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Background Mycobacterium abscessus (Mabs), a rapidly growing Mycobacterium species, is considered an MDR organism. Among the standard antimicrobial multi-drug regimens against Mabs, amikacin is considered as one of the most effective. Parenteral amikacin, as a consequence of its inability to penetrate inside the cells, is only active against extracellular mycobacteria. The use of inhaled liposomal amikacin may yield improved intracellular efficacy by targeting Mabs inside the cells, while reducing its systemic toxicity. Objectives To evaluate the colocalization of an amikacin liposomal inhalation suspension (ALIS) with intracellular Mabs, and then to measure its intracellular anti-Mabs activity. Methods We evaluated the colocalization of ALIS with Mabs in eukaryotic cells such as macrophages (THP-1 and J774.2) or pulmonary epithelial cells (BCi-NS1.1 and MucilAir), using a fluorescent ALIS and GFP-expressing Mabs, to test whether ALIS reaches intracellular Mabs. We then evaluated the intracellular anti-Mabs activity of ALIS inside macrophages using cfu and/or luminescence. Results Using confocal microscopy, we demonstrated fluorescent ALIS and GFP-Mabs colocalization in macrophages and epithelial cells. We also showed that ALIS was active against intracellular Mabs at a concentration of 32 to 64 mg/L, at 3 and 5 days post-infection. Finally, ALIS intracellular activity was confirmed when tested against 53 clinical Mabs isolates, showing intracellular growth reduction for nearly 80% of the isolates. Conclusions Our experiments demonstrate the intracellular localization and intracellular contact between Mabs and ALIS, and antibacterial activity against intracellular Mabs, showing promise for its future use for Mabs pulmonary infections.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis , to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis . In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus ; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in

Mycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surface-exposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

International audience

Free-living amoebae might represent an evolutionary niche. In order to get more insights into the potential amoebal training ground forMycobacterium abscessus, we characterized its full transcriptome in amoeba (Ac) and macrophages (Mφ), as well as theMycobacterium chelonaeintra-Ac transcriptome for comparison. Up-regulated genes in Ac allowedM. abscessusto resist environmental stress and induce defense mechanisms, as well as showing switch from carbohydrate carbon sources to fatty acid metabolism. Eleven genes implicated in the adaptation to intracellular stress, were mutated, with all but one confirmed to be involved inM. abscessusintra-Mφ survival. Cloning two of these genes inM. chelonaeincreased its intra-Mφ survival. One mutant was particularly attenuated in Mφ that corresponded to the deletion of an Eis N-acetyl transferase protein (MAB_4532c). Taken together,M. abscessustranscriptomes revealed the intracellular lifestyle of the mycobacteria, with Ac largely contributing to the enhancement ofM. abscessusintra-Mφ survival.

Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis. A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8(MAB), had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8(MAB) mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8(MAB). Systematic lipid analysis revealed that MmpL8(MAB) was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8(MAB) in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

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.

The coevolution of mycobacteria and amoebae seems to have contributed to shaping the virulence of nontuberculous mycobacteria in macrophages. We identified a pool of genes essential for the intracellular survival of Mycobacterium abscessus inside amoebae and macrophages and discovered a hot spot of transposon insertions within the orthologous ESX-4 T7SS locus. We generated a mutant with the deletion of a structural key ESX component, EccB4. We demonstrate rupture of the phagosomal membrane only in the presence of an intact eccB4 gene. These results suggest an unanticipated role of ESX-4 T7SS in governing the intracellular behavior of a mycobacterium. Because M. abscessus lacks ESX-1, it is tempting to speculate that ESX-4 operates as a surrogate for ESX-1 in M. tuberculosis.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555 , encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis . Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus . Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Mycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surface-exposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

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; In mycobacteria, various type VII secretion systems corresponding to different ESX (ESAT-6 secretory) types, are contributing to pathogenicity, iron acquisition, and/or conjugation. In addition to the known chromosomal ESX loci, the existence of plasmid-encoded ESX systems was recently reported. To investigate the potential role of ESX-encoding plasmids on mycobacterial evolution we analysed a large representative collection of mycobacterial genomes, including both chromosomal and plasmid-borne sequences. Data obtained for chromosomal ESX loci confirmed the previous 5 classical ESX types and identified a novel mycobacterial ESX-4-like type, termed ESX-4-bis. Moreover, analysis of the plasmid-encoded ESX loci showed extensive diversification, with at least 7 new ESX profiles, identified. Three of them (ESX-P clusters 1, 2 and 3) were found in multiple plasmids, while four corresponded to singletons. Our phylogenetic and gene-order-analyses revealed two main groups of ESX types: i) ancestral types, including ESX-4 and ESX-4-like systems from mycobacterial and non-mycobacterial actinobacteria, and ii) mycobacteria-specific ESX systems, including ESX-1-2-3-5 systems and the plasmid-encoded ESX types. Synteny analysis revealed that ESX-P systems are part of phylogenetic groups that derived from a common ancestor, which diversified and resulted in the different ESX types through extensive gene rearrangements. A converging body of evidence, derived from composition bias-, phylogenetic- and synteny analyses points to a scenario in which ESX-encoding plasmids have been a major driving force for acquisition and diversification of type VII systems in mycobacteria, which likely played (and possibly still play) important roles in the adaptation to new environments and hosts during evolution of mycobacterial pathogenesis.

