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ObjectiveTo describe the clinical characteristics and outcomes of coronavirus disease 2019 (COVID-19) among patients with myasthenia gravis (MG) and identify factors associated with COVID-19 severity in patients with MG.MethodsThe CO-MY-COVID registry was a multicenter, retrospective, observational cohort study conducted in neuromuscular referral centers and general hospitals of the FILNEMUS (Filière Neuromusculaire) network (between March 1, 2020, and June 8, 2020), including patients with MG with a confirmed or highly suspected diagnosis of COVID-19. COVID-19 was diagnosed based on a PCR test from a nasopharyngeal swab or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology, thoracic CT scan, or typical symptoms. The main outcome was COVID-19 severity based on location of treatment/management (home, hospitalized in a medical unit, or in an intensive care unit). We collected information on demographic variables, general history, and risk factors for severe COVID-19. Multivariate ordinal regression models were used to identify factors associated with severe COVID-19 outcomes.ResultsAmong 3,558 patients with MG registered in the French database for rare disorders, 34 (0.96%) had COVID-19. The mean age at COVID-19 onset was 55.0 ± 19.9 years (mean MG duration: 8.5 ± 8.5 years). By the end of the study period, 28 patients recovered from COVID-19, 1 remained affected, and 5 died. Only high Myasthenia Gravis Foundation of America (MGFA) class (≥IV) before COVID-19 was associated with severe COVID-19 (p = 0.004); factors that were not associated included sex, MG duration, and medium MGFA classes (≤IIIb). The type of MG treatment had no independent effect on COVID-19 severity.ConclusionsThis registry-based cohort study shows that COVID-19 had a limited effect on most patients, and immunosuppressive medications and corticosteroids used for MG management are not risk factors for poorer outcomes. However, the risk of severe COVID-19 is elevated in patients with high MGFA classes (odds ratio, 102.6 [4.4–2,371.9]). These results are important for establishing evidence-based guidelines for the management of patients with MG during the COVID-19 pandemic.

International audience; Despite the impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and /composition in response to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes in its structure, but no reports exist supportings this assumption. Herein, using [MS2] recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we identified found that the mycobacterial protein GlfH1 (Rv3096), which protein exhibits an exo-β-D-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents the a first step towards understanding the remodeling of mycobacterial AG.

Objective Chronic inflammatory demyelinating polyradiculoneuropathies (CIDP) with antibodies against neurofascin 155 (Nfasc155) or contactin-1 (CNTN1) have distinctive clinical features. Knowledge on their electrophysiological characteristics is still scarce. In this study, we are investigating whether these patients have specific electrophysiological characteristics. Methods The electrophysiological data from 13 patients with anti-Nfasc155 IgG4 antibodies, 9 with anti-CNTN1 IgG4 antibodies were compared with those of 40 consecutive CIDP patients without antibodies. Results All the patients with antibodies against Nfasc155 or CNTN1 fulfilled the EFNS/PNS electrodiagnostic criteria for definite CIDP. There was no electrophysiological difference between patients with anti-CNTN1 and anti-Nfasc155 antibodies. Nerve conduction abnormalities were heterogeneously distributed along nerves trunks and roots. They were more pronounced than in CIDP without antibodies. Motor conduction velocity on median nerve 7.4 ms were predictive of positive antibodies against the node of Ranvier with a sensitivity of 59% and a specificity of 93%. Conclusions Marked conduction abnormalities may suggest the presence of positive antibodies against the node of Ranvier. Significance Anti-Nfasc155 and anti-CNTN1 antibodies target the the paranodal axo-glial domain but are associated with nerve conduction abnormalities mimicking a “demyelinating” neuropathy.

Mycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-D-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3Msm) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3Msm expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3Msm was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium deoxycholate that favor solubilization and stabilization of MmpL3Msm in solution. In addition, we report a protocol that allows reconstitution of MmpL3Msm into peptidiscs.

Mycobacterium abscessusest une bactérie non tuberculeuse, environnementale, à croissance rapide, qui est responsable d’infections pulmonaires sévères, notamment chez les patients atteints de mucoviscidose. Le traitement actuel combine l’utilisation d’une b-lactamine et d’un aminoglycoside associés à un macrolide. Cette bactérie est polyrésistante à la plupart des antibiotiques utilisés en clinique. Les mécanismes de résistance, innés ou acquis, qu’elle a développés, conduisent fréquemment à des échecs thérapeutiques, ce qui limite considérablement les moyens de lutte disponibles pour le clinicien. Une compréhension globale des mécanismes de résistance de cette bactérie s’avère ainsi nécessaire pour contrer les infections pulmonaires qu’elle provoque.

