Julie Couston

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 (DHFR _Nad ). We show that overexpression of DHFR _Nad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFR _Nad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFR _Nad 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. Highlights: First biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFR _Nad induces high-level trimethoprim resistance in N. asteroides . Crystal structure of DHFR _Nad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC ₅₀ data confirm strong DHFR _Nad inhibition by trimethoprim and methotrexate

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. β‐lactam antibiotics are of particular interest in this context. In this study, we aimed to address the β‐lactam susceptibility profile of a clinical strain of N. cyriacigeorgica and assessed whether it correlated with the enzymatic activity of purified β‐lactamase of the strain. We herein established that the strain is highly susceptible to imipenem and ceftriaxone, moderately susceptible to meropenem and resistant to amoxicillin. The resistance could be counteracted by β‐lactamase inhibitors from two distinct chemical classes: vaborbactam, and avibactam while clavulanate was less potent. We demonstrated that the β‐lactam susceptibility of the strain is in direct line with the enzymatic activity of purified NCY‐1, a class A β‐lactamase. NCY‐1 was indeed only active with amoxicillin but displayed poor activity towards other classes of β‐lactams. The NCY‐1 activity could be inhibited in vitro by vaborbactam, clavulanate, and avibactam. We consolidated these data by determining the high‐resolution structure of NCY‐1 bound to avibactam. The structural analysis supported a conserved inhibitor binding site among other Nocardia class A β‐lactamases strongly suggesting a broad inhibition spectrum of avibactam across Nocardia species.

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. β-lactam antibiotics are of particular interest in this context. In this study, we aimed to address the β-lactam susceptibility profile of a clinical strain of N. cyriacigeorgica and assessed whether it correlated with the enzymatic activity of purified β-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 β-lactamase inhibitors from two distinct chemical classes: vaborbactam, and avibactam while clavulanate was less potent. We demonstrated that the β-lactam susceptibility of the strain is in direct line with the enzymatic activity of purified NCY-I, a class A β-lactamase. NCY-I was indeed only active with amoxicillin but displayed poor activity towards other classes of β-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 β-lactamases strongly suggesting a broad inhibition spectrum of avibactam across Nocardia species.

The mycobacterial outer membrane is composed of unusual lipids and glycolipids. Some of these lipids are exported to the cell envelope by resistance‐nodulation‐division (RND) transporters called mycobacterial membrane protein large (MmpL). While the oligomeric state of most RND transporters is well established, MmpL assembly remains unclear. Here, we investigated MmpL10, the trehalose polyphleate transporter. Biochemical data suggest that MmpL10 forms a homotrimer and that its oligomerization is driven by a coiled‐coil domain. Structural modeling and electron microscopy data reveal the presence of a tubular extension that spans the mycobacterial cell wall and reaches the mycomembrane. As most MmpL proteins possess this extension, oligomerization may be a common feature of this family of transporters, possibly involved in the transport of the MmpL cargo.

Nocardia are Gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered to be opportunist pathogens, as they often infect immunocompromised patients. Although their clinical incidence is low, many Nocardia species are now considered to be emerging pathogens. Primary sites of infection by 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 are tackled with multiple classes of antibiotics, in addition to the traditional treatment targeting the folate pathway. β-Lactams are often included in the regimen, but many Nocardia species present moderate or strong resistance to some members of this drug class. Genomic, microbiological and biochemical studies have reported the presence of class A β-lactamases (ABLs) in a handful of Nocardia species, but no structural investigation of Nocardia β-lactamases has yet been performed. In this study, the expression, purification and preliminary biochemical characterization of an ABL from an N. cyriacigeorgica (NCY-1) clinical strain are reported. The crystallization and the very high resolution crystal structure of NCY-1 are also described. The sequence and structural analysis of the protein demonstrate that NCY-1 belongs to the class A1 β-lactamases and show its very high conservation with ABLs 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. β-lactamases.

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. ß-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 ß-lactamases (ABL) in a handful of Nocardia species but no structural investigation of Nocardia ß-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 ß-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. ß-lactamases. Synopsis: The crystal structure at high resolution of a class A ß-lactamase from a clinical strain of Nocardia cyriacigeorgica is reported.

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two-component flavin-dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X-ray structures in apo, ligand-bound and cofactor-bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two-component flavin-dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogenMycobacterium abscessusis presented. The structure solved at high resolution displays high similarity to homologs fromRhodococcus,PaenibacillusandPseudomonasspecies.In silicodocking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two-component flavin-dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria.