RESUMEN
Staphylococcus epidermidis is normally a commensal colonizer of human skin and mucus membranes, but, due to its ability to form biofilms on indwelling medical devices, it has emerged as a leading cause of nosocomial infections. Bacteremia or bloodstream infection is a frequent and costly complication resulting from biofilm fouling of medical devices. Our goal was to develop a murine model of S. epidermidis infection to identify potential vaccine targets for the prevention of S. epidermidis bacteremia. However, assessing the contribution of adaptive immunity to protection against S. epidermidis challenge was complicated by a highly efficacious innate immune response in mice. Naive mice rapidly cleared S. epidermidis infections from blood and solid organs, even when the animals were immunocompromised. Cyclophosphamide-mediated leukopenia reduced the size of the bacterial challenge dose required to cause lethality but did not impair clearance after a nonlethal challenge. Nonspecific innate immune stimulation, such as treatment with a Toll-like receptor 4 (TLR4) agonist, enhanced bacterial clearance. TLR2 signaling was confirmed to accelerate the clearance of S. epidermidis bacteremia, but TLR2(-/-)mice could still resolve a bloodstream infection. Furthermore, TLR2 signaling played no role in the clearance of bacteria from the spleen. In conclusion, these data suggest that S. epidermidis bloodstream infection is cleared in a highly efficient manner that is mediated by both TLR2-dependent and -independent innate immune mechanisms. The inability to establish a persistent infection in mice, even in immunocompromised animals, rendered these murine models unsuitable for meaningful assessment of antibody-mediated therapies or vaccine candidates.
Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Bacteriemia/prevención & control , Modelos Animales de Enfermedad , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , Staphylococcus epidermidis/inmunología , Animales , Ciclofosfamida/toxicidad , Inmunidad Innata , Leucopenia/inducido químicamente , Ratones , Ratones Noqueados , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
Biofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream infection and systemic pathogen dissemination. Staphylococcal biofilms are the most common cause of central-line-associated bloodstream infections, and antibiotic resistance makes them difficult to treat. As an alternative to antibiotic intervention, we sought to identify anti-staphylococcal biofilm targets for the development of a vaccine or antibody prophylactic. A screening strategy was devised using a microfluidic system to test antibody-mediated biofilm inhibition under biologically relevant conditions of shear flow. Affinity-purified polyclonal antibodies to target antigen PhnD inhibited both Staphylococcus epidermidis and S. aureus biofilms. PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages, and deletion of phnD inhibited normal biofilm formation. We further adapted our microfluidic biofilm system to monitor the interaction of human neutrophils with staphylococcal biofilms and demonstrated that PhnD-specific antibodies also serve as opsonins to enhance neutrophil binding, motility, and biofilm engulfment. These data support the identification of PhnD as a lead target for biofilm intervention strategies performed either by vaccination or through passive administration of antibodies.
Asunto(s)
Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Biopelículas , Humanos , Neutrófilos/inmunología , Neutrófilos/microbiología , Fagocitosis/inmunología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Pneumolysin (PLY) is a cholesterol-binding, pore-forming protein toxin. It is an important virulence factor of Streptococcus pneumoniae and a key vaccine target against pneumococcal disease. We report a systematic structure-driven approach that solves a long-standing problem for vaccine development in this field: detoxification of PLY with retention of its antigenic integrity. Using three conformational restraint techniques, we rationally designed variants of PLY that lack hemolytic activity and yet induce neutralizing antibodies against the wild-type toxin. These results represent a key milestone toward a broad-spectrum protein-based pneumococcal vaccine and illustrate the value of structural knowledge in formulating effective strategies for antigen optimization.
