Mutational and transcriptional analyses of an avian pathogenic Escherichia coli ColV plasmid

<p>Abstract</p> <p>Background</p> <p>Previously we described a 184-kb ColV plasmid, pAPEC-O2-ColV, that contributed to the ability of an <it>E. coli </it>to kill avian embryos, grow in human urine, and colonize the murine kidney. Here, the roles of several genes encoded by this plasmid in virulence were assessed using mutational and transcriptional analyses.</p> <p>Methods</p> <p>Genes chosen for deletion were <it>iss</it>, <it>tsh</it>, <it>iutA</it>, <it>iroN</it>, <it>sitA</it>, and <it>cvaB</it>. In addition, a 35-kb region of the plasmid, containing <it>iss</it>, <it>tsh</it>, and the ColV and <it>iro </it>operons, along with a 15-kb region containing both the aerobactin and <it>sit </it>operons, were deleted. Mutants were compared to the wild-type (APEC O2) for lethality to chick embryos and growth in human urine. Expression of the targeted genes was also assessed under these same conditions using RT-PCR</p> <p>Results</p> <p>No significant differences between the mutants and the wild-type in these phenotypic traits were detected. However, genes encoding known or predicted iron transport systems were up-regulated during growth in human urine, as compared to growth in LB broth, while <it>iss</it>, <it>hlyF</it>, and <it>iroN </it>were strongly up-regulated in chick embryos.</p> <p>Conclusion</p> <p>While no difference was observed between the mutant strains and their wild-type parent in the phenotypic traits assayed, we reasoned that some compensatory virulence mechanism, insensitivity of the virulence assays, or other factor could have obscured changes in the virulence of the mutants. Indeed we found several of these genes to be up-regulated in human urine and/or in the chick embryo, suggesting that certain genes linked to ColV plasmids are involved in the establishment of avian extraintestinal infection.</p

Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens

Murine and Bovine γδ T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα−/−, and GMCSF−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections