Peptidoglycan Cross-Linking Activity of l , d -Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams

Abstract

ABSTRACT In most bacteria, the essential targets of β-lactam antibiotics are the d , d -transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l , d -transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l , d -transpeptidase paralogues Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 , which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt Cd2 and Ldt Cd3 catalyzed the formation of 3→3 cross-links ( l , d -transpeptidase activity), the hydrolysis of the C-terminal d -Ala residue of the disaccharide-tetrapeptide substrate ( l , d -carboxypeptidase activity), and the exchange of the C-terminal d -Ala for d -Met. Ldt Cd1 displayed only l , d -carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt Cd1 and Ldt Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt Cd1 and Ldt Cd2 was higher for the carbapenems (480 to 6,600 M −1 s −1 ) than for ampicillin and ceftriaxone (3.9 to 82 M −1 s −1 ). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt Cd1 and Ldt Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt Cd1 and Ldt Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.