Background Due to their health condition, patients with neuromuscular diseases (NMD) are at greater risk of developing serious complications with COVID-19. The objective of this study was to analyze the prevalence of COVID-19 among NMD patients and the risk factors for its impact and severity during the first wave of the pandemic. Clinical data were collected from NMD-COVID-19 patients, between March 25, 2020 and May 11, 2020 in an anonymous survey carried out by expert physicians from the French Health Care Network Filnemus. Results Physicians reported 84 patients, including: 34 with myasthenia gravis, 27 with myopathy and 23 with neuropathy. COVID-19 had no effect on NMD for 48 (58%) patients and 48 (58%) patients developed low COVID-19 severity. COVID-19 caused the death of 9 (11%) NMD patients. Diabetic patients were at greater risk of dying. Patients with diabetes, hypertension or severe forms of NMD had a higher risk of developing a moderate or severe form of COVID-19. In our cohort, corticosteroids and other immunosuppressants were not significantly associated with higher COVID-19 severity for acquired NMD. Conclusion During this period, a small percentage of French NMD patients was affected by COVID-19 compared to the general French population and COVID-19 had a limited short-term effect on them. Diabetes, hypertension and a severe degree of NMD were identified as risk factors of unfavorable outcome following COVID-19. Conversely, in our cohort of patients with acquired NMD, corticosteroids or other immunosuppressants did not appear to be risk factors for more severe COVID-19.

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis., Author summary Current antimycobacterial drugs are inadequate to handle the increasing number of non-tuberculous mycobacteria infections that eclipse tuberculosis infections in many developed countries. Of particular importance for cystic fibrosis patients, Mycobacterium abscessus is notoriously difficult to treat where patients spend extended time on antibiotics with cure rates comparable to extreme drug resistant M. tuberculosis. Here, we identified epetraborole (EPT) with in vitro and in vivo activities against M. abscessus. We showed that EPT targets the editing domain of the leucyl-tRNA synthetase (LeuRS) and that escape mutants lost LeuRS editing activity, making these mutants susceptible to misaminoacylation with leucine mimics. Most importantly, combination therapy of EPT and norvaline limited the rate of EPT resistance in both M. abscessus and M. tuberculosis, and this was the first study to demonstrate improved in vivo efficacy of EPT and norvaline compared to EPT in a murine model of M. abscessus pulmonary infection. The demonstration of norvaline adjunct therapy with EPT for M. abscessus infections is promising for cystic fibrosis patients and could translate to other mycobacterial infections, such as tuberculosis.

Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper.

Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug-resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N-acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high-resolution crystal structures of Mabs_Eis2 in its apo- and cofactor-bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high-resolution crystal structures presented here may pave the way to the design of Eis2-specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. DATABASE: Structural data are available in the PDB database under the accession numbers: 6RFY, 6RFX and 6RFT.

International audience; This manuscript discloses the design and synthesis of a series of C-4 functionalized 1,8-naphthalimide-heterocyclic hydrazide conjugates along with their anti-mycobacterial and cytotoxic evaluation. The present work assumes significance as it describes the first report on the amalgamation of C-4 substituted naphthalimides with various heterocyclic hydrazides. However, contrary to the rationale behind the synthesis of the conjugates, none of them inhibited the growth of Mycobacterium tuberculosis at the tested concentrations though these were non-cytotoxic towards the Vero kidney epithelial cell line.

