RESUMEN
Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.
Asunto(s)
Biomasa , Contaminación de ADN , Feto , Microbiota , Animales , Femenino , Humanos , Embarazo , Líquido Amniótico/inmunología , Líquido Amniótico/microbiología , Mamíferos , Microbiota/genética , Placenta/inmunología , Placenta/microbiología , Feto/inmunología , Feto/microbiología , Reproducibilidad de los ResultadosRESUMEN
In recent work we identified genes that confer the slow-growing and antibiotic-resistant small-colony variant (SCV) form of Staphylococcus aureus, as associated with the amount of capsule the bacteria produce. In this study we isolated a triclosan-resistant SCV (tr-SCV) and demonstrated that it produces significantly less capsule, an effect that appears to be mediated at the transcriptional stage. As with other SCVs, we found that the tr-SCV produces less toxins, and when compared to both a capsule and an Agr mutant we found the tr-SCV to be significantly attenuated in an insect model of infection.
Asunto(s)
Infecciones Estafilocócicas , Triclosán , Humanos , Triclosán/farmacología , Staphylococcus aureus/genética , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Staphylococcus aureus bacteraemia (SAB) is a major cause of blood-stream infection (BSI) in both healthcare and community settings. While the underlying comorbidities of a patient significantly contributes to their susceptibility to and outcome following SAB, recent studies show the importance of the level of cytolytic toxin production by the infecting bacterium. In this study we demonstrate that this cytotoxicity can be determined directly from the diagnostic MALDI-TOF mass spectrum generated in a routine diagnostic laboratory. With further development this information could be used to guide the management and improve the outcomes for SAB patients.
Asunto(s)
Bacteriemia , Infecciones Estafilocócicas , Bacteriemia/diagnóstico , Bacteriemia/microbiología , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureusRESUMEN
Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus. By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance-associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing small colony variant (SCV) phenotype of S. aureus, and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion for the ability of S. aureus to cause disease, we believe that these findings explain the role of the mpsB gene as a mortality-associated locus during human disease.
Asunto(s)
Proteínas Bacterianas/metabolismo , Evasión Inmune , Staphylococcus aureus/patogenicidad , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriemia/microbiología , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Humanos , Potenciales de la Membrana , Mutación , Percepción de Quorum , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiologíaRESUMEN
Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Antibacterianos/metabolismo , Antibacterianos/farmacología , Estudio de Asociación del Genoma Completo , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Vitamina K 3/metabolismo , Vitamina K 3/farmacologíaRESUMEN
Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Antibacterianos/farmacología , Genotipo , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/genéticaRESUMEN
Staphylococcus aureus is a major human pathogen, and the emergence of antibiotic-resistant strains is making all types of S. aureus infections more challenging to treat. With a pressing need to develop alternative control strategies to use alongside or in place of conventional antibiotics, one approach is the targeting of established virulence factors. However, attempts at this have had little success to date, suggesting that we need to better understand how this pathogen causes disease if effective targets are to be identified. To address this, using a functional genomics approach, we have identified a small membrane-bound protein that we have called MspA. Inactivation of this protein results in the loss of the ability of S. aureus to secrete cytolytic toxins, protect itself from several aspects of the human innate immune system, and control its iron homeostasis. These changes appear to be mediated through a change in the stability of the bacterial membrane as a consequence of iron toxicity. These pleiotropic effects on the ability of the pathogen to interact with its host result in significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and sepsis model of infection. Given the scale of the effect the inactivation of MspA causes, it represents a unique and promising target for the development of a novel therapeutic approach.
Asunto(s)
Bacteriemia/microbiología , Evasión Inmune , Infecciones Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Factores de Virulencia/genética , Células A549 , Animales , Bacteriemia/inmunología , Bacteriemia/patología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Eritrocitos/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hemo/inmunología , Hemo/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Homeostasis/inmunología , Humanos , Hierro/inmunología , Hierro/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Fagocitosis , Proteómica/métodos , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patología , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/patología , Toxoide Estafilocócico/genética , Toxoide Estafilocócico/inmunología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Células THP-1 , Virulencia , Factores de Virulencia/inmunología , Factores de Virulencia/toxicidad , alfa-Defensinas/genética , alfa-Defensinas/inmunologíaRESUMEN
Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Péptidos Cíclicos/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/genética , Staphylococcus aureus/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Citoplasma/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Percepción de Quorum , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
The secretion of proteins that damage host tissue is well established as integral to the infectious processes of many bacterial pathogens. However, recent advances in our understanding of the activity of toxins suggest that the attributes we have assigned to them from early in vitro experimentation have misled us into thinking of them as merely destructive tools. Here, we will discuss the multifarious ways in which toxins contribute to the lifestyle of bacteria and, by considering their activity from an evolutionary perspective, demonstrate how this extends far beyond their ability to destroy host tissue.
Asunto(s)
Adenilil Ciclasas/metabolismo , Bacterias/metabolismo , Toxinas Bacterianas/metabolismo , Evolución Biológica , Interacciones Huésped-Patógeno/inmunología , Animales , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/toxicidad , Humanos , Superantígenos/inmunología , Superantígenos/metabolismoRESUMEN
Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.
Asunto(s)
Bacteriemia/microbiología , Evolución Biológica , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Biopelículas , Trampas Extracelulares/fisiología , Genómica , Humanos , Péptido Hidrolasas/metabolismo , Polimorfismo Genético , Staphylococcus aureus/enzimología , alfa-DefensinasRESUMEN
Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.
