Methicillin-resistant Staphylococcus aureus (MRSA) has acquired an inducible resistance mechanism to ?- lactam antibiotics that encompasses essentially all members of the antibiotic class. This resistance is conferred by a set of genes that encode an antibiotic sensor/signal transducer protein, gene repressor and two resistant determinants, a class A ?-lactamases and a special penicillin-binding protein (PBP) referred to as PBP2a. We have documented that the antibiotic sensor/signal transducer protein BlaR1 experiences covalent modification by ?-lactam antibiotics in its membrane-surface domain, which through a unique process that we have termed "lysine N-decarboxylation switch" activates the protein for signal transduction across the membrane. Subsequent to this event, the cytoplasmic domain of BlaR1 experiences phosphorylation, all within the time frame relevant to induction of resistance. The elucidation of the importance of this BlaR1 phosphorylation to the antibiotic resistance events is the subject of study under Specific Aim 1. PBP2a performs cross-linking of the cell wall in MRSA, a function that is indispensible to its survival. PBP2a is not inhibited well by ?-lactam antibiotics as it has a closed active site, hence its function in resistance. We have elucidated an allosteric site on this protein that is triggered to facilitate opening of the active site for the physiological role of the protein. The allosteric site is an Achiles'Heel of PBP2a, since its triggering for the opening of the active site would leave the protein (and MRSA) vulnerable to ?-lactam antibiotics that have met their obsolescence in treatment of infections by MRSA.
In Specific Aim 2 we propose to investigate how this protein performs its physiological role and how its processes can be subverted in devising new strategies in treatment of MRSA infections. Furthermore, we propose to study antibiotic resistance mechanisms that arise by alterations in the allosteric site.

Public Health Relevance

Methicillin-resistant Staphylococcus aureus (MRSA) has devised elaborate and highly effective strategies to evade the existing armamentarium of antibiotics that are available to us. Its resistance to -lactam antibiotics (such as penicillins;cephalosporins; carbapenems; etc.) is essentially overencompassing. Understanding of the details of these molecular events is critical for development of strategies to keep MRSA; as a problematic bacterial strain; in check in the clinical setting.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI104987-02
Application #
8600959
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Huntley, Clayton C
Project Start
2013-01-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
2
Fiscal Year
2014
Total Cost
$424,367
Indirect Cost
$145,178
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Frederick, Thomas E; Wilson, Brian D; Cha, Jooyoung et al. (2014) Revealing cell-surface intramolecular interactions in the BlaR1 protein of methicillin-resistant Staphylococcus aureus by NMR spectroscopy. Biochemistry 53:10-2
Fishovitz, Jennifer; Hermoso, Juan A; Chang, Mayland et al. (2014) Penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus. IUBMB Life 66:572-7
Blázquez, Blas; Llarrull, Leticia I; Luque-Ortega, Juan R et al. (2014) Regulation of the expression of the ?-lactam antibiotic-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA). Biochemistry 53:1548-50
Fishovitz, Jennifer; Rojas-Altuve, Alzoray; Otero, Lisandro H et al. (2014) Disruption of allosteric response as an unprecedented mechanism of resistance to antibiotics. J Am Chem Soc 136:9814-7
Otero, Lisandro H; Rojas-Altuve, Alzoray; Llarrull, Leticia I et al. (2013) How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function. Proc Natl Acad Sci U S A 110:16808-13