RESUMEN
In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by ß-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the ß-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Animales , Ratones , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Ácido Palmítico/metabolismo , Ácidos Grasos/metabolismo , Fosfolípidos/metabolismoRESUMEN
Resistance-nodulation-division (RND) superfamily efflux pumps promote antibiotic resistance in Gram-negative pathogens, but their role in Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) is undocumented. However, recent in vitro selections for resistance of S. aureus to an antimicrobial fatty acid, linoleic acid, and an antibiotic, rhodomyrtone, identified H121Y and C116R substitution variants, respectively, in a TetR family regulator, FarR, promoting increased expression of the RND pump FarE. Hypothesizing that in vivo selection pressures have also promoted the emergence of FarR variants, we searched available genome data and found that strains with FarRH121Y from human and bovine hosts have emerged sporadically in clonal complexes (CCs) CC1, CC30, CC8, CC22, and CC97, whereas multiple FarR variants have occurred within CC5 hospital-associated (HA)-MRSA. Of these, FarRE160G and FarRE93EE were exclusive to CC5, while FarRC116Y, FarRP165L, and FarRG166D also occurred in nonrelated CCs, primarily from bovine hosts. Within CC5, FarRC116Y and FarRG166D strains were polyphyletic, each exhibiting two emergence events. FarRC116Y and FarRE160G were individually sufficient to confer increased expression of FarE and enhanced resistance to linoleic acid (LA). Isolates with FarRE93EE were most closely related to S. aureus N315 MRSA and exhibited increased resistance independently of FarRE93EE. Accumulation of pseudogenes and additional polymorphisms in FarRE93EE strains contributed to a multiresistance phenotype which included fosfomycin and fusidic acid resistance in addition to increased linoleic acid resistance. These findings underscore the remarkable adaptive capacity of CC5 MRSA, which includes the polyphyletic USA100 lineage of HA-MRSA that is endemic in the Western hemisphere and known for the acquisition of multiple resistance phenotypes.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Animales , Bovinos , Humanos , Staphylococcus aureus/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Ácido Linoleico/farmacología , Ácido Linoleico/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Staphylococcus aureus is the leading cause of infections worldwide, and methicillin-resistant strains (MRSA) are emerging. New strategies are urgently needed to overcome this threat. Using a cell-based screen of ~45,000 diverse synthetic compounds, we discovered a potent bioactive, MAC-545496, that reverses ß-lactam resistance in the community-acquired MRSA USA300 strain. MAC-545496 could also serve as an antivirulence agent alone; it attenuates MRSA virulence in Galleria mellonella larvae. MAC-545496 inhibits biofilm formation and abrogates intracellular survival in macrophages. Mechanistic characterization revealed MAC-545496 to be a nanomolar inhibitor of GraR, a regulator that responds to cell-envelope stress and is an important virulence factor and determinant of antibiotic resistance. The small molecule discovered herein is an inhibitor of GraR function. MAC-545496 has value as a research tool to probe the GraXRS regulatory system and as an antibacterial lead series of a mechanism to combat drug-resistant Staphylococcal infections.
