DOI: 10.14457/tu.the.2020.856
title: Preliminary study of secretome analysis from Helicobacter pylori dual resistance to clarithromycin and metronidazole strain

description: An untreated Helicobacter pylori (H. pylori) infection is the major cause of gastritis which can develop into stomach cancer. Eradication of H. pylori with antibiotics is a simple, non-invasive, inexpensive method that makes it an accessible treatment for the public. Therefore, in the current gastric cancer prevention, this treatment is an effective treatment for gastritis that has been proven to be associated with H. pylori. An up-to-date standard treatment for H. pylori eradication is in the form of triple therapy: two antibiotics (clarithromycin and metronidazole) and a proton pump inhibitor. However, current antibiotic treatments for H. pylori infection are less effective due to increased rates of drug resistance. This occurs due to an unnecessary overuse of antibiotics, antibiotics abuse of patients, and taking unnecessary medications for too long. To date, the prevalence of drug resistance from clarithromycin, metronidazole, as well as concomitant resistance to clarithromycin and metronidazole (Cla-R, Mtz-R, Cla/Mtz-R, respectively) in the eradication of H. pylori remains high worldwide. For a better understanding and exploration of a new protein relevant to treatment options for H. pylori bacteria, this research, hence, aimed to study proteins secreted from Cla/Mtz-R, Cla-R, Mtz-R, H. pylori-associated gastritis strains. By working with proteomic study (in-solution digestion combined with nanoLC-MS/MS), 592 expression proteins were detected, of which 590, 583, 582, and 578 proteins were identified from Cla/Mtz-R, Cla-R, Mtz-R, and Sensitive strains (Sen-S), respectively. After identifying the biological activity of proteins through protein database searches (PANTHER software version 15.0), These proteins are responsible for the cellular processes such as the metabolic process (41%), cellular process (27%), process localization (10%), etc. Based on the Venn diagram, there are 23 different proteins that overlap between the four strains. In addition, by analyzing the heat maps of these overlapping proteins, the results showed that the Cla/Mtz-R strains exhibited the most differentiated protein expressions, while the less different expressions were found in the Sen-S strains. When considering the overlapped proteins based on the Sen-S strains, nine proteins were identified. In addition, the prediction between proteins-proteins and proteins-antibiotics (metronidazole and clarithromycin) interaction through the STITCH database has been studied. The results revealed two possible central role proteins: rpoBC (found in Cla/Mtz-R, Cla-R, and Sen-S strains, but not found in Mtz-R strains) and FBPAII (found in Sen-S strains only). To quantitatively determine the mRNA expression level of rpoB and fba genes, quantitative reverse transcription PCR analysis was then performed. Their primers were designed from H. pylori 26695 template DNA by Primer 3 web version 4.1.0. Compare with the Sen-S strain, the result showed a down-regulation of rpoB from all three antibiotics strains, though its secreted protein was identified in Cla/Mtz-R, Cla-R, and Sen-S strains, but not in Mtz-R strains. This suggests the relationship between rpoB expression and metronidazole sensitivity. On the other hand, an up-regulation of fba gene mRNA expression level was observed from all three strains used in this study. This suggests inconsistent expression levels between protein (FBPAII) and mRNA (fba) that may be involved in the mechanism of antibiotic susceptibility and resistance to H. pylori. In conclusion, this is the first report that demonstrates the secretome analysis from clarithromycin, metronidazole, and dual resistance H. pylori-associated gastritis compared to the sensitive strain. Moreover, it also shows the association of rpoBC and FBPAII with antibiotic susceptible H. pylori-associated gastritis. However, the exact mechanisms of the two proteins involved in keeping the pathogens susceptible to antibiotics remain to be studied in the future.