RESUMEN
Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endocarditis Bacteriana/microbiología , Enterococcus faecalis/patogenicidad , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos , Animales , Válvula Aórtica/microbiología , Válvula Aórtica/patología , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Plásmidos , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Eliminación de Secuencia , VirulenciaRESUMEN
We determined vaginal Staphylococcus aureus superantigens. Staphylococci were quantified from tampons/diaphragms in 2003 to 2005, with counts compared to those determined in 1980 and 1981. In 2003 to 2005, more women were colonized than in 1980 and 1981 (23 versus 12%). Enterotoxins G and I and enterotoxin-like superantigens M and N declined, but enterotoxin-like superantigens K, L, and Q increased.
Asunto(s)
Antígenos Bacterianos/genética , Staphylococcus aureus/inmunología , Superantígenos/genética , Vagina/microbiología , Factores de Virulencia/genética , Dispositivos Anticonceptivos Femeninos/microbiología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Productos para la Higiene Menstrual/microbiología , Minnesota , Staphylococcus aureus/aislamiento & purificación , Factores de TiempoRESUMEN
Staphylococcus aureus is an important cause of pulmonary infections. The role of S. aureus alpha-toxin as a virulence factor is unclear. We hypothesized that airway epithelium is a target of S. aureus alpha-toxin and that exposure of airway epithelium to alpha-toxin results in damage to the airway epithelium. To examine the hypothesis that alpha-toxin is capable of independently producing airway epithelium damage as measured by permeability and morphometry, an isolated whole mouse trachea test apparatus was developed. In vitro epithelial permeability (P) was calculated and digital micrographs were analyzed morphometrically. Purified S. aureus alpha-toxin produced a significant increase in tracheal epithelial P (P < 0.05). Morphometric analysis revealed the ratio of adherent tracheal epithelium attached to the basement membrane divided by the total length of the basement membrane decreased in a dose-dependent manner with 1 microg/ml alpha-toxin and 10 microg/ml alpha-toxin (P < 0.05). We developed a novel isolated whole mouse trachea test apparatus for the measurement of tracheal epithelium damage. Increased P and separation of the tracheal epithelium from the basement membrane occurred after S. aureus alpha-toxin exposure. We conclude that mammalian airway epithelium is a target of S. aureus alpha-toxin.
Asunto(s)
Toxinas Bacterianas/toxicidad , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proteínas Hemolisinas/toxicidad , Mucosa Respiratoria/metabolismo , Staphylococcus aureus , Tráquea/metabolismo , Animales , Dextranos/farmacocinética , Modelos Animales de Enfermedad , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patología , Exotoxinas/toxicidad , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Masculino , Ratones , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Tráquea/efectos de los fármacos , Tráquea/patología , Traqueítis/metabolismo , Traqueítis/microbiología , Traqueítis/patologíaRESUMEN
Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Membrana Celular/metabolismo , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Endocarditis Bacteriana/microbiología , Enterotoxinas/genética , Histidina Quinasa , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , ARN sin Sentido/genética , ARN Bacteriano/genética , Conejos , Proteínas Represoras/genética , Infecciones Estafilocócicas/microbiología , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Superantígenos/genética , Transcripción Genética , VirulenciaRESUMEN
Toxic shock syndrome (TSS) may be mediated by superantigen-activated T cells, a theory we tested in rabbits, which are more susceptible to the lethal effects of superantigens, such as TSS toxin-1 (TSST-1), than are mice. Rabbits exposed to 10 cGy of total body irradiation exhibited T cell deficiency, with profound depletion of splenic lymphocytes and circulating CD4(+) lymphocytes, as well as an inability to manifest delayed-type hypersensitivity. Nevertheless, these rabbits remained completely susceptible to TSST-1, indicating that TSS can occur in the setting of marked immunosuppression.
