High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population^1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide³. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA^4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells^5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks^5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the “pilus”⁵. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface^9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.     

Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that encodes for a type IV secretion system

CagZ, a 23 kDa protein encoded by the cagZ gene (HP0526) of the cag pathogenicity island of Helicobacter pylori, has been cloned, over-expressed, purified and its three-dimensional structure determined. The protein consists of a single compact L-shaped domain, composed of seven alpha-helices including about 70% of the total residues. Three-dimensional homology searches did not reveal structural homologues, and CagZ can be considered representative of a new protein fold. The presence of a disordered C-terminal tail and the nature of the molecular surface suggest that CagZ may participate in the interaction of effector proteins with one or more components of the H.pylori type IV secretion system on the cytoplasmic side of the inner membrane.     

Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells

Background

Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).

Results

The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.

Conclusion

Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.     

Instructions for authors

Although Helicobacter pylori infects 50% of the total human population, only a small fraction of the infected people suffer from severe diseases like peptic ulcers and gastric adenocarcinoma. H. pylori strains, host genotypes and environmental factors play important role in deciding the extent and severity of the gastroduodenal diseases. The bacteria has developed a unique set of virulence factors to survive in the extreme ecological niche of human stomach. Together these virulence factors make H. pylori one of the most successful human pathogenic bacteria colonizing more than half of the human population. Understanding the mechanism of action of the major H. pylori virulence factors will shed light into the molecular basis of its pathogenicity.     

羊种布鲁氏菌链霉素耐受基因筛选及鉴定

【背景】布鲁氏菌（Brucella spp.）是一种兼性胞内寄生菌,能够引起世界范围内的人兽共患流行病——布鲁氏菌病（以下简称布病）。链霉素是治疗布病的推荐药物,但我国已有链霉素耐受分离株（根据CLSI推荐的耐药折点）。【目的】筛选并鉴定羊种布鲁氏菌（Brucella melitensis）链霉素耐受的新基因。【方法】利用转录组筛选羊种布鲁氏菌链霉素耐受新基因,并利用同源重组、分子对接等技术预测并鉴定相关基因的功能。【结果】羊种布鲁氏菌（M5疫苗株）在含2×MIC浓度链霉素培养基上,12 h后通过耐受恢复增殖能力。转录组分析表明,细胞膜组成成分在低浓度链霉素耐受中发挥重要作用,核糖体通路相关基因表达量显著性增加[|log₂FC|≥2.0, P<0.05],群体感应通路、Ⅳ型分泌系统相关基因表达量显著性降低（|log₂FC|≥2.0, P<0.05）。利用同源重组技术缺失组成Ⅳ型分泌系统的元件,发现缺失virB3和virB5基因后,M5疫苗株链霉素MIC值增高,回补后与亲本株差异不显著。分子对接预测发现,VirB3和VirB5能够通过氢键与链霉素结合。【结论】链霉素主要影响羊种布鲁氏菌细胞膜组分涉及的相关通路。羊种布鲁氏菌通过降低Ⅳ型分泌系统组成元件virB3和virB5基因的表达耐受链霉素。本研究为布鲁氏菌耐药株研究提供了新思路,为布鲁氏菌新药研发提供了候选靶点。     

Insights from the molecular docking of curcumin to the virulent factors of Helicobacter pylori

The domains of virulent (Ureα/β, VacA-p55, and CagA) factors of Helicobacter pylori play a pivotal role in developmentalprocesses of numerous diseases including gastric cancer. The pharmacological role of curcumin indicates that it could regulate thesignaling of virulent factors by interacting with active domains. However, the controlling mechanism of the curcumin interactionsand the binding diversity on structural basis of virulent (Ureα/β, VacA-p55, and CagA) factors are unknown. Curcumin astherapeutic agent was filtered by using Lipinski rule׳s five and the druglikeness property for assessment of pharmacologicalproperties. Here outcome of molecular docking presented the 3-D structure of curcumin complex, that interacted with especiallyconserved residues of target domains. The structure revealed that the curcumin complexation with domains of these proteinsprovided structural insight into the diverse nature of proteins (Ureα/β, VacA-p55, and CagA) recognition. In silico studyelucidated that the broad specificity of curcumin was achieved by multiple binding mode mechanisms such as distinct hydrogenand hydrophobic interactions with involvement of binding energy. The higher score of curcumin in complexation with bothsubunits Ureα/β showed the stable binding, and less stability with VacA-p55 complexation with lower score. Curcumin exhibitedgood interaction with these targeted virulent factors, although extensive interactions of curcumin with Ureα/β subunits could havean important implication to prevent survival and colonisation of H. pylori in stomach.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones

ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.     

Membrane-associated Activation of Cholesterol ��-Glucosyltransferase, an Enzyme Responsible for Biosynthesis of Cholesteryl-��-d-Glucopyranoside in Helicobacter pylori Critical for Its Survival

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

A novel Helicobacter pylori cell-surface polysaccharide

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.     

Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.     

The CagA protein of Helicobacter pylori suppresses the functions of dendritic cell in mice

CagA protein is the most assessed effecter molecule of Helicobacter pylori. In this report, we demonstrate how CagA protein regulates the functions of dendritic cells (DC) against H. pylori infection. In addition, we found that CagA protein was tyrosine-phosphorylated in DC. The responses to cagA-positive H. pylori in DC were reduced in comparison to those induced by cagA-negative H. pylori. CagA-overexpressing DC also exhibited a decline in the responses against LPS stimulation and the differentiation of CD4⁺ T cells toward Th1 type cells compared to wild type DC. In addition, the level of phosphorylated IRF3 decreased in CagA-overexpressing DC stimulated with LPS, indicating that activated SHP-2 suppressed the enzymatic activity of TBK1 and consequently IRF3 phosphorylation. These data suggest that CagA protein negatively regulates the functions of DC via CagA phosphorylation and that cagA-positive H. pylori strains suppress host immune responses resulting in their chronic colonization of the stomach.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.