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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.

International audience; Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.

Physical forces and mechanical properties have critical roles in cellular function, physiology and disease. Over the past decade, atomic force microscopy (AFM) techniques have enabled substantial advances in our understanding of the tight relationship between force, mechanics and function in living cells and contributed to the growth of mechanobiology. In this Primer, we provide a comprehensive overview of the use of AFM-based force spectroscopy (AFM-FS) to study the strength and dynamics of cell adhesion from the cellular to the single-molecule level, spatially map cell surface receptors and quantify how cells dynamically regulate their mechanical and adhesive properties. We first introduce the importance of force and mechanics in cell biology and the general principles of AFM-FS methods. We describe procedures for sample and AFM probe preparations, the various AFM-FS modalities currently available and their respective advantages and limitations. We also provide details and recommendations for best usage practices, and discuss data analysis, statistics and reproducibility. We then exemplify the potential of AFM-FS in cellular and molecular biology with a series of recent successful applications focusing on viruses, bacteria, yeasts and mammalian cells. Finally, we speculate on the grand challenges in the area for the next decade. Atomic force microscopy-based force spectroscopy can probe the strength and dynamics of cell adhesion to understand how physical forces influence cellular function, physiology and disease. Here, Dufrene and colleagues discuss the ability of this technology to work as an ultra-sensitive force sensor to study the adhesion and elasticity of complex biological systems including viruses, bacteria, yeasts and mammalian cells.

This study focuses on the synthesis of 1,7- and 3,4-indole-fused lactones via a simple and efficient reaction sequence. The functionalization of these “oxazepino-indole” and “oxepino-indole” tricycles is carried out by palladium catalysed C[sbnd]C coupling, nucleophilic substitution or 1,3-dipolar cycloaddition. The evaluation of their activity against Mycobacterium tuberculosis shows that the “oxazepino-indole” structure is a new inhibitor of M. tuberculosis growth in vitro.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading – at a velocity of ∼150 nm s−1 – indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In Caulobacter crescentus, this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As Caulobacter assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.

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.

Adhesion to extracellular matrix proteins is an important first step in host invasion, employed by many bacterial pathogens. In mycobacteria, the secreted Ag85 complex proteins, involved in the synthesis of the cell envelope, are known to bind to fibronectin (Fn) through molecular forces that are currently unknown. In this study, single-molecule force spectroscopy is used to study the strength, kinetics and thermodynamics of the Ag85-Fn interaction, focusing on the multidrug-resistant Mycobacterium abscessus species. Single Ag85 proteins bind Fn with a strength of ~75 pN under moderate tensile loading, which compares well with the forces reported for other Fn-binding proteins. The binding specificity is demonstrated by using free Ag85 and Fn peptides with active binding sequences. The Ag85-Fn rupture force increases with mechanical stress (i.e., loading rate) according to the Friddle&ndash, Noy&ndash, de Yoreo theory. From this model, we extract thermodynamic parameters that are in good agreement with previous affinity determinations by surface plasmon resonance. Strong bonds (up to ~500 pN) are observed under high tensile loading, which may favor strong mycobacterial attachment in the lung where cells are exposed to high shear stress or during hematogenous spread which leads to a disseminated infection. Our results provide new insight into the pleiotropic functions of an important mycobacterial virulence factor that acts as a stress-sensitive adhesin.

Pathogenic bacteria use a variety of cell surface adhesins to promote binding to host tissues and protein-coated biomaterials, as well as cell-cell aggregation. These cellular interactions represent the first essential step that leads to host colonization and infection. Atomic force microscopy (AFM) has greatly contributed to increase our understanding of the specific interactions at play during microbial adhesion, down to the single-molecule level. A key asset of AFM is that adhesive interactions are studied under mechanical force, which is highly relevant as surface-attached pathogens are often exposed to physical stresses in the human body. These studies have identified sophisticated binding mechanisms in adhesins, which represent promising new targets for antiadhesion therapy.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.

International audience; Mycobacterium abscessus, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the fmt gene encoding a putative O-methyltransferase was deleted in the M. abscessus S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the Δfmt mutant, and this defect was rescued upon complementation with a functional fmt gene. In contrast, the introduction of fmt derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that fmt encodes an O-methyltransferase. Unexpectedly, phenotypic analyses showed that Δfmt was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by Δfmt was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt O-methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of M. abscessus as well as in the early steps of the interaction between the bacilli and macrophages.

Mycobacterium abscessus is a human pathogen responsible for severe respiratory infections, particularly in patients with underlying lung disorders. Notorious for being highly resistant to most antimicrobials, new therapeutic approaches are needed to successfully treat M. abscessus-infected patients. Clofazimine (CFZ) and bedaquiline (BDQ) are two antibiotics used for the treatment of multidrug-resistant tuberculosis and are considered alternatives for the treatment of M. abscessus pulmonary disease. To get insights into their mechanisms of resistance in M. abscessus, we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the MAB_2300-MAB_2301 genes, encoding an MmpS-MmpL efflux pump. Here, in silico studies identified a second mmpS-mmpL (MAB_1135c-MAB_1134c) target of MAB_2299c. A palindromic DNA sequence upstream of MAB_1135c, sharing strong homology with the one located upstream of MAB_2300, was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of MAB_1135c-1134c in a wild-type strain led to increased susceptibility to both CFZ and BDQ. In addition, deletion of these genes in a CFZ/BDQ-susceptible mutant lacking MAB_2299c as well as MAB_2300-MAB_2301 further exacerbated the sensitivity of this strain to both drugs in vitro and inside macrophages. Overall, these results indicate that MAB_1135c-1134c encodes a new MmpS-MmpL efflux pump system involved in the intrinsic resistance to CFZ and BDQ. They also support the view that MAB_2299c controls the expression of two separate MmpS-MmpL efflux pumps, substantiating the importance of MAB_2299c as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.

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; 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.

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.

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

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.

Malonyl-coenzyme A (CoA) is a crucial extender unit for the synthesis of mycolic and other fatty acids in mycobacteria, generated in a reaction catalyzed by acetyl-CoA carboxylase. We previously reported on the essentiality of accD6Mtb encoding the functional acetyl-CoA carboxylase subunit in Mycobacterium tuberculosis. Strikingly, the homologous gene in the fast-growing, non-pathogenic Mycobacterium smegmatis - (accD6Msm) appeared to be dispensable, and its deletion did not influence the cell lipid content. Herein, we demonstrate that, despite the difference in essentiality, accD6Msm and accD6Mtb encode proteins of convergent catalytic activity in vivo. To identify an alternative, AccD6-independent, malonyl-CoA synthesis pathway in M. smegmatis, a complex genetic approach combined with lipid analysis was applied to screen all five remaining carboxyltransferase genes (accD1-accD5) with respect to their involvement in mycolic acid biosynthesis and ability to utilize acetyl-CoA as the substrate for carboxylation. This approach revealed that AccD1Msm, AccD2Msm and AccD3Msm are not essential for mycolic acid biosynthesis. Furthermore, we confirmed in vivo the function of AccD4Msm as an essential, long-chain acyl-CoA carboxyltransferase, unable to carboxylate short-chain substrate. Finally, our comparative studies unambiguously demonstrated between-species difference in in vivo ability of AccD5 carboxyltransferase to utilize acetyl-CoA that influences AccD6 essentiality in pathogenic and non-pathogenic mycobacteria.

Mycobacterium abscessusis a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF).Mycobacterium abscessusis known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show thatM. abscessusS and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion,M. abscessusS exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

International audience; 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.

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the β-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a high-resolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors.

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.