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J. Biol. Chem., Vol. 283, Issue 19, 12769-12776, May 9, 2008

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Restoration of Susceptibility of Methicillin-resistant Staphylococcus aureus to β-Lactam Antibiotics by Acidic pH

ROLE OF PENICILLIN-BINDING PROTEIN PBP 2a^*

Sandrine Lemaire¹, Cosimo Fuda, Françoise Van Bambeke², Paul M. Tulkens, and Shahriar Mobashery

From the Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, B-1200 Brussels, Belgium and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of β-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by β-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) β-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.

Received for publication, January 4, 2008 , and in revised form, March 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants AI33170 and GM 61629 in the United States. This work was also supported in Belgium by Fonds de la Recherche Scientifique Médicale Grant 3.4.597.06 [EC] , Fonds de la Recherche Scientifique travel fellowships (to S. L.), and Belgian Federal Science Policy Office Research Project P5/33 (research action P5). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. SP1.

² Maitre de Recherches of the Fonds de la Recherche Scientifique.

¹ Boursière of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture. To whom correspondence should be addressed: Unité de pharmacologie cellulaire et moléculaire, Université catholique de Louvain, UCL 73.70 Ave. E. Mounier 73, B-1200 Brussels, Belgium. Tel.: 32-2-7647375; Fax: 32-2-7647373; E-mail: sandrine.lemaire{at}facm.ucl.ac.be.

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