H. PYLORI AND C. JEJUNI

Question 1

1. How can Helicobacter infections be diagnosed and what is the most common therapy?

Answer 1

For the diagnosis, these are the most common approaches:
- Biopsy check using endoscopy. It is a really quick urease test. It consists on an histological examination + microbial culture.
- Carbon urea breath. 14C or 13C-labelled urea is metabolized into CO2
- Blood antibody test
- Stool antigen test.
For the treatment:
- Triple therapy
o Proton pump inhibitor to stop acid production (omeprazole, lansoprazole, esomeprazole, pantoprazole).
o Clarithromycin
o Amoxicillin
- Quadruple therapy + bismuth subsalicylate (pepto-bismol; coats ulcers).
But be careful because the resistance is increasing. This can become a major problem. It is inside the priority list for R&D of new antibiotics.

Question 2

2. Please list three factors/mechanisms that are important for virulence and colonization of the human stomach by Helicobacter pylori?

Answer 2

1. The production of urease to catalyze the hydrolysis of urea into carbon dioxide and ammonia to neutralize the acidic environment of the stomach.
2. The expression of the adhesin BabA and SabA to adhere to host cells. BabA adhesin binds fucosylated blood groups antigens in early stages of colonization, while SabA binds sialylated Lewis X antigens. The adhesion is acid sensitive.
3. Secretion system. Most Helicobacter pylori strains that cause disease contain the cag pathogenicity island (PAI), a chromosomal region comprising approximately 37,000 base pairs and 29 genes. Most of the cag genes are probably involved in the assembly of the type IV secretion system that translocates the protein CagA into the cytoplasm of gastric epithelial cells. CagA is an oncoprotein. • CagA acts as non-physiological scaffold/hub protein by interacting with multiple host signaling molecules • CagA and CagA~P cause numerous cell alterations: affects cell structure polarity & motility; cell scattering & proliferation; activation of pro-oncogenic protein tyrosine phosphatase SHP2

Question 3

3. Which enzyme facilitates survival of H. pylori in the acidic environment of the human stomach and what is it underlying molecular mechanism?

Answer 3

Urease. Catalyzes the hydrolysis of urea into carbo dioxide and ammonia. It neutralizes the acid of the human stomach.
Mechanism: Urea crosses the outer membrane (OM) and then the inner membrane (IM) through the H+ gated urea channel, UreI. Cytoplasmic urease hydrolyzes urea 2NH3 `H2CO3, and the latter is converted to CO2 by cytoplasmatic beta-carbonic anhydrase (β-CA). These gases cross the IM and the CO2 is converted to HCO3 − by the membrane bound α-carbonic anhydrase (α-CA), thereby maintaining periplasmic pH at ~6.1. Exiting NH3 neutralizes the H+ that is produced by carbonic anhydrase as well as the entering H+, and can also exit the OM to alkalize the medium. This allows maintenance of a periplasmic pH that is much higher than the medium.

Question 4

4. VacA of H. pylori is a key example for a multifunctional toxin. Please list two mechanisms/roles of VacA during H. pylori virulence.

Answer 4

VacA is a pore forming toxin (vacuolation). It has some roles during H. pylori virulence.

• Alter mitochondrial membrane permeability.
• Interferes with the activation and proliferation of T lymphocytes.
• It stimulates pro inflammatory signaling

Question 5

5. What are the most common sources of Campylobacter spp. infections?

Answer 5

More than 90% of sporadic human cases are because of consumption of contaminated poultry, beef or milk.

Question 6

6. Please list two differences in colonization of human and chicken hosts by Campylobacter jejuni.

Answer 6

In humans C. jejuni circumvents the mucus layer and interacts with the intestinal epithelial layer. By contrast, C. jejuni resides primarly in the mucosal layer in chickens’ intestines, without interacting with epithelial cells.
Interaction of C. jejuni with human epithelial cells causes IL-8 production, which causes recruitment of dendritic cells (DC), macrophages and neutrophils. This leads to inflammation, diarrhoea and clearance. In chicken it has been observed in vitro that IL-1b and IL-6 are produced, but this host response does not lead to inflammatory diarrhoea in chickens.
Chicken: 41-45°C, human 37°C: body temperature as potential signal for host-specific infection.

Question 7

7. Campylobacter jejuni lacks a classical secretion system for injection of virulence factors into host cells. How are proteins secreted by Campylobacter jejuni ? What type of secretion system does the responsible cellular structure resemble?

Answer 7

The functional flagellar apparatus acts as a secretion machinery using cia and Fed proteins.
It resembles the T3SS: It is composed of an MS ring (dark blue) and C ring (orange) that surround the flagellar type III secretion system (fT3SS) core in the inner membrane, a rod and hook structure (dark blue) that transverses the periplasm and outer membrane and an extracellular flagellar filament (red).

Question 8

8. Please describe the composition and mechanism of action of the cytolethal distending toxin (CDT) of Campylobacter jejuni.

Answer 8

It is composed of three subunits: CdtA, CdtB and CdtC.
CdtA and CdtC are thought to bind to an unknown receptor on the host cell surface. CDT is taken up into host cells by way of clathrin-coated pits. Following internalization, nuclear localization signals on CdtB probably lead to its active transport into the nucleus through the classical nuclear-import cycle. Once in the nucleus, the toxin leads to double-strand DNA breaks and cell-cycle arrest. Whether or not CdtB acts as a DNase to cause the DNA damage directly, as opposed to this being an indirect effect of some other CdtB enzymatic activity, has yet to be established. This toxin also induces IL-8 in the intestine causing inflamation

Question 9

9. Campylobacter and Helicobacter have numerous „homopolymeric simple sequence repeats“in their genomes. Please describe two mechanisms how such repeats can lead to differential gene expression.

Answer 9

A feature of both eukaryotic and bacterial SSRs is that they are prone to high rates of mutation through slipped strand mispairing.
o Intergenic SSR: in promoter elements, can change spacing between -10 box and -35 box, affecting transcription.
o Intragenic SSR: can cause frame-shift mutations, affecting translation

Question 10

10. What are the three surface structures important for virulence of Campylobacter jejuni? What is the molecular basis for the Guillan-Barré Syndrom that can occur as a sequela during Campylobacter jejuni infection?

Answer 10

Surface structures: Lipopolysaccharyde (LOS), capsule and flagella.
Guillan-Barré syndrome and the Miller Fisher syndrome are autoimmune disorders caused by the human neuronal ganglioside. The LOS of C. jejuni is a lipid A linked polysaccharide found in the outer leaflet of the outer membrane. It lacks the O-specific polysaccharide of other Gram-negative bacteria. This LOS is highly variable. It presents molecular mimicry of human neuronal ganglioside.