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To the editor:S. aureus pneumonia causes mortality in healthy individuals or hos-pital settings1, and recent increases in morbidity are attributed to the rapid spread of community-associated, methicillin-resistant strains (CA-MRSA)2,3. We previously described a mouse model of staphylococ-cal pneumonia that mimics clinicopathologic features of human disease and identified α-hemolysin (Hla) as an essential virulence factor in S. aureus Newman, a human clinical isolate4. Labandeira-Rey et al.5 used the laboratory strain S. aureus RN6390 to identify Panton-Valentine leuko-cidin (PVL) as another virulence factor essential for lung infections. Hla and PVL are members of the family of β-channel pore-forming toxins (β-CFT), which assemble into heptameric structures that penetrate cell membranes6. In contrast to Hla, the gene for which is expressed by almost all staphylococcal isolates, PVL is secreted only by strains lysogenized with a bacteriophage carrying lukS-PV and lukF-PV, the structural genes for PVL2,7. Here we examined the contribution of β-CFTs to the patho-genesis of lung infections with clinically relevant CA-MRSA strains, LAC (USA300) and MW2 (USA400)3. (Animal experiments were reviewed, approved and conducted in accordance with guidelines set by the Institutional Animal Care and Use Committee at The University of Chicago.)

Replacement of hla with the hla::erm allele completely abolished the ability of S. aureus LAC to cause lung infections in C57BL/6J mice (Fig. 1a), whereas deletion of lukS-PV and lukF-PV (∆pvl) in S. aureus LAC or MW2 caused no difference in the overall mortality of animals with staphylococcal pneumonia (Fig. 1b). Deletion of lukS-PV and lukF-PV in either of the two CA-MRSA strains did not affect bacterial growth in the lungs of mice (Fig. 1c). Lung sections revealed pathologic evidence of pneumonia manifested by immune cell infiltration, hemorrhage, loss of alveolar architecture with consolidation of lung parenchyma, and bacte-rial infiltrates; these features were indistinguishable in animals infected with strains that did or did not secrete PVL (Fig. 1d). Importantly, dele-tion of lukS-PV and lukF-PV did not alter staphylococcal secretion of Hla (Fig. 1e).

We wondered whether possible contributions of PVL to staphylococ-cal pneumonia may be masked by the dominant role of Hla. We trans-formed S. aureus Newman with a plasmid encoding lukS-PV and lukF-PV (ppvl; see Supplementary Methods online). We observed no alteration in pneumonia-related mortality of experimental mice infected with S. aureus Newman harboring either vector alone or ppvl (Fig. 1f), in spite of the high level of PVL expression from the ppvl plasmid (Fig. 1g). Additionally, neither recovery of staphylococci from the lungs of infected mice nor histopathologic evidence of disease was altered by expression of PVL (data not shown). As previously reported4, loss of hla expression in S. aureus Newman completely abrogated staphylococcal virulence and mortality in lung infection; this defect could not be reversed by transfor-mation of the hla::erm mutant with ppvl (Fig. 1f). In contrast with these results, transformation with phla, a plasmid that promotes expression

Poring over pores: α-hemolysin and Panton-Valentine leukocidin in Staphylococcus aureus pneumonia

of Hla, resulted in a complete restoration of the virulence phenotype, with all of the infected mice succumbing to pneumonia within 24 h

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Figure 1 Hla is a virulence factor for CA-MRSA pneumonia. (a) Comparison of mortality from staphylococcal pneumonia in animals infected with CA-MRSA strain LAC and its Hla-deficient isogenic variant hla::erm (P < 0.0007). Comparison of animal mortality (b), bacterial replication (c) and H&E–stained lung tissues (d; scale bars, 120µm) in animals infected with CA-MRSA strains LAC and MW2 or their isogenic ∆pvl mutants. Statistical analysis did not reveal significant differences in either mortality or bacterial replication in each isogenic pair examined. (e) Immunoblot analysis of LukS-PV, LukF-PV and Hla in culture supernatants from LAC and MW2 strains. A cross-reactive species is marked with an asterisk (*). (f) Examination of the effects of plasmid-mediated overexpression of PVL (ppvl) compared to vector alone in strain Newman (P = 0.27), or compared to vector alone and plasmid-encoded Hla (phla) in the Newman hla::erm strain (P = 0.00004). (g) Immunoblot analysis of culture supernatants derived from wild-type S. aureus Newman (WT) or S. aureus Newman hla::erm harboring ppvl, phla or vector.