Background Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune-mediated inflammatory disease of the peripheral nervous system characterized by a response directed against certain myelin proteins and for which therapies are limited. Previous studies have suggested a beneficial role of FTY720, a sphingosine 1-phosphate (S1P) receptor agonist, known to deplete lymphocytes from the peripheral blood by sequestering them into lymph nodes, in the treatment of experimental autoimmune neuritis (EAN). Therefore, we investigated whether FTY720 is also beneficial in chronic experimental autoimmune neuritis (c-EAN), a recently developed rat model mimicking human CIDP. Methods c-EAN was induced in Lewis rats by immunization with S-palm P0(180–199) peptide. Rats were treated with FTY720 (1 mg/kg) or vehicle intraperitoneally once daily from the onset of clinical signs for 18 days; clinical signs were assessed daily until 60 days post-immunization (dpi). Electrophysiological and histological features were examined at different time points. We also evaluated the serum levels of different pro- and anti-inflammatory cytokines by ELISA or flow cytometry at 18, 40, and 60 dpi. Results Our data demonstrate that FTY720 decreased the severity and abolished the chronicity of the disease in c-EAN rats. Therapeutic FTY720 treatment reversed electrophysiological and histological anomalies, suggesting that myelinated fibers were subsequently preserved, it inhibited macrophage and IL-17+ cell infiltration in PNS, and it significantly reduced circulating pro-inflammatory cytokines. Conclusions FTY720 treatment has beneficial effects on c-EAN, a new animal model mimicking human CIDP. We have shown that FTY720 is an effective immunomodulatory agent, improving the disease course of c-EAN, preserving the myelinated fibers, attenuating the axonal degeneration, and decreasing the number of infiltrated inflammatory cells in peripheral nerves. These data confirm the interest of testing FTY720 or molecules targeting S1P in human peripheral neuropathies. Electronic supplementary material The online version of this article (10.1186/s12974-019-1441-4) contains supplementary material, which is available to authorized users.

International audience; Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 μg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 μg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.

International audience; Purpose: Diagnosis of inherited ataxia and related diseases represents a real challenge given the tremendous heterogeneity and clinical overlap of the various causes. We evaluated the efficacy of molecular diagnosis of these diseases by sequencing a large cohort of undiagnosed families.Methods: We analyzed 366 unrelated consecutive patients with undiagnosed ataxia or related disorders by clinical exome-capture sequencing. In silico analysis was performed with an in-house pipeline that combines variant ranking and copy-number variant (CNV) searches. Variants were interpreted according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines.Results: We established the molecular diagnosis in 46% of the cases. We identified 35 mildly affected patients with causative variants in genes that are classically associated with severe presentations. These cases were explained by the occurrence of hypomorphic variants, but also rarely suspected mechanisms such as C-terminal truncations and translation reinitiation.Conclusion: A significant fraction of the clinical heterogeneity and phenotypic overlap is explained by hypomorphic variants that are difficult to identify and not readily predicted. The hypomorphic C-terminal truncation and translation reinitiation mechanisms that we identified may only apply to few genes, as it relies on specific domain organization and alterations. We identified PEX10 and FASTKD2 as candidates for translation reinitiation accounting for mild disease presentation.

Several infectious pathologies in humans, such as tuberculosis or SARS-CoV-2, are responsible for tissue or lung damage, requiring regeneration. The regenerative capacity of adult mammals is limited to few organs. Critical injuries of non-regenerative organs trigger a repair process that leads to a definitive architectural and functional disruption, while superficial wounds result in scar formation. Tissue lesions in mammals, commonly studied under non-infectious conditions, trigger cell death at the site of the injury, as well as the production of danger signals favouring the massive recruitment of immune cells, particularly macrophages. Macrophages are also of paramount importance in infected injuries, characterized by the presence of pathogenic microorganisms, where they must respond to both infection and tissue damage. In this review, we compare the processes implicated in the tissue repair of non-infected versus infected injuries of two organs, the skeletal muscles and the lungs, focusing on the primary role of macrophages. We discuss also the negative impact of infection on the macrophage responses and the possible routes of investigation for new regenerative therapies to improve the recovery state as seen with COVID-19 patients.

International audience; A series of 4-aminoquinoline-isoindoline-dione-isoniazid triads were synthesized and assessed for their anti-mycobacterial activities and cytotoxicity. Most of the synthesized compounds exhibited promising activities against the mc26230 strain of M. tuberculosis with MIC in the range of 3.125-12.5 µg/mL and were non-cytotoxic against Vero cells. The conjugates lacking either isoniazid or quinoline core in their structural framework failed to inhibit the growth of M. tuberculosis; thus, further strengthening the proposed design of triads in the present study.

