RESUMEN
Staphylococcus aureus produces numerous factors that facilitate survival in the human host. S. aureus coagulase (Coa) and von Willebrand factor-binding protein (vWbp) are known to clot plasma through activation of prothrombin and conversion of fibrinogen to fibrin. In addition, S. aureus clumping factor A (ClfA) binds fibrinogen and contributes to platelet aggregation via a fibrinogen- or complement-dependent mechanism. Here, we evaluated the contribution of Coa, vWbp and ClfA to S. aureus pathogenesis in a rabbit model of skin and soft tissue infection. Compared to skin abscesses caused by the Newman wild-type strain, those caused by isogenic coa, vwb, or clfA deletion strains, or a strain deficient in coa and vwb, were significantly smaller following subcutaneous inoculation in rabbits. Unexpectedly, we found that fibrin deposition and abscess capsule formation appear to be independent of S. aureus coagulase activity in the rabbit infection model. Similarities notwithstanding, S. aureus strains deficient in coa and vwb elicited reduced levels of several proinflammatory molecules in human blood in vitro. Although a specific mechanism remains to be determined, we conclude that S. aureus Coa, vWbp and ClfA contribute to abscess formation in rabbits.
Asunto(s)
Absceso/microbiología , Coagulasa/metabolismo , Infecciones de los Tejidos Blandos/microbiología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Absceso/patología , Animales , Proteínas Bacterianas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Conejos , Infecciones de los Tejidos Blandos/patología , Infecciones Cutáneas Estafilocócicas/patologíaRESUMEN
Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.
Asunto(s)
Coinfección/microbiología , Coinfección/virología , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Interacciones Microbianas , Infecciones por Orthomyxoviridae/complicaciones , Neumonía Estafilocócica/complicaciones , Staphylococcus aureus/crecimiento & desarrollo , Animales , Coinfección/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/patología , Macaca fascicularis , Masculino , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Neumonía Estafilocócica/microbiología , Neumonía Estafilocócica/patologíaRESUMEN
Bacterial skin and soft tissue infections are abundant worldwide and many are caused by Staphylococcus aureus. Indeed, S. aureus is the leading cause of skin and soft tissue infections in the USA. Here, we describe a mouse model of skin and soft tissue infection induced by subcutaneous inoculation of S. aureus. This animal model can be used to investigate a number of factors related to the pathogenesis of skin and soft tissue infections, including strain virulence and the contribution of specific bacterial molecules to disease, and it can be employed to test the potential effectiveness of antibiotic therapies or vaccine candidates.
Asunto(s)
Piel/patología , Infecciones Cutáneas Estafilocócicas/patología , Staphylococcus aureus/patogenicidad , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Piel/microbiología , Infecciones Cutáneas Estafilocócicas/microbiologíaRESUMEN
BACKGROUND: Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo. METHODS: We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models. RESULTS: Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain. CONCLUSIONS: Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.
Asunto(s)
Exotoxinas/toxicidad , Inflamación/inducido químicamente , Staphylococcus aureus/patogenicidad , Factores de Virulencia/toxicidad , Animales , Modelos Animales de Enfermedad , Exotoxinas/administración & dosificación , Humanos , Inflamación/inmunología , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Ratones , Neutrófilos/inmunología , Neutrófilos/microbiología , Conejos , Infecciones Estafilocócicas/patología , Factores de Virulencia/administración & dosificaciónRESUMEN
CA-MRSA infections are often caused by strains encoding PVL, which can cause lysis of PMNs and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations, PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce O(2)(-) in response to fMLF, but unlike priming by LPS, this response did not require TLR signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL--a finding consistent with priming. Importantly, PMNs primed with PVL had an enhanced ability to bind/ingest and kill Staphylococcus aureus. Priming of PMNs with other agonists, such as IL-8 or GM-CSF, altered the ability of PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up-regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL-mediated priming of PMNs enhances the host innate immune response.
