We have discovered a novel mechanism of resistance to β-lactams that is independent of penicillinase and the low affinity penicillin bindin protein (PBP), PBP2a, the two known mechanisms of β-lactam resistance in Staphylococcus aureus. This new type of resistance was identified during experiments in which methicillin- susceptible S. aureus strains were passaged in the presence of each of the two so-called "fifth generation" anti-MRSA cephalosporins, ceftobiprole and ceftaroline. Whole genome sequencing of a ceftobiprole- passage mutant revealed mutations in genes encoding PBP4, a non-essential, low-molecular weight PBP; GdpP, a putative signaling protein; and AcrB, a putative transporter. Ceftaroline also selected for PBP4 and GdpP mutants, but not AcrB mutants, indicating the primary importance of the former two proteins. We hypothesize 1) that a gain of transpeptidase function by mutant PBP4 accounts for high-level β-lactam resistance; and 2) that GdpP contributes to resistance via a signaling pathway that up-regulates expression of pbp4. Two specific aims are proposed to test these hypotheses. Aim 1: To determine the mechanism by which mutations in pbp4 confer high-level β-lactam resistance. pbp4 missense mutations will be repaired in mutants or introduced into parent strains by allelic replacement mutagenesis. Isogenic strains will be tested for β-lactam resistance to identify mutations of importance. PBP binding assays and analyses of peptidoglycan structure will be performed to determine the effect of mutations on PBP binding and to test for functional changes in carboxypeptidase or transpeptidase activities. Binding and enzymatic activity assays, including β-lactamase, also will be conducted with model substrates for recombinant wild-type and mutant proteins. X-ray crystallography will be used to identify the structural basis of functional changes, particularly those associated with transpeptidase activity. Aim 2: To determine the role of gdpP in mediating response to β-lactam antibiotics. GdpP is a putative signaling protein that has phosphodiesterase activity against cyclic diadenosine monophosphate (c-di-AMP), a recently identified second messenger. Mutations in gdpP were associated with increased expression of pbp4 and with resistance to β-lactams. We hypothesize that these mutations lead to intracellular accumulation c-di-AMP through loss of GdpP phosphodiesterase activity. To test this hypothesis intracellular concentrations of c-di-AMP will be manipulated by mutation of gdpP or by inhibition of expression of dacA, which encodes the diadenylate cyclase that generates c-di-AMP, and effects on pbp4 expression determined. As GdpP is a signaling molecule, microarray studies will be conducted to identify potential downstream proteins in its regulatory pathway. Recombinant GdpP also will be purified and analyzed by x-ray crystallography to identify its critical structural properties. Achieving these aims will increase knowledge of β-lactam antibiotic effects and mechanisms of resistance.