Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.     

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.     

HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.     

The HopX (AvrPphE) family of Pseudomonas syringae type III effectors require a catalytic triad and a novel N-terminal domain for function

Many gram-negative plant pathogenic bacteria employ type III secretion systems to deliver effector proteins directly into the host cell during infection. On susceptible hosts, type III effectors aid pathogen growth by manipulating host defense pathways. On resistant hosts, some effectors can activate specific host disease resistance (R) genes, leading to generation of rapid and effective immune responses. The biochemical basis of these processes is poorly understood. The HopX (AvrPphE) family is a widespread type III effector among phytopathogenic bacteria. We determined that HopX family members are modular proteins composed of a conserved putative cysteine-based catalytic triad and a conserved potential target/cofactor interaction domain. HopX is soluble in host cells. Putative catalytic triad residues are required for avirulence activity on resistant bean hosts and for the generation of a cell-death response in specific Arabidopsis genotypes. The putative target/cofactor interaction domain is also required for these activities. Our data suggest that specific interaction with and modification of a cytosolic host target drives HopX recognition in resistant hosts and may contribute to virulence in susceptible hosts. Surprisingly, the Legionella pneumophila genome was found to contain a protein with similarity to HopX in sequence and domain arrangement, suggesting that these proteins might also contribute to animal pathogenesis and could be delivered to plant and animal hosts by diverse secretion systems.     

Identification of S-nitrosylation of proteins of Helicobacter pylori in response to nitric oxide stress

Innate and adaptive immune responses are activated in humans when Helicobacter pylori invades the gastric mucosa. Nitric oxide (NO) and reactive nitrogen species are important immune effectors, which can exert their functions through oxidation and S-nitrosylation of proteins. S-nitrosoglutathione and sodium nitroprus-side were used as NO donors and H. pylori cells were incubated with these compounds to analyze the inhibitory effect of NO. The suppressing effect of NO on H. pylori has been shown in vitro. Furthermore, the proteins modified by S-nitrosylation in H. pylori were identified through the biotin switch method in association with matrix-assisted laser desorption ionization/time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Five S-nitrosylated proteins identified were a chaperone and heat-shock protein (GroEL), alkyl hydroperoxide reductase (TsaA), urease alpha subunit (UreA), HP0721, and HP0129. Importantly, S-nitrosylation of TsaA and UreA were confirmed using purified recombinant proteins. Considering the importance of these enzymes in antioxidant defenses, adherence, and colonization, NO may exert its antibacterial actions by targeting enzymes through S-nitrosylation. Identification of protein S-nitrosylation may contribute to an understanding of the antibacterial actions of NO. Our findings provide an insight into potential targets for the development of novel therapeutic agents against H. pylori infection.     

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.     

Biochemical characterization of protein complexes from the Helicobacter pylori protein interaction map: strategies for complex formation and evidence for novel interactions within type IV secretion systems

We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H. pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H. pylori type IV secretion systems. Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization. We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H. pylori interactome, because 76% of the interactions tested were confirmed biochemically. Of the interactions involving type IV secretion proteins, three could be confirmed. One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively. Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion. Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496.     

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.     

幽门螺杆菌cag PAI编码的Ⅳ型分泌系统

幽门螺杆菌(Helicobacter pylori,H.pylori)是定植于人胃部特定的病原菌,感染呈全球分布,感染率高达50%以上。现已证实它是轻度胃炎,消化性溃疡及胃癌的主要病因。Ⅰ型H.pylori菌株含有一个约40kb的特殊基因片段,即cag致病岛(cytotoxin associated gene pathogenicity island,cag PAI),该片段只出现于致病相关菌株,基因呈高密度分布并编码一个分泌转运系统称为Ⅳ型分泌系统(type Ⅳ secretion system,TFSS),通过转运相关毒素而参与H.pylori诱导上皮细胞细胞内的酪氨酸磷酸化、细胞骨架重排、基垫结构形成、活化核转录因子NF-κB、诱导促炎细胞因子白细胞介素-8的表达等,故在H.pylori的致病中起着关键作用。近年来,研究者们致力于研究Ⅳ型分泌系统的功能,但是对于这个装置是如何转运蛋白进入宿主细胞的确切机制还是知之甚少,因此,对Ⅳ型分泌系统的研究将有助于进一步明确H.pylori致病机制,并为临床诊断和治疗提供新的靶点。     

