RESUMO
Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.
Assuntos
Polissacarídeos Bacterianos , Staphylococcus aureus , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Acetilgalactosamina/análise , Óperon , Escherichia coli/genética , Expressão Gênica , Parede Celular/químicaRESUMO
Production of capsular polysaccharides in Staphylococcus aureus is transcriptionally regulated by a control region of the cap operon that consists of SigA- and SigB-dependent promoters. A large number of regulators have been shown to affect cap gene expression. However, regulation of capsule is only partially understood. Here we found that SarZ was another regulator that activated the cap genes through the SigA-dependent promoter. Gel electrophoresis mobility shift experiments revealed that SarZ is bound to a broad region of the cap promoter including the SigA-dependent promoter but mainly the downstream region. We demonstrated that activation of cap expression by SarZ was independent of MgrA, which also activated capsule through the SigA-dependent promoter. Our results further showed that oxidative stress with hydrogen peroxide (H2O2) treatments enhanced SarZ activation of cap expression, indicating that SarZ is able to sense oxidative stress to regulate capsule production. IMPORTANCE Expression of virulence genes in Staphylococcus aureus is affected by environmental cues and is regulated by a surprisingly large number of regulators. Much is still unknown about how virulence factors are regulated by environment cues at the molecular level. Capsule is an antiphagocytic virulence factor that is highly regulated. In this study, we found SarZ was an activator of capsule and that the regulation of capsule by SarZ was affected by oxidative stress. These results provide an example of how a virulence factor could be regulated in response to an environmental cue. As the host oxidative defense system plays an important role against S. aureus, this study contributes to a better understanding of virulence gene regulation and staphylococcal pathogenesis.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Imunoglobulina A Secretora/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismoRESUMO
Secreted bacterial proteins are difficult to identify directly from an infection site due to a limited amount of bacteria and presence of a large quantity of host proteins. Here we describe a rat model of orthopedic implant that allows us to harvest bacterial biofilm materials sufficient for identification of bacterial proteins in the biofilm matrix by liquid chromatography-tandem MS (GeLC-MS/MS) analysis.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/fisiologia , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Masculino , Próteses e Implantes/microbiologia , Ratos , Staphylococcus aureus/metabolismo , Espectrometria de Massas em TandemRESUMO
Staphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (Pcap) upstream of the cap operon. Pcap, which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the Pcap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of Pcap, mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases.IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus, an important human pathogen.
Assuntos
Cápsulas Bacterianas/química , Imunoglobulina A Secretora/fisiologia , Polissacarídeos Bacterianos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Staphylococcus aureus/fisiologia , Fatores de Virulência/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Immunoblotting , Imunoglobulina A Secretora/genética , Mutação , Polissacarídeos Bacterianos/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transcrição Reversa , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.
Assuntos
Adesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Adesinas Bacterianas/genética , Cápsulas Bacterianas/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Polissacarídeos Bacterianos/metabolismo , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Virulência/genéticaRESUMO
The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.
Assuntos
Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/fisiologia , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Toxinas Bacterianas/genética , Ratos , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
Capsule is one of many virulence factors produced by Staphylococcus aureus, and its expression is highly regulated. Here, we report the repression of capsule by direct interaction of XdrA and CodY with the capsule promoter region. We found, by footprinting analyses, that XdrA repressed capsule by binding to a broad region that extended from upstream of the -35 region of the promoter to the coding region of capA, the first gene of the 16-gene cap operon. Footprinting analyses also revealed that CodY bound to a large region that overlapped extensively with that of XdrA. We found that repression of the cap genes in the xdrA mutant could be achieved by the overexpression of codY but not vice versa, suggesting codY is epistatic to xdrA However, we found XdrA had no effect on CodY expression. These results suggest that XdrA plays a secondary role in capsule regulation by promoting CodY repression of the cap genes. Oxacillin slightly induced xdrA expression and reduced cap promoter activity, but the effect of oxacillin on capsule was not mediated through XdrA.IMPORTANCEStaphylococcus aureus employs a complex regulatory network to coordinate the expression of various virulence genes to achieve successful infections. How virulence genes are coordinately regulated is still poorly understood. We have been studying capsule regulation as a model system to explore regulatory networking in S. aureus In this study, we found that XdrA and CodY have broad binding sites that overlap extensively in the capsule promoter region. Our results also suggest that XdrA assists CodY in the repression of capsule. As capsule gene regulation by DNA-binding regulators has not been fully investigated, the results presented here fill an important knowledge gap, thereby further advancing our understanding of the global virulence regulatory network in S. aureus.