International audience; Suppressor of cytokine signaling (SOCS) proteins are key physiological regulators of both innate and adaptive immunity. These proteins belong to the three major classes of modulators of cytokines signaling. In the following article, we used porcine polarized intestinal cells to study early response to the protozoan, Entamoeba histolytica, and we identified by rapid amplification of cDNA ends (RACE) PCR porcine SOCS1, SOCS4, SOCS5 and SOCS6 encoding sequences. With more than 92% identity predicted porcine SOCS proteins are very similar to their human counterparts. Among SOCS transcripts, only SOCS2 mRNA was significantly induced in epithelial intestinal cells in response to the cytolytic activity of the parasite. The transcriptomic profile obtained after 3h of co-culture of polarized intestinal cells with E. histolytica was clearly oriented toward inflammation and the recruitment of neutrophils. These transcriptomic data have been normalized with accuracy by the utilisation of multiple validated reference genes. The analysis offers a first set of reference genes useful for future studies in porcine intestinal cells. Our data shed light on the understanding of the early response of polarized intestinal cells to E. histolytica and identified a potential involvement of SOCS2 in the parasite regulation of the host response.

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.

Mycobacteria possess type VII secretion systems (T7SS), also known as ESX. Recently, our laboratory has demonstrated that the ESX-4 system of M. abscessus is an essential actor in the intracellular survival of M. abscessus Furthermore, mutation of the gene coding for a structural protein of ESX-4 leads to a decrease in the secretion of several proteins, two belonging to the esx-4 locus, EsxU and EsxT. For this project, we studied the role of EsxU and EsxT proteins in M. abscessus. We first demonstrated the localization of these proteins in M. abscessus, EsxU and EsxT are associated with the bacterial membrane and are also secreted. Furthermore, EsxU and EsxT form a complex characteristic of proteins secreted by mycobacterial T7SS. EsxU and EsxT proteins are able to interact with artificial membranes to form pore-like structures in vitro. In an intramacrophage context, esxU and esxT absence does not influence the intracellular behaviour of the ΔesxUT mutated strain, but prevents the disruption of the phagosomal membrane, keeping M. abscessus ΔesxUT in an unacidified phagosome. Finally, we identify a hypervirulent phenotype associated with ΔesxUT deletion characterized by increased animal mortality and bacterial growth in mice and zebrafish. All our results suggest that the absence of both substrates improves the survival and persistence of M. abscessus in animals. We also wanted to study the immunogenic potential of these proteins. The T cell response to synthetic peptides of the EsxU and EsxT proteins did not identify epitopes that induce a T response. This suggests that EsxU and T are not immunogenic after subcutaneous inoculation of M. abscessus in the mouse model suggesting that EsxU and EsxT are not good markers of infection. The secretome and a bioinformatics analysis based on the search for secretion patterns identified other potential substrates of SST7 in M. abscessus. Two proteins with a proline-glutamate (PE) an d proline-glutamate-glutamate (PPE) motif have peptides that induce a Th1-type immune response. However, further experiments are needed to determine whether these proteins represent good diagnostic targets in patients and whether they are able to discriminate M. abscessus infections from infections caused by other mycobacteria., Les mycobactéries possèdent des systèmes de sécrétion de type VII (SST7) aussi nommé ESX. Récemment, notre laboratoire a démontré que le système ESX-4 de M. abscessus est un acteur essentiel à sa survie intracellulaire. De plus, la mutation du gène codant pour une protéine structurale de l’ESX-4 entraîne une diminution de la sécrétion de plusieurs protéines dont deux appartenant au locus esx-4, EsxU et EsxT. Pour ce projet, nous avons étudié le rôle des protéines EsxU et EsxT chez M. abscessus. Nous avons d'abord démontré la localisation de ces protéines chez M. abscessus. EsxU et EsxT sont associées avec la membrane bactérienne et secrétées dans le milieu extérieur. De plus, elles forment un complexe caractéristique des protéines sécrétées par les SST7 des mycobactéries et sont capables in vitro d’interagir avec des membranes artificielles pour former des structures semblables à des pores. Dans les macrophages, leur absence n'influence pas le comportement intracellulaire de la souche mutée ΔesxUT, mais empêche la rupture de la membrane phagosomale, maintenant M. abscessus ΔesxUT dans un phagosome non acidifié. Enfin, nous identifions un phénotype hypervirulent associé à ΔesxUT, caractérisé par une augmentation de la mortalité et de la croissance bactérienne chez la souris et le poisson zèbre. L’ensemble de nos résultats suggèrent que l'absence des deux substrats améliore la survie et la persistance de M. abscessus dans ces modèles animaux. Nous avons aussi voulu étudier le potentiel immunogène de ces protéines. La réponse lymphocytaire T, vis-à-vis de peptides synthétiques des protéines EsxU et EsxT n’a pas permis d’identifier d’épitopes induisant une réponse T. Ceci suggère qu’EsxU et T ne sont pas immunogènes après inoculation de M. abscessus par voie sous-cutanée dans le modèle souris ce qui laisse penser qu’EsxU et EsxT ne représentent pas de bons marqueurs de l’infection. Le sécrétome ainsi qu’une analyse bio-inform atique basée sur la recherche de motifs de sécrétion ont permis d’identifier d’autres substrats potentiels de SST7 chez M. abscessus. Deux protéines avec un motif proline-glutamate (PE) et proline-glutamate-glutamate (PPE) ont des peptides qui induisent une réponse immunitaire Th1. Néanmoins des expériences supplémentaires sont nécessaires pour déterminer si ces protéines représentent de bonnes cibles de diagnostic chez les patients et si elles sont capables de discriminer les infections à M. abscessus des infections causées par d’autres mycobactéries.