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Antibacterianos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Piperidinas/farmacología , Piridinas/farmacología , Resistencia betalactámica/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Larva/microbiología , Lepidópteros/microbiología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Células RAW 264.7 , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Factores de Virulencia/antagonistas & inhibidoresRESUMEN
Although the GraS sensor kinase of Staphylococcus aureus is known for the sensing of and resistance to cationic antimicrobial peptides (CAMPs), we recently established that it also signals in response to acidic pH, which is encountered on human skin concurrently with CAMPs, antimicrobial unsaturated free fatty acids (uFFA), and calcium. We therefore evaluated how these environmental signals would affect GraS function and resistance to antimicrobial uFFA. Growth at pH 5.5 promoted increased resistance of S. aureus USA300 to linoleic and arachidonic acids but not palmitoleic or sapienic acid. However, enhanced resistance to these C16:1 uFFA was achieved by supplementing acidic medium with 0.5 mM calcium or subinhibitory CAMPs. Enhanced resistance to uFFA at acidic pH was dependent on GraS and GraS-dependent expression of the lysyl-phosphatidylglycerol synthase enzyme MprF, through a mechanism that did not require the lysyl-transferase function of MprF. In addition to enhanced resistance to antimicrobial uFFA, acidic pH also promoted increased production of secreted proteases in a GraS-dependent manner. During growth at pH 5.5, downstream phenotypes of signaling through GraS, including resistance to uFFA, MprF-dependent addition of positive charge to the cell surface, and increased production of secreted proteases, all occurred independently of acidic amino acids in the extracytoplasmic sensor loop of GraS that were previously found to be required for sensing of CAMPs. Cumulatively, our data indicate that signaling through GraS at acidic pH occurs through a mechanism that is distinct from that described for CAMPs, leading to increased resistance to antimicrobial uFFA and production of secreted proteases.IMPORTANCEStaphylococcus aureus asymptomatically colonizes 30% of humans but is also a leading cause of infectious morbidity and mortality. Since infections are typically initiated by the same strain associated with asymptomatic colonization of the nose or skin, it is important to understand how the microbe can endure exposure to harsh conditions that successfully restrict the growth of other bacteria, including a combination of acidic pH, antimicrobial peptides, and antimicrobial fatty acids. The significance of our research is in showing that acidic pH combined with antimicrobial peptide or environmental calcium can signal through a single membrane sensor protein to promote traits that may aid in survival, including modification of cell surface properties, increased resistance to antimicrobial fatty acids, and enhanced production of secreted proteases.
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Ácidos Grasos Insaturados/química , Proteínas Quinasas/genética , Transducción de Señal , Staphylococcus aureus/enzimología , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Farmacorresistencia Bacteriana , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lisina/genética , Pruebas de Sensibilidad Microbiana , Fosfatidilgliceroles/genética , Staphylococcus aureus/genéticaRESUMEN
Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the -10 element of the P farR promoter, (ii) in palindrome PAL1 immediately in the 3' direction of P farR , or (iii) within PAL2 upstream of the predicted P farE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in P farR Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded.IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.
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Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Ácido Linoleico/metabolismo , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Regiones Promotoras Genéticas , Unión Proteica , Staphylococcus aureus/genéticaRESUMEN
Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.
Asunto(s)
Toxinas Bacterianas/metabolismo , Degranulación de la Célula , Dermatitis Atópica/microbiología , Mastocitos/citología , Staphylococcus aureus/patogenicidad , Animales , Toxinas Bacterianas/farmacología , Señalización del Calcio/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Dermatitis Atópica/inmunología , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Femenino , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Interleucina-4/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Staphylococcus aureus/metabolismo , Quinasa SykRESUMEN
Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection.
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Queratinocitos/microbiología , Neuropéptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Staphylococcus aureus/patogenicidad , Proteína de Unión al GTP rac1/metabolismo , Animales , Separación Celular , Epidermis/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Citometría de Flujo , Gentamicinas/química , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Microbiota , Microscopía Fluorescente , Fagocitosis , Proteínas Recombinantes/metabolismo , Piel/microbiología , Infecciones Estafilocócicas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
UNLABELLED: Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H121Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farR-dependent manner. Second, an H121Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 µM linoleic acid, increased accumulation of [(14)C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. IMPORTANCE: Staphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes, farR and farE, that constitute a dedicated regulator and effector of S. aureus resistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression of farE, which encodes an efflux pump, is induced in an farR-dependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Proteínas Bacterianas/metabolismo , Ácido Linoleico/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Humanos , Polimorfismo de Nucleótido Simple , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid.
