be important for
delivery of CagA (Ishijima et al. 2011).
We analyzed secretor status in biopsies and saliva by Ulex staining,
determination of Lewis status by immunofluorescence, and by PCR for the
G428A mutation in the secretor fucosyltransferase FUT2. We could only
detect a correlation of BabA with Ulex staining of biopsies, not with secretor
status determined from saliva. It was also not correlated with Lewis

30

phenotypes of either gastric biopsies or saliva or with the FUT2 G428A

polymorphism.
To test how BabA-mediated adherence is influenced by the phenotype of the
tissues, we applied FITC-labeled 17875/Leb to sections from 61 of the gastric
biopsies. A significantly higher binding was observed between Lewis
phenotypes a-b- (probably secretor) and a-b+ (secretor) compared to
a+b- (non-secretor) and a+b+ (weak secretor), i.e. Lea staining suggested that
the secretor expression is reduced. Significantly higher binding was also
detected in those tissues that were strongly positive for Ulex staining and
normal/heterozygous for the FUT2 G428A polymorphism, the marker for
secretor positive phenotype.
In conclusion, we have confirmed a functional correlation between BabA,
CagA, and VacA, and most importantly, BabA-positive strains demonstrated
higher binding to tissues of positive secretor phenotype. Thus, we
demonstrated that secretors are more likely to be infected with a BabApositive strain that enables closer contact with epithelial cells and thus
causes more of tissue damage.

Paper IV
Studies of Lewis antigens and H. pylori adhesion in CHO cell lines
engineered to express Lewis b determinants
In this study, we constructed Leb-expressing recombinant Chinese hamster
ovary (CHO) cells. We further analyzed how these cells presented Leb and if
recombinant Leb could support BabA-mediated adhesion of H. pylori.
CHO cells were transfected with expression vectors containing four different
genes that are necessary for Leb expression on core 3 O-glycans, GlcNAcand Gal-transferases for the backbone and the secretor- and Lewis-fucosyl
transferases for addition of the fucose. Two Leb-expressing clones (1c5 and
2c2) were selected for further analysis. Flow cytometry revealed differences
in expression of Leb: 2c2 with high levels of both Leb and Ley expression and
1c5 having lower levels of Leb expression and no Ley expression. However,
both clones express Lea. By co-expressing the reporter proteins PSGL1/mIgG2b and AGP/mIgG2b that are glycosylated by N- and O-glycans,
respectively, we detected Leb on O-glycans on the surface of both clones but
lower expression in 1c5. The 2c2 clone also expressed Leb and Ley in Nglycans. In addition, 2c2 demonstrated Leb and Ley expression also in
glycolipids whereas 1c5 was low in Leb and with no Ley glycolipid.

31

We further investigated whether the Leb expression of these cells was

suitable to support BabA-mediated adherence of H. pylori. We applied FITClabeled 17875/Leb and specific adherence was detected to the 2c2 and 1c5
clones, 7600 and 2200 H. pylori were found, respectively, compared to
400 for non-recombinant CHO cells.
To summarize these results, we could detect a distinct difference between the
CHO cells clones in terms of Leb expression. The 1c5 clone expressed Leb on
O-glycans and a very poorly in glycolipids. In contrast, the 2c2 clone
displayed strong expression in both N- and O-glycans and in addition in
glycolipids. 2c2 also expressed Ley both on N-glycans and in glycolipids. The
binding results demonstrate that O-glycan expression of Leb supports BabAmediated adherence to Leb. These results, however, do not reveal if Nglycans or glycolipids could support adherence. However, biochemical tests
of fractionated glyco-conjugates might provide for information about the
receptor proportion of N-glycans and glycolipids.

