Biology subjects

Couston, J.

Publications and source records attributed to Couston, J..

3 recordsLinked to original sources

bioRxiv2026

Biochemical, structural, and functional characterization of the Nocardia asteroides dihydrofolate reductase: a primary target of anti-nocardiosis treatment

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

microbiology↗

bioRxiv2025

NCY-I beta-lactamase activity correlates with antimicrobial susceptibility of a clinical strain of Nocardia cyriacigeorgica

Nocardiosis is an infectious disease caused by several Nocardia species, among which Nocardia cyriacigeorgica is one of the most frequently isolated species in the clinic. Albeit most isolates of this species are susceptible to standard treatment combining trimethoprim and sulfamethoxazole, resistance has been reported, necessitating alternative or combination therapies. {beta}-lactam antibiotics are of particular interest in this context. In this study, we aimed to address the {beta}-lactam susceptibility profile of a clinical strain of N. cyriacigeorgica and assessed whether it correlated with the enzymatic activity of purified {beta}-lactamase of the strain. We herein established that the strain is highly susceptible to imipenem and ceftriaxone, moderately sensitive to meropenem and resistant to amoxicillin. The resistance could be counteracted by {beta}-lactamase inhibitors from two distinct chemical classes: vaborbactam, and avibactam while clavulanate was less potent. We demonstrated that the {beta}-lactam susceptibility of the strain is in direct line with the enzymatic activity of purified NCY-I, a class A {beta}-lactamase. NCY-I was indeed only active with amoxicillin but displayed poor activity towards other classes of {beta}-lactams. The NCY-I activity could be inhibited in vitro by vaborbactam, clavulanate and avibactam. We consolidated these data by determining the high-resolution structure of NCY-I bound to avibactam. The structural analysis supported a conserved inhibitor binding site among other Nocardia class A {beta}-lactamases strongly suggesting a broad inhibition spectrum of avibactam across Nocardia species.

microbiology↗

bioRxiv2023

Crystal structure of a Class A beta-lactamase from Nocardia cyriacigeorgica

Nocardia are gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered opportunist pathogens as they often infect immunocompromised patients. Albeit, their clinical incidence is low, many Nocardia species are nowadays considered emerging pathogens. Primary sites of infection of Nocardia are the skin or the lungs but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus, tackled with multiple classes of antibiotics, on top of the traditional treatment targeting the folate pathway. {beta}-lactams are often included in the regimen but many Nocardia species present moderate or strong resistance to some of this drug class. Genomic, microbiological, and biochemical studies have reported the presence of class A {beta}-lactamases (ABL) in a handful of Nocardia species but no structural investigation of Nocardia {beta}-lactamases has been performed yet. In this study, we report the expression, purification, and preliminary biochemical characterization of the ABL from a Nocardia cyriacigeorgica (NCY-1) clinical strain. We describe, as well, the crystallization and the very high-resolution crystal structure of NCY-1. The protein sequence and structural analysis demonstrate that NCY-1 belongs to {beta}-lactamase of class A1 and attest to its very high conservation with ABL from other human pathogenic Nocardia. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target Nocardia spp. {beta}-lactamases. SynopsisThe crystal structure at high resolution of a class A {beta}-lactamase from a clinical strain of Nocardia cyriacigeorgica is reported.

biochemistry↗