International audience; Enhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5-related N-acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In non-tuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multi-drug resistant NTM, possesses two functionally distinct Eis homologues, Eis1 Mab and Eis2 Mab. While Eis2 Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1 Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1 Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1 Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl-CoA. The structure revealed that Eis1 Mab has a typical homohexameric Eis-like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1 Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate-binding pocket to these compounds in Eis1 Mab. Overall, this study underscores the molecular determinants explaining functional differences between Eis1 Mab and Eis2 Mab, especially those inherent to their capacity to modify aminoglycosides.

International audience

Background: Data are needed on long-term effect of natalizumab (NTZ) in relapsing-remitting multiple sclerosis (RRMS). Objectives: To evaluate the time of onset of secondary progressive phase in patients with an RRMS treated with NTZ and to investigate predictive factors. Methods: TYSTEN is an observational study. Patients starting NTZ between 2007 and 2012 were included and followed up until October 2018. Relapses, Expanded Disability Status Scale (EDSS) scores, and results of brain magnetic resonance imaging (MRI) were collected each year. Data were used to estimate the cumulative probability of several poor outcomes such as secondary progressive multiple sclerosis (SPMS) conversion, EDSS worsening, EDSS 4.0, and EDSS 6.0. Results: 770 patients were included. The mean follow-up duration was 97 months and the mean time exposure to NTZ was 66 months. At 10 years, the cumulative probability of SPMS was 27.7%. Predictive factors for poor outcomes were a ⩾1-point increase in EDSS score from baseline, new T2 lesion or T1 gadolinium-enhancing lesion, the occurrence of relapse at 1 or 2 years and No Evidence of Disease Activity (NEDA-3; no relapse, no new T2 or T1 gadolinium-enhancing lesions, no progression) was a protective factor. Conclusion: In our cohort of patients treated with NTZ, poor outcomes were infrequent and are driven by disease activity.

International audience; In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an "oxazepino-indole" structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.

Summary: Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses. : Bernut et al. investigate the mechanism by which cystic fibrosis patients are vulnerable to Mycobacterium abscessus infection. Using zebrafish, they show that dysfunction of CFTR reduces both macrophage bactericidal activity and neutrophil recruitment to the forming protective granulomas. Together, this leads to hypersusceptibility to M. abscessus infection and larval death. Keywords: cystic fibrosis, Mycobacterium abscessus, CFTR, NADPH oxidase, zebrafish, pathogenesis, innate immunity

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics, such as bedaquiline and imipenem, although in vitro data have questioned this combination. We report that the efficacy of bedaquiline-imipenem combination treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus . The addition of imipenem contributed to clearing the infection in the spleen.

Non‐tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus, are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis, the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus. Structure‐activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD‐88 as a new promising active analogue against M. abscessus. Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half‐life of 3.2 hours., Molecular modelling: Molecular representation of the interaction of PIPD1 and M. abscessus MmpL3 predictive model. On one side, PIPD1 is hydrogen‐bound to the carboxylic acid group of D618 through its amino moiety. On the other side, its hydroxyl group is H‐bound to the hydroxyl of Y219 and a potent interaction with the carboxylic acid group of D258 can occur. A stabilization occurred through π‐π interactions between the toluene moiety of PIPD1 and phenyl rings of F262 and F622.

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments., info:eu-repo/semantics/published

International audience; Macrolides are the cornerstone of Mycobacterium abscessus multidrug therapy, despite that most patients respond poorly to this class of antibiotics due to the inducible resistance phenotype that occurs during drug treatment. This mechanism is driven by the macrolide-inducible ribosomal methylase encoded by erm(41), whose expression is activated by the transcriptional regulator WhiB7. However, it has been debated whether clarithromycin and azithromycin differ in the extent to which they induce erm(41)-mediated macrolide resistance. Herein, we show that macrolide resistance is induced more rapidly in various M. abscessus isolates upon exposure to azithromycin than to clarithromycin, based on MIC determination. Macrolide-induced expression of erm(41) was assessed in vivo using a strain carrying tdTomato placed under the control of the erm(41) promoter. Visualization of fluorescent bacilli in infected zebrafish demonstrates that azithromycin and clarithromycin activate erm(41) expression in vivo. That azithromycin induces a more rapid expression of erm(41) was confirmed by measuring the ␤-galactosidase activity of a reporter strain in which lacZ was placed under the control of the erm(41) promoter. Shortening the promoter region in the lacZ reporter plasmid identified DNA elements involved in the regulation of erm(41) expression, particularly an AT-rich motif sharing partial conservation with the WhiB7-binding site. Mutation of this motif abrogated the macrolide-induced and WhiB7-dependent expression of erm(41). This study provides new mechanistic information on the adaptive response to macrolide treatment in M. abscessus.

