RESUMEN
Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired. This suggested that ris-dependent products are required for the intracellular survival of bacteria. In this study we demonstrate that ris-regulated products play a role in B. bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.
Asunto(s)
Proteínas Bacterianas , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/fisiología , Macrófagos Peritoneales/microbiología , Receptores de Superficie Celular/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Antibacterianos/farmacología , Bordetella bronchiseptica/efectos de los fármacos , Recuento de Colonia Microbiana , Femenino , Depuradores de Radicales Libres/metabolismo , Genes Bacterianos/fisiología , Genes Reguladores/fisiología , Macrólidos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de Superficie Celular/genéticaRESUMEN
The phosphoglucomutase (PGM)-encoding gene of Bordetella bronchiseptica is required for lipopolysaccharide (LPS) biosynthesis. An insertion mutant of the wild-type B. bronchiseptica strain BB7865 which disrupted LPS biosynthesis was created and characterized (BB7865pgm). Genetic analysis of the mutated gene showed it shares high identity with PGM genes of various bacterial species and forms part of an operon which also encompasses the gene encoding phosphoglucose isomerase. Functional assays for PGM revealed that enzyme activity is expressed in both bvg-positive and bvg-negative strains of B. bronchiseptica and is substantially reduced in BB7865pgm. Complementation of the mutated PGM gene with that from BB7865 restored the wild-type condition for all phenotypes tested. The ability of the mutant BB7865pgm to survive within J774. A1 cells was significantly reduced at 2 h (40% reduction) and 24 h (56% reduction) postinfection. BB7865pgm was also significantly attenuated in its ability to survive in vivo following intranasal infection of mice, being effectively cleared from the lungs within 4 days, whereas the wild-type strain persisted at least 35 days. The activities of superoxide dismutase, urease, and acid phosphatase were unaffected in the PGM-deficient strain. In contrast, the inability to produce wild-type LPS resulted in a reduced bacterial resistance to oxidative stress and a higher susceptibility to the antimicrobial peptide cecropin P.
Asunto(s)
Bordetella bronchiseptica/enzimología , Bordetella bronchiseptica/patogenicidad , Lipopolisacáridos/biosíntesis , Péptidos , Fosfoglucomutasa/metabolismo , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Bordetella bronchiseptica/genética , Farmacorresistencia Microbiana , Femenino , Genes Bacterianos , Prueba de Complementación Genética , Glucosa-6-Fosfato Isomerasa/genética , Pulmón/microbiología , Macrófagos/citología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Insercional , Operón , Paraquat/farmacología , Fenotipo , Fosfoglucomutasa/genética , Homología de Secuencia de AminoácidoRESUMEN
Dendritic cells (DCs) play a central role in the generation of acquired immunity to infections by pathogenic microorganisms. Salmonella enterica serotype Typhimurium is known to survive and proliferate intracellularly within macrophages and nonphagocytic cells, but no data exist on how this pathogen interacts with DCs. In this report, we show the capacity of serotype Typhimurium to survive within the established mouse DC line CB1. In contrast to the case for the macrophage model, the compartments of DCs containing serotype Typhimurium are devoid of lysosomal membrane glycoproteins and the PhoPQ two-component regulatory system is not essential for pathogen intracellular survival.
Asunto(s)
Antígenos CD/metabolismo , Proteínas Bacterianas/metabolismo , Células Dendríticas/microbiología , Glicoproteínas de Membrana/metabolismo , Salmonella enterica/metabolismo , Animales , Proteínas Bacterianas/genética , Compartimento Celular , Línea Celular , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Proteínas de Membrana de los Lisosomas , Ratones , Salmonella enterica/genética , Salmonella enterica/crecimiento & desarrollo , SerotipificaciónRESUMEN
Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.
Asunto(s)
Fosfatasa Ácida/biosíntesis , Proteínas Bacterianas , Bordetella bronchiseptica/patogenicidad , Estrés Oxidativo , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bordetella bronchiseptica/genética , Línea Celular , Células Dendríticas/citología , Células Dendríticas/microbiología , Genes Bacterianos , Genes Reguladores , Pulmón/microbiología , Macrófagos/citología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Especies Reactivas de Oxígeno , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The invasion of group B streptococci (GBS) in HEp-2 epithelial cells was analyzed by electron microscopy and a quantitative antibiotic survival assay. Invasion of GBS involved intimate attachment of streptococcal chains, engulfment of the adherent bacteria by cellular protrusions, entry of the bacteria in a 'polar' fashion and formation of membrane-bound vacuoles in which most of the intracellular streptococci resided. At later stages of infection bacteria were also found free in the cytoplasm. Efficient uptake of streptococci by HEp-2 cells occurred within 20 min and live intracellular bacteria were detectable up to 48 h post-infection. Invasion of GBS required activation of the eukaryotic actin microfilament system involving, at least partially, protein kinase signal transduction pathways. Invasion was inhibited in a dose-dependent manner by decreasing extracellular Ca2+ levels as well as by substances known to interfere with eukaryotic calcium regulatory systems. These results suggest that GBS invade HEp-2 cells by triggering calcium-dependent phagocytosis-like internalization mechanisms and persist intracellularly both in vacuoles and free in the cytoplasm.