Biology subjects

Calvopina, K.

Publications and source records attributed to Calvopina, K..

4 recordsLinked to original sources

bioRxiv2025

Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis

{beta}-Lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimisation of the classical {beta}-lactam scaffolds or development of new or non-{beta}-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of {beta}-lactams include L,D-transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the {beta}-lactam antibacterial mechanisms are not understood. We used fluorogenic substrate mimics to investigate the effects of {beta}-lactams and reported LdtMt2 inhibitors on Mycobacterium smegmatis (Msm), assessing their impacts on the transpeptidase activities of Ldts and PBPs in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; though under the tested conditions a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, inhibition of both PBPs and Ldts was observed in all active inhibitors, though {beta}-lactams manifest increased potency of PBP inhibition. The combination of the {beta}-lactams meropenem and faropenem with selected LdtMt2 inhibitors manifested an additive inhibitory effect against Msm. Our results highlight the importance of further optimising PBPs and Ldt transpeptidase inhibition, particularly of Ldts, to improve {beta}-lactam efficacy versus mycobacteria.

biochemistry↗

bioRxiv2024

Structure and dynamics of Burkholderia pseudomallei OXA-57, a distinctive low-efficiency class D β-lactamase with carbapenem-hydrolyzing activity

The Gram-negative bacterium Burkholderia pseudomallei causes the severe disease melioidosis. {beta}-Lactams, including carbapenems, are the primary treatment, but are susceptible to chromosomal {beta}-lactamases, including the class D enzyme OXA-57. Here we show recombinant OXA-57 is active towards penicillins and first-generation cephalosporins, slowly hydrolyzes carbapenems including imipenem and meropenem, but is inactive towards oxyimino-cephalosporins (e.g., ceftazidime). Unlike many OXA enzymes, OXA-57 is sensitive to the mechanism-based inhibitor clavulanic acid, but less so to the diazabicyclooctane avibactam and not to the cyclic boronate vaborbactam. Crystal structures of covalent OXA-57:avibactam and OXA-57:meropenem complexes reveal limited hydrogen- bonding interactions with bound ligands. In molecular dynamics simulations, bound meropenem is mobile, while the water necessary for deacylation has only limited active site access. These observations are consistent with the low level of meropenem turnover, supporting proposals that OXA {beta}-lactamases generally possess limited carbapenemase activity, and highlight the potential importance of OXA-57 in B. pseudomallei {beta}-lactam resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/630153v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@d635d7org.highwire.dtl.DTLVardef@168d67borg.highwire.dtl.DTLVardef@1d95733org.highwire.dtl.DTLVardef@1ebeabb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Novel mechanisms of efflux-mediated levofloxacin resistance and reduced amikacin susceptibility in Stenotrophomonas maltophilia.

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multi-factorial, but the most significant factor is production of efflux pumps, particularly SmeDEF. Here we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVR, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed as SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVR causing over-production of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Over-production of one ABC transporter, Smlt1651-4 (renamed as SmaCDEF) causes levofloxacin resistance in K M6 LEVR. Over-production of the other ABC transporter, Smlt2642/3 (renamed SmaAB) and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVR. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia, and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

microbiology↗

bioRxiv2019

TonB dependent uptake of β-lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia.

The {beta}-lactam antibiotic ceftazidime is one of only a handful of drugs with proven clinical efficacy against the opportunistic human pathogen Stenotrophomonas maltophilia, Here, we show that mutations in the energy transducer TonB, encoded by smlt0009 in S. maltophilia, confer ceftazidime resistance. This breaks the dogma that {beta}-lactams enter Gram-negative bacteria only by passive diffusion through outer membrane porins. By confirming cross-resistance of Smlt0009 mutants with a siderophore-conjugated lactivicin antibiotic, we reveal that attempts to improve penetration of antimicrobials into Gram negative bacteria by conjugating them with TonB substrates is likely to select {beta}-lactam resistance in S. maltophilia, increasing its clinical threat. Furthermore, we show that S. maltophilia clinical isolates that have an Smlt0009 mutation already exist. Remarkably, therefore, {beta}-lactam use is already eroding the potential utility of currently experimental siderophore-conjugated antimicrobials against this species.

microbiology↗