Infections caused by non-tuberculous mycobacteria (NTM) are increasing globally and are notoriously difficult to treat due to intrinsic resistance of these bacteria to many common antibiotics. NTM are diverse and ubiquitous in the environment, with only a few species causing serious and often opportunistic infections in humans, including Mycobacterium abscessus. This rapidly growing mycobacterium is one of the most commonly identified NTM species responsible for severe respiratory, skin and mucosal infections in humans. It is often regarded as one of the most antibiotic-resistant mycobacteria, leaving us with few therapeutic options. In this Review, we cover the proposed infection process of M. abscessus, its virulence factors and host interactions and highlight the commonalities and differences of M. abscessus with other NTM species. Finally, we discuss drug resistance mechanisms and future therapeutic options. Taken together, this knowledge is essential to further our understanding of this overlooked and neglected global threat. Non-tuberculous mycobacteria, such as Mycobacterium abscessus, are an increasing global health burden, in part due their extensive drug resistance. In this Review, Johansen, Herrmann and Kremer discuss the infection process, host interactions, mechanisms of drug resistance and drug development.

New drugs or therapeutic combinations are urgently needed against Mycobacterium abscessus . Previously, we demonstrated the potent activity of indole-2-carboxamides 6 and 12 against M. abscessus . We show here that these compounds act synergistically with imipenem and cefoxitin in vitro and increase the bactericidal activity of the β-lactams against M. abscessus . In addition, compound 12 also displays synergism with imipenem and cefoxitin within infected macrophages.

International audience; The Mycobacterium fortuitum complex comprises several closely related species, causing pulmonary and extra-pulmonary infections. However, there is very limited knowledge about the disease pathogenesis involved in M. fortuitum infections, particularly due to the lack of suitable animal models. Using the zebrafish model, we show that embryos are susceptible to M. fortuitum infection in a dose-dependent manner. Furthermore, zebrafish embryos form granulomas from as early as 2 days post-infection, recapitulating critical aspects of mycobacterial pathogenesis observed in other pathogenic species. The formation of extracellular cords in infected embryos highlights a previously unknown pathogenic feature of M. fortuitum. The formation of large corded structures occurs also during in vitro growth, suggesting that this is not a host-adapted stress mechanism deployed during infection. Moreover, transient macrophage depletion led to rapid embryo death with increased extracellular cords, indicating that macrophages are essential determinants of M. fortuitum infection control. Importantly, morpholino depletion of the cystic fibrosis transmembrane conductance regulator (cftr) significantly increased embryo death, bacterial burden, bacterial cords and abscesses. There was a noticeable decrease in the number of cftr-deficient infected embryos with granulomas as compared to infected controls, suggesting that loss of CFTR leads to impaired host immune responses and confers hypersusceptiblity to M. fortuitum infection. Overall, these findings highlight the application of the zebrafish embryo to study M. fortuitum and emphasizes previously unexplored aspects of disease pathogenesis of this significant mycobacterial species.

ObjectiveTo report the association of JC virus infection of the brain (progressive multifocal encephalopathy [PML]) during the course of sarcoidosis and the challenging balance between immune reconstitution under targeted cytokine interleukin 7 (IL7) therapy for PML and immunosuppression for sarcoidosis.MethodsOriginal case report including deep sequencing (whole-exome sequencing) to exclude a primary immunodeficiency (PID) and review of the literature of cases of PML and sarcoidosis.ResultsWe report and discuss here a challenging case of immune reconstitution with IL7 therapy for PML in sarcoidosis in a patient without evidence for underling PID or previous immunosuppressive therapy.ConclusionsNew targeted therapies in immunology and infectiology open the doors of more specific and more specialized therapies for patients with immunodeficiencies, autoimmune diseases, or cancers. However, before instauration of these treatments, the risk of immune reconstitution inflammatory syndrome and potential exacerbation of an underlying disease must be considered. It is particularly true in case of autoimmune disease such as sarcoidosis or lupus.

