RESUMEN

Group A Streptococcus (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD+-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease. NADase can be translocated into host cells by a SLO-dependent mechanism, and cytosolic NADase has been assigned multiple properties such as protection of intracellularly located GAS bacteria and induction of host cell death through energy depletion. Here, we used a set of isogenic GAS mutants and a macrophage infection model and report that streptococcal NADase inhibits the innate immune response by decreasing inflammasome-dependent interleukin 1ß (IL-1ß) release from infected macrophages. Regulation of IL-1ß was independent of phagocytosis and ensued also under conditions not allowing SLO-dependent translocation of NADase into the host cell cytosol. Thus, our data indicate that NADase not only acts intracellularly but also has an immune regulatory function in the extracellular niche.IMPORTANCE In the mid-1980s, the incidence and severity of invasive infections caused by serotype M1 GAS suddenly increased. The results of genomic analyses suggested that this increase was due to the spread of clonal bacterial strains and identified a recombination event leading to enhanced production of the SLO and NADase toxins in these strains. However, despite its apparent importance in GAS pathogenesis, the function of NADase remains poorly understood. In this study, we demonstrate that NADase inhibits inflammasome-dependent IL-1ß release from infected macrophages. While previously described functions of NADase pertain to its role upon SLO-mediated translocation into the host cell cytosol, our data suggest that the immune regulatory function of NADase is exerted by nontranslocated enzyme, identifying a previously unrecognized extracellular niche for NADase functionality. This immune regulatory property of extracellular NADase adds another possible explanation to how increased secretion of NADase correlates with bacterial virulence.

Asunto(s)

Interacciones Huésped-Patógeno , Inflamasomas/metabolismo , Interleucina-1beta/antagonistas & inhibidores , NAD+ Nucleosidasa/metabolismo , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/inmunología , Factores de Virulencia/metabolismo , Animales , Células Cultivadas , Humanos , Evasión Inmune , Macrófagos/microbiología , Ratones Endogámicos C57BL , Streptococcus pyogenes/genética

RESUMEN

UNLABELLED: Group A Streptococcus (GAS, Streptococcus pyogenes) is an ongoing threat to human health as the agent of streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. In animal models of infection, macrophages have been shown to contribute to host defense against GAS infection. However, as GAS can resist killing by macrophages in vitro and induce macrophage cell death, it has been suggested that GAS intracellular survival in macrophages may enable persistent infection. Using isogenic mutants, we now show that the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase) mediate GAS intracellular survival and cytotoxicity for macrophages. Unexpectedly, the two toxins did not inhibit fusion of GAS-containing phagosomes with lysosomes but rather prevented phagolysosome acidification. SLO served two essential functions, poration of the phagolysosomal membrane and translocation of NADase into the macrophage cytosol, both of which were necessary for maximal GAS intracellular survival. Whereas NADase delivery to epithelial cells is mediated by SLO secreted from GAS bound to the cell surface, in macrophages, the source of SLO and NADase is GAS contained within phagolysosomes. We found that transfer of NADase from the phagolysosome to the macrophage cytosol occurs not by simple diffusion through SLO pores but rather by a specific translocation mechanism that requires the N-terminal translocation domain of NADase. These results illuminate the mechanisms through which SLO and NADase enable GAS to defeat macrophage-mediated killing and provide new insight into the virulence of a major human pathogen. IMPORTANCE: Macrophages constitute an important element of the innate immune response to mucosal pathogens. They ingest and kill microbes by phagocytosis and secrete inflammatory cytokines to recruit and activate other effector cells. Group A Streptococcus (GAS, Streptococcus pyogenes), an important cause of pharyngitis and invasive infections, has been shown to resist killing by macrophages. We find that GAS resistance to macrophage killing depends on the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase). GAS bacteria are internalized by macrophage phagocytosis but resist killing by secreting SLO, which damages the phagolysosome membrane, prevents phagolysosome acidification, and translocates NADase from the phagolysosome into the macrophage cytosol. NADase augments SLO-mediated cytotoxicity by depleting cellular energy stores. These findings may explain the nearly universal production of SLO by GAS clinical isolates and the association of NADase with the global spread of a GAS clone implicated in invasive infections.