The changes of proteomes components of Helicobacter pylori in response to acid stress without urea

Acid stress is the most obvious challenge Helicobacter pylori encounters in human stomach. The urease system is the basic process used to maintain periplasmic and cytoplasmic pH near neutrality when H. pylori is exposed to acidic condition. However, since the urea concentration in gastric juice is approximately 1 mM, considered possibly insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis may contribute to the acid adaptation in H. pylori. In order 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 30 min 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. The functions of these proteins included 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.     

Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.     

Carbohydrate-dependent inhibition of Helicobacter pylori colonization using porcine milk

Breast milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. In a previous study, we showed that pigs express the Helicobacter pylori receptors, sialyl Lewis x (Le x) and Le b, on various milk proteins. Here, we investigate the pig breed- and individual-specific expression of these epitopes, as well as the inhibitory capacity of porcine milk on H. pylori binding and colonization. Milk proteins from three different pig breeds were analysed by western blotting using antibodies with known carbohydrate specificity. An adhesion assay was used to investigate the capacity of pig milk to inhibit H. pylori binding to neoglycoproteins carrying Le b and sialyl-di-Le x. alpha1,3/4-fucosyltransferase transgenic FVB/N mice, known to express Le b and sialyl Le x in their gastric epithelium, were colonized by H. pylori and were subsequently treated with Le b- and sialyl Le x-expressing or nonexpressing porcine milk, or water (control) only. The degree of H. pylori colonization in the different treatment groups was quantified. The expression of the Le b and sialyl Le x carbohydrate epitopes on pig milk proteins was breed- and individual specific and correlated to the ability of porcine milk to inhibit H. pylori adhesion in vitro and H. pylori colonization in vivo. Milk from certain pig breeds may have a therapeutic and/or prophylactic effect on H. pylori infection.     

Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.     

Identification of surface proteins of Helicobacter pylori by selective biotinylation,affinity purification,and two-dimensional gel electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.     

应用生物信息学方法筛选幽门螺杆菌疫苗候选抗原

目的：应用生物信息学分析方法筛选幽门螺杆菌新的疫苗候选抗原。方法：从TIGRCMR下载幽门螺杆菌26695和J99株全基因组序列,应用生物信息学SignalP、PredTMBB、LipoP、TMHMM、Phobius、PSORT-B和SubLoc等分析软件,筛选幽门螺杆菌新的外膜蛋白和分泌蛋白疫苗候选抗原。结果：从幽门螺杆菌26695株筛选得到54个编码β-桶型跨膜蛋白、脂蛋白或分泌表达蛋白的疫苗候选蛋白抗原,从幽门螺杆菌J99株得到61个呈现上述表达方式的疫苗候选蛋白抗原;且这2株细菌的疫苗候选蛋白呈现良好的交集状况,即有43个候选疫苗蛋白是相同的。结论：用生物信息学分析方法可以从全基因组范围内快速筛选到保守的分泌或表面暴露的疫苗候选抗原,为疫苗抗原的快速筛选与鉴定奠定了基础。     

幽门螺杆菌适应性定植蒙古沙鼠前、后的蛋白质组学研究

为比较研究幽门螺杆菌(Hp)适应性定植蒙古沙鼠前后的蛋白表达谱变化,将Hp临床分离株感染沙鼠,并体内连续传代,驯化一株高适应性菌株,然后采用双向电泳和质谱技术对适应性变化前后两株菌的差异蛋白进行鉴定。结果表明,随着Hp临床株在沙鼠体内的连续传代,感染率逐渐升高,第10代后,感染率稳定在90%以上。适应性定植后,Hp蛋白谱发生了变化。在选择的5个候选差异蛋白点中,共鉴定出4个蛋白,分别为Hp菌的功能未知的HP0318编码蛋白、氢化酶表达/形成蛋白(hypB)、异柠檬酸脱氢酶(icd)、ADP-L-D-甘露庚糖表异构酶(rfaD)。上述鉴定蛋白可能与Hp适应性定植具有很大的相关性。     

Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling

To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.