Contribution of Panton-Valentine leukocidin in community-associated methicillin-resistant Staphylococcus aureus pathogenesis

Abstract

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.

Figure 1. PVL does not alter global gene and protein expression profiles.

Clinical strains of USA300 (LAC and SF8300) and USA400 (MW2) and their respective isogenic Δpvl mutant strains were cultured to mid-exponential or stationary phases of growth in TSB or CCY media. (A) TaqMan real-time RT-PCR for comparison of fold changes in transcript levels of selected Agr-regulated genes in wild type and Δpvl mutant strains. See also Table 2 for additional data derived from in vitro growth to exponential phase and stationary phase in CCY or TSB media. agrA, accessory global regulator; hla, alpha-toxin; hlgA, gamma-haemolysin component A; splA, serine protease; spa, protein A; sdrD, serine aspartate repeat protein; clfB, clumping factor B. (B) Cell extracts separated by 12% SDS-PAGE (Protean II gel, Bio-Rad) using cultures grown to stationary phase. (C) Culture supernatants, prepared from growth in TSB or CCY media, were separated by gradient 10-20% SDS-PAGE. PVL subunits were identified by automated-direct infusion tandem mass spectrometry . (D, E, F) Western immunoblot analysis of supernatants and cell extracts from cultures grown to stationary or mid-exponential phase. Proteins were detected with rabbit polyclonal antibodies specific for LukF-PV, Hla (α-toxin), or Spa. The immunoblots in panel E were exposed on the same film for equal times or using a longer exposure for MW2 (inset), which produced less Spa. Protein samples for SDS-PAGE presented in panels B and C were prepared in a manner identical to those shown in panels E and F.