Helicobacter pylori A model organism for understanding bacterial pathogenesis

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Helicobacter pylori A model organism for
understanding bacterial pathogenesis

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Objectives

• To understand the molecular mechansms by which
  Helicobacter pylori causes human disease
• To use Helicobacter pylori as a model for
  understanding bacterial-host interactions

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Discovery of H. pylori

• Early 1900s - bacteria were detected by
  microscopy in human gastric tissue
• 1983 successful culture of a previously
  unrecognized bacterial organism from human
  gastric tissue
• Initial name Campylobacter pyloridis
• Revised name Helicobacter pylori

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Characteristics of H. pylori

• Gram-negative curved rod
• Microaerophilic
• Colonies visible after 48-72 hours
• Strong enzymatic activities urease, oxidase,
  catalase

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Representative Helicobacter species

• Organism
• H. pylori
• H. heilmannii
• H. felis
• H. mustelae
• H. nemestrinae
• H. acinonyx
• H. cinaedi
• H. fennelliae
• H. muridarum
• H. Canis
• H. pullorum
• H. bilis
• H. hepaticus

• Host and site
• Human stomach
• Many mammals, stomach
• Cat or dog stomach
• Ferret stomach
• Macaque stomach
• Cheetah stomach
• Rodent or human intestine
• Human intestine
• Rodent intestine
• Dog intestine
• Chicken intestine
• Mouse intestine or liver
• Mouse intestine or liver

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Whole genome analysis of two different H. pylori
strains

• Two H. pylori strains selected for genomic
  sequence analysis (strains 26695 and J99)
• Number of predicted ORFs is about 1500
• Number of ORFs present only in
• strain 26695 117
• Number of ORFs present only in
• strain J99 89
• Nature 1999397176-180

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Genetic diversity among different H. pylori
strains
Burucoa et al., J Clin Microbiol 1999374071-4080
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Phylogeny of vacA alleles from different H.
pylori strains
Gottke et al., J Infect Dis 20001811674-1681
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H. pylori epidemiology

• The human stomach is the main reservoir.
• H. pylori is found in humans throughout the
  world.
• Infection is typically acquired early in life.
• Infection usually persists for decades if not
  treated.
• Person-to-person transmission is likely.
• H. pylori is present in about 30 of the U.S.
  population.

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Helicobacter pylori on a global scale

• Global human population 6 billion
• Proportion infected with H. pylori 60
• Global number of H. pylori-infected humans about
  3.6 billion

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Localization of H. pylori in the human stomach

• H. pylori lives in the mucus layer overlying
  gastric mucosa (non-invasive bacterium).
• H. pylori only colonizes the mucus layer
  overlying gastric-type epithelium.
• H. pylori can colonize the duodenum in regions of
  gastric metaplasia.

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Ingestion of H. pylori by a human volunteer

• Baseline normal gastric histology
• Day 0 ingestion of organism
• Day 2 Epigastric pain, nausea, vomiting
• Day 5 Neutrophilic antral gastritis, gastric pH
  1.2
• Day 8 Gastric pH 7.6
• Day 10 Resolution of symptoms
• Day 30 Persistence of gastritis and H. pylori

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H. pylori and gastric inflammation

• Inflammation always accompanies H. pylori
  infection.
• Termed chronic superficial gastritis
• Lymphocytes, monocytes, neutrophils in lamina
  propria
• Usually asymptomatic
• Resolves following eradication of H. pylori

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H. pylori infection is a risk factor for duodenal
ulcer disease

• gt90 of patients with idiopathic duodenal
  ulcers are infected with H. pylori
• Prior H. pylori infection is associated with an
  increased risk for duodenal ulcer
• H. pylori infection causes gastric damage in
  animal models
• Eradication of H. pylori results in decreased
  rates of recurrence

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H. pylori infection is a risk factor for gastric
carcinoma

• Case-control studies based on serologic analysis
  of stored sera
• Experimental H. pylori infection of Mongolian
  gerbils results in gastric tumors
• Prospective study of patients who have a high
  risk for developing gastric cancer

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Geographic variation in the incidence of gastric
cancer
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Prospective study of the development of gastric
cancer

