| Helicobacter pylori is a spiral-shaped, microaerophilic, Gram-negative bacterium that infects the stomachs of more than 50% of the world's population. It has been demonstrated that Helicobacter pylori infection is strongly associated with some gastrointestinal diseases, such as gastritis, peptic ulcers, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Epidemiological studies show that fecal-oral route is thought to be the primary manner of transmission of H. pylori. In order to establish an infection and to persist in the stomach, H. pylori must overcome the constantly changing environments such as nutrient deficiency, oxygen tension, and low pH in the stomach and so on. The regulatory mechanisms of H. pylori to allow bacterial survival under these environmental stresses are of interest.The sigma factor is an essential dissociable subunit of prokaryotic RNA polymerase that confers promoter recognition specificity on RNA polymerase in the initiation of transcription. Bacteria can use alternative sigma factors, which direct the RNA polymerase holoenzyme to a specific class of promoters, to adapt to environmental changes. The association of appropriate alternative sigma factors with core RNA polymerase provides an effective mechanism for simultaneously regulating large numbers of prokaryotic genes. In gram-negative bacteria, RpoS (σS) has been recognized as a general stress responsive sigma factor that contributes to survival in stationary-phase and other stress conditions such as osmotic shock, heat, and low pH. However, as the small genome of H. pylori, relatively few transcriptional regulators for gene expression have been annotated, including just three sigma factorsσ70 (RpoD), a54 (RpoN), and a28 (FliA), whereasσs which is typically associated with various stress responses in many gram-negative bacteria is lacked in H. pylori. However, a54 does not share a common role among all pathogens, as a54 dependent genes described to date control a wide diversity of processes. We are interested in whether a54 of H. pylori could regulate bacterial survival when H. pylori encounters environmental stresses.In the present study, we found that the expression of a54 is induced when H. pylori enters into stationary phase, in which stage the nutrient becomes exhausted and harmful substance is accumulated. Additonally, the rpoN mutant showed a marked decrease in viability in late stationary phase compared with wild-type H.pylori. These results indicatedσ54 played an important role when H.pylori is subjected to unfavorable situations. However, the mechnism by which a54 regulate H. pylori survival in starved environment remains unclear. Thus proteomic analysis was used to compare the protein expression profiles of the H. pylori 26695 and its rpoN null mutant in early stationary phase.Many clinical evidences show that eradication of H. pylori results in significant remission from these diseases. Widely used triple therapy, consisting of a proton pump inhibitor and two antibiotics such as metronidazole, amoxicillin, or clarithromycin, yields a high eradication rate. However, eradication failure often occurs, which is associated with undesirable side effects of these drugs, poor patient compliance and high cost of combination therapy. An additional reason that should be emphasized is the increasing resistance of H. pylori to antibiotics. For example, strains of H. pylori resistant to metronidazole and clarithromycin have been reported. Thus, it becomes highly necessary to search for an efficacious antibacterial agent to overcome the above clinical problems. Garlic probably has the potential to fulfill these requirements.Garlic, a natural food in diet, has some extraordinary advantages as an antibacterial agent, including easy accessibility, low cost and negligible side effects with moderate consumption. Garlic is even active against antibiotic-resistant organisms. Garlic extracts in combination with antibiotics can lead to total or partial synergism. Garlic can also suppress toxin production by bacteria.It has been shown that garlic constituents can inhibit the growth of H. pylori in vitro. Moreover, garlic in combination with omeprazole showed a synergic effect in vitro in a concentration-dependent manner. Several epidemiological studies suggested that a decreased risk of gastric cancer is associated with an increasing consumption of allium vegetables, possibly due to an effect on H. pylori, as this organism is associated with gastric cancer.Thus, it is very important to study the antibacterial mode of action of garlic constituents because of the high incidences of H. pylori-related diseases throughout the world. Although previous studies have revealed that the antimicrobial effect of garlic is mainly due to its chemical reaction with thiol groups of various enzymes, such as alcohol dehydrogenase and thioredoxin reductase, a detailed analysis is lacking of the global molecular responses induced by garlic and its derivatives, and a proteomic strategy is required to globally profile the cellular responses at the protein level under defined conditions.The contents of my research and main results are as follows:1. The regulation ofσ54 on Helicobacter pylori survival of stationary phaseDuring the process of infection and transmission, Helicobacter pylori should be confronted with unfavorable enviroments and the bacterium requires rapid alterations in gene expression to allow itself survival under stress conditions. However, the mechanism underlining this regulation remains unknown. We set the stationary phase as a model to study the survival regulation of H. pylori in response to adverse enviroments. Our results that the expression ofσ54 is significantly induced in stationary phase and the rpoN mutant showed a significantly lower viability than the wild-type H. pylori in late stationary phase, suggesting that a54 is involved in H. pylori survival under adverse enviroments. Proteomic analysis identified 11 proteins were positively regulated and 10 proteins were negatively regulated by a54 in stationary phase. Our data revealed that when H. pylori enters into stationary phase,σ54 can decrease the need for proliferation through negatively regulating the genes involved in energy metabolism and biosynthesis, and enhance stress-resistant ability through positively regulating the genes involved in protein fate and redox reaction, which would result in prolonged bacterial survival under adverse environment. Our investigations will shed new light on the adaptive regulation of H. pylori under stress conditions.2. The mode of action of allitridi against Helicobacter pylori Eradication of Helicobacter pylori with traditional therapy often fails in clinical treatment. As a result, a novel efficacious therapeutic agent is strongly needed. Allitridi, a proprietary garlic derivative, has been successfully used to treat both systemic fungal and bacterial infections in China. Our previous study has shown a dose-dependent inhibitory effect of allitridi on H. pylori growth. However, the antibacterial mode of action of allitridi is still unclear. Proteomic analysis was used to study the global protein alterations induced by allitridi. A total of 21 protein spots were identified to be differentially expressed. Our results indicated that the bacteriostatic mechanism of allitridi in H. pylori can be attributed to its multitarget inhibitory effects in energy metabolism and biosynthesis including amino acid biosynthesis, protein synthesis, mRNA synthesis and fatty acid biosynthesis. Allitridi can also disturb the expression of antioxidant proteins and decrease the production of virulence factors. Western blot analysis showed that allitridi at subinhibitory concentrations can potently suppress the production of CagA and VacA. Our investigations on the antibacterial mode of action of allitridi provide an insight into the potential use of allitridi as a therapeutic agent against H. pylori infection.
|
PDF Full Text Request