Mycobacterium kansasii is a slow-growing nontuberculous mycobacteria responsible for coinfections particularly in patients with human immunodeficiency virus. To date, our knowledge of M. kansasii infection has been hampered owing to the lack of an effective animal model to study pathogenesis. In the current study, we showed that the zebrafish embryo is permissive to M. kansasii infection, resulting in chronic infection and formation of granulomas. On macrophage depletion, we identified M. kansasii forms extracellular cords, resulting in acute infection and rapid larval death. These findings highlight the feasibility of zebrafish for studying M. kansasii pathogenesis and for the first time identify extracellular cords in this species.

International audience; The prevalence of pulmonary infections due to non-tuberculous mycobacteria such as Mycobacterium abscessus, has been increasing and surpassing tuberculosis (TB) in some industrialized countries. Due to intrinsic resistance to most antibiotics that drastically limits conventional chemotherapeutic treatment options, new anti-M. abscessus therapeutics are urgently needed against this emerging pathogen. Extensive screening of a library of benzimidazole derivatives that were previously shown to be active against Mycobacterium tuberculosis led to the identification of a lead compound exhibiting very potent in vitro activity against a wide panel of M. abscessus clinical strains. Designated EJMCh-6, this compound, a 2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole), also exerted a very strong activity against intramacrophage-residing M. abscessus. Moreover, treatment of infected zebrafish embryos with EJMCh-6 correlated with a significantly increased embryo survival and a decrease in the bacterial burden as compared to untreated fish. Insights into the mechanism of action were inferred from the generation of spontaneous benzimidazole-resistant strains and the identification of a large set of missense mutations in MmpL3; the mycolic acid transporter in mycobacteria. Over-expression of the mutated mmpL3 alleles in a susceptible M. abscessus strain was associated with high resistance levels to EJMCh-6 and to other known MmpL3 inhibitors. Mapping the mutations conferring resistance on a MmpL3 three-dimensional homology model defined a potential EJMCh-6-binding cavity. These data emphasize a yet unexploited chemical structure class against M. abscessus with promising translational developments for the treatment of M. abscessus lung diseases.

International audience; Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (tAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize tAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote tAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that tAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis. Upon infection with Mycobacterium tuberculosis (M. tb), the causative agent of TB, less than 5% of infected people will progress towards active infection, whereas 95% remain classified as latently infected without any symptoms or signs of illness 1. In these asymptomatic individuals, bacteria persist within granulomatous lesions in a non-replicating or "dormant" state, from which they may reactivate to induce active TB. It is hypothesized that these persistent bacterial sub-populations are characterized by both low metabolic activity and the presence of large lipid droplets (LD), referred to as intracytoplasmic lipid inclusions (ILI) that are filled with triacylglycerol (TAG) 2,3. Interestingly, TAG synthesis is a conserved feature of several bacterial species belonging to the Actinobacteria phylum, consisting largely of soil dwelling bacteria and including the Mycobacterium genus 4-6. The accumulated TAG constitute a major source of carbon and energy, sustaining survival of intra-and extracellular mycobacteria, and is also directly linked to division arrest, loss of acid-fastness and to increased drug tolerance 7-11. In recent years, a large number of in vitro and ex vivo models have been developed to mimic the ILI-inducing environment encountered by mycobacteria within their hosts 12. It has been demonstrated that within differentiated foamy macrophages (FM), the TAG content of lipid bodies (LB) can be hydrolysed and processed by intraphagosomal mycobacteria, thus leading to the formation of ILI 2,7,8,13. Moreover, several studies have reported that ILI can also be synthesized by extracellular bacteria during stressful conditions and may be considered as a metabolic strategy employed by prokaryotic cells to survive under harsh environments 4,5,14,15. While mycobacterial ILI formation has been observed in slow-and fast-growing mycobacteria such as species from the M. tb complex 9,16-21 , M. leprae 22

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria. 294;46

Twelve new Cyclophostin and Cyclipostins analogues (CyC19-30) were synthesized, thus extending our series to 38 CyCs. Their antibacterial activities were evaluated against four pathogenic mycobacteria (Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium bovis BCG, and Mycobacterium tuberculosis) and two Gram negative bacteria. The CyCs displayed very low toxicity toward host cells and were only active against mycobacteria. Importantly, several CyCs were active against extracellular M. abscessus (CyC17/CyC18β/CyC25/CyC26) or intramacrophage residing mycobacteria (CyC7(α,β)/CyC8(α,β)) with minimal inhibitory concentrations (MIC50) values comparable to or better than those of amikacin or imipenem, respectively. An activity-based protein profiling combined with mass spectrometry allowed identification of the potential target enzymes of CyC17/CyC26, mostly being involved in lipid metabolism and/or in cell wall biosynthesis. Overall, these results strengthen the selective activity of the CyCs against mycobacteria, including the most drug-resistant M. abscessus, through the cumulative inhibition of a large number of Ser- and Cys-enzymes participating in key physiological processes.

