The Proteomics Of Stress Responses In Helicobacter Pylori

As one of the most important human pathogenic bacteria, Helicobacter pylori(H.pylori) colonize mainly in human stomach. It has been recognized as the aetiological agent of gastritis and peptic ulcer disease. Moreover, long-term infection with this bacterium is closely related to the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. In 1994 international cancer institute had determined it being type I carcinogen. As shown by epidemiology, the mechanism of transmission of H.pylori is thought to be primarily by the fecal-oral route. It is necessarily inhibited by kinds of adverse conditions during growth in vivo and transmission in vitro. Only by starting a series of adaptive regulatory mechanisms, H. pylori can resist these stressing factors to survive and induce diseases.H.pylori has a unique feature which is its ability to survive in the extremely acidic environment of human stomach. The pH of human stomach content ranges from 1.0 during starvation to 5.0 in the digestive phase. When migrating from acidic lumen into gastric mucus layer, H.pylori invariably faces pH fluctuations. One of the best-studied processes in H pylori to resist acid stress is the urease system. However, the urea in gastric juice is approximately 1 mM. which sometimes may be insufficient to ensure the survival of H.pylori. Thus it is assumed that additional mechanisms of pH homeostasis may be required for the acid adaptation in H.pylori.The reflux of bile into the stomach post-prandially is a normal occurrence, especially in patients with gastroduodenal disease. Many studies support that faecal-oral is one important route of H.pylori entering the digestive tract. During transmission, H.pylori must tolerate and resist bile stress when traversing the bile-rich intestinal tract. Furthermore, H.pylori has been detected in bile, gall-bladder tissue and liver samples through polymerase chain reaction and Southern blot hybridization. All these points indicate that the ability of H.pylori to tolerate bile is likely to be very important for their colonization and survival in the gastrointestinal tract of humans. Although it has been established that H. pylori is bile-sensitive, the mechanism allowing it to colonize a bile-containing environment is unclear.Morphologic change is an important adaptive ways for bacteria to resist various unfavourable conditions. The viable but nonculturable coccoid form of H.pylori can be induced by many environmental stresses such as O₂ stress, pH alteration or exposure to antibiotics. Transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism. When stressed, bacteria can enter various nondividing states, which are medically important. It has been shown that the filaments of bacteria are formed when cell division is inhibited while their growth continues. Previous studies found that H.pylori changes into filamentous form in a high-salt level medium at early stationary phase, aztreonam can also induce pronounced filamentation in H.pylori. The study on filamentous H.pylori is helpful to identify possible cell division checkpoints in this bacterium.Genomic and proteomic technologies are both integrative and high-throughput technologies whose rapid development led to a burst across all branches of the life sciences. Proteins are the direct executors of vital activities and mRNA level is not consistently reliable to predict protein abundance due to the posttranslational and posttranscriptional modification. Proteomics is to analyze the structure and function of the whole proteins expressed by genome in a cell, tissue or organism at global level. Furthermore, proteomic technologies have been proven to be particularly useful to study the physiological responses of bacteria to various environmental stresses. The genomic sequencing of two H.pylori strains (H.pylori26695 and H.pyloriJ99) has been completed. In order to efficiently exploit this information, we analyzed the protein expression changes of H.pylori under acid and bile stress and filamentous H.pylori using comparative proteomics. The aim is to reveal the possible survival mechanisms underlying in this important human pathogen. The research contents and main results are as follows:1. The changes of proteomes components of Helicobacter pylori in response to acid stress without urea.Acid stress is the most obvious challenge Helicobacter pylori (H. pylori) encounters in human stomach. The urease system is the basic process to maintain periplasmic and cytoplasmic pH near neutrality when H.pylori is exposed to acidity. However, since the urea concentration in gastric juice is approximately 1 mM, which may be insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis seem to contribute to the acid adaptation in H.pylori. To identify the acid-related proteins other than the urease system we have compared the proteome profiles of H.pylori strain 26695 exposed to different levels of external pH (7.4, 6.0, 5.0, 4.0, 3.0 and 2.0) for 30min in the absence of urea using 2-DE. Differentially expressed proteins were identified by MALDI-TOF-TOF-MS analysis, which turned out to be 36 different proteins with diverse functions, including ammonia production, molecular chaperones, energy metabolism, cell envelope, response regulator and some proteins with unknown function. SOM analysis indicated that H.pylori responds to acid stress through multi-mechanisms involving many proteins, which depend on the levels of acidity the cells encounter.2. Helicobacter pylori proteins response to human bile stress.The ability of H.pylori to tolerate bile is likely to be very important for its colonization and survival in the gastrointestinal tract of humans. Since bile can be acidified after reflux into the low pH of human stomach, we first tested the inhibitory effect of fresh normal-appearing human bile on H.pylori before and after acidification. The result showed that the acidification of bile attenuated its inhibitory activity on H.pylori. Next, the protein profiles of H.pylori under human bile and acidified bile stress were obtained by two-dimensional electrophoresis. Protein spots with differential expression were identified using tandem matrix assisted laser desorption ionzation time of flight mass spectrometry (MALDI-TOF-TOF). The results showed that the changes of proteomic profiles under bile and acidified bile are similar when compared with the normal H.pylori. The expression of 28 proteins was found to be modulated with the majority being induced both duing bile or acidified bile exposure. These proteins include molecular chaperones, proteins involved in iron storage, chemotaxis protein, enzymes related to energy metabolism and flagellar protein. It indicates that H.pylori responds to bile and acidified bile stress through multi-mechanisms involving many signal pathways. And furthermore, to confirm the function of some proteins in H.pylori response to bile stress, we constructed the deficiency mutant of H pylori predicted coding region Hp0721 and flagellar motor switch protein and found they have lower resistant ability to bile stress. This suggests that these two proteins are likely to play a role in bile resistant response of H.pylori.3. Analysis of aztreonam-inducing proteome changes in nondividing filamentous Helicobacter pyloriWhen stressed, bacteria can enter various nondividing states, which are medically important. In the present study, nondividing filamentous form in H.pylori was induced by aztreonam, which can block cell division by inhibiting FtsI, a transpeptidase required for cross-linking of the peptidoglycan cell wall during division. Realtime-PCR results showed that FtsI has a lower expression level in filamentous H.pylori while the expression of other several penicillin-binding proteins (Hp0597, Hp1372, Hp1373 and Hp1565) did not display obvious difference when H.pylori becomes to nondividing filamentous form. In order to find other possible cell division checkpoints in H.pylori, 2-DE was used to compare the proteomic profile of nondividing filamentous H.pylori with its spiral form. Differentially expressed protein spots in 2-DE map of filamentous H.pylori were identified by MALDI-TOF-TOF analysis. These turned out to be twenty-one different proteins that are involved in various cellular processes. Out of them there is one protein, cell division inhibitor (MinD), related to cell division was induced by aztreonam. Sequence comparison showed that MinD of H.pylori and that of E.coli share 50% identical residues. We then constructed the depletion mutant of MinD in H.pylori26695. Scanning electron microscope observation showed that the depletion of this protein provoked some bacteria change into minicell-shaped and the growth and viability of the mutant is lower than that of the wild type. This may be due to the minicells induced by depletion of MinD are almostly anucleated cells and do not give rise to viable colony forming units.