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Amin Talebibezminabadi

Tuesday 12 November 2013

Virulence Factors and Treatment of Helicobacter pylori infections

Promotor: Prof. dr. J. Wagenaar
Defence: 12 November 2013

Helicobacter pylori (H. pylori) is a bacterium that can colonize the epithelial mucosa of the human stomach. With approximately 50% of the world’s population infected by H. pylori, this makes it the most common cause of chronic gastritis, gastroduodenal ulcer disease, and also gastric adenocarcinoma. It is not clear why some people with H. pylori get disease symptoms while most infected individuals never show any symptoms in spite of suffering from serious chronic gastritis. Currently, the main topics for studies on H. pylori are antibiotic resistance and the presence of virulence factors and their association with specific clinical outcomes. To date, different virulence factors such as babA, iceA and vacA have been suggested; however, there is no clue how these factors can affect the disease outcomes associated with H. pylori infection. Another major topic with H. pylori is that over the last years, the efficacy of therapeutic regimens seems to be decreasing due to the development of antibiotic resistance. This is an alarming issue with regard to treatment efficacy.

Virulence Factors
Among the H. pylori virulence factors, cagA is the most important determinant and it has been shown to be undeniably associated with serious H. pylori induced disorders. The cagA gene is located at one end of the cag pathogenicity island (PAI), which is an approximately 40 kbp region that is incorporated into the H. pylori genome by horizontal transfer from an unknown source. In chapter 2 the frequency of cagA gene in H. pylori strains isolated from symptomatic patients were investigated. Of 128 strains, 84 (65.6%) were cagA positive. Much to our surprise no significant association was observed between specific disease types and cagA positive isolates (P>0.05). A lack of correlation between the cagA and more severe diseases has been reported before and was attributed to geographical differences in the studied regions. The duodenal ulcer-promoting gene (dupA) was recently reported as a novel H. pylori virulence factor associated with an increased rate of occurrence of duodenal ulcer (DU) and a decreased risk for gastric cancer (GC). As described in chapter 3, dupA could serve as independent predictor for the clinical outcome of H. pylori infection, as was suggested previously. While our association study provides only circumstantial evidence for an obvious role of dupA as a disease causing factor it cannot prove that it is a true virulence factor. This would e.g. require additional infection experiments with isogenic mutants. In an attempt to explain this we tested the growth of DupA positive and negative strains at various pH conditions. We observed that DupA positive strains are more resistant to low pH, making it likely that DupA positive strains reside in the lower parts of the stomach (antrum vs corpus). This difference in the distribution of the gastric colonization would explain with the different patient populations.
The Helicobacter outer membrane (Hom) adhesion molecules constitute a paralogous family of proteins that contain a signal sequence at the C terminus and several alternating hydrophobic/hydrophilic motifs, which are typical markers of outer membrane proteins. In 2009, it has been suggested that homB can be a good biomarker to predict the presence of gastric adenocarcinoma in H. pylori infected individuals. The authors indicated that more studies with higher sample size in different geographical regions are required to confirm their findings. In the Northern parts of Iran there is a high risk of getting gastric cancer as a result of H. pylori infection. Our results in chapter 4 showed that the homB gene can be a valuable biomarker for the prediction of gastric cancer in H. pylori infected patients (P<0.001). Moreover, our study was the first to confirm this association in an Asian country. But again, this is an association study only and to proof that homB is a true virulence factor one would need to further investigate the exact biological mechanisms.
Presumably, the type and location of the H. pylori colonization in the gastric mucosal epithelium plays a critical role with regards to the clinical symptoms of the H. pylori infection. Thus it is no surprise that the presence/absence of certain H. pylori adhesions is associated with the occurrence of various digestive disorders. In chapter 5, we confirmed the previously reported association between the presence of the adhesion factor encoding babA2 and iceA1 genes and disease outcome. Also in the tested North Iranian population both genes are more prevalent in patients suffering from gastric cancer than in the other patients groups. To date, only a few studies have investigated the possible association of babA2 and clinical outcome. In most Asian countries studies the prevalence of babA2is around 90%. Interestingly we found that in the Iranian population the prevalence of babA2 in H. pylori strains is 40%. Due to this more even distribution between babA2 positive and negative strains in our relatively small study population, we were able to draw statistically significant conclusions on the association between the presence of this adhesion factor encoding gene and disease outcome. However, we need more studies in different geographical regions to confirm current findings. Also here further experiments with higher number of samples in different geographic population are needed to substantiate the general validity of our findings.
In chapter 6 we studied the recently proposed H. pylori virulence factors jhp0652, tnpA and tnpB. The results from our study confirm the significant association of the cagA gene and gastric cancer, and a weak, correlation was observed between the presence of the cagA gene and duodenal ulcer. For the tnpA gene an association with GC was observed, but no significant association was observed for tnpB and gastroduodenal diseases. The exact biological role these genes play in virulence is currently still unclear. Recently, it was suggested that jhp0562 is responsible for encoding a glycosyltransferase involved in LPS biosynthesis. Thus it is tempting to speculate that the presence of the jhp0562gene affects the LPS composition and thereby the colonization properties of the bacterium. The H. pylori tnpA and tnpB genes encode a transpose that can induce deletions in the cag region. As this cag region encodes several virulence factors that are known to affect disease outcome, the disruption of this region is likely to affect the virulence of this gastric pathogen.

As mentioned above in chapter 3 we have used previously published PCR assays to confirm that the presence of the dupA gene is higher in duodenal ulcers patients and lower in gastric cancer patients compared to gastritis only patients. An in silico analysis of all available dupA genes sequences from the public DNA databases revealed significant mismatches of various available dupA primers. In chapter 7, we designed new primers against more conserved dupA sequences and showed that our newly designed primer set (AF-dupA) found more H. pylori isolates positive for the dupA gene than the old primers as designed by others (and used in chapter 3 by ourselves). While we did show that the dupA gene is more prevalent than was previously estimated based on the old PCR assays, we do not know if and how this would affect the associations with the various patient populations as we do not know the clinical symptoms for the patients that we isolated our strains from. Hence this is an aspect that definitely would warrant further testing.

With H. pylori the increase in antibiotic resistance has become an alarming issue that seriously affects the efficacy of H. pylori treatment. While monitoring the H. pylori resistance does not deal with the consequences of this issue, it does aid the physicians in their rational choice of therapeutic formulations. In chapters 8 and 9 we showed that the prevalence of antibiotic resistance is increasing. Unfortunately, resistance against moxifloxacine is also already 4.6% in the tested strains. Frequently used antibiotics such as clarithromycin in respiratory diseases and also metronidazole for gastrointestinal parasites can be an explanation for having such high rate of resistance among the H. pylori strains. However, as we found in chapter 8, moxifloxacine can be an alternative in current therapeutic regimens. Additionally, the next intervention might be considering the use of non-invasive tests such as screening the H. pylori DNA isolated from feacal samples for the resistance of the infecting H. pylori strains.