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In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC [7966.sub.T] of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4[degrees]C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37[degrees]C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses ([LD.sub.50]). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same [LD.sub.50] dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.

Abstract Background Salmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine. Results The protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Naïve mice receiving either cells or sera from immunized mice were partially protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-γ, and IL-10 cytokines in splenocytes induced with STM cell lysate. Conclusions Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.

Background Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry. Methods and results To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the [DELA]stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in [DELA]stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in [DELA]stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Conclusions These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry. Keywords: Salmonella, Adhesion, StdA, Poultry, Author(s): Daniel C Shippy[sup.1] , Nicholas M Eakley[sup.1] , Dareen M Mikheil[sup.1] and Amin A Fadl[sup.1] Background Salmonella is a significant foodborne bacterium associated with enteric disease outbreaks in humans [...]

A total of 227 isolates from Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains were analyzed to determine the presence of various virulence factors. The study results provide first evidence for the successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene ( hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine β synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence. [ABSTRACT FROM AUTHOR]

Abnormal production of inflammatory cytokines and chemokines is a key feature of bacterial endotoxin, lipopolysaccharide (LPS)-induced inflammation, and cytotoxicity; however, the mechanisms regulating production of inflammatory markers remain unclear. Herein, we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR; AKR1B3) modulates NF-κB-dependent activation of inflammatory cytokines and chemokines in mouse serum, liver, heart, and spleen. Pharmacological inhibition or small interfering RNA ablation of AR prevented the biosynthesis of tumor necrosis factor-α, interleukin 1β, interleukin-6, macrophage-chemoattractant protein-1, and cyclooxygenase-2 and prostaglandin E2 in LPS-activated RAW264.7 murine macrophages. The AR inhibition or ablation significantly attenuated LPS-induced activation of protein kinase C (PKC) and phospholipase C (PLC), nuclear translocation of NF-κB, and phosphorylation and proteolytic degradation of IκBa in macrophages. Further- more, treatment of macrophages with 4-hydroxy-trans-2-nonenal (HNE), and cell-permeable esters of glutathionyl-4-hydro-xynonanal (GS-HNE) and glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF-κB and PLC/PKC. Pharmacological inhibition or antisense ablation of AR that catalyzes the reduction of GS-HNE to GS-DHN prevented PLC, PKC, IKKκ/β, and NF-κB activation caused by HNE and GS-HNE, but not by GS-DHN, suggesting that reduced GS-lipid aldehydes catalyzed by AR propagate LPS-induced production of inflammatory markers. Collectively, these data provide evidence that inhibition of AR may be a significant therapeutic approach in preventing bacterial endotoxin-induced sepsis and tissue damage. [ABSTRACT FROM AUTHOR]