Open AccessInsights into the l , d -Transpeptidases and d , d -Carboxypeptidase of Mycobacterium abscessus: Ceftaroline, Imipenem, and Novel Diazabicyclooctane Inhibitors
Author(s) -
Khalid M Dousa,
Sebastian Kurz,
Magdalena A. Taracila,
Tracey L. Bonfield,
Christopher R. Bethel,
Melissa D. Barnes,
Suresh B. Selvaraju,
Ayman M. Abdelhamed,
Barry N. Kreiswirth,
W. Henry Boom,
Shan Kasperbauer,
Charles L. Daley,
Robert A. Bonomo
Publication year - 2020
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.00098-20
Subject(s) - imipenem , avibactam , mycobacterium abscessus , microbiology and biotechnology , chemistry , cephalosporin , antibiotics , mycobacterium , biochemistry , biology , bacteria , klebsiella pneumoniae , antibiotic resistance , genetics , escherichia coli , gene
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 β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (Bla Mab ). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC 50 ≤ 0.25 μg/ml; MIC 90 ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, Bla Mab exhibited a lower K i app (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam ( k 2 / K = 3.4 × 10 5 ± 0.4 × 10 5 M -1 s -1 for avibactam and 6 × 10 2 ± 0.6 × 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 μM -1 s -1 ) than for imipenem (0.056 ± 0.006 μM -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 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that Ldt Mab2 readily accommodates imipenem, but the active site must widen to ≥8 Å 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 β-lactam combination in M. abscessus infections.