Date of Degree
Access restricted until 02/23/2018
PhD (Doctor of Philosophy)
Alexander R. Horswill
The Staphylococcus genus comprises a diverse group of Gram-positive bacteria that are opportunistic pathogens of humans and other mammals. S. epidermidis and S. aureus are the most common human pathogens of the staphylococci, causing a variety of infections including biofilm-based medical device infections, skin infections, and pneumonia. Both of these organisms produce proteases whose functions in virulence are not fully characterized. In S. epidermidis, protein-mediated biofilm formation requires a cell wall-anchored adhesin called Aap that must be proteolytically processed in order to allow intercellular adhesion. The S. epidermidis protease(s) responsible for cleaving Aap were unknown. Chapter II describes our findings that the secreted metalloprotease SepA is required for Aap-mediated biofilm formation and cleaves Aap at two different sites. Further, this protease is negatively regulated by the global regulator SarA.
Chapter III discusses studies of the S. aureus Spl (serine protease-like) proteases. Although they are produced in vivo, their substrates and role in virulence are unknown. We found that in a rabbit model of pneumonia, a mutant lacking the spl protease operon caused more localized disease compared to wild type S. aureus. Proteomics studies of the secreted and surface proteins in wild type compared to spl mutant S. aureus revealed several changes. We also found that the SplA protease cleaves human Mucin-16, the first identification of a biological substrate of the Spls.
Finally, we found that the animal-associated species S. caprae produces an autoinducing peptide (AIP) that is a potent inhibitor of S. aureus quorum sensing. We identified the S. caprae AIP structure as an 8-residue thiolactone ring. A synthetic version of the peptide inhibits S. aureus virulence and quorum sensing induction in a murine skin infection model. This is a novel example of quorum sensing cross talk between staphylococcal quorum sensing systems. These studies are described in Appendix A.
On the whole, this work identified two substrates of S. aureus proteases and demonstrated their importance in biofilm formation and infection. We also characterized a novel inhibitor of S. aureus quorum sensing that attenuates virulence. These findings shed light on the importance of staphylococcal secreted proteases and quorum sensing cross talk in the modulation of virulence factor production and the ability to cause disease.
bacterial pathogenesis, biofilm, protease, staphylococci
Copyright © 2016 Alexandra E. Paharik