We have identified a novel mode of high-level, broad-spectrum ?-lactam resistance in S. aureus that is not mediated by PBP2a, the penicillin-binding protein (PBP) that confers methicillin resistance. PBP4, a non- essential PBP, and GdpP, the only known phosphodiesterase (PDE) that mediates cyclic-di-adenosine-mono- phosphate (CDA) degradation, have critical roles in this type of resistance. Mutations that enhance PBP4's ability to make a highly cross-linked bacterial cell wall and loss-of-function mutations in GdpP are the genetic basis responsible for this uncanonical resistance. The highly cross-linked cell wall formation is driven either independently or cooperatively by two distinct biochemical features of PBP4, a) structural changes in its protein due to missense mutations and b) its overexpression due to mutations in its promoter region. The loss-of-function mutations in GdpP result in elevated concentrations of CDA in bacterial cells. CDA is a newly discovered cell-signaling second messenger in bacteria which acts as an allosteric regulator by binding to its effectors (proteins and RNAs). CDA broadly affects gene expression and controls GdpP related ?-lactam resistant phenotypes in a concentration dependent manner, suggesting that it is the deterministic factor in resistance. However, the precise role(s) of CDA in mediating ?-lactam resistance as well as other vital processes of S. aureus is currently unknown. These functional alterations of PBP4 and GdpP likely come at the cost of bacterial virulence due to depletion of cell wall associated proteins and attenuated production of cytolysins, respectively. This indicates a unique yin-yang relationship between two key pathogenic factors of S. aureus, ?-lactam resistance and virulence. We will investigate the fundamental basis of the functional changes in PBP4 and GdpP that lead to resistance and their impact on bacterial virulence.
Aim 1 : To determine the mechanism of PBP4-mediated ?-lactam resistance and the role of PBP4 in cell wall composition. The relative contribution of PBP4 missense and promoter mutations on cell wall synthesis will be evaluated biochemically and structurally. The mechanism(s) that control pbp4 expression will be investigated to identify regulator(s) and to determine how they confer PBP4-mediated ?-lactam resistance. PBP4's role on bacterial cell surface associated virulence factor expression will be determined.
Aim 2 : To define the role of cyclic-di-adenosine-mono-phosphate (CDA) signaling in S. aureus. Genetic and chemical proteomic approaches will be taken to identify CDA mediator/s in the bacteria that are responsible for ?-lactam resistance and virulence defect. Finally, our preliminary data suggest the presence of a novel CDA specific phosphodiesterase in S. aureus besides GdpP. We will identify this novel phosphodiesterase. The proposed research will advance knowledge of basic cellular processes in S. aureus.

Public Health Relevance

Staphylococcus aureus is an important bacterial pathogen that causes significant morbidity and mortality among humans worldwide. Resistance of S. aureus to ?-lactams, the drugs of choice to treat S. aureus, is widespread. We have identified new candidates that lead to high-level resistance to ?-lactams. The proposed research will investigate the biology of this novel resistance phenomenon. Results obtained through our research will advance knowledge of ?-lactam resistance mechanisms and could serve to identify new targets for better therapy in the future.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Huntley, Clayton C
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University of Maryland Baltimore
Schools of Dentistry/Oral Hygn
United States
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Basuino, Li; Jousselin, Ambre; Alexander, J Andrew N et al. (2018) PBP4 activity and its overexpression are necessary for PBP4-mediated high-level ?-lactam resistance. J Antimicrob Chemother 73:1177-1180
Alexander, J Andrew N; Chatterjee, Som S; Hamilton, Stephanie M et al. (2018) Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in Staphylococcus aureus. J Biol Chem 293:19854-19865
da Costa, Thaina M; de Oliveira, Carolina R; Chambers, Henry F et al. (2018) PBP4: A New Perspective on Staphylococcus aureus ?-Lactam Resistance. Microorganisms 6:
Chatterjee, Som S; Chen, Liang; Gilbert, Aubre et al. (2017) PBP4 Mediates ?-Lactam Resistance by Altered Function. Antimicrob Agents Chemother 61:
Hamilton, Stephanie M; Alexander, J Andrew N; Choo, Eun Ju et al. (2017) High-Level Resistance of Staphylococcus aureus to ?-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4). Antimicrob Agents Chemother 61:
Nigo, Masayuki; Diaz, Lorena; Carvajal, Lina P et al. (2017) Ceftaroline-Resistant, Daptomycin-Tolerant, and Heterogeneous Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Causing Infective Endocarditis. Antimicrob Agents Chemother 61:
Chan, Liana C; Gilbert, Aubre; Basuino, Li et al. (2016) PBP 4 Mediates High-Level Resistance to New-Generation Cephalosporins in Staphylococcus aureus. Antimicrob Agents Chemother 60:3934-41
Greninger, Alexander L; Chatterjee, Som S; Chan, Liana C et al. (2016) Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Resistant to Fifth-Generation Cephalosporins Reveals Potential Non-mecA Mechanisms of Resistance. PLoS One 11:e0149541
Chambers, Henry F (2013) Cellulitis, by any other name. Clin Infect Dis 56:1763-4