32

Discussion and Concluding Remarks

The glycosylated mucin layer that lines the entire gastro-intestinal tract is
one of the first lines of defense against gastrointestinal pathogens and,
therefore, displays individual diversity in its carbohydrate repertoire. This
polymorphism is presumably the net result of an arms race between the
pathogens and their host. Changes of a pathogen in response to a new
receptor, and potentially a new niche, demand that the host follow with
additional changes to avoid being overtaken by the pathogen (Bishop &
Gagneux 2007). In addition, diversity among individuals in carbohydrate
patterns limits the horizontal spread of infections, such as that seen for the
resistance against disease by Norovirus among non-secretors.
For most enteropathogens, the acidity of the stomach is lethal. However,
H. pylori has cleverly adapted its tissue tropism to this unique niche in many
ways and is, therefore, the (almost) exclusive persistent pathogen of human
stomach. The quasi-panmictic population structure i.e. resembling an
unstructured population, of H. pylori facilitates persistence of the infection
by ensuring that a subpopulation of at least a single clone is capable of
surviving and pursuing infection in response to environmental changes. The
persistence of a bacterial clone is highly dependent on adherence capability.
A good example of an adhesin that is capable of adaptation is the E. coli
FimH protein. From 133 sequenced fimH genes there are 63 unique alleles
(Sokurenko 2004). Such a high diversity of the FimH adhesin would be
beneficial as it can shift in binding affinity and target other mono or
multivalent mannose receptors. In comparison, BabA is even more
polymorphic and each gene represents a unique allele. H. pylori strains are
very different in BabA-mediated Leb binding and display a >1500-fold
spectra of high binding affinities (Aspholm-Hurtig et al. 2004). Moreover, as
it has been shown in the present study, some strains are very low in binding
affinity and many strains dont bind at all and have lost all affinity even
though they still express BabA. This indicates that there is a selective
pressure on BabA to retain the ability to bind the ABO/Leb blood group
antigens. The reason for this is not known but probably reflects the narrow
niche in which H. pylori is the only permanent inhabitant. The selection for
binding to the ABO/Leb structures is probably also due to the high
prevalence of these receptors in the human gastric epithelium that increases
the chances for H. pylori to find a suitable host cell for cross talk. Some
diversity in receptor specificity for BabA is seen between generalist and
specialist strains, but because of the high diversity it is still not known how
BabA binds to the ABO/Leb structures or which amino acids promote

33

binding. The binding affinity is not the only selection that drives diversity of
BabA. Because of the outer membrane localization of BabA, the diversity of
BabA is probably also a consequence of a continuous process to evade
neutralizing immune responses.
The first study that demonstrated the correlation of babA with CagA and
VacA virulence factors in disease development analyzed the prevalence of the
babA2, which was considered to be expressed due to an intact signal peptide
sequence (Gerhard et al. 1999). However, owing to the polymorphic
characteristics of BabA expression, in which not all genes express a protein
and not all proteins are functional, it is interesting to speculate on actual
implications of such correlation. Could merely BabA expression itself be
enough to trigger a higher immune response and consequently cause more
inflammation and disease? Infections of Leb transgenic mice resulted in
higher levels of inflammation compared to non-transgenic mice suggesting
that adherence stimulates mucosal inflammation. Since BabA is not much
immunogenic during chronic infection in man, this hypothesis seems
unlikely. Thus, it is reasonable to speculate that the adherence properties of
BabA are correlated to disease progression. Therefore, a more complete way
of correlating BabA in clinical strains to disease would be to appreciate Leb
binding by the bacteria as a first indication of a BabA-positive phenotype.
Consequently, in Paper III, we considered biopsies to be BabA-positive by
immunofluorescence with a BabA-specific antibody. Although this does not
necessarily indicate a functional BabA, it is closer to in vivo expression of
BabA compared to inventory of babA genotype.
In Paper III, we found BabA to correlate with CagA and VacAs1. The
functional role of BabA in disease progression has not been entirely
elucidated, though new results point towards a role for BabA in the delivery
of VacA and CagA to the host cells (Ishijima et al. 2011). Tight adherence
with immediate access to cellular junctions would not only promote more
efficient delivery of VacA, but would also allow for the bacteria to swim
towards the basal membrane to deliver CagA where the integrin receptors
are i.e. the presumed docking station for the T4SS machinery. Many gastric
cell lines, such as the AGS cells, are available for signaling studies with H.
pylori, but they are variable or low in Leb expression and this makes it
challenging to investigate BabA-mediated signaling. To overcome this, we
constructed CHO cells with defined carbohydrate repertoires (Paper IV).
Unfortunately, these hamster cells are resistant to delivery of CagA (personal
observation), which might relate to differences in 1-integrins. Despite the
lack of CagA delivery into the host cells, it would be interesting to study if the
BabA-Leb interaction itself could trigger a signaling cascade in the cells. The
functional receptor for BabA in the human gastric mucosa is not known.

34

However, it is known that BabA interacts with both the MUC5AC and MUC1
mucins, and might interact with non-mucin glycosylated proteins in the host
cell membrane. In addition, binding mediated by glycolipid receptors could
also promote signaling that could aid in promoting loosening of the cellular
junctions and function as a complement to VacA during infection.
Gastric cancer is the second most common cancer worldwide and often with
poor outcome because the cancer is usually detected at an advanced stage
where treatment is difficult and the mortality rate is high, hence denoted the
silent killer. Though we have gained some knowledge about the steps in
cancer development, there are still many important questions to address.
What factors promote a symptomatic disease? Why do some people develop
cancer vs. peptic ulcer disease? Are there any benefits in H. pylori infection
or should we aim to eradicate all infections? Do we need to eradicate
infections to prevent disease or would it be enough to merely suppress the
infectious load e.g. 10-100 fold? In this perspective, it is important to
consider whether we need sterilizing vaccines or whether it would be enough
if vaccines could reduce infection 100-fold. Many scientists are studying the
important roles of CagA and VacA in disease development, but additionally
factors must be of importance, especially since the majority of clinical
isolates express CagA and VacA. If additional virulence factors could be
identified that would predict those strains that are prone to develop disease,
more focused treatments with antibiotics could be an option, in particular in
combination with a vaccine that would prevent re-infection. Such
combination would make a difference for people all around the globe.

