Giovanni Suarez

A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a sigma(54) activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila, namely, a Delta act mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Delta act Delta vasH mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the vgrG1 gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and Escherichia coli, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of A. hydrophila possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.

Expression of B7-H1 on Gastric Epithelial Cells: Its Potential Role in Regulating T Cells during Helicobacter pylori Infection

Helicobacter pylori infection is associated with gastritis, ulcers, and gastric cancer. The infection becomes chronic as the host response is unable to clear it. Gastric epithelial cells (GEC) play an important role during the host response, and their expression of class II MHC and costimulatory molecules such as CD80 and CD86 suggests their role in local Ag presentation. Although T cells are recruited to the infected gastric mucosa, they have been reported to be hyporesponsive. In this study, we detected the expression of B7-H1 (programmed death-1 ligand 1), a member of B7 family of proteins associated with T cell inhibition on GEC. Quantitative real-time RT-PCR revealed that B7-H1 expression increased significantly on GEC after H. pylori infection. Western blot analysis showed that B7-H1 expression was induced by various H. pylori strains and was independent of H. pylori virulence factors such as Cag, VacA, and Urease. The functional role of B7-H1 in the cross talk between GEC and T cells was assessed by coculturing GEC or H. pylori-infected GEC with CD4+ T cells isolated from peripheral blood. Using blocking Abs to B7-H1 revealed that B7-H1 was involved in the suppression of T cell proliferation and IL-2 synthesis, and thus suggested a role for B7-H1 on the epithelium as a contributor in the chronicity of H. pylori infection.

Iron deficiency accelerates Helicobacter pylori–induced carcinogenesis in rodents and humans

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.

N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout Delta ahyRI mutant. Protease production was substantially reduced in the Delta ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the Delta ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the Delta ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the Delta ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the Delta ahyRI/ahyR(+) complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Subepithelial Myofibroblasts are Novel Nonprofessional APCs in the Human Colonic Mucosa

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that α-smooth muscle actin+, CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.

The Helicobacter pylori urease B subunit binds to CD74 on gastric epithelial cells and induces NF-κB activation and interleukin-8 production

ABSTRACT The pathogenesis associated with Helicobacter pylori infection is the result of both bacterial factors and the host response. We have previously shown that H. pylori binds to CD74 on gastric epithelial cells. In this study, we sought to identify the bacterial protein responsible for this interaction. H. pylori urease from a pool of bacterial surface proteins was found to coprecipitate with CD74. To determine how urease binds to CD74, we used recombinant urease A and B subunits. Recombinant urease B was found to bind directly to CD74 in immunoprecipitation and flow cytometry studies. By utilizing both recombinant urease subunits and urease B knockout bacteria, the urease B-CD74 interaction was shown to induce NF-κB activation and interleukin-8 (IL-8) production. This response was decreased by blocking CD74 with monoclonal antibodies. Further confirmation of the interaction of urease B with CD74 was obtained using a fibroblast cell line transfected with CD74 that also responded with NF-κB activation and IL-8 production. The binding of the H. pylori urease B subunit to CD74 expressed on gastric epithelial cells presents a novel insight into a previously unrecognized H. pylori interaction that may contribute to the proinflammatory immune response seen during infection.

Functional plasticity in the type IV secretion system of Helicobacter pylori.

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and “tunes” the host inflammatory response so as to maximize persistent infection.

Braun Lipoprotein (Lpp) Contributes to Virulence of Yersiniae: Potential Role of Lpp in Inducing Bubonic and Pneumonic Plague

ABSTRACT Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 × 107 CFU of the Δlpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Δlpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD50). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Δlpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Δlpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Δlpp mutant than in those infected with WT CO92. Additionally, the Δlpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Δlpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Helicobacter pylori Binds to CD74 on Gastric Epithelial Cells and Stimulates Interleukin-8 Production

ABSTRACT The pathogenesis associated with Helicobacter pylori infection requires consistent contact with the gastric epithelium. Although several cell surface receptors have been suggested to play a role in adhesion, the bacterium-host interactions that elicit host responses are not well defined. This study investigated the interaction of H. pylori with the class II major histocompatibility complex (MHC)-associated invariant chain (Ii; CD74), which was found to be highly expressed by gastric epithelial cells. Bacterial binding was increased when CD74 surface expression was increased by gamma interferon (IFN-γ) treatment or by fibroblast cells transfected with CD74, while binding was decreased by CD74 blocking antibodies, enzyme cleavage of CD74, and CD74-coated bacteria. H. pylori was also shown to bind directly to affinity-purified CD74 in the absence of class II MHC. Cross-linking of CD74 and the engagement of CD74 were verified to stimulate IL-8 production by unrelated cell lines expressing CD74 in the absence of class II MHC. Increased CD74 expression by cells increased IL-8 production in response to H. pylori, and agents that block CD74 decreased these responses. The binding of H. pylori to CD74 presents a novel insight into an initial interaction of H. pylori with the gastric epithelium that leads to upregulation of inflammatory responses.

Characterization of a mouse model of plague after aerosolization of Yersinia pestis CO92

Yersinia pestis is a Gram-negative bacterium, and the causative agent of bubonic plague and pneumonic plague. Because of its potential use as a biological warfare weapon, the plague bacterium has been placed on the list of category A select agents. The dynamics of pneumonic infection following aerosolization of the highly virulent Y. pestis CO92 strain have been poorly studied; therefore, the purpose of this study was to determine the LD(50) dose, bacterial dissemination, cytokine/chemokine production and tissue damage in Swiss-Webster mice over a 72 h course of infection. We exposed mice in a whole-body Madison chamber to various doses of Y. pestis CO92 aerosolized by a Collison nebulizer, and determined that the LD(50) presented dose (Dp) of the bacterium in the lungs was 2.1 x 10(3) c.f.u. In a subsequent study, we infected mice at a Dp of 1.3 x 10(4) c.f.u., and harvested organs and blood at 1, 24, 48 and 72 h post-infection. Histopathological examination, in addition to measurement of bacterial dissemination and cytokine/chemokine analysis, indicated progressive tissue injury, and an increased number of animals succumbing to infection over the course of the experiment. Using these data, we were able to characterize the mouse plague model following aerosolization of Y. pestis CO92.