RESUMO
Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease.
Assuntos
Toxinas Bacterianas/toxicidade , Proteínas Hemolisinas/toxicidade , Infecções dos Tecidos Moles/patologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/fisiologia , Animais , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Recidiva , Infecções dos Tecidos Moles/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/metabolismoRESUMO
Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.
Assuntos
Aglutinação/fisiologia , Sepse/prevenção & controle , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Antitrombinas/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Coagulantes/metabolismo , Coagulase/imunologia , Coagulase/metabolismo , Modelos Animais de Doenças , Coração/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Imunização Passiva , Longevidade/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Ligação Proteica , Sepse/imunologia , Sepse/microbiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/ultraestrutura , Fator de von Willebrand/imunologia , Fator de von Willebrand/metabolismoRESUMO
Staphylococcus aureus is a frequent cause of skin infection and sepsis in humans. Preclinical vaccine studies with S. aureus have used a mouse model with intraperitoneal challenge and survival determination as a measure for efficacy. To appreciate the selection of protective antigens in this model, we sought to characterize the pathological attributes of S. aureus infection in the peritoneal cavity. Testing C57BL/6J and BALB/c mice, >10(9) CFU of S. aureus Newman were needed to produce a lethal outcome in 90% of animals infected via intraperitoneal injection. Both necropsy and histopathology revealed the presence of intraperitoneal abscesses in the vicinity of inoculation sites. Abscesses were comprised of fibrin as well as collagen deposits and immune cells with staphylococci replicating at the center of these lesions. Animals that succumbed to challenge harbored staphylococci in abscess lesions and in blood. The establishment of lethal infections, but not the development of intraperitoneal abscesses, was dependent on S. aureus expression of alpha-hemolysin (Hla). Active immunization with nontoxigenic Hla(H35L) or passive immunization with neutralizing monoclonal antibodies protected mice against early lethal events associated with intraperitoneal S. aureus infection but did not affect the establishment of abscess lesions. These results characterize a mouse model for the study of intraperitoneal abscess formation by S. aureus, a disease that occurs frequently in humans undergoing continuous ambulatory peritoneal dialysis for end-stage renal disease.
Assuntos
Abscesso/microbiologia , Toxinas Bacterianas/toxicidade , Proteínas Hemolisinas/toxicidade , Peritonite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Anticorpos Neutralizantes , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fibrina/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Nefropatias/microbiologia , Nefropatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/imunologiaRESUMO
Staphylococcus aureus is a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistant S. aureus [MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope of S. aureus is decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of V(H)3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpA(KKAA)), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the V(H)3(+) Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpA(KKAA) that, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpA(KKAA) MAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpA(KKAA) MAbs may be useful for the prevention and therapy of staphylococcal disease in humans.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/sangue , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Estafilocócicas/imunologia , Proteína Estafilocócica A/metabolismo , Abscesso/microbiologia , Abscesso/prevenção & controle , Animais , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Humanos , Nefropatias/microbiologia , Nefropatias/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/imunologiaRESUMO
During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.
Assuntos
Anticorpos Antibacterianos/imunologia , Coagulase/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Sítios de Ligação , Coagulase/genética , Coagulase/metabolismo , Feminino , Genoma Bacteriano , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Coelhos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/classificação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Fatores de TempoRESUMO
The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion.