Asunto(s)
Toxinas Bacterianas , Ciclosporina/farmacología , Enterotoxinas/efectos de la radiación , Enterotoxinas/toxicidad , Inmunosupresores/farmacología , Choque Séptico/mortalidad , Irradiación Corporal Total , Animales , Femenino , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos BALB C , Conejos , Choque Séptico/inmunología , Choque Séptico/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Superantígenos/efectos de la radiación , Superantígenos/toxicidad , Linfocitos TRESUMEN
Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR beta-chain (Vbeta). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central alpha helix adjacent to the N-terminal alpha helix and the beta7-beta9 loop as well as with two universally conserved SAG residues (Leu(137) and Tyr(144) in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly(16), Trp(116), Glu(132), His(135), Gln(136), and Gln(139)), each individually critical for functional activity as well as direct interaction with the human TCR Vbeta2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vbeta2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vbeta specificity of TSST-1.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Staphylococcus aureus/inmunología , Superantígenos/metabolismo , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Modelos Animales de Enfermedad , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/farmacología , Fiebre/inmunología , Fiebre/microbiología , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Mitógenos/genética , Mitógenos/metabolismo , Mitógenos/farmacología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Conejos , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Choque Séptico/inmunología , Choque Séptico/microbiología , Bazo/citología , Bazo/inmunología , Staphylococcus aureus/genética , Superantígenos/química , Superantígenos/genética , Superantígenos/farmacologíaRESUMEN
Recent genomic studies have revealed extensive variation in natural populations of many pathogenic bacteria. However, the evolutionary processes which contribute to much of this variation remain unclear. A previous whole-genome DNA microarray study identified variation at a large chromosomal region (RD13) of Staphylococcus aureus which encodes a family of proteins with homology to staphylococcal and streptococcal superantigens, designated staphylococcal exotoxin-like (SET) proteins. In the present study, RD13 was found in all 63 S. aureus isolates of divergent clonal, geographic, and disease origins but contained a high level of variation in gene content in different strains. A central variable region which contained from 6 to 10 different set genes, depending on the strain, was identified, and DNA sequence analysis suggests that horizontal gene transfer and recombination have contributed to the diversification of RD13. Phylogenetic analysis based on the RD13 DNA sequence of 18 strains suggested that loss of various set genes has occurred independently several times, in separate lineages of pathogenic S. aureus, providing a model to explain the molecular variation of RD13 in extant strains. In spite of multiple episodes of set deletion, analysis of the ratio of silent substitutions in set genes to amino acid replacements in their products suggests that purifying selection (selective constraint) is acting to maintain SET function. Further, concurrent transcription in vitro of six of the seven set genes in strain COL was detected, indicating that the expression of set genes has been maintained in contemporary strains, and Western immunoblot analysis indicated that multiple SET proteins are expressed during the course of human infections. Overall, we have shown that the chromosomal region RD13 has diversified extensively through episodes of gene deletion and recombination. The coexpression of many set genes and the production of multiple SET proteins during human infection suggests an important role in host-pathogen interactions.
Asunto(s)
Cromosomas Bacterianos , Evolución Molecular , Exotoxinas/genética , Genoma Bacteriano , Staphylococcus aureus/genética , Animales , Secuencia de Bases , Western Blotting , Exotoxinas/análisis , Exotoxinas/toxicidad , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Conejos , Proteínas Recombinantes/análisis , Infecciones Estafilocócicas/sangreRESUMEN
The cocrystal structure of streptococcal pyrogenic exotoxin C (SPE C) with HLA-DR2a (DRA*0101,DRB5*0101) revealed a zinc-dependent interaction site through residues 167, 201, and 203 on SPE C and residue 81 on the beta-chain of HLA-DR2a (DRA*0101,DRB5*0101). Mutation of these SPE C residues resulted in dramatically reduced biological activities. Thus, the zinc-dependent major histocompatibility complex II binding site is critical for maximal biological function of SPE C.
Asunto(s)
Proteínas Bacterianas , Exotoxinas/toxicidad , Antígeno HLA-DR2/metabolismo , Proteínas de la Membrana , Superantígenos/toxicidad , Zinc/farmacología , Animales , Sitios de Unión , Exotoxinas/química , Conejos , Relación Estructura-Actividad , Superantígenos/químicaRESUMEN
Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.
Asunto(s)
Toxinas Bacterianas/química , Pirógenos/química , Staphylococcus aureus/química , Superantígenos/química , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Filogenia , Pirógenos/genética , Pirógenos/aislamiento & purificación , Proteínas Recombinantes/química , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Superantígenos/genética , Superantígenos/aislamiento & purificación , VirulenciaRESUMEN
Infectious endocarditis is a microbial infection of the endothelial lining of the heart that typically occurs on damaged or prosthetic heart valves. The characteristic lesion seen with infective endocarditis, termed "the vegetation," is composed of bacteria surrounded by a platelet/fibrin layer attached to the underlying endothelium. The vegetation has long been believed to exclude or hinder host defenses from clearing bacteria, although formal demonstration of mechanisms by which this occurs are lacking. This study investigated the ability of the vegetation to exclude host antibodies specific for the bacterial surface protein aggregation substance in vivo during experimental endocarditis caused by Enterococcus faecalis. The results demonstrate that, once the vegetation encloses bacteria, they are no longer accessible to high-titer bacterial-specific host antibodies, establishing a mechanism by which the vegetation functions to protect the bacteria from the humoral immune response.