φSa3mw Prophage as a Molecular Regulatory Switch of Staphylococcus aureus β-toxin Production.

Phage regulatory switches (phage-RSs) are a newly described form of active lysogeny where prophages function as regulatory mechanisms for expression of chromosomally-encoded, bacterial genes. In Staphylococcus aureus, φSa3int is a widely-distributed family of prophages that integrate into the β-toxin structural gene hlb effectively inactivating it. However, β-toxin producing strains often arise during infections and are more virulent in experimental infective endocarditis and pneumonia infections. We present evidence that in S. aureus MW2, φSa3mw excision is temporally and differentially responsive to growth conditions relevant to S. aureus pathogenesis. PCR analyses of φSa3mw (integrated and excised) and of intact hlb showed that φSa3mw preferentially excises in response to hydrogen peroxide-induced oxidative stress and during biofilm growth. φSa3mw remains as a prophage when in contact with human aortic endothelial cells in culture. A criterion for a prophage to be considered a phage-RS is the inability to lyse host cells. MW2 grown under phage-inducing conditions did not release infectious phage particles by plaque assay or transmission electron microscopy, indicating that φSa3mw does not carry out a productive lytic cycle. These studies highlight a dynamic, and perhaps more sophisticated, S. aureus -prophage interaction where φSa3int prophages provide a novel regulatory mechanism for the conditional expression of virulence factors. IMPORTANCE β-toxin is a sphingomyelinase hemolysin that significantly contributes to Staphylococcus aureus pathogenesis. In most S. aureus isolates the prophage φSa3int inserts into the β-toxin gene hlb inactivating it. Yet, human and experimental infections give rise to β-toxin-producing variants. However, it remained to be established whether φSa3mw excises in response to specific environmental cues, restoring the β-toxin gene sequence. This is not only of fundamental interest but also critical when designing intervention strategies and therapeutics. We provide evidence that φSa3mw actively excises allowing the conditional expression of β-toxin. φSa3int prophages may play a novel and largely uncharacterized role in S. aureus pathogenesis as molecular regulatory switches that promote bacterial fitness and adaptation to the challenges presented by the mammalian host.