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Antibacterianos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Lipasa/metabolismo , Staphylococcus aureus Resistente a Meticilina/enzimología , Triglicéridos/metabolismo , Infecciones Comunitarias Adquiridas/microbiología , Humanos , Hidrólisis , Lipasa/química , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Peso Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
Staphylococcus aureus is a bacterial pathogen known to cause infections in epidemic waves. One such epidemic was caused by a clone known as phage-type 80/81, a penicillin-resistant strain that rose to world prominence in the late 1950s. The molecular underpinnings of the phage-type 80/81 outbreak have remained unknown for decades, nor is it understood why related S. aureus clones became epidemic in hospitals in the early 1990s. To better understand the molecular basis of these epidemics, we sequenced the genomes of eight S. aureus clinical isolates representative of the phage-type 80/81 clone, the Southwest Pacific clone [a community-associated methicillin-resistant S. aureus (MRSA) clone], and contemporary S. aureus clones, all of which are genetically related and belong to the same clonal complex (CC30). Genome sequence analysis revealed that there was coincident divergence of these clones from a recent common ancestor, a finding that resolves controversy about the evolutionary history of the lineage. Notably, we identified nonsynonymous SNPs in genes encoding accessory gene regulator C (agrC) and α-hemolysin (hla)--molecules important for S. aureus virulence--that were present in virtually all contemporary CC30 hospital isolates tested. Compared with the phage-type 80/81 and Southwest Pacific clones, contemporary CC30 hospital isolates had reduced virulence in mouse infection models, the result of SNPs in agrC and hla. We conclude that agr and hla (along with penicillin resistance) were essential for world dominance of phage-type 80/81 S. aureus, whereas key SNPs in contemporary CC30 clones restrict these pathogens to hospital settings in which the host is typically compromised.
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Bacteriófagos/clasificación , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/virología , Bacteriófagos/genética , Brotes de Enfermedades , Genoma Bacteriano , Genoma Viral , Humanos , Mutación , Filogenia , Polimorfismo de Nucleótido Simple , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , VirulenciaRESUMEN
The scpAB and sspABC operons of Staphylococcus aureus encode Staphopain cysteine proteases ScpA and SspB, and their respective Staphostatins ScpB and SspC, which are thought to protect against premature activation of Staphopain precursors during protein export. However, we found that the proSspB precursor was secreted and activated without detriment to S. aureus in the absence of SspC function. Our data indicate that this is feasible due to a restricted substrate specificity of mature SspB, a stable precursor structure and slow secretion kinetics. In contrast, mature ScpA had a broad substrate specificity, such that it was prone to autolytic degradation, but also was uniquely able to degrade elastin fibres. Modelling of proScpA relative to the proSspB structure identified several differences, which appear to optimize proScpA for autocatalytic activation, whereas proSspB is optimized for stability, and cannot initiate autocatalytic activation. Consequently, recombinant proSspB remained stable and unprocessed when retained in the cytoplasm of Escherichia coli, whereas proScpA initiated rapid autocatalytic activation, leading to capture of an activation intermediate by ScpB. We conclude that the status of sspBC in S. aureus, as paralogues of the ancestral scpAB genes, facilitated a different activation mechanism, a stable proSspB isoform and modified Staphostatin function.
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Cisteína Endopeptidasas/metabolismo , Precursores de Proteínas/metabolismo , Staphylococcus aureus/metabolismo , Secuencia de Aminoácidos , Citoplasma/metabolismo , Elastina/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
BACKGROUND: The accessory gene regulator (agr) is a quorum sensing cluster of genes which control colonization and virulence in Staphylococcus aureus. We evaluated agr function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against agr functional and dysfunctional HA-MRSA and CA-MRSA. METHODS: 40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of agr function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10(6) and 10(8) cfu/ml of agr functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model. RESULTS: 15% isolates were agr dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10(6) cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among agr functional and dysfunctional strains. However, against a high initial inoculum of 10(8) cfu/ml, killing activity was notably attenuated against agr dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log(10) CFU/ml for agr functional vs. 2.41 log(10) CFU/ml for agr dysfunctional MRSA (p = 0.0007). CONCLUSIONS: Dysfunction in agr was less common among CA-MRSA vs. HA-MRSA. agr dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.
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Antibacterianos/farmacocinética , Proteínas Bacterianas/metabolismo , Infecciones Comunitarias Adquiridas/microbiología , Infección Hospitalaria/microbiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Infecciones Estafilocócicas/microbiología , Transactivadores/metabolismo , Vancomicina/farmacocinética , Proteínas Bacterianas/genética , Canadá , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Transactivadores/genéticaRESUMEN
SUMMARY: The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.
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Proteínas Bacterianas/metabolismo , Metaloendopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Termolisina/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Elastasa Pancreática/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimología , Alineación de SecuenciaRESUMEN
Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.
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Purinas/biosíntesis , Staphylococcus aureus/patogenicidad , Animales , Proteínas Portadoras/metabolismo , Femenino , Fibronectinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Unión Proteica , Staphylococcus aureus/genética , Virulencia/genéticaRESUMEN
Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells.IMPORTANCES. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction.
