Helicobacter pylori A Gastric Pathogen MIMG C106 06-07-2005 Dr. Ken Bradley Office Hours For Final Week Wednesday, June 8 From Noon - 5pm MSB 2825a

Helicobacter pylori

Micro-aerophilic Micro-aerophilic,, Gram-negative, slow-growing, spiral-shaped, flagellated bacterium

Helicobacter pylori

n n n

Genome sequenced in 1997 Genome size – 1,667,867 base pairs 1590 predicted coding sequences

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H. pylori -Associated Diseases •Gastritis •Duodenal and gastric ulcers •Gastric adenocarcinomas •14th leading cause of death in the world •8th leading cause by 2010

*Infection in the lumen of the stomach is not accessible to immunocytes and macrophages

Ulcers

Previous medical paradigm was "no acid, no ulcer"

H. pylori - Etiologic Agent of Human Gastric Disease? 1881, 1893 - Spiral organisms found in stomach contents of dogs and other mammals 1938 - Spiral organisms first described in human gastric mucosa 1975 - First attempts to culture gastric bacteria

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The interesting discovery of H. pylori

Barry Marshall •First

cultured and shown to cause ulcers in 1982!

Serendipity! ….and then it took some guts!!!

H. pylori in the acidic stomach

A silver stain (Warthin Starry) of H.pylori (black wiggly lines) on gastric mucussecreting epithelial cells (x1000). This picture is of Dr. Marshall's stomach biopsy, taken 8 days after he drank a culture of H. pylori. This image is from the Helicobacter Foundation website: www.helico.com

Helicobacter pylori Infections

Most common chronic bacterial infection of humans (present in half of the world population)

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H. pylori and Human Ulcer Diseases • 90% of duodenal ulcers and 70% of gastric ulcers are H. pylori positive • Presence of gastritis is a risk factor for duodenal ulcers and ulcer relapse • Cure of H. pylori infection leads to a dramatic reduction in ulcer relapse rate • Addition of antibiotics to acid suppressive therapy increases speed of healing of acute ulcers

Transmission Ancient association with humans Believed to be present in human stomachs before human migration started ~100,000 years ago Overlap between genetically distinct H. pylori and human populations

Transmission Developing Countries

• 80% population colonized • Colonization during childhood • Parent-child transmission (genotyping) • Strains persist and reinfection rare • Very low rate of ulcers!

Industrialized Nations

• 20-50% population colonized • Colonization later in life • Transmission between spouses rare • Rare childhood infection rate ( pH 8.5 Some bacteria can transit stomach but do not persist (S. typhimurim, V. cholera, Y. enterocolitica, and some strains of E. coli) H. pylori is the only know organism to colonize and persist in the stomach

Keeping DNA Intact Under Acidic Environment • Highly active DNA proofreading and repair systems • Slow growth to give DNA repair a chance • Highly natural competence and high recombination rate - but counteracted by encoding over 20 pairs active restriction systems

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pH maintenance • Stomach is a hostile environment for microorganisms Stomach contents pH ~1.0 • Helicobacter pylori is a neutralophile • Requires pH of 4-8 to survive • Overcomes problem with UREASE activity to increase pH of environment through ammonia production ‡Urease important H. pylori virulence factor, Ureasenegative mutants are unable to colonize in animal models urease Urea

CO 2 + 2 Ammonia

H. pylori Urease



Produced in large quantities ß5-6% of total cell protein ßOptimal activity at pH 7, but bacterial-associated activity increases 10- to 20-fold as pH drops!



Urease gene cluster codes for seven genes ßureA and ureB are structural subunits ßureI is transporter



Active urease found in cytoplasm of log phase cells Necessary for survival in the stomach Urea derived from gastric secretions

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UreI and Urea Import ureI encodes a H+ gated urea channel in inner membrane Increases urea permeability 300-fold with pH drop

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Urease •BCECF fluorescent dye, fluorescent @ pH 5.5-8.0 •Fluorescence in the periplasm of H. pylori cultured at pH 5.5 upon addition of 5mM urea ‡gastric secretions =1-3 mM •Urease allows formation of a neutral pH microenvironment in H. pylori J. Clin. Invest. 106:339–347 (2000).

Cytotoxicity of Urease H. pylori ammonia production has little effect on its own metabolic activity Monochloramine production occurs in presence of leukocytes, urea and H. pylori urease Helicobacter urease may promote DNA damage in gastric epithelial cells May contribute to development of gastric cancer associated with chronic H. pylori infection

H. pylori was classified as a class 1 carcinogen in 1994

Survival in low pH environment SUMMARY Urease is crucial for H. pylori survival at acidic pH.