Asunto(s)

Macrófagos/efectos de los fármacos , NAD+ Nucleosidasa/farmacología , Fagosomas/metabolismo , Streptococcus pyogenes/efectos de los fármacos , Estreptolisinas/farmacología , Proteínas Bacterianas/farmacología , Línea Celular , Clonación Molecular , Citosol/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Microscopía Confocal , Fagocitosis/efectos de los fármacos , Streptococcus pyogenes/metabolismo

RESUMEN

The CsrRS two-component regulatory system of group A Streptococcus (GAS; Streptococcus pyogenes) responds to subinhibitory concentrations of the human antimicrobial peptide LL-37. LL-37 signaling through CsrRS results in upregulation of genes that direct synthesis of virulence factors, including the hyaluronic acid capsule and streptolysin O (SLO). Here, we demonstrate that a consequence of this response is augmented GAS resistance to killing by human oropharyngeal keratinocytes, neutrophils, and macrophages. LL-37-induced upregulation of SLO and hyaluronic acid capsule significantly reduced internalization of GAS by keratinocytes and phagocytic killing by neutrophils and macrophages. Because vitamin D induces LL-37 production by macrophages, we tested its effect on macrophage killing of GAS. In contrast to the reported enhancement of macrophage function in relation to other pathogens, treatment of macrophages with 1α,25-dihydroxy-vitamin D3 paradoxically reduced the ability of macrophages to control GAS infection. These observations demonstrate that LL-37 signals through CsrRS to induce a virulence phenotype in GAS characterized by heightened resistance to ingestion and killing by both epithelial cells and phagocytes. By inducing LL-37 production in macrophages, vitamin D may contribute to this paradoxical exacerbation of GAS infection. IMPORTANCE It remains poorly understood why group A Streptococcus (GAS) causes asymptomatic colonization or localized throat inflammation in most individuals but rarely progresses to invasive infection. The human antimicrobial peptide LL-37, which is produced as part of the innate immune response to GAS infection, signals through the GAS CsrRS two-component regulatory system to upregulate expression of multiple virulence factors. This study reports that two CsrRS-regulated GAS virulence factors-streptolysin O and the hyaluronic acid capsule-are critical in LL-37-induced resistance of GAS to killing by human throat epithelial cells and by neutrophils and macrophages. Vitamin D, which increases LL-37 production in macrophages, has the paradoxical effect of increasing GAS resistance to macrophage-mediated killing. In this way, the human innate immune response may promote the transition from GAS colonization to invasive infection.

Asunto(s)

Catelicidinas/farmacología , Streptococcus pyogenes/efectos de los fármacos , Vitamina D/farmacología , Péptidos Catiónicos Antimicrobianos , Línea Celular , Humanos , Queratinocitos/microbiología , Macrófagos/microbiología , Neutrófilos/microbiología

RESUMEN

The CsrRS (or CovRS) two component system controls expression of up to 15% of the genome of group A Streptococcus (GAS). While some studies have suggested that the sensor histidine kinase CsrS responds to membrane perturbations as a result of various environmental stresses, other data have implicated the human antimicrobial peptide LL-37 and extracellular Mg(2+) as specific signals. We now report that Mg(2+) and LL-37 have opposite effects on expression of multiple genes that are activated or repressed by the transcriptional regulator CsrR. Using a GAS isolate representative of the recently emerged and widely disseminated M1T1 clone implicated in severe invasive disease, we found marked up-regulation by CsrRS of multiple virulence factors including pyrogenic exotoxin A, DNase Sda1, streptolysin O, and the hyaluronic acid capsular polysaccharide, among others. Topology and surface protein labeling studies indicated that CsrS is associated with the bacterial cell membrane and has a surface-exposed extracellular domain accessible to environmental ligands. Replacement of a cluster of three acidic amino acids with uncharged residues in the extracellular domain of CsrS abrogated LL-37 signaling and conferred a hyporesponsive phenotype consistent with tonic activation of CsrS autokinase activity, an effect that could be overridden by mutation of the CsrS active site histidine. Both loss- and gain-of-function mutations of a conserved site in the receiver domain of CsrR established an essential role for lysine 102 in CsrS-to-CsrR signal transduction. These results provide strong evidence that Mg(2+) and LL-37 are specific signals that function by altering CsrS autokinase activity and downstream phosphotransfer to CsrR to modulate its activity as a transcriptional regulator. The representation of multiple antiphagocytic and cytotoxic factors in the CsrRS regulon together with results of in vitro phagocytic killing assays support the hypothesis that CsrRS mediates conversion of GAS from a colonizing to an invasive phenotype in response to signaling by host LL-37.

