Peptidoglycan cross-linking activity of l,d-transpeptidases from clostridium difficile and inactivation of these enzymes by β-lactams

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 4Å3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3Å3 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 LdtCd1, LdtCd2, and LdtCd3, 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. LdtCd2 and LdtCd3 catalyzed the formation of 3Å3 cross-links (L,Dtranspeptidase 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. LdtCd1 displayed only L,D-carboxypeptidase activity. Mass spectrometry analyses indicated that LdtCd1 and LdtCd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of LdtCd3 by these β-lactams was not detected. The acylation efficacy of LdtCd1 and LdtCd2 was higher for the carbapenems (480 to 6,600 Mβ1 sβ1) than for ampicillin and ceftriaxone (3.9 to 82 Mβ1 sβ1). In contrast, the efficacy of the hydrolysis of β-lactams by LdtCd1 and LdtCd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that LdtCd1 and LdtCd2 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.