Assuntos
Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas Repressoras/genética , Staphylococcus aureus/genética , Sítios de Ligação , Epistasia Genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Regiões Promotoras Genéticas , VirulênciaRESUMO
The matrix proteins of Staphylococcus aureus biofilm have not been well defined. Previous efforts to identify these proteins were performed using in vitro systems. Here we use a proteomic approach to identify biofilm matrix proteins directly from infected bone implants using a rat model of orthopedic implant-associated S. aureus infection. Despite heavy presence of host proteins, a total of 28 and 105 S. aureus proteins were identified during acute infection and chronic infection, respectively. Our results show that biofilm matrix contains mostly intracellular cytoplasmic proteins and, to a much less extent, extracellular and cell surface-associated proteins. Significantly, leukocidins were identified in the biofilm matrix during chronic infection, suggesting S. aureus is actively attacking the host immune system even though they are protected within the biofilm. The presence of two surface-associated proteins, Ebh and SasF, in the infected bone tissue during acute infection was confirmed by immunohistochemistry. In addition, a large number of host proteins were found differentially expressed in response to S. aureus biofilm formed on bone implants.
Assuntos
Artroplastia/efeitos adversos , Proteínas de Bactérias/metabolismo , Proteômica/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Biofilmes , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Leucocidinas/metabolismo , Proteínas de Membrana/metabolismo , Ratos , Infecções Estafilocócicas/etiologia , Staphylococcus aureus/metabolismoRESUMO
RNAIII, the effector of the agr quorum-sensing system, plays a key role in virulence gene regulation in Staphylococcus aureus, but how RNAIII transcriptionally regulates its downstream genes is not completely understood. Here, we show that RNAIII stabilizes mgrA mRNA, thereby increasing the production of MgrA, a global transcriptional regulator that affects the expression of many genes. The mgrA gene is transcribed from two promoters, P1 and P2, to produce two mRNA transcripts with long 5' UTR. Two adjacent regions of the mgrA mRNA UTR transcribed from the upstream P2 promoter, but not the P1 promoter, form a stable complex with two regions of RNAIII near the 5' and 3' ends. We further demonstrate that the interaction has several biological effects. We propose that MgrA can serve as an intermediary regulator through which agr exerts its regulatory function.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Percepção de Quorum/genética , RNA Bacteriano/metabolismo , Elementos Reguladores de Transcrição/genética , Staphylococcus aureus/enzimologia , Pareamento de Bases , Sequência de Bases , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Componentes do Gene , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Staphylococcus aureus capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the cap operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the -35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the cap promoter. To search for potential proteins which directly interact with the cap promoter region (Pcap), we directly analyzed the proteins interacting with the Pcap DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate cap gene expression by specifically binding to the cap promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that rbsR was directly controlled by SigB and that RbsR was a repressor of the rbsUDK operon, involved in ribose uptake and phosphorylation. The repression of rbsUDK by RbsR could be derepressed by D-ribose. However, D-ribose did not affect RbsR activation of capsule. IMPORTANCE: Staphylococcus aureus is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of rbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the rbs operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in S. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.
Assuntos
Cápsulas Bacterianas/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Ribose/metabolismo , Staphylococcus aureus/genética , Transcrição GênicaRESUMO
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.
Assuntos
Proteínas de Bactérias/genética , Proteínas Quinases/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Humanos , Fosforilação , Proteínas Quinases/biossíntese , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Fatores de TranscriçãoRESUMO
The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.
Assuntos
Biofilmes , Regulação Bacteriana da Expressão Gênica , Loci Gênicos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/genética , Desoxirribonucleases/metabolismo , Espaço Extracelular/metabolismo , Mutação , Peptídeo Hidrolases/biossíntese , Fenótipo , Sequências Reguladoras de Ácido Nucleico , Staphylococcus aureus/metabolismoRESUMO
Large-scale recombination events have led to the emergence of epidemic clones of several major bacterial pathogens. However, the functional impact of the recombination on clonal success is not understood. Here, we identified a novel widespread hybrid clone (ST71) of livestock-associated Staphylococcus aureus that evolved from an ancestor belonging to the major bovine lineage CC97, through multiple large-scale recombination events with other S. aureus lineages occupying the same ruminant niche. The recombination events, affecting a 329âkb region of the chromosome spanning the origin of replication, resulted in allele replacement and loss or gain of an array of genes influencing host-pathogen interactions. Of note, molecular functional analyses revealed that the ST71 hybrid clone has acquired multiple novel pathogenic traits associated with acquired and innate immune evasion and bovine extracellular matrix adherence. These findings provide a paradigm for the impact of large-scale recombination events on the rapid evolution of bacterial pathogens within defined ecological niches.
RESUMO
We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS(C)) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.
Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Peptídeo Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Animais , Bacteriemia/microbiologia , Bacteriemia/patologia , Proteínas de Bactérias/genética , Infecções Relacionadas a Cateter/microbiologia , Infecções Relacionadas a Cateter/patologia , Modelos Animais de Doenças , Camundongos , Fatores de Transcrição , VirulênciaRESUMO
Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections are often characterized by recalcitrance to antimicrobial therapy, which is a function not only of widespread antimicrobial resistance among clinical isolates, but also the capacity to form biofilms. Biofilms consist of ordered populations of bacterial colonies encased in a polysaccharide and/or proteinaceous matrix. This unique physiologic adaptation limits penetration of antimicrobial molecules and innate immune effectors to the infectious focus, increasing the likelihood of treatment failure and progression to chronic infection. Investigation of mechanisms of biofilm formation and dispersal, as well as the physiologic adaptations to the biofilm lifestyle, is therefore critical to developing new therapies to combat MRSA infections. In this chapter, we describe two in vitro methods for the investigation of staphylococcal biofilm formation, a microtiter plate-based assay of biofilm formation under static conditions and a flow cell-based assay of biofilm formation under fluid shear. We also detail an in vivo murine model of catheter-associated biofilm formation that is amenable to imaging and microbiologic analyses. Special consideration is given to the conditions necessary to support biofilm formation by clinical isolates of S. aureus.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Infecções Relacionadas a Cateter/etiologia , Infecções Relacionadas a Cateter/microbiologia , Catéteres/efeitos adversos , Técnicas de Cultura de Células/instrumentação , Modelos Animais de Doenças , Desenho de Equipamento , Humanos , Camundongos , Infecções Estafilocócicas/etiologiaRESUMO
BACKGROUND: Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection. METHODS: The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model. RESULTS: In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo. CONCLUSIONS: Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.
Assuntos
Proteínas de Bactérias/genética , Endocardite Bacteriana/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Mutação , Ratos , Ratos Sprague-Dawley , Deleção de Sequência , Staphylococcus aureus/patogenicidade , Transativadores/genética , Transativadores/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpC(trap)) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpC(trap) followed by gel electrophoresis combined with liquid chromatography-tandem mass spectrometry proteomics. We identified a total of 103 proteins that are potential substrates of ClpC in strain Newman. The direct protein-protein interaction of ClpC with a subset of the captured proteins was verified in a bacterial two-hybrid system. The captured proteins could be grouped into various functional categories, but most were related to proteins involved in the stress response. Several known ClpC substrates were captured, including ClpP, TrfA/MecA, ClpB, DnaK, DnaJ, GroL, RecA, and CodY, supporting the validity of our approach. Our results also revealed many new ClpC substrates, including AgrA, CcpA, RsbW, MurG, FtsA, SrtA, Rex, Atl, ClfA, and SbcC. Analysis of capsule production showed that three of the captured proteins, which were not previously known to be transcriptional regulators, did affect capsule production.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Alinhamento de Sequência , Staphylococcus aureus/genéticaRESUMO
Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developments of various genetic tools and molecular methods for staphylococcal research, which include reporter systems and vectors for controllable gene expression, gene inactivation, gene essentiality testing, chromosomal integration and transposon delivery. It is furthermore illustrated how mutant strain construction by homologous or site-specific recombination benefits from sophisticated counterselection methods. The underlying genetic components have been shown to operate in wild-type staphylococci or modified chassis strains. Finally, possible future developments in the field of applied Staphylococcus genetics are highlighted.
Assuntos
Genética Microbiana/métodos , Biologia Molecular/métodos , Staphylococcus/genéticaRESUMO
A major constituent of many Staphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or poly N-acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of the icaADBC operon, which is negatively regulated by the divergently transcribed icaR gene. We previously reported the identification of a gene, rbf, involved in the positive transcriptional regulation of icaADBC transcription by repressing icaR in S. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream of icaR or icaA, suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulates ica expression. Here we report that the unknown factor is SarX protein. Results from epistasis assays and genetic complementation analyses suggest that Rbf upregulates SarX, which then downregulates IcaR, thereby activating icaADBC. Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream of icaR within the icaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to the sarX promoter in S. aureus. These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation in S. aureus.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/fisiologia , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Epistasia Genética , Teste de Complementação Genética , Ligação Proteica , Staphylococcus aureus/genéticaRESUMO
The formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis is an important aspect of many staphylococcal infections, most notably endocarditis, osteomyelitis and infections associated with indwelling medical devices. The major constituents of staphylococcal biofilms are polysaccharides, such as poly N-acetyl glucosamine (PIA/PNAG), cell surface and secreted bacterial proteins, and extracellular DNA. The exact composition of biofilms often varies considerably between different strains of staphylococci and between different sites of infection by the same strain. PIA/PNAG is synthesized by the products of four genes, icaADBC, that are encoded in a single operon. A fifth gene, icaR, is a negative regulator of icaADBC. Expression of icaADBC is tightly regulated, but can often be induced in vitro by growing staphylococci in the presence of high salt, high glucose, or ethanol. Regulation of icaADBC is complex and numerous regulatory factors have been implicated in control of icaADBC. Many of these are well known global transcriptional regulatory factors like SarA and sigmaB, whereas other regulators, such as IcaR, seem to affect expression of relatively few genes. Here, we will summarize how various regulatory factors affect the production of PIA/PNAG in staphylococci.