Asunto(s)
Exotoxinas/fisiología , Leucocidinas/fisiología , Staphylococcus aureus Resistente a Meticilina/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Infecciones Estafilocócicas/inmunología , Toxinas Bacterianas/metabolismo , Células Cultivadas , Exotoxinas/metabolismo , Humanos , Leucocidinas/metabolismo , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Neutrófilos/efectos de los fármacos , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiologíaRESUMEN
The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus.
Asunto(s)
Toxinas Bacterianas/genética , Infección Hospitalaria/microbiología , Infección Hospitalaria/patología , Exotoxinas/genética , Leucocidinas/genética , Neumonía Estafilocócica/microbiología , Neumonía Estafilocócica/patología , Staphylococcus aureus/patogenicidad , Factores de Virulencia/genética , Adulto , Anciano , Anciano de 80 o más Años , Infección Hospitalaria/mortalidad , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Tipificación Molecular , Neumonía Estafilocócica/mortalidad , Medición de Riesgo , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
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.
Asunto(s)
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
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are frequently associated with strains harboring genes encoding Panton-Valentine leukocidin (PVL). The role of PVL in the success of the epidemic CA-MRSA strain USA300 remains unknown. Here we developed a skin and soft tissue infection model in rabbits to test the hypothesis that PVL contributes to USA300 pathogenesis and compare it with well-established virulence determinants: alpha-hemolysin (Hla), phenol-soluble modulin-alpha peptides (PSMα), and accessory gene regulator (Agr). The data indicate that Hla, PSMα, and Agr contribute to the pathogenesis of USA300 skin infections in rabbits, whereas a role for PVL could not be detected.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/patogenicidad , Infecciones de los Tejidos Blandos/microbiología , Infecciones de los Tejidos Blandos/patología , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Factores de Virulencia/metabolismo , Absceso/microbiología , Absceso/patología , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Exotoxinas/genética , Exotoxinas/metabolismo , Femenino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Histocitoquímica , Leucocidinas/genética , Leucocidinas/metabolismo , Microscopía , Conejos , Piel/microbiología , Piel/patología , Transactivadores/genética , Transactivadores/metabolismo , Virulencia , Factores de Virulencia/genéticaRESUMEN
Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Suero/microbiología , Staphylococcus aureus/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Exotoxinas/genética , Exotoxinas/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Ratones , Viabilidad Microbiana , Neutrófilos/citología , Neutrófilos/microbiología , Porosidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Virulencia/genéticaRESUMEN
A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates--those that comprise the epidemic USA300 clone--were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.
Asunto(s)
ADN Bacteriano/genética , Variación Genética , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Plásmidos , Infecciones Estafilocócicas/microbiología , Antibacterianos/farmacología , Análisis por Conglomerados , ADN Bacteriano/química , Farmacorresistencia Bacteriana Múltiple , Humanos , Metales Pesados/toxicidad , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.
Asunto(s)
Evasión Inmune/fisiología , Neutrófilos/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/inmunología , Staphylococcus epidermidis/patogenicidad , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Hemólisis/efectos de los fármacos , Hemólisis/genética , Humanos , Evasión Inmune/genética , Inmunidad Celular/fisiología , Datos de Secuencia Molecular , Neutrófilos/fisiología , Filogenia , Homología de Secuencia de Aminoácido , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismoRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alpha-hemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Deltahla) strains caused skin lesions in a mouse infection model that were significantly smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Deltahla strains. Passive immunization with Hla-specific antisera or active immunization with a nontoxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.
Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Proteínas Hemolisinas/antagonistas & inhibidores , Inmunización Pasiva/métodos , Inmunización/métodos , Infecciones Cutáneas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Proteínas Hemolisinas/deficiencia , Humanos , Ratones , Ratones Endogámicos BALB C , Piel/patología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidadRESUMEN
Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence.