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Interacciones Huésped-Patógeno , Macrófagos/microbiología , Fagosomas/química , Fagosomas/microbiología , Proteínas Quinasas/genética , Staphylococcus aureus/crecimiento & desarrollo , Aminoaciltransferasas/genética , Animales , Péptidos Catiónicos Antimicrobianos/deficiencia , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Carga Bacteriana , Proteínas Bacterianas/genética , Células Cultivadas , Farmacorresistencia Bacteriana , Concentración de Iones de Hidrógeno , Hígado/microbiología , Macrófagos/ultraestructura , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Proteínas Quinasas/deficiencia , Especies Reactivas de Oxígeno/metabolismo , Regulón , Staphylococcus aureus/efectos de los fármacosRESUMEN
Borderline oxacillin-resistant Staphylococcus aureus (BORSA) exhibit oxacillin MIC values of 1-8 microg ml(-1), but lack mecA, which encodes the low-affinity penicillin-binding protein (PBP)2a. The relationship of the BORSA phenotype with specific genetic backgrounds was assessed, as well as amino acid sequence variation in the normal PBP2. Among 38 BORSA, 26 had a common PFGE profile of genomic DNA, and were multilocus sequence type (ST)25. The other isolates were genetically diverse. Complete pbp2 sequences were determined for three BORSA, corresponding to ST25, ST1 and ST47, which were selected on the basis of lacking blaZ-encoded beta-lactamase. The essential transpeptidase-domain-encoding segment of pbp2 was also sequenced from seven additional ST25 isolates. Amino acid substitutions occurred in the transpeptidase domain of all BORSA, irrespective of clonal type. A Gln(629)-->Pro substitution was common to all ST25 BORSA, but most could be distinguished from one another by additional unique substitutions in the transpeptidase domain. The ST1 and ST47 isolates also possessed unique substitutions in the transpeptidase domain. Plasmid-mediated expression of pbp2 from an ST25 or ST1 isolate in S. aureus RN6390 increased its oxacillin MIC from 0.25 to 4 microg ml(-1), while pbp2 from a susceptible strain, ATCC 25923, had no effect. Therefore, different amino acid substitutions in PBP2 of diverse BORSA lineages contribute to borderline resistance. The predominant ST25 lineage was not related to any of the five clonal complexes that contain meticillin-resistant S. aureus (MRSA), suggesting that ST25 cannot readily acquire mecA-mediated resistance.
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Antibacterianos/farmacología , Proteínas Bacterianas/genética , Oxacilina/farmacología , Proteínas de Unión a las Penicilinas/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Sustitución de Aminoácidos , Canadá , Farmacorresistencia Bacteriana , Datos de Secuencia Molecular , Peptidil Transferasas/genética , Polimorfismo de Nucleótido Simple , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/químicaRESUMEN
Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16â¶1) and linoleic acid (C18â¶1) were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10-12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus.
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Antibacterianos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Interacciones Huésped-Patógeno , Staphylococcus aureus Resistente a Meticilina/fisiología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Piel/microbiología , Infecciones Cutáneas Estafilocócicas/transmisión , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Proteolisis , Piel/metabolismo , Infecciones Cutáneas Estafilocócicas/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Staphylococcus aureus clonal complex CC30 has caused infectious epidemics for more than 60 years, and, therefore, provides a model system to evaluate how evolution has influenced the disease potential of closely related strains. In previous multiple genome comparisons, phylogenetic analyses established three major branches that evolved from a common ancestor. Clade 1, comprised of historic pandemic phage type 80/81 methicillin susceptible S. aureus (MSSA), and Clade 2 comprised of contemporary community acquired methicillin resistant S. aureus (CA-MRSA) were hyper-virulent in murine infection models. Conversely, Clade 3 strains comprised of contemporary hospital associated MRSA (HA-MRSA) and clinical MSSA exhibited attenuated virulence, due to common single nucleotide polymorphisms (SNP's) that abrogate production of α-hemolysin Hla, and interfere with signaling of the accessory gene regulator agr. We have now completed additional in silico genome comparisons of 15 additional CC30 genomes in the public domain, to assess the hypothesis that Clade 3 has evolved to favor niche adaptation. In addition to SNP's that influence agr and hla, other common traits of Clade 3 include tryptophan auxotrophy due to a di-nucleotide deletion within trpD, a premature stop codon within isdH encoding an immunogenic cell surface protein involved in iron acquisition, loss of a genomic toxin-antitoxin (TA) addiction module, acquisition of S. aureus pathogenicity islands SaPI4, and SaPI2 encoding toxic shock syndrome toxin tst, and increased copy number of insertion sequence ISSau2, which appears to target transcription terminators. Compared to other Clade 3 MSSA, S. aureus MN8, which is associated with Staphylococcal toxic shock syndrome, exhibited a unique ISSau2 insertion, and enhanced production of toxic shock syndrome toxin encoded by SaPI2. Cumulatively, our data support the notion that Clade 3 strains are following an evolutionary blueprint toward niche-adaptation.