Ure(-) Ure(-) H. pylori cannot colonize But, following colonization, H. Pylori stays close to the surface of mucosal epithelium via its motility and chemotaxis

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Chemotaxis •H. Pylori shows chemotaxis toward urea, urea analogs and sodium bicarbonate (produced by cells of gastric mucosa) •Urease activity enhances chemotaxis •Possibly has chemotaxis receptor for each •May serve to locate urea for acid neutralization and locate gastric epithelium for colonization UreB +

UreB -

PV P 10 mM urea No PVP No urea

INFECTION AND IMMUNITY, Oct. 1998, p. 4832–4837 Vol. 66, No. 10

Motility of H. pylori H. Pylori possess unipolar flagella contained in a sheath contiguous with outer membrane that prevents damage • Consists of FlaA and FlaB • expressed in different amounts Spiral morphology allows H. pylori to penetrates mucus layer like a corkscrew • Motile at 200 cP vs. E. coli 20 cP

Motility Flagella powered by proton motive force • Increased motility at low pH allow quick transit of stomach • Urease activity may contribute to proton gradient, but not necessary for motility

7% motile

66% motile

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Summary - Route of infection Oral ingestion Transit of gastric environment • Adult stomach pH ~ 1-2 • Infection occurs in early childhood, higher stomach pH • Urease activity to neutralize acid Penetrating gastric mucus • Thick, sloughing • Flagella allow for effective penetration of viscous mucus Attachment • Adhesins enable attachment to gastric mucosal surface • Allows bacteria to persist in stomach No competition for H. pylori once established on the mucosal surface

Other H. Pylori virulence factors 1.Cag-PAI – 29 genes coding for type IV secretion system and other genes 2. VacA

– Vacuolating cytotoxin

3. BabA – Adhesin

cag pathogenicity island 29 genes with different GC content from rest of genome Presence of cag-PAI associated with: • More severe inflammatory response • Peptic ulcer and gastric cancer in western populations • Most strains in developing world contain cag-PAI Contains sequences for type IV secretion apparatus • Deliver bacterial products directly into gastric epithelium • Modulates signal transduction pathways • Activates inflammatory cascades • CagA

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Type III secretion system

Type IV secretion system

cag-PAI Encodes a Type IV Secretion System ÿ cag-PAI contains virB/D genes, homologous to the type IV secretion system of Agrobacterium tumefaciens. ÿ CagA is translocated into epithelial cells by this secretion system. ÿ Translocated CagA is tyrosine phosphorylated by a host kinase. Backert et al. 2000

CagA ÿ cagA localizes at the right border of cag-PAI. ÿ CagA protein is ~120-135 kD; strain variation exists in the C-terminus. ÿ Contains 4 potential phosphorylation sites. Phosphorylation takes place on Tyr972 in strain 26695. ÿ Highly hydrophilic and has no transmembrane domains. Backert et al. 2001

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CagA

•Directly injected into gastric epithelial cells •Phosphorylated by Src tyrosine kinases ßActivates host signaling pathways, including Ras/MEK/Erk •CagA localizes to plasma membrane and interacts with SHP-2, a host tyrosine phosphatase ßSHP-2 is involved in regulation of spreading, migration and adhesion of cells •Interaction of CagA and SHP-2 induces abnormal proliferation and movement of gastric epithelial cells •CagA also shown to interact with intracellular junctions and disrupts their function

Amieva et al Science. 2003;300(5624):1430-4

VacA • Vacuolating toxin is present in almost all H. pylori strains • Multimeric exotoxin binds to and enters gastric epithelial cells • Produces endosomal acid vacuoles in mammalian cells and induces cell death through unknown pathways • Strain expressing toxin are associated with increased risk of ulcer • Purified toxin produces ulcer-like lesions in absence of bacteria

How Does VacA Function? • Three hypotheses: – VacA functions as a pore-forming toxin – VacA functions by disrupting cell signaling from the cell surface – VacA functions as an A-B intracellularly acting toxin

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VacA-Induced Vacuolation control cells

+ VacA

BabA Adhesin Interacts with blood group antigens Lewis b on gastric epithelial cells • BabA2 + strains may be associated with risk of peptic ulcers, and distal gastric adenocarcinoma • Prevents removal from the stomach by gastric emptying • Enhances H. pylori colonization and pathogenicity • May allow for efficient delivery of virulence factors through increased adhesion • Relatively low incidence in clinical samples from Asian populations

Two Types of H. pylori Strains ÿ Type I: More virulent • VacA---- Vacuolating cytotoxin • CagA---- Cytotoxin associated gene A • cag-PAI---- cag Pathogenecity Island ÿ Type II: Resemble commensal bacteria

• Secrete little VacA • Devoid of cag-PAI

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Good News! You Have a Bacterial Infection. Antibiotic Therapy Maintenance Therapy Ulcer Surgery

17 days $995 187 days $11,186 307 days $17,661

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