Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) infections are a growing problem in the United States with over 18,000 deaths reported annually. There is no vaccine and as S. aureus becomes more drug resistant the number of treatment options is diminishing. New strategies and approaches are urgently needed to combat the rise in S. aureus infections. The ability of S. aureus to cause disease is due, in part, to an extensive repertoire of secreted and cell wall associated proteins. These virulence factors include adhesins, toxins, exoenzymes, and superantigens. Typically these virulence factors are made inside the bacterial cell and are transported across the cell membrane in a denatured state. Once outside the cell they refold into their active form. Peptidyl-prolyl cis-trans isomerases (PPIases) are enzymes that assist in the folding of proteins. They catalyze the isomerization between the cis and trans form of proline peptide bonds, thus accelerating the rate of refolding in proteins containing prolines. The absence of PPIase activity can lead to delayed/incorrect folding and a loss of protein activity. In bacteria PPIases have been shown to assist in the folding of secreted virulence factors and in doing so contribute to virulence, however their role in S. aureus remains unknown. Previous work on Staphylococcal nuclease (Nuc), a secreted virulence factor, has demonstrated that the isomerization state of a single proline bond (K116-P117) controls the rate of Nuc refolding. The addition of a PPIase can accelerate refolding, however the identity of the Staphylococcal PPIase involved is unknown. In this proposal we identify a Staphylococcal PPIase (PrsB) that is required for Nuc activity. We demonstrate that equal amounts of Nuc are secreted in a prsB mutant and wild-type strain, however the secreted Nuc is less active when secreted from the prsB mutant. Consequently, we hypothesize that PrsB is assisting Nuc folding and in doing so contributes to S. aureus pathogenesis via its PPIase activity.
In aim 1 of this proposal we will test this hypothesis by first determining the amino acid residues important for PrsB PPIase activity. Using this information we will construct a PrsB PPIase-inactive strain (prsB?PPIase) by introducing amino acid substitutions that disrupt PPIase activity onto the S. aureus chromosome. The resulting strain will be used in an abscess model of infection to determine the specific contribution of PrsB PPIase activity to virulence.
In aim 2 we will investigate direct protein targets of PrsB. We will perform in vitro Nuc refolding assays to determine the direct contribution of PrsB to Nuc refolding, and identify S. aureus proteins that directly interact with and are co-purified with PrsB by immunoprecipitation. We hypothesize that the co-purified proteins represent additional PrsB targets and may be responsible for a decrease in cytotoxicity observed in the culture supernatants of the prsB mutant. The data generated from this proposal will serve as the foundation for future studies to explore the molecular mechanism through which PrsB affects the virulence of S. aureus and identify potential PrsB inhibitors, which may represent a novel anti-virulence therapy to combat S. aureus infection.
New treatments and therapies are urgently needed to combat infections caused by antibiotic resistant bacteria, including methicillin resistant Staphylococcus aureus (MRSA). The proposed research project will investigate how MRSA toxins are activated after they are generated and secreted by the bacteria. Understanding this process could lead to the development of strategies to block activation of these toxins and decrease the severity of MRSA infection.