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Adaptación Biológica , Evolución Molecular , Staphylococcus aureus/genética , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Biología Computacional , Genoma Bacteriano , Islas Genómicas , Genotipo , Humanos , Secuencias Repetitivas Esparcidas , Recombinación Genética , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Bacterial infection with Staphylococcus aureus is a common complication of atopic dermatitis (AD). The incidence of community-acquired methicillin-resistant S. aureus infection (MRSA) in the AD population is unknown. OBJECTIVES: This study aimed to assess the prevalence of S. aureus and MRSA in pediatric patients with AD, to compare disease severity, and to characterize the clonal diversity of the isolates. METHODS: We carried out a prospective, cross-sectional study of 200 patients with AD. The severity of AD was defined as mild, moderate, or severe depending on a composite AD severity score. A swab was taken from the nares of each patient and another from affected skin or folds. Genotyping of all S. aureus isolates was conducted by polymerase chain reaction (PCR) amplification of the S. aureus protein A (spa) gene. RESULTS: According to the severity score, 66.5% of subjects were ranked as having mild AD, 29.5% as having moderate and 4% as having severe AD. Staphylococcus aureus colonization was seen in 61.5% of all patients, represented by 43.7% of skin swabs and 48% of nares swabs. Only one of the isolations represented MRSA. Older age and higher AD severity scores were associated with S. aureus colonization (P = 0.03 and P < 0.001, respectively). No significant associations were noted for attendance at day care, family members with frequent skin infections, or family members working in healthcare. Isolates from spa CC015 were cultured in 19.2% of patient samples. The single MRSA culture showed a new spa type that belonged to CC127. CONCLUSIONS: The results of this study confirm a high rate of S. aureus colonization of pediatric patients with AD. The low rate of MRSA requires further proof from larger prospective studies.
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Dermatitis Atópica/microbiología , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Cutáneas Estafilocócicas/epidemiología , Proteínas Bacterianas/genética , Canadá/epidemiología , Niño , Preescolar , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Estudios Transversales , Femenino , Variación Genética , Humanos , Lactante , Masculino , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Prevalencia , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Infecciones Cutáneas Estafilocócicas/microbiologíaRESUMEN
Staphylococcus aureus promotes the onset and severity of atopic dermatitis (AD), which is exacerbated by superantigen toxins SEB and SEC. The genetic identity of these isolates, and their relationship to common hospital- or community-associated methicillin resistant S. aureus (HA-MRSA and CA-MRSA) has not been defined. We conducted spa typing, partial multi-locus sequence typing (MLST), and toxin profiling (seb, sec, lukS-PV) of S. aureus from 119 pediatric and 40 adult AD patients. MLST clonal complexes CC45, CC5, CC15, CC1, CC8 and CC30 accounted for 79% of isolates, representing the same major groups reported for nosocomial S. aureus in hospital intensive care units. The highest disease severity was associated with CC1, which was significantly greater relative to CC15 (p = 0.017) or CC30 (p = 0.040), but with no significant difference relative to CC45, CC5 or CC8. Although there were two few lukS-PV, seb or sec isolates to infer a role in disease severity, CC45 was identified as a source of SEC producing strains, and lukS-PVL was associated with a small number of CC5 pediatric isolates. CC1 harbored the only CA-MRSA that was identified, and was a source of isolates that expressed both seb and sec, and closely resembled the USA400 strain of CA-MRSA.