• Population 1526 Japanese patients
• 1246 H. pylori-positive
• 445 with non-ulcer dyspepsia
• 297 with gastric ulcer
• 229 with gastric hyperplastic polyps
• 275 with duodenal ulcer
• 280 H. pylori-negative
• Mean followup period 7.8 years
• NEJM 2001345784-789

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Development of gastric cancer related to
endoscopic findings

• No.
  () with
  
  gastric cancer
• H. pylori-positive (n1246) 36
  (2.9)
• NUD (n445)
  21 (4.7)
• Gastric ulcer (n297)
  10 (3.4)
• Gastric polyps (n229)
  5 (2.2)
• Duodenal ulcer (n275)
  0
• H. pylori-negative (n280) 0

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Gastric non-Hodgkins lymphoma

• Monoclonal B-cell proliferation
• Spectrum of severity
• MALT mucosal-associated lymphoid tissue
• Eradication of H. pylori is associated with tumor
  regression

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Infectious diseases with carcinogenic potential

• Viral
• Hepatitis viruses
• Papilloma virus
• Bacterial
• H. pylori
• Parasite
• Clonorchis sinensis
• Schistosomiasis

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Pathology or disease states associated with H.
pylori infection

• Chronic superficial gastritis
• Gastric ulcer
• Duodenal ulcer
• Atrophic gastritis
• Gastric adenocarcinoma
• Gastric MALT lymphoma
• Gastric non-Hodgkins lymphoma

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PATHOGENESIS OF H. pylori INFECTION
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Stages in a typical bacterial infection

• Bacterial residence in a natural reservoir
• Bacterial encounter with the host
• Attachment to cells (adherence)
• Entry into tissue (invasion)
• Bacterial replication
• Evasion of host defenses
• Return to original reservoir or transmission to
  new hosts

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Stages in a typical bacterial infection

• Bacterial residence in a natural reservoir
• Bacterial encounter with the host
• Attachment to cells (adherence)
• Entry into tissue (invasion)
• Bacterial replication
• Evasion of host defenses
• Return to original reservoir or transmission to
  new hosts

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Bacterial virulence determinants

• Specific bacterial components (virulence
  determinants) may be required for various stages
  of the infectious process.
• Adhesins mediate bacterial adherence to host
  cells.
• Bacterial surface proteins (e.g. invasins)
  mediate entry into host cells.
• Bacterial secreted toxins contribute to tissue
  damage or modulation of host cell function.
• Bacterial surface components or secreted factors
  mediate resistance to host defenses.

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Important questions relevant to H. pylori

• What are the mechanisms that allow H. pylori to
  colonize the human stomach, whereas other
  bacteria cannot?
• How does H. pylori interact with the gastric
  mucosa?
• How does H. pylori persistently colonize the
  stomach without being eradicated by host
  defenses?
• Why does H. pylori specifically colonize the
  human stomach?
• Why are there multiple possible clinical outcomes
  of H. pylori infection, and what are the factors
  that determine clinical outcome?

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How does H. pylori survive in the acidic gastric
environment?
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H. pylori adaptations for life in an acidic
environment

• Motility (flagella)
• Localization in the gastric mucus layer
• Urease activity
• Acid-induced changes in H. pylori gene expression
• Modulation of gastric acid physiology

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How does H. pylori interact with gastric
epithelial cells?
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Binding of H. pylori to Leb antigens on human
gastric mucosa
A C Leb-positive tissue B D Leb-negative
tissue
Boren et al., Science 19932621892
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Anti-Lewis b inhibits binding of H. pylori to
human gastric mucosa
Boren et al., Science 19932621892
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Binding of H. pylori to gastric epithelial cells
via multiple adhesin-receptor interactions

• BabA adhesin binds to Lewis b on surface of cells
• SabA adhesin binds to sialyl-dimeric Lewis x on
  surface of cells

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Cell vacuolation induced by Helicobacter pylori
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Alteration of cell morphology by Helicobacter
pylori
AGS cells alone
AGS cells plus H. pylori
Segal et al., PNAS
19999614559-64
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Purification of a vacuolating toxin from H.
pylori broth culture supernatant
Cover et al., J Biol Chem 199226710570-5
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Analysis of VacA toxin oligomeric structure by
deep-etch EM
Cover et al., J Cell Biol 1997138759-769
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Multiple effects of VacA on gastric epithelial
cells