Asunto(s)
Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Estreptolisinas/inmunología , Estreptolisinas/metabolismo , Animales , Antígenos Bacterianos , Proteínas Bacterianas/efectos adversos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Rastreo Diferencial de Calorimetría , Células Cultivadas , Dicroismo Circular , Hemólisis/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Ovinos , Estreptolisinas/efectos adversos , Estreptolisinas/genéticaRESUMEN
The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Asunto(s)
Antibacterianos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Benzofuranos/uso terapéutico , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Pironas/uso terapéutico , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Administración Oral , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Benzofuranos/farmacología , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Infecciones Comunitarias Adquiridas/microbiología , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Esquema de Medicación , Enoil-ACP Reductasa (NADH)/metabolismo , Femenino , Humanos , Cinética , Ratones , Pruebas de Sensibilidad Microbiana , Pironas/farmacología , Sepsis/microbiología , Sepsis/mortalidad , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , Staphylococcus aureus/crecimiento & desarrollo , Tasa de SupervivenciaRESUMEN
Proteins of the Bcl-2 family are important regulators of cell fate. The role of these proteins in controlling mitochondrial apoptotic processes has been extensively investigated, although exact molecular mechanisms are incompletely understood. However, mounting evidence indicates that these proteins also function at the endoplasmic reticulum and other locations within the cell. Both pro- and anti-apoptotic Bcl-2 family members can regulate endoplasmic reticulum calcium, cellular pH and endoplasmic reticulum resident proteins. In this review, we discuss the activities and potential targets of Bcl-2 family members at the endoplasmic reticulum and other cellular locations.
Asunto(s)
Apoptosis , Retículo Endoplásmico/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Calcio/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Mitocondrias/fisiología , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ-producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b(+)CD103(-) dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b(-)CD103(+) DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (T(RM) cells). Optimal Ct clearance required both T(RM) seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.
Asunto(s)
Vacunas Bacterianas/inmunología , Infecciones por Chlamydia/prevención & control , Chlamydia trachomatis/inmunología , Memoria Inmunológica , Células TH1/inmunología , Útero/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos CD/inmunología , Vacunas Bacterianas/administración & dosificación , Antígenos CD11/inmunología , Linfocitos T CD8-positivos/inmunología , Chlamydia trachomatis/efectos de la radiación , Células Dendríticas/inmunología , Femenino , Cadenas alfa de Integrinas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Membrana Mucosa/inmunología , Nanopartículas/administración & dosificación , Subgrupos de Linfocitos T/inmunología , Rayos Ultravioleta , Vacunación/métodos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC(90) values of ≤0·015 µg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (â¼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Asunto(s)
Antibacterianos/sangre , Antibacterianos/farmacología , Benzofuranos/sangre , Benzofuranos/farmacología , Proteínas Sanguíneas/metabolismo , Pironas/sangre , Pironas/farmacología , Animales , Daptomicina/sangre , Daptomicina/farmacología , Perros , Gentamicinas/sangre , Gentamicinas/farmacología , Semivida , Humanos , Masculino , Meticilina/sangre , Meticilina/farmacología , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Surfactantes Pulmonares/metabolismo , Ratas , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
The Bcl-2 family member Bax is an apoptosis-promoting protein that normally resides in an inactive state within the cytoplasm of healthy cells. Upon induction of apoptosis by diverse stimuli, Bax undergoes a conformational change and translocates to mitochondria, where it oligomerizes and forms pores that allow the release of cytochrome c and other cytotoxic factors. Protein-protein interactions between Bax and other Bcl-2 family members are strongly implicated in Bax activation, but a compelling case has recently been made for the involvement of lipids in this process as well. Here we report that purified Bax undergoes a reversible conformational change upon incubation with lipid vesicles in the absence of other proteins. This Bax-liposome interaction does not depend on a specific lipid composition. Changes in Bax conformation were observed by immunoprecipitation with the conformation-specific antibody 6A7, circular dichroism spectroscopy, and differential scanning calorimetry. Although liposomes induced Bax to become 6A7-reactive (a feature normally associated with the onset of apoptosis), the protein did not insert into membranes, become oligomeric, or form pores, clearly indicating that other triggers are required for Bax to achieve its final pro-apoptotic state. Indeed, the lipid-induced Bax conformational change is shown to be required for tBid-induced Bax oligomerization and pore formation, putting it upstream of tBid activity in this molecular pathway to Bax activation. These data demonstrate that Bax is sensitized to activation by transient interaction with lipid membrane surfaces and provide evidence that Bax activation proceeds in a stepwise fashion, with multiple triggers and potential levels of regulation.