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).

RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3 domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3 domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3 domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop-loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3 domain in establishing a network of S. aureus virulence factors.[Keywords: Regulatory RNA; translational repression; antisense regulation; RNase III; virulence; Staphylococcus aureus]Supplemental material is available at http://www.genesdev.org.

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell-wall-associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII-mediated inhibition of translation and degradation of the stable spa mRNA by the double-strand-specific endoribonuclease III (RNase III). The 3' end domain of RNAIII, partially complementary to the 5' part of spa mRNA, efficiently anneals to spa mRNA through an initial loop-loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-Hemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here, we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-Hemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2+ cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.

The K. kingae-specific real-time PCR places K. kingae as the leading cause of OAI in children at our hospital.

RNAIII, a 514-nt RNA molecule, regulates the expression of many Staphylococcus aureus genes encoding exoproteins and cell-wall-associated proteins. We have studied the structure of RNAIII in solution, using a combination of chemical and enzymatic probes. A model of the secondary structure was derived from experimental data with the help of computer simulation of RNA folding. The model contains 14 hairpin structures connected by unpaired nucleotides. The data also point to three helices formed by distant nucleotides that close off structural domains. This model was generally compatible with the results of in vivo probing experiments with dimethylsulfate in late exponential-phase cultures. Toe-printing experiments revealed that the ribosome binding site of hld, which is encoded by RNAIII, was accessible to the Escherichia coli 30S ribosomal subunit, suggesting that the in vitro structure represented a translatable form of RNAIII. We also found that, within the 39 end of RNAIII, the conserved hairpin 13 and the terminator form an intrinsic structural domain that exerts specific regulatory activity on protein A gene expression.

We examined the capacity of Staphylococcus aureus strains to release Panton-Valentine leukocidin (PVL) in the presence of antibiotics. No PVL was detected when S. aureus was incubated at inhibitory concentrations, while subinhibitory concentrations of oxacillin enhanced the PVL level; clindamycin, linezolid, and fusidic acid were inhibitory; and vancomycin had roughly no effect.Staphylococcus aureus is an important human pathogen. It expresses a variety of exoproteins, including Panton-Valentine leukocidin (PVL) (31). While Voyich et al. could not establish clear differences in virulence between isogenic pairs of PVLpositive/negative strains (29), Labandeira-Rey et al. clearly demonstrated the role of PVL as a major determinant of virulence in an acute pneumonia mouse model using other sets of isogenic strains for PVL (13) and thus confirmed the results of the princeps experiments showing that PVL is a virulence factor (15). The apparent discrepancy between these studies basically comes from the choice of the experimental models and the choice of the strains.

SummaryStaphylococcus aureus is a major pathogen responsible for both nosocomial and communityacquired 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-1b release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1b release and inflammasome activation in primary human macrophages. The cytolytic peptides, d-haemolysin and PSMa3; the pore-forming toxins, g-haemolysin and LukDE; and b-haemolysin synergize with PVL to amplify IL-1b 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-1b release in human alveolar macrophages. Furthermore, IL-1b 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.