RESUMEN
The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa/metabolismo , Sepsis/microbiología , Infecciones Estafilocócicas/microbiología , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Femenino , Histidina Quinasa/genética , Ratones , Fosforilación , Regulón , Sepsis/metabolismo , Transducción de Señal , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/patogenicidad , Virulencia , Factores de Virulencia/genéticaRESUMEN
The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus/genética , Transcripción Genética , Secuencia de Bases , Biopelículas/crecimiento & desarrollo , División Celular , Membrana Celular/metabolismo , Genes Bacterianos , Sitios Genéticos , Datos de Secuencia Molecular , Mutación/genética , Operón/genética , Filogenia , Regiones Promotoras Genéticas/genética , Unión Proteica , Transporte de Proteínas , Staphylococcus aureus/citología , Staphylococcus aureus/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
The WalKR two-component system is essential for the viability of Staphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR in S. aureus through a phosphomimetic amino acid replacement (WalR(c), D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb, emp, fnbA, and fnbB), cytolysis (hlgACB, hla, and hlb), and innate immune defense evasion (scn, chp, and sbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalR(c) are strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively active walR(c) allele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Inflamación/metabolismo , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Citocinas/metabolismo , Huella de ADN , Desoxirribonucleasa I , Escherichia coli K12/clasificación , Escherichia coli K12/metabolismo , Citometría de Flujo , Humanos , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/fisiología , Factores de Virulencia/genéticaRESUMEN
Uropathogenic Escherichia coli (UPEC) strains are a leading cause of infections in humans, but the mechanisms governing host colonization by this bacterium remain poorly understood. Previous studies have identified numerous gene clusters encoding proteins involved in sugar transport, in pathogen-specific islands. We investigated the role in fitness and virulence of the vpe operon encoding an EII complex of the phosphotransferase (PTS) system, which is found more frequently in human strains from infected urine and blood (45%) than in E. coli isolated from healthy humans (15%). We studied the role of this locus in vivo, using the UPEC E. coli strain AL511, mutants, and complemented derivatives in two experimental mouse models of infection. Mutant strains displayed attenuated virulence in a mouse model of sepsis. A role in kidney colonization was also demonstrated by coinfection experiments in a mouse model of pyelonephritis. Electron microscopy examinations showed that the vpeBC mutant produced much smaller amounts of a capsule-like surface material than the wild type, particularly when growing in human urine. Complementation of the vpeBC mutation led to an increase in the amount of exopolysaccharide, resistance to serum killing, and virulence. It was therefore clear that the loss of vpe genes was responsible for all the observed phenotypes. We also demonstrated the involvement of the vpe locus in gut colonization in the streptomycin-treated mouse model of intestinal colonization. These findings confirm that carbohydrate transport and metabolism underlie the ability of UPEC strains to colonize the host intestine and to infect various host sites.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Escherichia coli Uropatógena/enzimología , Escherichia coli Uropatógena/patogenicidad , Animales , Técnicas Bacteriológicas , Metabolismo de los Hidratos de Carbono , Proteínas de Escherichia coli/genética , Femenino , Fermentación , Eliminación de Gen , Humanos , Intestinos/microbiología , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Orina/microbiología , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismo , VirulenciaRESUMEN
The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.
Asunto(s)
Viabilidad Microbiana , Peptidoglicano/metabolismo , Multimerización de Proteína/fisiología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Metabolismo de los Hidratos de Carbono/genética , Secuencia de Carbohidratos/fisiología , Pared Celular/enzimología , Pared Celular/metabolismo , Endopeptidasas/metabolismo , Endopeptidasas/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Hidrolasas/genética , Hidrolasas/metabolismo , Hidrólisis , Viabilidad Microbiana/genética , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Organismos Modificados Genéticamente , Peptidoglicano/química , Staphylococcus aureus/genéticaRESUMEN
We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a DeltacymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the DeltacymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the DeltacymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host.
Asunto(s)
Cistina/metabolismo , Genes Bacterianos/fisiología , Macrófagos/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Animales , Línea Celular , Cobre/farmacología , Cistina/farmacología , Disulfuros/farmacología , Femenino , Eliminación de Gen , Homeostasis/fisiología , Peróxido de Hidrógeno/farmacología , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Staphylococcus aureus/patogenicidad , Telurio/farmacología , Regulación hacia Arriba/fisiología , VirulenciaRESUMEN
We have characterized the master regulator of cysteine metabolism, CymR, in Staphylococcus aureus. CymR repressed the transcription of genes involved in pathways leading to cysteine formation. Eight direct DNA targets were identified using gel-shift or footprinting experiments. Comparative transcriptome analysis and in vitro studies indicated that CysM, the OAS-thiol-lyase, was also implicated in this regulatory system. OAS, the direct precursor of cysteine, prevents CymR-dependent binding to DNA. This study has allowed us to predict sulphur metabolism functions for previously uncharacterized S. aureus genes. We show that S. aureus is able to grow on homocysteine as the sole sulphur source suggesting efficient MccA and MccB-dependent conversion of this compound into cysteine. We propose that SA1850 is a new thiosulphate transporter and that TcyP and TcyABC are l-cystine transporters. CymR directly controls the use of sulphur sources of human origin such as taurine and homocysteine. The cymR mutant also displayed a reduced capacity to form biofilms, indicating that CymR is involved in controlling this process in S. aureus via an ica-independent mechanism. These data indicate that fine-tuning of sulphur metabolism plays an important part in the physiology of this major pathogen and its adaptation to environmental conditions and survival in the host.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Proteínas Represoras/metabolismo , Staphylococcus aureus/genética , Azufre/metabolismo , Proteínas Bacterianas/genética , Cistina/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , ARN Bacteriano/genética , Proteínas Represoras/genética , Staphylococcus aureus/metabolismoRESUMEN
The food-borne pathogen Listeria monocytogenes is adapted to a diversity of environments, such as soil, food, body fluids, and the cytosol of eukaryotic cells. The transition between saprophytic and pathogenic life is mediated through complex regulatory pathways that modulate the expression of virulence factors. Here we examined the expression of inlJ, a recently identified gene encoding a protein of the LPXTG-internalin family and involved in pathogenesis. We show that inlJ expression is controlled neither by the major listerial regulator of virulence genes, PrfA, nor by AxyR, a putative AraC regulator encoded by a gene adjacent to inlJ and divergently transcribed. The InlJ protein is not produced by bacteria grown in vitro in brain heart infusion medium or replicating in the cytosol of tissue-cultured cells. In contrast, it is efficiently produced and localized at the surface of bacteria present in the liver and blood of infected animals. Strikingly, the expression of inlJ by a heterologous promoter in L. monocytogenes or L. innocua promotes bacterial adherence to human cells in vitro. Taken together, these results strongly suggest that InlJ acts as a novel L. monocytogenes sortase-anchored adhesin specifically expressed during infection in vivo.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/metabolismo , Factores de Virulencia/metabolismo , Adhesinas Bacterianas/genética , Animales , Línea Celular , Medios de Cultivo/química , Humanos , Listeria monocytogenes/patogenicidad , Listeriosis/sangre , Listeriosis/microbiología , Macrófagos/microbiología , Ratones , Transcripción Genética , Factores de Virulencia/genéticaRESUMEN
The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly, Staphylococcus aureus cell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two major S. aureus autolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved in S. aureus cell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example in S. aureus of a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.