Jérôme Feuillard

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.

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending of the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.