International audience; 1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis. To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequenc-ing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

International audience; Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.

International audience; A series of secondary amine-substituted isoindoline-1,3-dione-4-aminoquinolines were prepared viamicrowave heating and assayed for their anti-mycobacterial activities. The compound with a butyl chainas a spacer between the two pharmacophores and piperidine as the secondary amine component onthe isoindoline ring was the most potent and non-cytotoxic among the synthesized compounds,exhibiting a minimum inhibitory concentration (MIC99) of 6.25 mg mL1 against Mycobacteriumtuberculosis.

Slow growing pathogenic mycobacteria utilize host-derived lipids and accumulate large amounts of triacylglycerol (TAG) in the form of intracytoplasmic lipid inclusions (ILI), serving as a source of carbon and energy during prolonged infection. Mycobacterium abscessus is an emerging and rapidly growing species capable to induce severe and chronic pulmonary infections. However, whether M. abscessus, like Mycobacterium tuberculosis, possesses the machinery to acquire and store host lipids, remains unaddressed. Herein, we aimed at deciphering the contribution of the seven putative M. abscessus TAG synthases (Tgs) in TAG synthesis/accumulation thanks to a combination of genetic and biochemical techniques and a well-defined foamy macrophage (FM) model along with electron microscopy. Targeted gene deletion and functional complementation studies identified the MAB_3551c product, Tgs1, as the major Tgs involved in TAG production. Tgs1 exhibits a preference for long acyl-CoA substrates and site-directed mutagenesis demonstrated that His144 and Gln145 are essential for enzymatic activity. Importantly, in the lipid-rich intracellular context of FM, M. abscessus formed large ILI in a Tgs1-dependent manner. This supports the ability of M. abscessus to assimilate host lipids and the crucial role of Tgs1 in intramycobacterial TAG production, which may represent important mechanisms for long-term storage of a rich energy supply. This article is protected by copyright. All rights reserved.

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

International audience; The Xpert MTB/RIF Ultra assay has recently been launched to improve the detection of smear negative disease. This retrospective study compares the sensitivity of Xpert MTB/RIF Ultra with that of Xpert MTB/RIF tests and IS6110 real-time PCR in sputum. Diagnostic performance of three molecular tests was evaluated using 48 culture-positive clinical respiratory specimens diluted to obtain paucibacillary sputum specimens. Xpert MTB/RIF Ultra had the highest sensitivity of 100% compared to 42% (P

New therapeutic approaches are needed against Mycobacterium abscessus, a respiratory mycobacterial pathogen that evades efforts to successfully treat infected patients. Clofazimine and bedaquiline, two drugs used for the treatment of multidrug-resistant tuberculosis, are being considered as alternatives for the treatment of lung diseases caused by M. abscessus With the aim to understand the mechanism of action of these agents in M. abscessus, we sought herein to determine the means by which M. abscessus can develop resistance. Spontaneous resistant strains selected on clofazimine, followed by whole-genome sequencing, identified mutations in MAB_2299c, encoding a putative TetR transcriptional regulator. Unexpectedly, mutants with these mutations were also cross-resistant to bedaquiline. MAB_2299c was found to bind to its target DNA, located upstream of the divergently oriented MAB_2300-MAB_2301 gene cluster, encoding MmpS/MmpL membrane proteins. Point mutations or deletion of MAB_2299c was associated with the concomitant upregulation of the mmpS and mmpL transcripts and accounted for this cross-resistance. Strikingly, deletion of MAB_2300 and MAB_2301 in the MAB_2299c mutant strain restored susceptibility to bedaquiline and clofazimine. Overall, these results expand our knowledge with respect to the regulatory mechanisms of the MmpL family of proteins and a novel mechanism of drug resistance in this difficult-to-treat respiratory mycobacterial pathogen. Therefore, MAB_2299c may represent an important marker of resistance to be considered in the treatment of M. abscessus diseases with clofazimine and bedaquiline in clinical settings.