Asunto(s)
Genoma Bacteriano , Staphylococcus aureus Resistente a Meticilina/genética , Modelos Genéticos , Virulencia/genética , Estudio de Asociación del Genoma Completo , Mutación INDEL , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Polimorfismo de Nucleótido SimpleRESUMEN
The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for "latch" strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Fibrinógeno/química , Fibronectinas/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus aureus/genética , Resonancia por Plasmón de SuperficieRESUMEN
The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.
Asunto(s)
Autofagia/fisiología , Proteínas Bacterianas/metabolismo , Células Dendríticas/microbiología , Infecciones Estafilocócicas/inmunología , Transactivadores/metabolismo , Animales , Bacteriemia/metabolismo , Bacteriemia/microbiología , Western Blotting , Células de la Médula Ósea/microbiología , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunologíaRESUMEN
The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing cause for concern. These strains are more virulent than health care-associated MRSA (HA-MRSA) due to higher levels of toxin expression. In a previous study, we showed that the high-level expression of PBP2a, the alternative penicillin binding protein encoded by the mecA gene on type II staphylococcal cassette chromosome mec (SCCmec) elements, reduced toxicity by interfering with the Agr quorum sensing system. This was not seen in strains carrying the CA-MRSA-associated type IV SCCmec element. These strains express significantly lower levels of PBP2a than the other MRSA type, which may explain their relatively high toxicity. We hypothesized that as oxacillin is known to increase mecA expression levels, it may be possible to attenuate the toxicity of CA-MRSA by using this antibiotic. Subinhibitory oxacillin concentrations induced PBP2a expression, repressed Agr activity, and, as a consequence, decreased phenol-soluble modulin (PSM) secretion by CA-MRSA strains. However, consistent with other studies, oxacillin also increased the expression levels of alpha-toxin and Panton-Valentine leucocidin (PVL). The net effect of these changes on the ability to lyse diverse cell types was tested, and we found that where the PSMs and alpha-toxin are important, oxacillin reduced overall lytic activity, but where PVL is important, it increased lytic activity, demonstrating the pleiotropic effect of oxacillin on toxin expression by CA-MRSA.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/genética , Exotoxinas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas Hemolisinas/genética , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oxacilina/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/agonistas , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/biosíntesis , Infecciones Comunitarias Adquiridas/microbiología , Exotoxinas/agonistas , Exotoxinas/biosíntesis , Proteínas Hemolisinas/agonistas , Proteínas Hemolisinas/biosíntesis , Humanos , Leucocidinas/agonistas , Leucocidinas/biosíntesis , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/metabolismo , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Percepción de Quorum/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to ß-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to ß-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Contaminación de Equipos , Resistencia a la Meticilina/fisiología , Staphylococcus aureus Resistente a Meticilina/metabolismo , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Antibacterianos/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Masculino , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Ratones , N-Acetil Muramoil-L-Alanina Amidasa/genética , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Oxacilina/farmacología , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismoRESUMEN
Staphylococcus aureus produces a plethora of virulence factors critical to its ability to establish an infection and cause disease. We have previously characterized a small membrane protein, MspA, which has pleiotropic effects on virulence and contributes to S. aureus pathogenicity in vivo. Here we report that mspA inactivation triggers overaccumulation of the essential cell wall component, lipoteichoic acid (LTA), which, in turn, decreases autolytic activity and leads to increased cell size due to a delay in cell separation. We show that MspA directly interacts with the enzymes involved in LTA biosynthesis (LtaA, LtaS, UgtP, and SpsB), interfering with their normal activities. MspA, in particular, interacts with the type I signal peptidase SpsB, limiting its cleavage of LtaS into its active form. These findings suggest that MspA contributes to maintaining a physiological level of LTA in the cell wall by interacting with and inhibiting the activity of SpsB, thereby uncovering a critical role for the MspA protein in regulating cell envelope biosynthesis and pathogenicity.IMPORTANCEThe S. aureus cell envelope, comprising the cytoplasmic membrane, a thick peptidoglycan layer, and the anionic polymers lipoteichoic acid and wall teichoic acids, is fundamental for bacterial growth and division, as well as being the main interface between the pathogen and the host. It has become increasingly apparent that the synthesis and turnover of cell envelope components also affect the virulence of S. aureus. In this study, we show that MspA, an effector of S. aureus virulence, contributes to the maintenance of normal levels of lipoteichoic acid in the cell wall, with implications on cell cycle and size. These findings further our understanding of the connections between envelope synthesis and pathogenicity and suggest that MspA represents a promising target for the development of future therapeutic strategies.
Asunto(s)
Proteínas Bacterianas , Pared Celular , Lipopolisacáridos , Staphylococcus aureus , Ácidos Teicoicos , Ácidos Teicoicos/biosíntesis , Ácidos Teicoicos/metabolismo , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Factores de Virulencia/metabolismo , Virulencia , Infecciones Estafilocócicas/microbiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Regulación Bacteriana de la Expresión Génica , Animales , Ratones , Serina EndopeptidasasRESUMEN
The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Resistencia a la Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Animales , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Supervivencia Celular , Pared Celular/metabolismo , Infección Hospitalaria , Modelos Animales de Enfermedad , Expresión Génica , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ratones , Proteínas de Unión a las Penicilinas/metabolismo , Péptido Sintasas/metabolismo , Péptidos Cíclicos , Percepción de Quorum/genética , Sepsis/microbiología , Linfocitos T , Transactivadores/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.
RESUMEN
The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.