The Proteins S-Nitrosylation And Proteomics Of Helicobacter Pylori To Nitric Oxide Stress

Helicobacter pylori(H.pylori) is a Gram-negative,spiral and microphilic bacteria that selectively colonizes gastric mucosa,causes gastritis and gastric ulcers. This human pathogen is also strongly associated with gastric adenocarcinomas and mucosa-associated with lymphoid tissue lymphomas.In 1994,international cancer institute had determined it being typeâ… carcinogen.After colonizing the gastric mucosa,H.pylori can stimulate the human immune system,involving local and systemic antibody reaction,cell-mediated immune responses and so on.The current data from clinical tests and animal experiments shows that the host innate and adaptive immune reactions can protect against H.pylori to a certain degree,and their antibacterial actions will contribute to designing and constructing drugs to suppress the bacterial infection effectively.Nitric oxide(NO) is an important effector possessing antimicrobial activity and immunomodulatory effect.NO originates from the oxidative L-arginine pathway catalyzed by a family of enzymes termed nitric oxide synthase(NOS).There are two kinds of NOS in the organism:constitutive nitric oxide synthase(cNOS) and inducible nitric oxide synthase(iNOS).During the microbial infections,inflammation and some states of physiological stimulation,iNOS is expressed in various cells. When iNOS is expressed,a sustained production of NO can be formed.During microbial infections,excessive NO produced by iNOS plays important roles in the antimicrobial host defense.Thus,NO is one of the focuses in the research fields of current biology and medical science,especially in the microbiology field.It has become evident that the cysteine residues involve a multitude of protein functions.The process of NO binds to specific cysteine residues covalently is known as S-nitrosylation.This posttranslational modification participates in a broad range of physiological and pathological cellular events,such as smooth muscle relaxation, neurotransmission and immunoprotection.The most commonly used method for analysis of S-nitrosylation is biotin switch method(BSM).A combination of BSM with MS identification is used to analyze the S-nitrosylated proteins in different organisms.The antibacterial action of NO depends mainly on its ability to S-nitrosylate the cysteine residues.Therefore,BSM analysis is suitable to explore the specific targets of NO and RNS.Genomie and proteomic technologies are both integrative and high-throughput technologies whose rapid development has led to a burst across all branches of the life sciences.Gene is the carriers of genetic material,while proteins are the direct executors of vital activities,mRNA level is not consistently reliable to predict protein abundance due to the posttranslational and posttranscriptional modification. Proteomics is the large-scale study of proteins,particularly their structures and functions in the given time,space and distinct requirements.Furthermore,proteomic technologies have been proven to be particularly useful to study the adaptive responses of bacteria to various environmental stresses.The genomic sequencing of two H.priori strains(H.pylori26695 and H. pyloriJ99) has been completed.In order to efficiently exploit this information,we analyzed the S-nitrosylated proteins and described the protein expression changes of H.pylori under nitrosative stress using comparative proteomics.The aim is to reveal the specific targets of NO and RNS and characterize the protein response of H.pylori to the immune effectors.The research contents and main results are as follows:1.The proteins S-nitrosylation of Helicobacter pylori to nitric oxide stressThe human innate and adaptive immune responses are activated while Helicobacter pylori(H.pylori) invades the human gastric mucosa.Nitric oxide(NO) is an important innate immune effector,however the suppressing effect of NO and reactive nitrogen species(RNS) to H.pylori is not clear.The major action of RNS is protein oxidation and S-nitrosylation.However,the targets of RNS in H.pylori are unknown.Here,we described the strong inhibitory and killing effects of RNS to H. pylori and analyzed the S-nitrosylation of proteins in a whole organism after treating NO donor S-nitrosoglutathione GSNO.In this study,the H.priori protein was incubated in vitro with GSNO to induce protein S-nitrosylation,and the resulting S-nitrosylated proteins were purified by the biotin switch method(BSM),followed by gel electrophoresis separation and matrix-assisted laser desorption ionization/time of flight tandem mass spectrometry(MALDI-TOF-MS/MS) identification.5 S-nitrosylated proteins identified were chaperone and heat shock protein(GroEL), alkyl hydroperoxide reductase(TsaA),urease alpha subunit(UreA),HP0721 and HP0129.It can be concluded that targeting of enzymes related to antioxidant defenses, adherence and colonization through S-nitrosylation may partly explain the antibacterial actions of NO to H.pylori.2.The proteomies of Helicobacter pylori to nitric oxide stressHelicobacter pylori(H.pylori) is a highly pathogenic microorganism with various strategies to evade human immune responses.Nitric oxide(NO) and reactive nitrogen species(RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response.However,the mechanisms of H.pylori how to survive the nitrosative stress are not clear.Here the proteomic approach has been used to define the adaptive response of H.pylori to nitrosative stress.Proteomic analysis showed that 38 protein spots were regulated by NO donor,sodium nitroprusside(SNP).These proteins were involved in protein processing, antioxidation,general stress response and virulence,as well as some unknown functions.Particularly,some of them were participated in iron metabolism,potentially under the control of ferric uptake regulator(Fur).Real time PCR revealed that fur was induced under nitrosative stress,consistent with our deduction.One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase(TrxR). Inactivation of fur or trxR can lead to increased susceptivity to nitrosative stress respectively.These studies described the adaptive response of H.pylori to nitric oxide stress,and analyzed the relevant roles of Fur regulon and TrxR in nitrosative stress management.