Background: When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum. Materials and Methods: The impact of targeting repetitive IS6110 sequences on the PCR sensitivity was evaluated across mycobacterium strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing

The progression of mycobacterial diseases requires the development of new therapeutics. This study evaluated the efficacy and selectivity of a panel of Cyclophostin and Cyclipostins analogues (CyCs) against various bacteria and mycobacteria. The activity 26 CyCs was first assayed by the agar plate method. Compounds exhibiting 50–100% growth inhibition were then selected to determine their minimum inhibitory concentrations (MICs) by the resazurin microtiter assay (REMA). The best drug candidate was further tested against clinical mycobacterial isolates and bacteria responsible for nosocomial infections, including 6 Gram-negative bacteria, 5 Gram-positive bacteria, 29 rapid-growing mycobacteria belonging to the Mycobacterium chelonae–abscessus clade and 3 slow-growing mycobacteria ( Mycobacterium marinum , Mycobacterium bovis BCG and Mycobacterium tuberculosis ). Among the 26 CyCs tested, 10 were active and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate ( CyC 17 ) was further tested against 26 clinical strains and showed high selectivity for mycobacteria, with MICs ( M. abscessus infections. Together, these results support the fact that such CyCs represent a new family of potent and selective inhibitors against mycobacteria. This is of particular interest for future chemotherapeutic developments against mycobacterial-associated infections, especially against M. abscessus , the most drug-resistant mycobacterial species.

Evidence-based clinical practice guidelines are essential to ensure that childhood cancer survivors at risk of chronic health conditions receive effective long-term follow-up care. However, adult survivors of childhood cancer are not always engaged in recommended health promotion and follow-up practices, as many centres do not have a formal transition programme that prepares survivors and their families for successful transfer from child-centred to adult-oriented healthcare. The need for a specific pan-European guideline for the transition of care for childhood cancer survivors has been recognised. The first step is to define the concept of transition of care for survivors of childhood cancer based on existing evidence.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

International audience; Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

International audience; An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7β, CyC8β, and CyC17 bind covalently to the catalytic Ser124 residue in Ag85C; inhibit mycolyltransferase activity (i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8β disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.

International audience; Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.

International audience; It was recently shown that Mycobacterium tuberculosis produces cellulose which forms an integral part of its extracellular polymeric substances within a biofilm set-up. Using Mycobacterium smegmatis as a proxy model organism, we demonstrate that M. smegmatis biofilms treated with purified MSMEG\₆752 releases the main cellulose degradation-product (cellobiose), detected by using ionic chromatography, suggesting that MSMEG\₆752 encodes a cellulase. Its overexpression in M. smegmatis prevents spontaneous biofilm formation. Moreover, the method reported here allowed detecting cellobiose when M. smegmatis cultures were exposed to a subinhibitory dose of rifampicin. Overall, this study highlights the role of the MSMEG\₆752 in managing cellulose production induced during biofilm formation and antibiotic stress response.

Mycobacterium abscessus represents an important respiratory pathogen among the rapidly-growing non-tuberculous mycobacteria. Infections caused by M. abscessus are increasingly found in cystic fibrosis (CF) patients and are often refractory to antibiotic therapy. The underlying immunopathological mechanisms of pathogenesis remain largely unknown. A major reason for the poor advances in M. abscessus research has been a lack of adequate models to study the acute and chronic stages of the disease leading to delayed progress of evaluation of therapeutic efficacy of potentially active antibiotics. However, the recent development of cellular models led to new insights in the interplay between M. abscessus with host macrophages as well as with amoebae, proposed to represent the environmental host and reservoir for non-tuberculous mycobacteria. The zebrafish embryo has also appeared as a useful alternative to more traditional models as it recapitulates the vertebrate immune system and, due to its optical transparency, allows a spatio-temporal visualization of the infection process in a living animal. More sophisticated immunocompromised mice have also been exploited recently to dissect the immune and inflammatory responses to M. abscessus. Herein, we will discuss the limitations, advantages and potential offered by these various models to study the pathophysiology of M. abscessus infection and to assess the preclinical efficacy of compounds active against this emerging human pathogen.