RESUMEN
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the rRNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in Escherichia coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralog of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
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Proteínas de Escherichia coli , Escherichia coli , Staphylococcus aureus/genética , Proteínas de Escherichia coli/genética , ARN , Virulencia/genética , ARN Ribosómico 23S/genética , Metiltransferasas/genéticaRESUMEN
BACKGROUND: Severe sepsis, combining acute osteomyelitis and lung involvement, has been described increasingly in healthy children with the spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). METHODS: Outcomes (mortality, hematogenous spread, lung and bone involvements) of rabbit osteomyelitis caused by CA-MRSA LAC(WT) USA300 and its Panton-Valentine leukocidin (PVL)- and α-hemolysin (Hla)-negative isogenic derivatives (LACΔpvl and LACΔhla, respectively) were compared. RESULTS: Three days after inoculation (D3), all LAC(WT)- and LACΔpvl-, and 72% of LACΔhla-infected rabbits had no hematogenous spread and similar lung and bone bacterial densities. LACΔpvl and LACΔhla caused less severe histological lung lesions than LAC(WT) (P ≤ .01). Between D3 and D9, 10 (53%) LAC(WT)-, 11 (55%) LACΔpvl-, but no LACΔhla-infected rabbits (P < .005) died of severe sepsis with disseminated infection. Unlike deceased animals, most LAC(WT), LACΔpvl, and LACΔhla D14 survivors had no hematogenous spread (P < .001). LAC(WT) (88%) caused more bone abscesses than LACΔpvl (0, P = .001) or LACΔhla (30%, P = .01). CONCLUSION: In this model, both PVL and Hla seemed to be required for early lung involvement via hematogenous spread. Hla, but not PVL, significantly impacted severe sepsis-related mortality. PVL was the predominant factor determining late-stage bone abscesses.
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Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Leucocidinas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Osteomielitis/microbiología , Sepsis/microbiología , Infecciones Estafilocócicas/microbiología , Absceso/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/genética , Exotoxinas/genética , Femenino , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Hemolisinas/genética , Inmunoglobulina G/sangre , Leucocidinas/genética , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Staphylococcus aureus Resistente a Meticilina/genética , Mutación , Osteomielitis/mortalidad , Osteomielitis/patología , Conejos , Sepsis/complicaciones , Infecciones Estafilocócicas/mortalidadRESUMEN
Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1ß release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1ß release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and ß-haemolysin synergize with PVL to amplify IL-1ß release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1ß release in human alveolar macrophages. Furthermore, IL-1ß released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury.
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Toxinas Bacterianas/metabolismo , Quimiocinas/metabolismo , Células Epiteliales/inmunología , Exotoxinas/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Leucocidinas/metabolismo , Macrófagos/microbiología , Staphylococcus aureus/patogenicidad , Animales , Niño , Humanos , Pulmón/inmunología , Pulmón/patología , Macrófagos/inmunología , Neutrófilos/inmunología , Staphylococcus aureus/inmunología , Factores de Virulencia/metabolismo , Adulto JovenRESUMEN
Introduction: The bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins. Methods: We present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival. Results: We found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models. Discussion: These findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens.
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Infecciones Comunitarias Adquiridas , Staphylococcus aureus Resistente a Meticilina , Neumonía Estafilocócica , Infecciones Estafilocócicas , Humanos , Neumonía Estafilocócica/microbiología , Staphylococcus aureus , Factores de Virulencia/genética , Hemoptisis , Proteómica , Exotoxinas/genética , Infecciones Estafilocócicas/microbiología , Infecciones Comunitarias Adquiridas/microbiología , Staphylococcus , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/genéticaRESUMEN
Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in hlgCB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on hlgC promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. IMPORTANCE S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in pvl-negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of hlgCB mRNA induce the differential expression of hlgCB, drastically impacting hlgC mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.
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Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Conejos , Staphylococcus aureus/metabolismo , Leucocidinas/genética , Leucocidinas/metabolismo , Leucocidinas/farmacología , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Virulencia/genética , Exotoxinas/genética , Exotoxinas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
OBJECTIVES: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). METHODS: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. RESULTS: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. CONCLUSION: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream.
