| Staphylococcus aureus contains 4 native, membrane-bound penicillin-binding proteins (PBP1-4) which are responsible for critical peptidoglycan transglycosylation and transpeptidation reactions in the cell wall. Additionally, PBP2 and PBP4 also mediate the development and maintenance of beta-lactam drug resistance in both clinical and laboratory isolates of Community-Acquired strains of S. aureus (CA-MRSA). Despite their important roles in the growth and pathogenesis of S. aureus, the regulation of pbp2 and pbp4 is poorly understood. We have used solid phase promoter retention (SPPR) affinity chromatography to isolate potential regulators of these genes, and have identified 4 putative transcription factors. These regulators include the ferric uptake regulator (Fur), the staphylococcal accessory regulator A (SarA), the oxidative stess regulator Spx, and a hypothetical protein with low level sequence identity to Spx which we call SpxL. We have confirmed that SpxL functions like a classic Spx-family regulator based on its RNAP-dependent gene regulatory ability and role in peroxide stress response. Through in vitro DNA-binding studies, it was also confirmed that SarA binds directly to the pbp2 and pbp4 promoter regions. Likewise, Spx and SpxL form complexes with RNA polymerase, which increases the affinity of RNAP for both the pbp2 and pbp4 promoter regions. Gene expression studies show that SarA, Fur, and SpxL appear to repress the expression of pbp2 and pbp4, while Spx increases gene expression. These data indicate that the pbp genes are highly regulated, and their expression is likely to be influenced by factors other than beta-lactam antibiotics, such as iron homeostasis, virulence, and oxidative stress. These studies have begun to clarify the cellular responses of CA-MRSA upon exposure to beta-lactam drugs, which is critical for understanding how to circumvent antibiotic defense mechanisms in this organism. |