Asunto(s)

Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Proteínas Represoras/genética , Transducción de Señal/fisiología , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Fenotipo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factores de Virulencia , Catelicidinas

RESUMEN

The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 microM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s(-1). Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.

Asunto(s)

Bacillus anthracis/química , Manganeso/metabolismo , Proteínas Represoras/metabolismo , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Represoras/química

RESUMEN

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cadmio/metabolismo , Cationes Bivalentes/farmacología , Dicroismo Circular , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Cinética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Níquel/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica

RESUMEN

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.

Asunto(s)

Receptores de Superficie Celular/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Calorimetría , Corynebacterium diphtheriae/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Prolina/química , Conformación Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding. We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites. We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N- and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor. Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process. Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions.

Asunto(s)

Proteínas Bacterianas/genética , Corynebacterium diphtheriae/genética , Proteínas de Unión al ADN/genética , Alanina , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Proteínas de Unión al ADN/química , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transcripción Genética , beta-Galactosidasa/metabolismo

RESUMEN

The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae. We recently described (L. Sun, J. C. vanderSpek, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active. We demonstrate here that while DtxR(E175K) remains active in vivo in the presence of 300 micro M 2,2'dipyridyl, the purified repressor is, in fact, dependent upon low levels of transition metal ion to transit from the inactive apo form to the active metal ion-bound form of the repressor. Binding studies using 8-anilino-1-naphthalenesulfonic acid suggest that the E175K mutation stabilizes an intermediate of the molten-globule form of the repressor, increasing exposure of hydrophobic residues to solvent. We demonstrate that the hyperactive DtxR(E175K) phenotype is dependent upon an intact ancillary metal ion-binding site (site 1) of the repressor. These observations support the hypothesis that metal ion binding in the ancillary site facilitates the conversion of the inactive apo-repressor to its active, operator-binding conformation. Furthermore, these results support the hypothesis that the C-terminal src homology 3-like domain of DtxR plays an active role in the modulation of repressor activity.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Metales/metabolismo , Proteínas Represoras/metabolismo , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cationes/metabolismo , Corynebacterium diphtheriae/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Colorantes Fluorescentes/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Dominios Homologos src

RESUMEN

While the native diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion has allowed initial isolation and characterization of the diphtheria toxin repressor (DtxR), the low level of reporter gene expression has limited the detection and analysis of mutations affecting subtle changes in repressor-operator binding. In order to overcome this difficulty, we have constructed a novel hybrid promoter/operator-lacZ transcriptional fusion in which the "-35" and spacing of the tac promoter was fused to the "-10" and interrupted palindromic sequence of toxO. We show that the hybrid tacPtoxO is regulated by the transition metal ion-dependent DtxR and that lacZ expression is increased approximately 70-fold in the reporter strain Escherichia coli DH5alpha/lambdaRS45-tacPtoxO-lacZ relative to DH5alpha/lambdaRS45-toxPO-lacZ. In addition, we have constructed a transcriptional fusion between tacPtoxO and luc, pJL1. We have used pJL1 to program S30 extracts of E. coli in order to direct in vitro the coupled transcription and translation of luciferase. We demonstrate the utility of this in vitro system in providing a direct functional link between in vivo and in vitro observations with DtxR and mutants of DtxR, which display subtle changes in activity in a manner not previously possible.

Asunto(s)

Proteínas Bacterianas/genética , Sondas de ADN/genética , Proteínas de Unión al ADN/genética , Toxina Diftérica/genética , Regiones Operadoras Genéticas/genética , Regiones Promotoras Genéticas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sondas de ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Toxina Diftérica/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Transcripción Genética , beta-Galactosidasa/análisis