Investigation of bacterial outer membrane proteins in Helicobacter pylorı infection

Helicobacter pylori is one of the most prevalent infectious disease worldwide. This bacterium is closely associated with human diseases of the upper gastrointestinal tract such as chronic gastritis, peptic ulcer and gastric adenocarcinoma. The role of bacterial virulence factors and pathophysiological mechanisms involved in the development of H. pylori related gastric cancer still remain to be elucidated. Heterogeneity among H. pylori strains in expression of the CagA as well as outer membrane proteins (OMPs) is thought to be important in determining clinical outcome of patients with H. pylori infection. RT RT-PCR was used to study the frequencies and levels of the expression of H. pylori cagA and omp6, omp13 (oipA), omp18, omp20 (alpA) OMPs genes in patients with H. pylori infection in association with clinical, endoscopic and histopathologic findings and to evaluate the effectiveness of different culture media for isolation of H. pylori from gastric biopsy specimens and to develop stock and indicator culture media for isolated H. pylori strains. A total of 179 patients with upper gastrointestinal complaints who were admitted to Outpatient Gastroenterology Polyclinic and Endoscopy Unit at Dokuz Eylül University Hospital and referred to upper endoscopy were included in this study. Antral and/or corporal gastric biopsy specimens were taken from each patient. Rapid urease test (RUT), histopathologic examination as well as culture and clarithromycin susceptibility testing were performed. Culture media containing different supplements as well as indicator and stock culture media were used for isolation and optimal bacterial culture of H. pylori from gastric biopsy specimens of 59 patients.The gene expression status of H. pylori cagA and omp6, omp13 (oipA), omp18, omp20 (alpA) outer membrane protein genes were investigated in antrum and/or corpus gastric biopsy specimens of 101 patients by TaqMan RT RT-PCR. Total RNA from each antrum and corpus was extracted and cDNA was synthesized to determine the gene expression frequencies and levels of H. pylori omp6, omp13, omp18, omp20 and cagA target genes as well as two house-keeping genes by TaqMan RT RT-PCR. Statistical analyses were performed on the data. H. pylori infection was found in 48 (40%) of 120 patients based on both positive RUT and histopathology. Seventy nine (66%) patients were positive for H. pylori by RUT. HLO was found positive in 63 (72%) of 88 patients examined by histopathology. Considering only culture method, gastric antrum and/or corpus biopsy specimens of 104 patients were cultured and 50 (48%) patients were culture positive for H. pylori. Among 48 patients with H. pylori infection, 30 (62.5%) patients were culture positive. The sensitivity and specificity of culture were 86.0% and 90.0% respectively. The endoscopic, histopathologic and demographic findings of patients were evaluated in associaton with H. pylori infection. There was not found statistically significant difference between male and female patients as well as mean age in terms of incidence of H. pylori infection (p=0.324, p=0.529). H. pylori infection was found statistically significant and associated with infiltration of polymorphonuclear (PNL), mononuclear leukocytes (MNL) and high density of H. pylori (HLO) in the antrum and corpus (p=0.006, p=0.001), (p=0.001, p=0.010), (p<0.001) but not associated with intestinal metaplasia (IM) (p=0.616, p=0.870). Of the 101 patients 77 (76.2%) patients were RT RT-PCR positive determined by Ct (threshold cycle) values obtained for both H. pylori 16S rRNA and ureA house-keeping genes. The sensitivity and specificity of RT RT-PCR were 94.0% ve 60.0% respectively. The Ct values of cagA, omp6, omp13 (oipA), omp18, omp20 (alpA), 16S rRNA and ureA genes were determined in gastric biopsy specimens as well as H. pylori NCTC 11637 subcultures. The Ct values were lower in H. pylori standard strain subcultures compared to gastric biopsy specimens. H. pylori gene expression frequencies of cagA, omp6, omp13, omp18 and omp20 in 45 (93.8%) out of 48 patients with H. pylori infection were 30 (66.7%), 35 (77.8%), 37 (82.2%), 45 (100.0%) and 37 (82.2%) respectively. cagA gene expression was positive in 30 out of 45 patients. This rate was lower than those of omp6, omp13, omp18 and omp20 genes. omp18 was expressed in gastric biopsy specimens of all 45 (100%) patients. Endoscopic findings of 48 patients were gastric ulcer/erosion (n=6), duodenal ulcer/erosion (n=6), erosive gastritis (n=11) and non-erosive gastritis (n=25). There was not found any significant association between gene expression frequencies of five studied genes with endoscopic findings (p>0.05). However, the relative expression level (ΔCt) of omp13 gene (gene transcripts) was found statistically significant (p=0.005, p=0.045) between patients with gastric ulcer/erosion and non-erosive gastritis as well as patients with erosive and non-erosive gastritis which was lower in patients with gastric ulcer/erosion and erosive gastritis compared to non-erosive gastritis. There was no significant association between gene expression frequencies/levels of five studied genes with histopathologic (H. pylori density, PNL, MNL) findings (p>0.05). The mean Ct values of five studied genes in antrum were lower than those of corpus. The relative expression levels (ΔCt) of omp6 and omp13 genes (gene transcripts) were higher in corpus than in antrum (p=0.014, p=0.026). H. pylori strains were isolated from gastric biopsy specimens in Columbia Blood agar supplemented with 7% defibrinated horse blood and H. pylori selective supplement (DENT) as well as TTC supplemented indicator medium. H. pylori isolates showed translucent and grey colonies on Columbia Blood agar but golden appearance on the indicator medium supplemented with TTC which was specific and facilitated the identification of isolated H. pylori strains. Detection and determination of the gene expression frequencies and levels of H. pylori virulence gene cagA and genes of some outer membrane proteins omp6, omp13, omp18 and omp20 by RT RT-PCR method revealed that neither cagA nor OMPs genes expression status had a statistically significant impact on patients' clinical outcome in our hospital. omp13 gene which encodes an inflammatory protein or adhesin might play a role in the pathogenesis of H. pylori infection in our hospital. The difference in omp6 and omp13 gene expression levels between the antrum and corpus may be clinically important. RT RT-PCR method was also found a reliable method and can be used in H. pylori pathogenesis related gene expression studies. However studies with larger sample size and more advanced molecular methods should be considered. Key words: Helicobacter pylori, oute rmembrane proteins, cagA, gene expression, RT RT-PCR