Asunto(s)
Infecciones Comunitarias Adquiridas/inmunología , Staphylococcus aureus Resistente a Meticilina/inmunología , Neutrófilos/metabolismo , Fagosomas/ultraestructura , Infecciones Estafilocócicas/inmunología , Apoptosis/genética , Regulación de la Expresión Génica/inmunología , Humanos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Análisis por Micromatrices , Microscopía Electrónica de Transmisión , Necrosis/genética , Neutrófilos/inmunología , Neutrófilos/microbiología , Neutrófilos/patología , Fagocitosis , Especificidad de la Especie , Infecciones Estafilocócicas/microbiología , Factores de VirulenciaRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Toxinas Bacterianas/toxicidad , Exotoxinas/toxicidad , Leucocidinas/toxicidad , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Neumonía Estafilocócica/etiología , Lesión Pulmonar Aguda/microbiología , Lesión Pulmonar Aguda/patología , Animales , Toxinas Bacterianas/genética , Modelos Animales de Enfermedad , Exotoxinas/genética , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Humanos , Técnicas In Vitro , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/genética , Neutrófilos/patología , Neumonía Estafilocócica/microbiología , Neumonía Estafilocócica/patología , Conejos , Virulencia/genéticaRESUMEN
Panton-Valentine leukocidin (PVL) is a cytolytic toxin associated with severe community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. However, the relative contribution of PVL to host cell lysis during CA-MRSA infection remains unknown. Here we investigated the relative contribution of PVL to human polymorphonuclear leukocyte (PMN) plasma membrane permeability and lysis in vitro by using culture supernatants from wild-type and isogenic lukS/F-PV negative (Deltapvl) USA300 and USA400 strains. Using S. aureus culture conditions that favor selective high production of PVL (CCY medium), there was on average more PMN plasma membrane permeability and cell lysis caused by supernatants derived from wild-type strains compared with those from Deltapvl strains. Unexpectedly, plasma membrane permeability did not necessarily correlate with ultimate cell lysis. Moreover, the level of pore formation caused by culture supernatants varied dramatically (e.g., range was 0.32-99.09% for wild-type USA300 supernatants at 30 min) and was not attributable to differences in PMN susceptibility to PVL among human blood donors. We conclude that PMN pore formation assays utilizing S. aureus culture supernatants have limited ability to estimate the relative contribution of PVL to pathogenesis (or cytolysis in vitro or in vivo), especially when assayed using culture media that promote selective high production of PVL.
Asunto(s)
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Leucocidinas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Neutrófilos/metabolismo , Análisis de Varianza , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Medios de Cultivo , Interacciones Huésped-Patógeno , Humanos , Leucocidinas/genética , Neutrófilos/microbiología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Methicillin-resistant Staphylococcus aureus is problematic both in hospitals and in the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S 2-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate that saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis revealed that SaeR/S of MW2 influences expression of a wide variety of genes with diverse biological functions. These data provide new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease.
Asunto(s)
Proteínas Bacterianas/genética , Inmunidad Innata/genética , Proteínas Quinasas/genética , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Animales , Modelos Animales de Enfermedad , Ratones , Mutagénesis , Neutrófilos/microbiología , Neutrófilos/fisiología , Fagocitosis , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis/microbiología , Eliminación de Secuencia , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/patogenicidad , Factores de Transcripción , VirulenciaRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged worldwide. The United States, in particular, is experiencing a serious epidemic of CA-MRSA that is almost entirely caused by an extraordinarily infectious strain named USA300. However, the molecular determinants underlying the pathogenic success of CA-MRSA are mostly unknown. To gain insight into the evolution of the exceptional potential of USA300 to cause disease, we compared the phylogeny and virulence of USA300 with that of closely related MRSA clones. We discovered that the sublineage from which USA300 evolved is characterized by a phenotype of high virulence that is clearly distinct from other MRSA strains. Namely, USA300 and its progenitor, USA500, had high virulence in animal infection models and the capacity to evade innate host defense mechanisms. Furthermore, our results indicate that increased virulence in the USA300/USA500 sublineage is attributable to differential expression of core genome-encoded virulence determinants, such as phenol-soluble modulins and alpha-toxin. Notably, the fact that the virulence phenotype of USA300 was already established in its progenitor indicates that acquisition of mobile genetic elements has played a limited role in the evolution of USA300 virulence and points to a possibly different role of those elements. Thus, our results highlight the importance of differential gene expression in the evolution of USA300 virulence. This finding calls for a profound revision of our notion about CA-MRSA pathogenesis at the molecular level and has important implications for design of therapeutics directed against CA-MRSA.
