Quantitative analysis of mRNA expression of protease genes among different Helicobacter pylori strains by Reverse transcriptase-PCR

Background and Aim : Helicobacter pylori is the main cause of several gastroduodenal diseases in Humans. Among various virulence factors of H. pylori, proteases may also be involved in its pathogenicity. More than ۲۰ putative proteases were described in H. pylori strains. To show any possible role for these proteases, this study aimed to evaluate mRNA expression of seven protease genes, including htrA (hp۱۰۱۹), clpP (hp۰۷۹۴), collagenase (hp۰۱۶۹), pqqE (hp۱۰۱۲), metalloprotease (hp۰۳۸۲), PspA (hp۱۴۳۵), and protease ۱۳۵۰ (hp۱۳۵۰) among different H. pylori strains by reverse transcriptase PCR (RT-PCR).Methods : This study was conducted on ۱۹ H. pylori strains isolated from patients with gastrointestinal disorders referred to Taleghani hospital in Tehran, Iran. The presence of seven protease genes in all strains was identified using PCR. To determine mRNA gene expression, each strain was inoculated with ۲ McFarland concentrations on BHI broth supplemented with horse serum and iron chloride and were incubated at ۳۷ °C for ۳ days under microaerophilic atmosphere. Total RNA was extracted using the RNeasy Plus Mini Kit and then was reverse transcribed into DNA using a cDNA synthesis kit. Prepared cDNA was used as a template in PCR using the specific primers of proteases. Finally, to evaluate the quantitative mRNA expression of protease genes, the gel images were analyzed using UVIgeltec software.Results : While mRNA expression was not observed in ۱۰.۵۲%, ۵.۲۶%, and ۵.۲۶% of the pqqE, collagenase, and clpP encoding strains, respectively, in the cases of other proteases, including htrA, metalloprotease, pspA and protease ۱۳۵۰, there was a complete link between the expression of protease genes and their positive genotype. Besides, what was seen about the expression of protease genes was their high expression diversity among different strains.Conclusion : Our results showed a great variety in the mRNA expression of protease genes. Since not all strains encoding protease genes showed the levels of the mRNA expression, the association between positive protease genotypes and their expression was not confirmed.

Keywords:

Helicobacter pylori , protease , mRNA expression , RT-PCR