Insights into the SaeR/S-mediated pathogenesis of Staphylococcus aureus
Nygaard, Tyler Kenji.
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Staphylococcus aureus is an ubiquitous and versatile Gram-positive bacterium capable of producing a wide range of diseases in humans. Within the last ten years there has been a surge of infections caused by hyper-virulent strains of community-associated methicillin-resistant S. aureus (CA-MRSA) distinct from previously characterized healthcare-associated MRSA (HA-MRSA). In particular, pulsed-field gel electrophoresis (PFGE) type USA300 has emerged as the prominent circulating strain of CA-MRSA in the United States. How S. aureus recognizes environmental signals during infection and responds to promote bacterial survival is clearly an important aspect of pathogenesis but remains incompletely defined. It is believed S. aureus recognizes extracellular signals and subsequently alters gene transcription in response via the collective influence of 16 bacterial two-component gene-regulatory systems. This investigation examines the role of one of these two-component systems, saeR/S, in USA300 pathogenesis. An isogenic deletion mutant of saeR/S generated in USA300 (USA300 Delta saeR/S) demonstrated this two-component system is critical to USA300 infection by upregulating transcription of numerous virulence genes. 5' RACE analysis identified transcript start sites for genes strongly regulated by saeR/S. Sequence alignment upstream from transcript start sites revealed a conserved partially palindromic 17 bp sequence specific to promoter regions under strong saeR/S regulation and EMSAs confirmed direct binding of recombinant SaeR His to this conserved sequence. In particular, strong saeR/S dependent transcription of the pore-forming hla toxin and robust SaeR His binding to the hla promoter prompted further studies examining the importance of this virulence gene during USA300 pathogenesis. An isogenic deletion mutant of hla in USA300 (USA300 Delta hla) was generated and used to demonstrate saeR/S-dependent transcription of hla is critical to USA300 pathogenesis during murine models of soft-tissue infection. Though hla did not influence saeR/S-mediated plasma membrane damage on polymorphonuclear leukocytes, this toxin induced pore formation on human T cells and promoted rapid human T cell proliferation. Collectively this study demonstrates saeR/Sdependent transcription of virulence factors is critical to USA300 pathogenesis. In particular, the saeR/S-mediated regulation of the potent pore-forming hla toxin is a primary effector of USA300 pathogenesis that causes human T cell plasma membrane damage and induces rapid human T cell proliferation.