The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.
- MeSH
- delece genu * MeSH
- faktory virulence analýza MeSH
- Francisella tularensis enzymologie imunologie patogenita MeSH
- glyceraldehyd-3-fosfátdehydrogenasy nedostatek metabolismus MeSH
- krevní proteiny metabolismus MeSH
- mikrobiální viabilita MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- proteindisulfidisomerasy nedostatek MeSH
- proteom analýza MeSH
- salmonelová infekce u zvířat mikrobiologie patologie MeSH
- vazba proteinů MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study we were interested in the vaccine potential of two attenuated mutants of Salmonella enterica serovar Enteritidis for poultry. The first mutant was attenuated by the removal of the whole Salmonella Pathogenicity Island 1 (SPI1) and the second mutant was devoid of the whole SPI2. These 2 mutants were used for oral vaccination of 2 chicken lines; Lohmann Brown and ISA Brown. Chickens were vaccinated orally on day 1 of life, revaccinated on day 21 and challenged on day 42. The challenge was performed either orally or intravenously. Despite a slightly different response between the two chicken lines, both the mutants gave protection to poultry against S. Enteritidis challenge as documented by findings such as the bacterial counts in tissues, spleen weight, antibody production and cytokine response (namely IL-17 and IL-22). When the 2 mutants were compared, vaccination with the SPI1 mutant proved to be more effective in the protection of poultry against S. Enteritidis challenge than the vaccination with the SPI2 mutant. On the other hand, vaccination with the SPI2 mutant stimulated a slightly higher antibody production and such a mutant might therefore be a better choice if Salmonella is used as a vector for the delivery of heterologous antigens with a desired stimulation of the humoral part of the immune system.
- MeSH
- aplikace orální MeSH
- atenuované vakcíny aplikace a dávkování genetika imunologie MeSH
- bakteriální nálož MeSH
- cytokiny sekrece MeSH
- genomové ostrovy MeSH
- kur domácí MeSH
- leukocyty mononukleární imunologie MeSH
- nemoci drůbeže imunologie prevence a kontrola MeSH
- protilátky bakteriální krev MeSH
- Salmonella enteritidis imunologie patogenita MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie patologie prevence a kontrola MeSH
- salmonelové vakcíny aplikace a dávkování genetika imunologie MeSH
- sekundární imunizace metody MeSH
- slezina patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.
- MeSH
- alantoin metabolismus MeSH
- drůbež MeSH
- kyselina močová metabolismus MeSH
- mnohočetná bakteriální léková rezistence MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nemoci drůbeže mikrobiologie patologie MeSH
- Salmonella enteritidis genetika metabolismus patogenita MeSH
- Salmonella typhimurium genetika metabolismus patogenita MeSH
- salmonelová infekce u zvířat mikrobiologie patologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.
- MeSH
- bakteriální geny genetika MeSH
- glykosyltransferasy genetika MeSH
- leukocyty mikrobiologie ultrastruktura MeSH
- mutace genetika MeSH
- nemoci prasat imunologie mikrobiologie patologie MeSH
- prasata MeSH
- Salmonella enteritidis enzymologie genetika imunologie MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie patologie MeSH
- salmonelové vakcíny imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- B-lymfocyty imunologie metabolismus patologie MeSH
- cékum imunologie patologie MeSH
- finanční podpora výzkumu jako téma MeSH
- kur domácí MeSH
- Salmonella enteritidis fyziologie patologie MeSH
- salmonelová infekce u zvířat etiologie imunologie patologie MeSH
- slezina imunologie patologie MeSH
- T-lymfocyty - podskupiny metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
