Differential Requirements for COPI Coats in Formation of Replication Complexes among Three Genera of Picornaviridae

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, ß-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain ß-COP. In contrast, ß-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells ß-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes

Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis

Preservation of hlicobacter pylori CagA translocation and host cell proinflammatory responses in the face of CagL hypervariability at amino acid residues 58/59

Carriage of the CagA oncoprotein by the human gastric cancer-associated pathogen Helicobacter pylori is significantly associated with this typically benign chronic infection advancing to a potentially fatal outcome. However it remains to be elucidated why only a small subset of individuals infected with H. pylori CagA-positive strains develops gastric cancer. H. pylori translocates CagA into host cells using a type IV secretion apparatus that interacts with host integrin receptors via a three-amino-acid-residue RGD motif on the H. pylori protein CagL. The RGD motif of CagL also plays a major role in the induction of proinflammatory responses. Upstream of this motif is a conserved glycine flanked by four hypervariable amino acid residues (residues 58, 59, 61 and 62). Certain amino acid polymorphisms at 58 and 59 are significantly prevalent in strains from gastric cancer patients in particular geographic regions; Y58E59 is seen in Taiwan and D58K59 in India. In light of the seemingly contradictory findings of recent CagL mutagenesis studies, we have examined the contribution of sequence promiscuity specifically at CagL residues 58 and 59 to CagA translocation and H. pylori-mediated proinflammatory responses of gastric epithelial cells. Using isogenic mutants of H. pylori strains P12 and 26695 with amino acid substitutions at CagL residues 58 and 59, we determined that carriage of the polymorphisms Y58E59, D58K59, D58E59, N58E59 or N58K59 did not significantly alter the capacity of H. pylori to translocate CagA into, or induce IL-8 secretion in, host cells. Our findings, together with other recently published data, suggest that the variation at CagL residues 58 and 59 does not influence type IV secretion system function in isolation, but rather may work in concert with particular polymorphisms elsewhere in CagL to modulate disease progression

The middle fragment of <i>Helicobacter pylori </i>CagA induces Actin rearrangement and triggers its own uptake into gastric epithelial cells

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α5β1. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613–636) and a putative β1 integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) β1 integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway

Helicobacter pylori Type IV Secretion System and Its Adhesin Subunit, CagL, Mediate Potent Inflammatory Responses in Primary Human Endothelial Cells

The Gram-negative bacterium, Helicobacter pylori, causes chronic gastritis, peptic ulcers, and gastric cancer in humans. Although the gastric epithelium is the primary site of H. pylori colonization, H. pylori can gain access to deeper tissues. Concurring with this notion, H. pylori has been found in the vicinity of endothelial cells in gastric submucosa. Endothelial cells play crucial roles in innate immune response, wound healing and tumorigenesis. This study examines the molecular mechanisms by which H. pylori interacts with and triggers inflammatory responses in endothelial cells. We observed that H. pylori infection of primary human endothelial cells stimulated secretion of the key inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8). In particular, IL-8, a potent chemokine and angiogenic factor, was secreted by H. pylori-infected endothelial cells to levels ~10- to 20-fold higher than that typically observed in H. pylori-infected gastric epithelial cells. These inflammatory responses were triggered by the H. pylori type IV secretion system (T4SS) and the T4SS-associated adhesin CagL, but not the translocation substrate CagA. Moreover, in contrast to integrin α5β1 playing an essential role in IL-8 induction by H. pylori upon infection of gastric epithelial cells, both integrin α5β1 and integrin αvβ3 were dispensable for IL-8 induction in H. pylori-infected endothelial cells. However, epidermal growth factor receptor (EGFR) is crucial for mediating the potent H. pylori-induced IL-8 response in endothelial cells. This study reveals a novel mechanism by which the H. pylori T4SS and its adhesin subunit, CagL, may contribute to H. pylori pathogenesis by stimulating the endothelial innate immune responses, while highlighting EGFR as a potential therapeutic target for controlling H. pylori-induced inflammation

Reductive evolution in outer membrane protein biogenesis has not compromised cell surface complexity in Helicobacter pylori

Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM). Here we have assessed the complexity of the OMP C-terminal β-barrel domains employed by H. pylori, and characterized the H. pyloriBAM complex. Around 50 Helicobacter-specific OMPs were assessed with predictive structural algorithms. The data suggest that H. pylori utilizes a unique β-barrel architecture that might constitute H. pylori-specific Type V secretions system. The structural and functional diversity in these proteins is encompassed by their extramembrane domains. Bioinformatic and biochemical characterization suggests that the low β-barrel-complexity requires only minimalist assembly machinery. The H. pylori proteins BamA and BamD associate to form a BAM complex, with features of BamA enabling an oligomerization that might represent a mechanism by which a minimalist BAM complex forms a larger, sophisticated machinery capable of servicing the outer membrane proteome of H. pylori

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori

Abstract The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ΔmodH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility

Helicobacter pylori type IV secretion system and its adhesin subunit, CagL, mediate potent inflammatory responses in primary human endothelial cells

The Gram-negative bacterium, Helicobacter pylori, causes chronic gastritis, peptic ulcers, and gastric cancer in humans. Although the gastric epithelium is the primary site of H. pylori colonization, H. pylori can gain access to deeper tissues. Concurring with this notion, H. pylori has been found in the vicinity of endothelial cells in gastric submucosa. Endothelial cells play crucial roles in innate immune response, wound healing and tumorigenesis. This study examines the molecular mechanisms by which H. pylori interacts with and triggers inflammatory responses in endothelial cells. We observed that H. pylori infection of primary human endothelial cells stimulated secretion of the key inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8). In particular, IL-8, a potent chemokine and angiogenic factor, was secreted by H. pylori-infected endothelial cells to levels ~10- to 20-fold higher than that typically observed in H. pylori-infected gastric epithelial cells. These inflammatory responses were triggered by the H. pylori type IV secretion system (T4SS) and the T4SS-associated adhesin CagL, but not the translocation substrate CagA. Moreover, in contrast to integrin α5β1 playing an essential role in IL-8 induction by H. pylori upon infection of gastric epithelial cells, both integrin α5β1 and integrin αvβ3 were dispensable for IL-8 induction in H. pylori-infected endothelial cells. However, epidermal growth factor receptor (EGFR) is crucial for mediating the potent H. pylori-induced IL-8 response in endothelial cells. This study reveals a novel mechanism by which the H. pylori T4SS and its adhesin subunit, CagL, may contribute to H. pylori pathogenesis by stimulating the endothelial innate immune responses, while highlighting EGFR as a potential therapeutic target for controlling H. pylori-induced inflammation. Introductio