Asunto(s)
Brotes de Enfermedades , Evolución Molecular , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Filogenia , Virulencia/genética , Animales , Regulación Bacteriana de la Expresión Génica , Humanos , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Estados UnidosRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Delta pvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis.
Asunto(s)
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Leucocidinas/metabolismo , Resistencia a la Meticilina/genética , Staphylococcus aureus/metabolismo , Animales , Bacteriemia/diagnóstico , Bacteriemia/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Granulocitos/citología , Resistencia a la Meticilina/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica/métodos , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/patogenicidad , Factores de VirulenciaRESUMEN
Emerging and re-emerging infectious diseases, especially those caused by drug-resistant bacteria, are a major problem worldwide. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) appeared rapidly and unexpectedly in the United States, resulting in an epidemic caused primarily by isolates classified as USA300. The evolutionary and molecular underpinnings of this epidemic are poorly understood. Specifically, it is unclear whether there has been clonal emergence of USA300 isolates or evolutionary convergence toward a hypervirulent phenotype resulting in the independent appearance of similar organisms. To definitively resolve this issue and understand the phylogeny of USA300 isolates, we used comparative whole-genome sequencing to analyze 10 USA300 patient isolates from eight states in diverse geographic regions of the United States and multiple types of human infection. Eight of 10 isolates analyzed had very few single nucleotide polymorphisms (SNPs) and thus were closely related, indicating recent diversification rather than convergence. Unexpectedly, 2 of the clonal isolates had significantly reduced mortality in a mouse sepsis model compared with the reference isolate (P = 0.0002), providing strong support to the idea that minimal genetic change in the bacterial genome can have profound effects on virulence. Taken together, our results demonstrate that there has been recent clonal expansion and diversification of a subset of isolates classified as USA300. The findings add an evolutionary dimension to the epidemiology and emergence of USA300 and suggest a similar mechanism for the pandemic occurrence and spread of penicillin-resistant S. aureus (known as phage-type 80/81 S. aureus) in the 1950s.
Asunto(s)
Infecciones Comunitarias Adquiridas/microbiología , Brotes de Enfermedades , Farmacorresistencia Bacteriana Múltiple , Variación Genética , Resistencia a la Meticilina , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Animales , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Infecciones Comunitarias Adquiridas/epidemiología , Modelos Animales de Enfermedad , Genoma Bacteriano , Humanos , Resistencia a la Meticilina/genética , Ratones , Polimorfismo de Nucleótido Simple/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificación , Estados Unidos , VirulenciaRESUMEN
In recent years, there has been a dramatic increase in the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. MW2 (pulsed-field type USA400), the prototype CA-MRSA strain, is highly virulent and has enhanced ability to evade killing by neutrophils. Although progress has been made, the molecular basis for enhanced virulence of CA-MRSA remains incompletely defined. To that end, we studied resistance of MW2 to key microbicides of human neutrophils. Hydrogen peroxide (H2O2), hypochlorous acid, and azurophilic granule proteins had significant bacteriostatic but limited staphylocidal activity toward MW2 under the conditions tested. An MW2-specific microarray revealed common changes in S. aureus gene expression following exposure to each microbicide, such as up-regulation of transcripts involved in gene regulation (e.g., saeRS and kdpDE) and stress response. Azurophilic granule proteins elicited the greatest number of changes in MW2 transcripts, including up-regulation of mRNAs encoding multiple toxins and hemolysins (e.g., hlgA, hlgB, hlgC, hla, lukS-PV, lukF-PV, sec4, and set17-26). Notably, H2O2 triggered up-regulation of transcripts related to heme/iron uptake (e.g., isdA, isdB, and isdCDEFsrtBisdG), and an isogenic isdAB-negative strain of MW2 had increased susceptibility to H2O2 (p<0.001) and human neutrophils (p<0.05) compared with the wild-type parental strain. These findings reveal a S. aureus survival response wherein Iron-regulated surface determinant (Isd) proteins are important for resistance to innate host defense. Collectively, the data provide an enhanced view of the mechanisms used by S. aureus to circumvent destruction by the innate immune system.