Insights into the L,D-Transpeptidases and D,D-Carboxypeptidase of Mycobacterium abscessus: Ceftaroline, Imipenem, and Novel Diazabicyclooctane Inhibitors

Mycobacterium abscessus is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating, with a focus on cell wall synthesis proteins (M. abscessus L,D-transpeptidases 1 to 5 [Ldt(Mab1) to Ldt(Mab5)] and D,D-carboxypeptidase) that are targeted by beta-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded beta-lactamase (Bla(Mab)). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC50 <= 0.25 mu g/ml; MIC90 <= 0.5 mu g/ml). Combining ceftaroline and imipenem with a beta-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the beta-lactams alone. In steady-state kinetic assays, Bla(Mab) exhibited a lower K-i app (0.30 +/- 0.03 mu M for avibactam and 136 +/- 14 mu M for relebactam) and a higher acylation rate for avibactam (k(2)/K= 3.4 x 10(5) +/- 0.4 x 10(5) M-1 s(-1) for avibactam and 6 x 10(2) +/- 0.6 x 10(2) M-1 s(-1) for relebactam). The k(cat)/K-m was nearly 10-fold lower for ceftaroline fosamil (0.007 +/- 0.001 mu M-1 s(-1)) than for imipenem (0.056 +/- 0.006 mu M-1 s(-1)). Timed mass spectrometry captured complexes of avibactam and Bla(Mab), Ldt(Mab1), Ldt(Mab2), Ldt(Mab4), and D,D-carboxypeptidase, whereas relebactam bound only Bla(Mab), Ldt(Mab1), and Ldt(Mab2). Interestingly, Ldt(Mab1), Ldt(Mab2), Ldt(Mab4), Ldt(Mab5), and D,D-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for Ldt(Mab1), Ldt(Mab2), Ldt(Mab4), and Ldt(Mab5) and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to Ldt(Mab1), and Ldt(Mab2) (e.g., K-i (app) or K-m of Ldt(Mab1), = 0.01 +/- 0.01 mu M for imipenem versus 0.73 +/- 0.08 mu M for ceftaroline fosamil). Molecular modeling indicates that Ldt(Mab2) readily accommodates imipenem, but the active site must widen to >= 8 angstrom for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (L,D-transpeptidases and D,D-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual beta-lactam combination in M. abscessus infections.