Assuntos
Divisão Celular , Proteínas de Membrana/metabolismo , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/citologia , Motivos de Aminoácidos , Genes Bacterianos , Teste de Complementação Genética , Mutação INDEL , Proteínas de Membrana/genética , Mutagênese Insercional , Mutação , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteína Estafilocócica A/genética , Staphylococcus aureus/genéticaRESUMO
The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Positivas/metabolismo , Proteínas de Membrana/metabolismo , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Biologia Computacional , Cisteína Endopeptidases , Fímbrias Bacterianas/metabolismo , Bactérias Gram-Positivas/enzimologia , Conformação Proteica , Transporte ProteicoRESUMO
Staphylococcus aureus encodes the specialized secretion system Ess (ESAT-6 secretion system). The ess locus is a cluster of eight genes (esxAB, essABC, esaABC) of which esxA and esxB display homology to secreted ESAT-6 proteins of Mycobacterium tuberculosis. EsxA and EsxB require EssA, EssB and EssC for transport across the staphylococcal envelope. Herein, we examine the role of EsaB and EsaC and show that EsaB is a negative regulator of EsaC. Further, EsaC production is repressed when staphylococci are grown in broth and increased when staphylococci replicate in serum or infected hosts. EsaB is constitutively produced and remains in the cytoplasm whereas EsaC is secreted. This secretion requires an intact Ess pathway. Mutants lacking esaB or esaC display only a small defect in acute infection, but remarkably are unable to promote persistent abscesses during animal infection. Together, the data suggest a model whereby EsaB controls the production of effector molecules that are important for host pathogen interaction. One such effector, EsaC, is a secretion substrate of the Ess pathway and implements its pathogenic function during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Abscesso/metabolismo , Abscesso/microbiologia , Animais , Proteínas de Bactérias/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Família Multigênica , Transporte Proteico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Immunomodulatory cytotoxins are prominent virulence factors produced by Staphylococcus aureus, a leading cause of bacterial sepsis, skin infection, and pneumonia. S. aureus α-toxin is a pore-forming toxin that utilizes a widely expressed receptor, ADAM10, to injure the host epithelium, endothelium, and immune cells. As each host tissue is characterized by a unique composition of resident cells and recruited immune cells, the outcome of α-toxin-mediated injury may depend on the infected tissue environment. Utilizing myeloid lineage-specific Adam10 knockout mice, we show that α-toxin exerts tissue-specific effects on innate immunity to staphylococcal infection. Loss of ADAM10 expression exacerbates skin infection, yet affords protection against lethal pneumonia. These diverse outcomes are not related to altered immune cell recruitment, but rather correlate with a defect in toxin-induced IL-1ß production. Extension of these studies through analysis of ADAM10 double-knockout mice affecting both the myeloid lineage and either the skin or lung epithelium highlight the prominence of toxin-induced injury to the epithelium in governing the outcome of infection. Together, these studies provide evidence of tissue specificity of pore-forming cytotoxin action in the modulation of host immunity, and illustrate that the outcome of infection is a collective manifestation of all effects of the toxin within the tissue microenvironment.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Pulmão/patologia , Proteínas de Membrana/metabolismo , Pele/patologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Toxinas Bacterianas/metabolismo , Células Cultivadas , Microambiente Celular , Perfilação da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Humanos , Imunidade Inata/genética , Interleucina-1beta/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Pele/imunologia , Pele/microbiologia , Infecções Estafilocócicas/transmissão , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismoRESUMO
Staphylococcus aureus is an important human pathogen that causes skin and soft tissue abscesses. Abscess formation is not unique to staphylococcal infection and purulent discharge has been widely considered a physiological feature of healing and tissue repair. Here we present a different view, whereby S. aureus deploys specific virulence factors to promote abscess lesions that are distinctive for this pathogen. In support of this model, only live S. aureus is able to form abscesses, requiring genes that act at one or more of four discrete stages during the development of these infectious lesions. Protein A and coagulases are distinctive virulence attributes for S. aureus, and humoral immune responses specific for these polypeptides provide protection against abscess formation in animal models of staphylococcal disease.
Assuntos
Abscesso/microbiologia , Abscesso/patologia , Infecções dos Tecidos Moles/microbiologia , Infecções dos Tecidos Moles/patologia , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Animais , Coagulase/metabolismo , Humanos , Proteína Estafilocócica A/metabolismo , Virulência , Fatores de Virulência/metabolismoRESUMO
Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.