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(Fig. 1f). Immunoblot analysis revealed the presence of LukS-PV, LukF-PV and Hla in cultures derived from the various strains (Fig. 1e,g), and mouse mortality from S. aureus pneumonia directly correlated with the level of Hla expression in each of the strains examined (data not shown).

As PVL is encoded by a bacteriophage, we examined whether expression of PVL from its native genomic locus can influence disease. A lukS-PV and lukF-PV lysogen of S. aureus Newman was generated with bacterio-phage φSa2mw (isolated from S. aureus MW2, ref. 8; Fig. 2a). Insertion of φSa2mw at nucleotide 1565379 of the S. aureus Newman chromosome led to LukS-PV and LukF-PV secretion (Fig. 2b). After we infected mice with S. aureus Newman and its isogenic φSa2mw lysogen, both strains replicated with equal efficiency in lung (Fig. 2c). No differences were observed in the overall mortality of mice with pneumonia from S. aureus Newman or its φSa2mw lysogen (Fig. 2d), indicating that PVL secretion through phage lysogeny does not affect staphylococcal virulence during lung infection. Despite the presence of the PVL bacteriophage, insertional

disruption of hla in S. aureus Newman φSa2mw (hla::erm) abolished the ability of this strain to cause lung infection (Fig. 2e).

To assess the effect of β-CFTs on human lung tissue, we analyzed the injury induced by infection with S. aureus LAC, MW2 or Newman in human A549 alveolar epithelial cells by measuring the release of lactate dehydrogenase. Disruption of the pvl locus did not diminish the cytotoxic effects of S. aureus LAC or MW2, a finding that is in agreement with the reported specificity of PVL for granulocytes and mononuclear phagocytes9 (Fig. 2f). In contrast, S. aureus Newman variants lacking Hla were unable to injure A549 cells, a defect that was complemented by phla and readily visualized by microscopy of infected cells (Fig. 2f,g).

The epidemiologic association of PVL bacteriophage with S. aureus strains from patients with necrotizing pneumonia led us to the hypothesis that PVL is a virulence factor involved in disease pathogenesis2. Labandeira-Rey et al.5 generated support for this theory in lung infections of BALB/c mice; however, these experiments used a laboratory strain overexpress-ing plasmid-encoded lukS-PV and lukF-PV to measure disease-associated mortality. They observed histopathologic evidence of lung disease after mouse infections with S. aureus RN6390 harboring PVL bacteriophage, but lysogenization had no effect on mortality5. We examined the contribution of β-CFTs to S. aureus lung infection with clinically relevant CA-MRSA iso-lates and S. aureus Newman PVL lysogens in C57BL/6J mice. Here, Hla, but not PVL, was essential for the pathogenesis of staphylococcal pneumonia. Future work is needed to examine whether all clinical S. aureus pneumonia isolates require hla to cause lung infections, whether other important viru-lence factors contribute to the pathogenesis of lung infections and whether the parameters established for mouse infection are broadly applicable to human pneumonia caused by S. aureus.

Juliane Bubeck Wardenburg1,2, Taeok Bae3, Michael Otto4, Frank R DeLeo4 & Olaf Schneewind1

1Departments of Microbiology and 2Pediatrics, University of Chicago, Chicago, Illinois 60637, USA. 3Department of Microbiology and Immunology, Indiana University School of Medicine Northwest, Gary, Indiana 46408, USA. 4Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA.e-mail: oschnee@bsd.uchicago.edu

Note: Supplementary information is available on the Nature Medicine web-site.

1. Lowy, F.D. N. Engl. J. Med. 339, 520–532 (1998).2. Gillet, Y. et al. Lancet 359, 753–759 (2002).3. Voyich, J.M. et al. J. Infect. Dis. 194, 1761–1770 (2006).4. Bubeck Wardenburg, J., Patel, R.J. & Schneewind, O. Infect. Immun. 75, 1040–1044

(2007).5. Labandeira-Rey, M. et al. Science 315, 1130–1133 (2007).6. Gouaux, E., Hobaugh, M. & Song, L. Protein Sci. 6, 2631–2635 (1997).7. Kaneko, J., Kimura, T., Narita, S., Tomita, T. & Kamio, Y. Gene 215, 57–67 (1998).8. Baba, T. et al. Lancet 359, 1819–1827 (2002).9. Gladstone, G.P. & van Heyningen, W.E. Br. J. Exp. Pathol. 38, 123–137 (1957).

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