Mycobacterium abscessus is a rapidly growing mycobacterium, causing opportunistic infections in humans, and notably pulmonary infections. M. abscessus is able to multiply inside macrophages (MФ) and environmental amoebae. Here we demonstrate that M. abscessus undergoes adaptations in amoebae allowing its survival in MФ. Intracellular adaptations of M. abscessus to amoebae and MФ were assessed by RNAseq. We observed a significant enrichment of biological pathways reflecting adaptations to oxidative stress and metabolic adaptations illustrated by the consumption of fatty acids and activation of the sulfate assimilation pathway. These adaptations have been described in intramacrophagic Mycobacterium tuberculosis, a strictly pathogenic mycobacteria infecting the lung of humans and causing tuberculosis. Among the set of genes induced by M. abscessus during the amoebal co-culture are genes implicated in polyamine transport, MoCo (molybdopterin cofactor) biosynthesis and iron-sulfur (Fe-S) cluster assembly. The induction of such genes, described as virulence factors from intracellular bacteria, might enhance M. abscessus virulence and thus allow its survival in MФ. Forty-five genes are highly induced along the amoebal co-culture. In comparison, the amoebal co-culture with Mycobacterium chelonae, a mycobacterium that belongs to the same genomic complex as M. abscessus and causing solely extrapulmonary infections, does not elicit the same adaptations; potentially explaining M. chelonae inability to persist in macrophages. Five operons, representing a total of 10 genes, were deleted from M. abscessus genome by homologous recombineering. These genes are required for both M. abscessus survival in amoebae and MФ. Overexpression of two of these genes in M. chelonae, MAB_1517c and MAB_2649, encoding a TcmP (tetracenomycin polyketide synthesis O-methyltransferase) protein and an MmpS (mycobacterial membrane protein small) protein respectively, enhances M. chelonae survival in MФ, suggesting that the induction of these genes favors M. abscessus survival in MФ. Analyses of M. abscessus transcriptome in MФ also shed light on adaptations specific to the bacterium intramacrophagic life. Several genes highly induced in macrophages are implicated in biological pathways known to contribute to bacteria virulence, including proline metabolism, protein secretion by the type II secretion system and the MEP (methylerythritol phosphate) pathway. Among the set of induced genes selected according to their level of induction and their biological activity, N-acetylation and redox activities, bounding to oxygen and detoxification from nitric oxide by dioxygenases are significantly enriched. Among operons from this set of genes, it appears that M. abscessus eis2 gene (MAB_4542c), encoding a N-acetyltransferase, is essential for M. abscessus survival in MФ.In addition, a complementary approach to RNAseq, the screening of a transposon (Tn) mutant library of M. abscessus inside amoebae, revealed important roles of the mmpL8 gene encoding a mycobacterial membrane protein large belonging to a family of proteins implicated in lipid biosynthesis and export to the cell surface. When this protein was no longer produced by M. abscessus, a lower amount of a new glycolipd family (GDND, glycosyl diacetylated nonadecyl diol) was observed as well as a deleterious phenotype in MФ.To conclude, our work has shown a fundamental role of amoebae in triggering the virulence of M. abscessus, further allowing its survival in macrophages. Besides, three genes that have been studied more extensively – mmpL8, eis2 and eccB4 (revealed by the Tn library screening) – are required for M. abscessus survival in macrophages and confirmed its pathogenic behavior., La mycobactérie à croissance rapide Mycobacterium abscessus est un pathogène opportuniste de l’homme, particulièrement des poumons, capable de se multiplier au sein de macrophages (MФ) mais aussi d’amibes environnementales. Nous avons pu montrer que l’environnement amibien est propice à l’adaptation de M. abscessus à cette survie intramacrophagique. Par des analyses transcriptomiques comparant les adaptations de M. abscessus au sein d’amibes ou de MФ, nous observons des enrichissements de voies biologiques démontrant des adaptations au stress oxydatif et des adaptations métaboliques telle que la béta-oxydation des lipides et l’assimilation des sulfates. