RESUMO
Staphylococcus aureus is the most pathogenic member of the Staphylococcaceae. While it acquired an arsenal of canonical virulence determinants that mediate pathogenicity, it has also metabolically adapted to thrive at sites of inflammation. Notably, it has evolved to grow in the presence of nitric oxide (NO·). To this end, we note that the Rex regulon, composed of genes encoding dehydrogenases, metabolite transporters, and regulators, is much larger in S. aureus than other Staphylococcus species. Here, we demonstrate that this expanded Rex regulon is necessary and sufficient for NO· resistance. Preventing its expression results in NO· sensitivity, and the closely related species, Staphylococcus simiae, also possesses an expanded Rex regulon and exhibits NO· resistance. We hypothesize that the expanded Rex regulon initially evolved to provide efficient anaerobic metabolism but that S. aureus has co-opted this feature to thrive at sites of inflammation where respiration is limited. One distinguishing feature of the Rex regulon in S. aureus is that it contains the srrAB two-component system. Here, we show that Rex blocks the ability of SrrA to auto-induce the operon, thereby preventing maximal SrrAB expression. This results in NO·-responsive srrAB expression in S. aureus but not in other staphylococci. Consequently, higher expression of cytochromes and NO· detoxification are also observed in S. aureus alone, allowing for continued respiration at NO· concentrations beyond that of S. simiae. We therefore contend that the intersection of the Rex and SrrAB regulons represents an evolutionary event that allowed S. aureus to metabolically adapt to host inflammatory radicals during infection. IMPORTANCE Pathogens must evolve virulence potential to improve transmission to new hosts as well as evolve metabolically to thrive within their current host. Staphylococcus aureus has achieved both of these, and here, we show that one such metabolic adaptation was the expansion of the Rex regulon. First, it affords S. aureus with efficient respiration-independent growth critical to surviving the inflammatory environment replete with respiration-inhibiting immune radicals. Second, it includes the srrAB operon encoding a two-component system critical to maximizing respiratory capacity in the face of host nitric oxide (NO·), a potent respiratory inhibitor. This second facet is only apparent in S. aureus and not in other closely related species. Thus, evolutionarily, it must have occurred relatively recently. The intertwining of the Rex and SrrAB regulons represents an important evolutionary event that affords S. aureus the metabolic flexibility required to thrive within inflamed tissue and cause disease.
Assuntos
Evolução Molecular , Óxido Nítrico/imunologia , Regulon , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/genética , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismoRESUMO
To achieve maximum fitness, pathogens must balance growth with tissue damage, coordinating metabolism and virulence factor expression. In the gram-positive bacterium Streptococcus pyogenes, the DNA-binding transcriptional regulator Carbon Catabolite Protein A (CcpA) is a master regulator of both carbon catabolite repression and virulence, suggesting it coordinates growth/damage balance. To examine this, two murine models were used to compare the virulence of a mutant lacking CcpA with a mutant expressing CcpA locked into its high-affinity DNA-binding conformation (CcpAT307Y). In models of acute soft tissue infection and of long-term asymptomatic mucosal colonization, both CcpA mutants displayed altered virulence, albeit with distinct growth/damage profiles. Loss of CcpA resulted in a diminished ability to grow in tissue, leading to less damage and early clearance. In contrast, constitutive DNA-binding activity uncoupled the growth/damage relationship, such that high tissue burdens and extended time of carriage were achieved, despite reduced tissue damage. These data demonstrate that growth/damage balance can be actively controlled by the pathogen and implicate CcpA as a master regulator of this relationship. This suggests a model where the topology of the S. pyogenes virulence network has evolved to couple carbon source selection with growth/damage balance, which may differentially influence pathogenesis at distinct tissues.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Infecções Estreptocócicas/genética , Streptococcus pyogenes/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Virulência/genéticaRESUMO
Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.
Assuntos
Proteínas de Bactérias/genética , Imunidade Inata/imunologia , Óxido Nítrico/farmacologia , Infecções Cutâneas Estafilocócicas/imunologia , Staphylococcus aureus/efeitos dos fármacos , Virulência/imunologia , Animais , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
We report the complete genome assemblies of the group A Streptococcus pyogenes serotype emm6 strain D471 and its streptomycin-resistant derivative JRS4. Both of these well-studied laboratory strains have been extensively characterized over the past three decades and have been instrumental in the discovery of multiple aspects of streptococcal pathogenesis.
RESUMO
The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI(-) mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP(-), MaeK(-), and MaeR(-) mutants were attenuated for virulence, whereas a MaeE(-) mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Malatos/metabolismo , Infecções dos Tecidos Moles/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/genética , Transporte Biológico , Feminino , Deleção de Genes , Concentração de Íons de Hidrogênio , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Camundongos , Camundongos Pelados , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Óperon , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Infecções dos Tecidos Moles/patologia , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , VirulênciaRESUMO
Streptococcus pyogenes causes a greater diversity of human disease than any other bacterial pathogen. Here, we present the complete genome sequence of the emm type 14 S. pyogenes strain HSC5. This strain is a robust producer of the cysteine protease SpeB and is capable of producing infection in several different animal models.
RESUMO
The plasmidic chromate resistance genes chrBAC from Shewanella sp. strain ANA-3 were transferred to Escherichia coli. Expression of chrA alone, on a high- or low-copy number plasmid, conferred increased chromate resistance. In contrast, expression of the complete operon chrBAC on a high-copy number plasmid did not result in a significant increase in resistance, although expression on a low-copy number plasmid made the cells up to 10-fold more resistant to chromate. The chrA gene also conferred increased chromate resistance when expressed in Pseudomonas aeruginosa. The chrR gene from the P. aeruginosa chromosome was necessary for full chromate resistance conferred by chrA. A diminished chromate uptake in cells expressing the chrA gene suggests that chromate resistance is due to chromate efflux.