DOI: 10.14457/tu.the.2020.853
title: Virulence gene expression of helicobacter pylori under adverse conditions

description: Helicobacter pylori, a dimorphic gram-negative microaerophilic bacterium, is a major cause of dyspepsia and a carcinogenic agent of gastric cancer. H. pylori is capable to survive in an adverse environment by transforming its spiral shape to coccoid form. It was previously reported that the coccoid H. pylori retained its ability to produce some virulence factors. For more understanding, the expression of major virulence genes (cagA, vacA, ureA, ureB, rocF, ggt, babA, sabA, napA, and oipA genes) of the coccoid induced by three different conditions; atmospheric oxygen, natural antibacterial herb and acidic pH were analyzed by the quantitative RT-PCR with SYBR green fluorescein method. H. pylori was converted into the coccoid by an incubation in the atmospheric oxygen and the expression of the coccoid virulence genes were compared with those of the spiral bacteria. After 24 hours of the oxygen exposure, approximately 85.52% of the coccoid-shape bacteria was observed. Of interest, all 10 virulence factor mRNAs of the ambient-oxygen treated sample were continuously produced. It was noted that most of the virulence factor mRNAs were transcribed in lower levels than those of the untreated sample. In contrast, the expression of the cagA gene was up-regulated over 2.4-fold. This data provides an insight into an awareness of the H. pylori coccoid form that could possibly be contaminated in an ambient environment and remains infectious.An increased antibiotic resistance of H. pylori leads to a number of explorations for new antibacterial compounds. Curcumin, the major bioactive ingredient of turmeric (Curcuma longa L.) was reported to inhibit the H. pylori growth and showed a therapeutic potential against H. pylori infection. Regarding to peptic ulcer, several effects of the curcumin were demonstrated on human gastric tissues, however, the effects on the H. pylori itself have been unclear. To gain more insight into the effects of the curcumin on these bacteria, the minimum inhibitory concentration of the curcumin against the H. pylori was determined (31.25 µg/ml). The bacteria were grown on the media containing the curcumin at subinhibitory concentration (15.63 µg/ml) and the expression level of the bacterial major virulence genes were examined. It was observed that the spiral-shape bacteria changed their morphology into the coccoid form in dose dependent manner, i.e., 10.89% at subinhibitory concentration (15.63 µg/ml) and 93.08% at minimum inhibitory concentration (31.25 µg/ml). It was noted that the curcumin-induced coccoid bacteria continuously expressed all of the virulence mRNAs with the topmost up-regulation of the ureA, ureB and babA genes. Data obtained in this study not only suggested another possible bacterial target of curcumin but also revealed the infectious ability of the H. pylori after exposed to antibacterial herb such as curcumin at subinhibitory concentration.In response to unfavorable environment as inside the human stomach, H. pylori can transform its shape from the spiral shape into coccoid form. Recent studies reported that even in an acidic environment, the pH-induced coccoid continuously produced some virulence factors. To extent our knowledge, the expression of the unreported ggt, babA and oipA genes and the previously published gene were studied in the acid stimulated coccoid form. The spiral H. pylori were converted into the coccoid form by growing on the culture media with acidic pH and the expression of the bacterial virulence genes were compared to those that were grown on the media with neutral pH. After a 3-day acid exposure, it was observed that 96.94%, 9.52% and 2.64% of the spiral-shape bacteria exposed to pH 4.0, 5.5 and 7.2, respectively, changed their morphology into the coccoid form. Despite in the coccoid form, the bacteria continuously expressed all major virulence protein mRNAs. Regarding to the NH3-producing enzymes, the up-regulation of the ureA, ureB and ggt genes but not the rocF gene were observed at acidic pH. The rocF gene encodes arginase, an enzyme in the final step of urea cycle that converts arginine to ornithine and urea. The resulted urea was subsequently used as a substrate for NH3 production. Unchanged expression of the rocF gene in this study indicated that the decreased pH may have less effect on the expression of this gene. In addition, the expression of the babA gene was also up-regulated.