Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant bacteria pathogen that has become a global clinical threat, accounting for nearly one-third of hospital-acquired infections. The MRSA threat is especially insidious due to its resistance to 2-lactam antibiotics (e.g. penicillins, carbapenems, cephalosporins), which remain the most widely used anti-infectives in the clinic. Elucidating the molecular basis for MRSA drug resistance is therefore imperative to develop strategies for its containment. A principal resistance mechanism of MRSA is the production of a ?-lactamase that hydrolytically destroys ?-lactam antibiotics. This production is triggered by the transmembrane sensor/transducer protein BlaR1. Exposure to a ?-lactam antibiotic triggers signal transduction by BlaR1, which leads to ?-lactamase production. Recent CD and IR studies demonstrate that the signal transduction entails conformational transitions in the extracellular sensor domain of BlaR1 (BlaRS henceforth) upon its binding of ?-lactam antibiotics. Defining these conformational transitions at the atomic level has become a critical goal in efforts to elucidate the signal transduction mechanism. Despite the availability of high-resolution protein structures, the atomic-level mechanism for the conformational transitions driving BlaR1 signal transduction remains obscure. We have begun studies of BlaRS, and our preliminary results point to a new set of molecular factors important for the BlaRS conformational transitions: protein conformational dynamics. Specifically, using solution Nuclear Magnetic Resonance (NMR), we observe that ?-lactam binding causes site-specific changes in local BlaRS flexibility. Hence, to elucidate the key events underlying BlaR1 signal transduction, we will investigate the functional consequences of BlaRS conformational dynamics. Accordingly, we propose three Specific Aims that will define how the interplay between protein dynamics and conformational change facilitates signal transduction on the part of BlaRS.
These Aims i nclude: (i) Comparing the site-specific changes in BlaRS flexibility upon activation by a series of different ?-lactam substrates;(ii) Defining the mechanism of interaction between BlaRS, and a peptide derived from the extra-cellular Loop 2 of the trans-membrane region of the BlaR1 receptor;(iii) Compare the effects of non-??-lactam inhibitors of BlaR1 versus ?-lactam substrates (antibiotics), on the dynamics and conformation of BlaRS. Our main experimental tool will be multi-dimensional NMR, which provides a uniquely powerful method for the atomic-level description of protein dynamics on a per- amino-acid-residue basis. Our studies will shed new light on the inner-workings of the signal transduction mechanism responsible for pernicious ?-lactam antibiotic resistance in MRSA.

Public Health Relevance

This proposal describes studies to elucidate the molecular mechanisms whereby the sensor transducer protein, BlaR1, facilitates antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA), currently a global clinical scourge. The results will advance our abilities to cope with acceleration of multi-drug resistance in MRSA and other bacterial pathogens.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Wehrle, Janna P
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University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
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Staude, Michael W; Frederick, Thomas E; Natarajan, Sivanandam V et al. (2015) Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus. Biochemistry 54:1600-10
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
Borbulevych, Oleg; Kumarasiri, Malika; Wilson, Brian et al. (2011) Lysine Nzeta-decarboxylation switch and activation of the beta-lactam sensor domain of BlaR1 protein of methicillin-resistant Staphylococcus aureus. J Biol Chem 286:31466-72