RESUMO
The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.
Assuntos
Proteínas de Bactérias/imunologia , Mutação , Plasmídeos/genética , Células Th1/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/patogenicidade , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Citocinas/análise , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Virulência , Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/microbiologiaRESUMO
Transcription of the K(+) transport operon kdp in Escherichia coli is induced during K(+)-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for new dke (named dke for decreased kdp expression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression. One dke mutation was shown to be in hns, encoding the nucleoid protein H-NS. Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also for trxA mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the kdp expression defect in trxB but not trxA mutants. Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes dsbC and dsbD did not affect kdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues. A trxB hns double mutant was even more severely affected for kdp expression than either single mutant. The dke mutations themselves had no effect on strength of the signal controlling kdp expression, and constitutive mutations in kdpDE were epistatic to hns and trxB. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for kdp transcription.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Potássio/metabolismo , Proteínas Quinases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Transporte de Íons/genética , Mutação , Oxirredução , Proteínas Quinases/metabolismo , Compostos de Sulfidrila/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Mutations in any one of three genes, kdpA, -B, or -C, in Escherichia coli abolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA'), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA' was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA' was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA' provides an incipient pathway for K+ translocation which can serve to transport K+ into the cells in response to the cytoplasmic membrane potential.