Peptidoglycan cross-linking activity of L,D-transpeptidases from Clostridium difficile and inactivation of theses enzymes by {beta}-lactams [PublishAheadOfPrint]

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 have purified the three putative C. difficile L,D-transpeptidases paralogues, Ldt_Cd1, Ldt_Cd2, and Ldt_Cd3, which have been previously identified by sequence analysis. The catalytic activity of the three proteins was assayed with a disaccharide-tetrapeptide purified form 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 by D-Met. Ldt_Cd1 only displayed 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 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 only inactivated by β-lactams of the carbapenem class due to a combination of rapid acylation coupled to the stability of the resulting covalent adducts.

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