Structure Based Annotation of Helicobacter pylori Strain 26695 Proteome

The availability of complete genome sequences of H. pylori 26695 has provided a wealth of information enabling us to carry out in silico studies to identify new molecular targets for pharmaceutical treatment. In order to construe the structural and functional information of complete proteome, use of computational methods are more relevant since these methods are reliable and provide a solution to the time consuming and expensive experimental methods. Out of 1590 predicted protein coding genes in H. pylori, experimentally determined structures are available for only 145 proteins in the PDB. In the absence of experimental structures, computational studies on the three dimensional (3D) structural organization would help in deciphering the protein fold, structure and active site. Functional annotation of each protein was carried out based on structural fold and binding site based ligand association. Most of these proteins are uncharacterized in this proteome and through our annotation pipeline we were able to annotate most of them. We could assign structural folds to 464 uncharacterized proteins from an initial list of 557 sequences. Of the 1195 known structural folds present in the SCOP database, 411 (34% of all known folds) are observed in the whole H. pylori 26695 proteome, with greater inclination for domains belonging to α/β class (36.63%). Top folds include P-loop containing nucleoside triphosphate hydrolases (22.6%), TIM barrel (16.7%), transmembrane helix hairpin (16.05%), alpha-alpha superhelix (11.1%) and S-adenosyl-L-methionine-dependent methyltransferases (10.7%).     

Nucleotide correspondence between protein-coding sequences of Helicobacter pylori 26695 and J99 strains

Comparison of open-reading frames (ORFs) H. pylori 26695 and J99 strains has been revealed prevalence of nucleotide replacements as transitions (more than 3%) above transversions (less than 1%). Prevalence of nucleotide transitions is caused by high speed of C : G to T : A transitions in a coding strand of DNA (3.5-5.3%) and not coding strand (2.9-3.9%). The correspondence rate of transversion (A --> C, A --> T, C --> A, C --> G, G --> C, G --> T, T --> A and T --> G) did not exceed 0.84%. The highest correspondence frequency between C and T was detected in ACGT-ATGT (28.3%) - the site of methylation by active methyltransferase M.Hpy99XI in H. pylori 26695 and J99. Thus one can speculate that predominant transition taking place in H. pylori is mutation of C into T, which is realized through cytosine methylation-deamination mechanism.     

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Helicobacter pylori strain 26695

Helicobacter pylori causes gastroduodenal disease, which is mediated in part by its outer membrane proteins (OMPs). To identify OMPs of H. pylori strain 26695, we performed a proteomic analysis. A sarcosine-insoluble outer membrane fraction was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 10 to 100 kDa, were visible on the gel in the alkaline pI regions (6.0 to 10.0). The proteome of the OMPs was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Of the 80 protein spots processed, 62 spots were identified; they represented 35 genes, including 16 kinds of OMP. Moreover, we identified 9 immunoreactive proteins by immunoblot analysis. This study contributes to the characterization of the H. pylori strain 26695 proteome and may help to further elucidate the biological function of H. pylori OMPs and the pathogenesis of H. pylori infection.     

Functional analysis of an acid adaptive DNA adenine methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N(6)-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3'and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co(2+), Mn(2+), and also Mg(2+) stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.     

Lipopolysaccharide structures of Helicobacter pylori wild-type strain 26695 and 26695 HP0826::Kan mutant devoid of the O-chain polysaccharide component

We describe a re-investigation of the structure of the lipopolysaccharide (LPS) from Helicobacter pylori genomic strain 26695 and its corresponding HP0826::Kan mutant lacking the O-chain component based on the in-depth NMR analysis of the oligosaccharide products obtained through the use of various degradation procedures performed on the purified LPS from both strains, as well as CE–MS data. New structural evidence indicates the presence of the linear arrangement of glucan and heptan portions of the LPS attached through -6-α-ddHep-3-α-l-Fuc-3-β-GlcNAc- fragment to the inner core dd-heptose residue. This structure differs from previously reported structures of the H. pylori 26695 LPS in several aspects.     

Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.     

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.     

Crystal structure of apo and copper bound HP0894 toxin from Helicobacter pylori 26695 and insight into mRNase activity

The toxin–antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and microorganism virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. In Helicobacter pylori, only two TA systems are known thus far. Our previous studies showed that HP0894–HP0895 acts as a TA system and that HP0894 exhibits intrinsic RNase activity. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remain unclear. Therefore, in an attempt to shed some light on the mechanism behind the TA system of HP0894–HP0895, here we present the crystal structures of apo- and metal-bound H. pylori 0894 at 1.28 Å and 1.89 Å, respectively. Through the combined approach of structural analysis and structural homology search, the amino acids involved in mRNase active site were monitored and the reorientations of different residues were discussed in detail. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself to exhibit this type of activity, acting as a general acid in an acid–base catalysis reaction, while His47 and His60 stabilize the transition state. Lys52, Glu58, Asp64 and Arg80 have phosphate binding and specific sequence recognition. Glu58 also acts as a general base, and substrate reorientation is caused by Phe88. Based on experimental findings, a model for antitoxin binding could be suggested.     

