Mycobacterium abscessus l , d -Transpeptidases Are Susceptible to Inactivation by Carbapenems and Cephalosporins but Not Penicillins

Abstract

As a growing number of clinical isolates of are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of β-lactams determines their activity against Here, we studied the interactions of β-lactams with two l,d-transpeptidases in, namely, Ldt and Ldt, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of β-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with β-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible and clinical isolates that are resistant to most antibiotics, which suggests that dual-β-lactam therapy has potential for the treatment of Finally, we solved the first crystal structure of an l,d-transpeptidase, Ldt, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and β-lactams, which provide an insight into the molecular basis for the relative efficacy of different β-lactams against .

Select β-lactam combinations exhibit synergy against Mycobacterium abscessus in vitro

“…If only one type of enzyme existed as the target for β-lactams, the pairs would likely exhibit additive activity rather than synergy. Variation in the level of inhibition of L,D-transpeptidases of Mab (21) and Mtb (20, 24, 41) by agents of this antibiotic class supports this hypothesis. We further hypothesize that, at the molecular level, the structures of β-lactams that exhibit synergy against Mab most effectively complement the structure of the binding sites available in the transpeptidases of this organism; thereby favoring initial binding and subsequent interaction to bring about effective inhibition of the enzymes.…”

“…Of these, 12 combinations achieved MIC reductions below presumed breakpoints for both drugs. Although a few studies have been published describing synergy between β-lactams and other antibiotic classes against Mab in vitro (32, 34, 39, 40), only one has assessed synergy between dual β-lactams (21). Our current study encompasses a broader array of combinations and offers a more comprehensive analysis of the synergistic activity of the two major β-lactam subclasses currently used to treat Mab infections; cephalosporins and carbapenems.…”

“…An initial survey of the Mab genome identified five putative LDT encoding genes (19). These LDTs are differentially susceptible to β-lactam subclasses, with most carbapenems exhibiting strong inhibitory activities, followed by cephalosporins, and only a select few penicillins exhibiting moderate inhibition of these enzymes (20, 21).…”

“…As LDT and DDT activities are required for synthesis of Mab peptidoglycan, simultaneous inhibition of both enzymes could be bactericidal. Since these enzymes exhibit differential susceptibilities to β-lactams (21, 23, 24), we hypothesize that a combination of β-lactam subclasses; one that optimally inhibits LDTs and another that specifically targets DDTs, will demonstrate synergy in killing Mab . In this study, we have tested this hypothesis by assessing potencies of combinatorial pairs against Mab using a panel of 16 β-lactams representing cephalosporins and carbapenems.…”

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

“…These representative sequences were used to build a phylogenetic tree using the maximum likelihood method (Figure 3A). Homologs of Tae5 STM clustered into 5 main clades: clade 1 (red, 6332 sequences), to which Tae5 STM belongs, is composed of proteins containing the uncharacterized DUF2778 domain (DUF2778 superfamily); clade 2 (blue, 44540 sequences) contains the L,D-transpeptidases from Bacillus subtilis (Ldt Bs , PDB 1Y7M) (Bielnicki et al, 2006); clade 3 (green, 44090 sequences) contains L,D-transpeptidases from Enterococcus faecium (Ldt fm , PDB 1ZAT) (Biarrotte-Sorin et al, 2006), Mycobacterium abscessus (LdtMa b , PDB 5UWV) (Kumar et al, 2017) and Mycobacterium tuberculosis (Ldt Mt1-3 , PDB 3TUR, 5DCC) (Erdemli et al, 2012; Bianchet et al, 2017); clade 4 (purple, 25242 sequences) contains an enzyme from Helicobacter pylori (Cds6, PDB 4XZZ) that has a catalytic domain resembling L,D-transpeptidases but with L,D-carboxypeptidase activity (Kim et al, 2015); clade 5 (gray, 25765 sequences) contains an L,D-transpeptidase from E. coli (YcbB, PDB 6NTW) (Caveney et al, 2019) and proteins recognized by the Pfam model YkuD_2.…”