• Cell vacuolation
• Formation of anion-selective membrane channels
• Alterations in endocytic trafficking
• Alterations in antigen presentation
• Extracellular release of lysosomal proteases
• Inhibition of cell proliferation
• Release of cytochrome c from mitochondria
• Increased permeability of epithelial monolayers
• Apoptosis
• Inhibition of T cell activation

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The H. pylori cag pathogenicity island
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THE H. pylori cag PATHOGENICITY ISLAND

• 40 kb chromosomal region present in some H.
  pylori strains but not others

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Tyrosine phosphorylation induced by Helicobacter
pylori
A- AGS cells alone B- AGS cells plus H. pylori C
D- AGS cells plus cag- H. pylori strains
Segal et al., PNAS 1997947595
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Translocation of H. pylori CagA into AGS cells
Green H. pylori Red CagA
Odenbreit et al., Science 20002871497-1500
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Tyrosine phosphorylation motifs in H. pylori CagA
Odenbreit et al., Science 20002871497-1500
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Intracellular activities of H. pylori CagA

• Translocated CagA protein undergoes
  phosphorylation on tyrosine residues, via
  eukaryotic cell kinases
• Phosphorylated CagA associates with SHP-2 (a
  tyrosine phosphatase) and stimulates phosphatase
  activity changes in cell morphology
• Non-phosphorylated Caga interacts with Grb2,
  leading to activation of Ras/MEK/ERK pathway
  cell scattering and proliferation

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The H. pylori cag pathogenicity island
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Bacterial type IV secretion systems
Christie et al., Mol Microbiol 200140294
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Model for structures formed by type IV secretion
systems
Covacci et al., Science 19992841328-1333
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Alterations of gene expression in AGS cells in
response to H. pylori
Guillemin et al., PNAS 20029915136
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Functions attributed to the cag pathogenicity
island

• Encodes CagA, a high-molecular-mass antigen that
  is translocated into epithelial cells and
  undergoes tyrosine phosphorylation
• Type IV secretion system for translocation of
  CagA into host cells
• Induction of cytokine expression in epithelial
  cells

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Alterations that occur following binding of H.
pylori to gastric epithelial cells

• Changes in cell shape and morphology
  (vacuolation, hummingbird phenotype)
• Activation of signal transduction pathways,
  leading to expression of cytokines
• Apoptosis

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WHY ARE THERE MULTIPLE POSSIBLE CLINICAL OUTCOMES
OF H. pylori INFECTION?

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Why are there multiple possible clinical outcomes
of H. pylori infection?

• Heterogeneity among H. pylori strains
• Heterogeneity among humans
• Heterogeneity in environmental influences

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H. pylori factors that contribute to
heterogeneity in clinical outcomes

• Presence/absence of cag pathogenicity island
• Allelic variation in vacA
• Type s2 vacA alleles encode a VacA protein that
  is less cytotoxic than type s1 alleles
• Strains containing the cag pathogenicity island
  and type s1 vacA alleles are associated with
  increased risk for peptic ulcer disease and
  gastric cancer.

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Host factors that contribute to heterogeneity in
clinical outcomes

• Variations in acid-secretory capacity of the
  stomach are relevant to clinical outcome
• Interleukin-1-beta is a strong inhibitor of
  gastric acid secretion
• Certain IL-1-beta genetic polymorphisms are
  associated with an increased risk for
  hypochlorhydria and gastric cancer.

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Factors influencing development of clinical
disease

• Bacterial factors
• cag pathogenicity island
• vacuolating toxin
• Host factors
• Gastric acidity
• Immune responses
• Environmental factors
• Gender
• Smoking

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Comparisons between H. pylori and other
bacterial pathogens

• Non-invasive versus invasive organisms
• Organisms that cause extensive tissue damage
  versus those that cause minimal damage
• Organisms that cause an acute transient infection
  versus those that establish chronic colonization

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Key points

• H. pylori represents a model for understanding
  the process by which bacteria colonize humans and
  cause disease
• Striking features of H. pylori include its
  capacity to establish persistent infection and
  its role in the development of gastric carcinoma.
• Each bacterial pathogen has its own unique set of
  virulence factors. However, there are many
  recurrent themes in the pathogenic mechanisms
  used by different pathogens.