The l,d-Transpeptidase Ldt_Ab from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture

Abstract

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3–3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A. baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 β-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance

“…Other beta-lactams such as cephems (cephalosporin) are poor inhibitors since acylation is slow and the thioester bond formed in the enzyme–antibiotic adduct is prone to hydrolysis ( 15 ). This has been shown for model organisms including E. faecium ( 15 ), Mycobacterium tuberculosis ( 16 ), and C. difficile Ldts ( 8 ) and is true for most Ldts studied to date despite some exceptions for M. tuberculosis Ldt Mt5 ( 16 ), Acinetobacter baumanii Ldt Ab ( 17 ), and C. difficile Ldt Cd3 ( 8 ), which are not inhibited by carbapenems. The contribution of Ldts to beta-lactam resistance has been documented in E. faecium ( 18 ), M. tuberculosis ( 19 ), Mycobacterium smegmatis ( 20 ), and A. baumanii ( 17 ).…”

“…This has been shown for model organisms including E. faecium ( 15 ), Mycobacterium tuberculosis ( 16 ), and C. difficile Ldts ( 8 ) and is true for most Ldts studied to date despite some exceptions for M. tuberculosis Ldt Mt5 ( 16 ), Acinetobacter baumanii Ldt Ab ( 17 ), and C. difficile Ldt Cd3 ( 8 ), which are not inhibited by carbapenems. The contribution of Ldts to beta-lactam resistance has been documented in E. faecium ( 18 ), M. tuberculosis ( 19 ), Mycobacterium smegmatis ( 20 ), and A. baumanii ( 17 ). In C. difficile , the inactivation of two of the three Ldts did not lead to a change in beta-lactam resistance ( 6 ).…”

Collateral Changes in Cell Physiology Associated with ADC-7 β-Lactamase Expression in Acinetobacter baumannii

“…ADC-7 cleaved between the d -Ala- d -Ala of the pentapeptide substrate, but not between the l -Lys- d -Ala of the tetrapeptide substrate, indicating that ADC-7 displays weak d,d -carboxypeptidase function. This suggests that ADC-7 may be spatially or temporally associated with LdtJ, the recently discovered l,d- transpeptidase responsible for the formation of 3,3-cross-links in A. baumannii ( 55 – 57 ).…”

Ferramentas de bioinformática aplicadas a busca racional por moléculas contra Acinetobacter baumannii

“…Das dez melhores moleculas contra a PBP, nenhuma apresenta em sua estrutura algum anel semelhante ao anel β-lactâmico, que é responsável pelo mecanismo de ação contra PBP's. Esse fenômeno pode ser explicado pelo fato de que das 295 moléculas obtidas do virtual screening baseado no ligante, nenhuma apresenta de fato um anel β-lactâmico, mas sim estruturas similares menos eletrofílicas, o que as torna menos relevante para interação com a PBP quando comparadas em termos de eletrofilicidade ao anel β-lactâmico (Toth et al, 2022).…”