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Bacteriemia , Endocarditis Bacteriana , Endocarditis , Infecciones Estafilocócicas , Animales , Conejos , Ratones , Estudio de Asociación del Genoma Completo , Bacteriemia/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Endocarditis Bacteriana/microbiología , Endocarditis/microbiologíaRESUMEN
Staphylococcus aureus infective endocarditis (SaIE) is a severe complication of S. aureus bacteremia (SAB) occurring in up to 22% of patients. Bacterial genetic factors and host conditions for SaIE have been intensely studied before; however, to date no study has focused on predisposing host genetic factors to SaIE. The present study aimed to identify genetic polymorphisms associated with SaIE by a Genome-Wide Association Study (GWAS) of 67 patients with definite native valve SaIE (cases) and 72 matched native valve patients with SAB but without IE (controls). All patients were enrolled in the VIRSTA cohort (Le Moing et al., 2015) study. Four single nucleotide polymorphisms (SNPs) located on chromosome 3 were associated with SaIE (P < 1 × 10-5) without reaching conventional genome-wide significance. For all, the frequency of the minor allele was lower in cases than in controls, suggesting a protective effect of the minor allele against SaIE. The same association was observed using an independent Danish verification cohort of SAB with (n = 57) and without (n = 123) IE. Ex vivo analysis of aortic valve tissues revealed that SaIE associated SNPs mentioned above were associated with significantly higher mRNA expression levels of SLC7A14, a predicted cationic amino acid transporter protein. Taken together, our results suggest an IE-protective effect of SNPs on chromosome 3 during the course of SAB. The effects of protective minor alleles may be mediated by increasing expression levels of SLC7A14 in valve tissues. We conclude that occurrence of SaIE may be the combination of a well-adapted bacterial genotype to a susceptible host.
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Proteínas Bacterianas/genética , Exotoxinas/fisiología , Leucocidinas/fisiología , Neumonía Estafilocócica/microbiología , Staphylococcus aureus/patogenicidad , Transactivadores/genética , Factores de Virulencia/fisiología , Animales , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Mutación Puntual , Staphylococcus aureus/genética , Transactivadores/metabolismo , VirulenciaRESUMEN
INTRODUCTION: In bone and joint infections (BJIs), bacterial toxins are major virulence factors: Panton-Valentine leukocidin (PVL) expression leads to severe local damage, including bone distortion and abscesses, while α-hemolysin (Hla) production is associated with severe sepsis-related mortality. Recently, other toxins, namely phenol-soluble modulins (PSMs) expressed by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300 (LAC WT) were shown to have ex vivo intracellular cytotoxic activity after S. aureus invasion of osteoblasts, but their in vivo contribution in a relatively PVL-sensitive osteomyelitis model remains poorly elucidated. MATERIALS AND METHODS: We compared the outcomes of experimental rabbit osteomyelitises induced with pvl+hla+psms+ LAC WT and its isogenic Δpsm derivatives (LAC Δpsmα and LAC Δpsmαßhld) using an inoculum of 3 × 108 CFUs. Mortality, hematogenous spread (blood culture, spleen and kidney), lung and bone involvements were assessed in two groups (non-survivors of severe sepsis and survivors sacrificed on day (D) 14). RESULTS: Severe sepsis-related mortality tended to be lower for Δpsm derivatives (Kaplan-Meier curves, P = .06). Non-survivors' bone LAC-Δpsmα (6.9 log10 CFUs/g of bone, P = .04) or -Δpsmαßhld (6.86 log10 CFUs/g of bone, P = .014) densities were significantly higher than LAC WT (6.43 log10 CFUs/g of bone). Conversely, lung Δpsmαßhld CFUs were significantly lower than LAC WT (P = .04). LAC Δpsmα, Δpsmαßhld and WT induced similar bone damage in D14 survivors, with comparable bacterial densities (respectively: 5.89, 5.91, and 6.15 log10 CFUs/g of bone). Meanwhile, pulmonary histological scores of inflammation were significantly higher for LAC Δpsmα- and Δpsmαßhld-infected rabbits compared to LAC WT (P = .04 and .01, respectively) but with comparable lung bacterial densities. CONCLUSION: Our experimental results showed that deactivating PSM peptides significantly limited bacterial dissemination from bone during the early phase of infection, but did not affect local severity of USA300 rabbit osteomyelitis.