基因组尺度分析和代谢工程策略

代谢工程是近年来发展起来的新技术,随着各种组学技术的发展,高通量数据整合方法用于分析细胞的代谢网络,改造代谢途径,以提高目标产物的产量。本文就代谢工程的发展状况,基因组尺度的分析技术,以及代谢工程策略进行了综述。分析了生物信息学和系统生物学方法在代谢途径构建和代谢网络分析中的作用,并就存在的问题和可能的解决途径进行了阐述。     

Analysis of automatically generated peptide mass fingerprints of cellular proteins and antigens from Helicobacter pylori 26695 separated by two-dimensional electrophoresis

Helicobacter pylori is a causative agent of severe diseases of the gastric tract ranging from chronic gastritis to gastric cancer. Cellular proteins of H. pylori were separated by high resolution two-dimensional gel electrophoresis. A dataset of 384 spots was automatically picked, digested, spotted, and analyzed by matrix-assisted laser desorption ionization mass spectrometry peptide mass fingerprint in triple replicates. This procedure resulted in 960 evaluable mass spectra. Using a new version of our data analysis software MS-Screener we improved identification and tested reliability of automatically generated data by comparing with manually produced data. Antigenic proteins from H. pylori are candidates for vaccines and diagnostic tests. Previous immunoproteomics studies of our group revealed antigen candidates, and 24 of them were now closely analyzed using the MS-Screener software. Only in three spots minor components were found that may have influenced their antigenicities. These findings affirm the value of immunoproteomics as a hypothesis-free approach. Additionally, the protein species distribution of the known antigen GroEL was investigated, dimers of the protein alkyl hydroperoxide reductase were found, and the fragmentation of gamma-glutamyltranspeptidase was demonstrated.     

全基因组范围代谢网络的构建和最小基因组研究

全基因组范围代谢网络（genome-scale metabolic network,GSMN）的构建是合成生物学研究的一个重要研究手段。通过整合各种组学数据和借助计算机进行模拟分析,将基因型与表型的关系进行定量关联,从而为从全局的角度探索和揭示生物代谢机制,进而对生物进行合理的重新设计和工程改造提供了有效的框架。该方法在最小基因组研究中也有着突出的优势,通过计算机辅助的基因组最小化模拟与分析,能够系统鉴定微生物基因组基因的必需性。迄今为止,已有近百个基因组范围的代谢网络发表,覆盖的生物包括原核生物、真核生物和古生生物,并广泛应用于医药、能源、环境、工业和农业等多个领域,展现出了广阔的应用前景。将对全基因组范围代谢网络构建的方法、应用,特别是其在最小基因组研究中的应用作简要的综述。     

Helicobacter pylori catalase

Helicobacter pylori is the major aetiological agent of gastroduodenitis in humans. Due to the potential importance of catalase in the growth and survival of Helicobacter pylori on the surface of inflamed mucosae, we have characterized catalase from H. pylori as a prelude to further studies on the function of the enzyme in vivo. The catalase activity of H. pylori was significantly affected by the presence of blood, serum or erythrocytes in the growth medium: the greatest activity was expressed when the bacterium was grown on medium containing serum. H. pylori catalase is a tetramer with a subunit Mr of 50,000. The enzyme had a pI of 9.0-9.3, was active over a broad pH range and was stable at 56 degrees C. It was non-competitively inhibited by sodium azide, and had no detectable peroxidase activity. The Km for the purified catalase was measured as 43 +/- 3 mM-H2O2 and the V as 60 +/- 3 mmol H2O2 min-1 (mg protein)-1. The native catalase has absorption maxima at 280 nm and 405 nm with further minor shoulders or peaks at 510 nm, 535 nm and 625 nm, consistent with the presence of an iron-porphyrin prosthetic group.     

Helicobacter pylori motility

Motility is essential for Helicobacter pylori colonization. This review discusses the biochemistry, genetics and genomics of the H. pylori flagellum, and compares these features with well-characterized bacteria.     

Lipopolysaccharide structures of Helicobacter pylori genomic strains 26695 and J99, mouse model H. pylori Sydney strain, H. pylori P466 carrying sialyl Lewis X, and H. pylori UA915 expressing Lewis B classification of H. pylori lipopolysaccharides into glycotype families.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.     

Diagnosis of Helicobacter pylori

The different invasive and noninvasive diagnostic tests for Helicobacter pylori have been applied mainly in emerging countries. Molecular methods have been developed, especially a test for detection of H. pylori and its clarithromycin resistance directly from stools. The long-term effects of eradication on histologic lesions have been studied in a meta-analysis and the prognostic value of post-treatment in gastric mucosa-associated lymphoid tissue lymphoma has been assessed. An operating link for gastritis assessment (the OLGA staging) has also been published. Attempts to simplify the urea breath test protocol have been made, and new stool antigen tests have been proposed and compared to those previously available.     

Transmission of Helicobacter pylori

Helicobacter gastroduodenitis is a serious chronic infectious disease that is responsible for widespread morbidity and mortality. An understanding of the way in which it spreads is fundamentally important when considering measures for its control. Its prevalence is highest in the developing world and in individuals with a disadvantaged socio-economic childhood. The disease is believed to be contracted during the early years of life.A faeco-oral mode of transmission is considered by many to be the most likely mode of spread, however, the organism is difficult to culture both from faeces and from the environment and unlike other enteric organisms Helicobacter does not give rise to a diarrhoeal illness that would facilitate its transmission. An orooral route of spread has also been suggested, however, Helicobacter cannot be cultured from saliva, and if it was spread orally there is no reason why childhood should be the most frequent age for its acquisition.A third possibility is that the bacterium is transmitted gastro-orally. In favor of this hypothesis, the infection is easily acquired following gastric intubation with inadequately disinfected equipment. Children have a greater tendency to vomit than adults, and tend to explore with their fingers and place foreign objects in their mouths. Initial Helicobacter infection causes a dyspeptic illness characterised by mucousy vomiting, which may provide a vehicle for transmission. Furthermore, during the acute infection the organism induces achlorhydria in the host, possibly enabling the organism to survive longer in vomited mucus in the absence of acid. This theory fits best with the epidemiological data. Those most at risk are children living in an overcrowded environment who share beds with one another and live in houses that do not possess a fixed hot water supply (thus making cleaning up of vomit more difficult). It is also commoner in institutionalized children and is associated with school catchment areas.