35

Acknowledgements
Det har varit en underbar och spnnande resa som doktorand, frn brjan
till slut. Som med mycket annat s r inte detta ngot man gr alldeles
sjlv, man behver std och hjlp av andra mnniskor ocks!
Jag frmst tacka min handledare Thomas! Din grnslsa klla till ider
samt frmga att komma p nya experiment inspirerar mig. Det har varit en
spnnande tid i din grupp och jag r tacksam fr din generositet med bde
lngvga resor och tid till att utforska mina egna intressen, det har verkligen
ftt mig att vxa som person, tack! Ett stort tack ven till min bitrdande
handledare Anna. Jag uppskattar din frisprkighet och att du sger vad du
tycker, tack fr alla stunder nr jag har behvt prata ur mig.
Kristoffer, vr biacoreguru! Tack fr trevligt sllskap i labbet under den
perioden som biacorens underbara ljud frgyllde tillvaron och fr all hjlp
med bde mina frsk till pH-manuskriptet och 812-projektet. Ola och
Susann, tack fr ett bra samarbete och hjlp med att klura ut vad min stam
egentligen binder till! Maria Azevedo, Leonor David, Jonas Lfling
and Jan Holgersson, thanks for nice collaborations!
The time I have spent at the department of Medical Biochemistry and
Biophysics has really been a great, and I would like to thank everyone that
contributes to the nice, open atmosphere. Ett speciellt tack till Ingrid, Clas,
Anna, Lola och Elisabet! Om ni inte vore s fantastiskt bra p det ni gr
s skulle jag aldrig f ngon forskning gjord!
Melissa, thank you so much for your help with the last experiments of My
manuscript and for our scientific, and sometimes not so scientific ,
discussions that we have had in our small office. They have really
enlightened this last year! Jeanna, thanks for all the help with protein
purification, discussions about microscopy and for bringing some culture
(not the bacterial one) into My life. Anna S, thank you for help with RIA, for
your amazing cakes and for talks about life. Rolf! jag har saknad dig sedan
du gick i pension. Lena, thanks for our nice conversations in the office,
sorry Im not as in to politics as you are. Alexej och Gran, tack fr all
hjlp med att lra mig PCR, kloning och uttryck. sa, Jenny, Marie and
Jalal, My best wishes for you!
Annelie, Var ska jag brja? Att f dela min doktorandtid med dig har varit
fantastiskt roligt. Utan dig hade jag varit tvungen att sjlv ka p alla

36

konferenser, lra Maria och Melissa att ka lngdskidor, prova vin i Napa
Valley och mycket mycket annat. Tack fr allt och lycka till med din
disputation! Pr, jag beundrar din ambition och humor, du kan vara en
doktor! Anna V, tack fr att du lrde mig vvnadssnitten och mikroskopets
underbara vrld och fr allt annat kul vi haft bde i och utanfr jobbet.
Anna , Carina och Crister, tack fr trevliga stunder p labbet!
Mina vnner utanfr Helicobacter-vrlden, Rima, tack fr ditt alltid s
glada och smittsamma humr, och fr alla block du snittat t mig. Emma,
min underbara och ordningsamma vn! det har varit kul att ntligen f ha
dig p samma institution igen s vi hunnit trffas varje dag (nstan iaf ).
Lisa, tack fr all optimism! Alla borde ha en vn som du, som hellre ser
saker som spnnande n jobbiga. Nu hoppas jag att vi alla tre hinner trffas
oftare igen fr fika och promenader. Anna och Mattias, tack fr hrliga
middagar (och ta ute) mitt i veckan som en paus frn vardagen, vet inte hur
jag skulle verlevt utan!
Jan och Rose-Marie, mina underbara svrfrldrar. Ett speciellt tack till
Jan som alltid hittar tid i sitt fullspckade pensionrsschema fr vara
barnvakt. Johan och Elin fr trevliga stunder med god mat!
Ett stort tack till min hela min familj, mamma, pappa, Anna & Johan,
Daniel och Maria som stdjer mig i det mesta jag gr, ven om det
innebr att vi inte hunnit trffas s mycket senaste tiden, men det blir nog
bttre efter det hr. Ett extra stort tack till Maria som mnga gnger ftt
rycka in som barnvakt nr tiden kniper.
David, min underbara lskade make som alltid hjlpt och stttat mig i
mnga r, men som gjort det lite extra den senaste tiden. Ida, min lskade
dotter. Den livsgldjen du sprider omkring dig har gjort detta skrivande
mycket lttare och om jag bara hade haft hlften av din energi hade denna
det hr varit gjort p ett nafs. Jag lskar er bda otroligt mycket!

37

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