The psmα locus regulates production of Staphylococcus aureus alpha-toxin during infection

Abstract

Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active pore-forming cytolysin alpha-toxin (Hla) and the amphipathic α-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.

FIG 1

An S. aureus Δpsmα psmβ hld strain has decreased virulence in an in vitro cytotoxicity assay. Shown are data for intoxication of A549 cells with the indicated dilutions (A) or a 1:2.5 dilution (B) of the S. aureus USA300 WT, Δhla, or Δpsm strain for 2 1/2 h. Cell death was measured by LDH release into the supernatant. Data shown are averages ± standard errors of the means of technical replicates and are representative of at least three independent experiments. ****, P < 0.0001.

FIG 2

An S. aureus Δpsmα psmβ hld strain has a defect in Hla transcription and protein production. (A) Bacterial growth of S. aureus WT and Δpsmα psmβ hld strains. OD₆₆₀, optical density at 660 nm. (B) Immunoblot of precipitated S. aureus USA300 supernatants harvested after growth for 3 or 6 h at 37°C. Immunoblotting with a polyclonal antibody against Hla is shown, and results are representative of three independent experiments. (C) Hla transcript levels measured by quantitative reverse transcription-PCR after 3 h of growth. The data represent the averages of three independent biological replicates. (D) Immunoblot of precipitated S. aureus USA400 MW2 supernatants harvested after growth for 3 or 6 h at 37°C. Quantification of immunoblots was performed with a Li-Cor Odyssey imager. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

An S. aureus Δpsmα psmβ hld strain has an in vivo defect in Hla production during S. aureus skin infection. C57BL/6J mice received subcutaneous inoculation of 2.5 × 10⁷ CFU of the S. aureus USA300 WT, Δhla, or Δpsmα psmβ hld strain. (A and B) Skin measurements of abscess area (A) and dermonecrosis area (B) of infected mice. (C) CFU recovery from skin abscesses harvested at 30 h postinfection. (D) Quantification of total Hla levels present in skin abscesses by an ELISA. All data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 4

An S. aureus Δpsmα psmβ hld strain exhibits an in vivo defect in Hla production during S. aureus pneumonia. C57BL/6J mice received intranasal inoculation with 3.3 × 10⁸ CFU of the S. aureus USA300 WT, Δhla, or Δpsmα psmβ hld strain. (A) Gross pathology of the left lung at 24 h postinfection. (B) CFU recovery from lung homogenates at 24 h postinfection. (C) Quantification of total Hla levels present in lung homogenates as determined by an ELISA. All data are representative of three independent experiments. ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

FIG 5

Plasmid-driven overexpression of hla restores virulence of an S. aureus Δpsmα psmβ hld mutant. (A, top) Intoxication of A549 cells with the S. aureus USA300 WT strain; the Δhla or Δpsmα psmβ hld mutant; or the Δpsmα psmβ hld strain complemented with the empty pOS1 vector or pOS1 containing the hla locus with its native promoter. Cell death was measured by LDH release into the supernatant. Data are averages ± standard errors of the means of technical replicates and are representative of at least three independent experiments. (Bottom) Anti-Hla immunoblot of bacterial supernatants harvested after 3 h of growth. The immunoblot is representative of two independent experiments. (B) Gross pathology of C57BL/6J mice infected subcutaneously with 2 × 10⁷ CFU of the S. aureus USA300 WT, Δhla, Δpsmα psmβ hld, or Δpsmα psmβ hld phla strain in 50 μl of PBS. (C) Quantification of total Hla levels present in skin abscesses as determined by an ELISA. Data are representative of two independent experiments. (D) Survival curve of C57BL/6J mice infected intranasally with 3.3 × 10⁸ CFU of the S. aureus USA300 WT, Δhla, Δpsmα psmβ hld, or Δpsmα psmβ hld phla strain. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.