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Toxinas Bacterianas , Staphylococcus aureus Resistente a Meticilina , Osteomielitis , Sepsis , Infecciones Estafilocócicas , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Osteomielitis/genética , Osteomielitis/metabolismo , Osteomielitis/microbiología , Osteomielitis/patología , Conejos , Sepsis/genética , Sepsis/metabolismo , Sepsis/microbiología , Sepsis/patología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patologíaRESUMEN
Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.
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Neoplasias del Colon/genética , Ligasas/genética , Complejos de Ubiquitina-Proteína Ligasa , Ciclosoma-Complejo Promotor de la Anafase , Antineoplásicos/farmacología , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase , Neoplasias de la Mama/genética , Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Neoplasias del Colon/tratamiento farmacológico , Ciclina B/genética , Ciclina B/metabolismo , Ciclina B1 , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Humanos , Ligasas/metabolismo , Neoplasias Hepáticas/genética , Melanoma/genética , Mitosis , Mutación , Neuroblastoma/genética , Nocodazol/farmacología , Neoplasias Ováricas/genética , Reacción en Cadena de la Polimerasa/métodos , Subunidades de Proteína , Células Tumorales CultivadasRESUMEN
OBJECTIVES: Community-acquired Staphylococcus aureus necrotizing pneumonia is a life-threatening disease. Panton Valentine Leukocidin (PVL) has been associated with necrotizing pneumonia. PVL triggers inflammasome activation in human macrophages leading to IL-1ß release. IL-1ß activates lung epithelial cells to release IL-8. This study aimed to assess the relevance of this inflammatory cascade in vivo and to test the potential of an IL-1 receptor antagonist (IL-1Ra/Kineret) to decrease inflammation-mediated lung injury. METHODS: We used the sequential instillation of Heat-killed S. aureus and PVL or S. aureus infection to trigger necrotizing pneumonia in rabbits. In these models, we investigated inflammation in the presence or absence of IL-1Ra/Kineret. RESULTS: We demonstrated that the presence of PVL was associated with IL-1ß and IL-8 release in the lung. During PVL-mediated sterile pneumonia, Kineret/IL-1Ra reduced IL-8 production indicating the relevance of the PVL/IL-1/IL-8 cascade in vivo and the potential of Kineret/IL-1Ra to reduce lung inflammation. However, Kineret/IL-1Ra was ineffective in blocking IL-8 production during infection with S. aureus. Furthermore, treatment with Kineret increased the bacterial burden in the lung. CONCLUSIONS: Our data demonstrate PVL-dependent inflammasome activation during S.aureus pneumonia, indicate that IL-1 signaling controls bacterial burden in the lung and suggest that therapy aimed at targeting this pathway might be deleterious during pneumonia.
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Toxinas Bacterianas/toxicidad , Exotoxinas/toxicidad , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Leucocidinas/toxicidad , Neumonía Estafilocócica/tratamiento farmacológico , Animales , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Neumonía Estafilocócica/etiología , Neumonía Estafilocócica/metabolismo , ConejosRESUMEN
Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.
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Toxinas Bacterianas/toxicidad , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/microbiología , Staphylococcus aureus Resistente a Meticilina/fisiología , Osteoblastos/citología , Osteoblastos/microbiología , Carga Bacteriana , Muerte Celular/efectos de los fármacos , Infecciones Comunitarias Adquiridas/microbiología , Atención a la Salud , Regulación Bacteriana de la Expresión Génica , Humanos , Osteoblastos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la EspecieRESUMEN
The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall-anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).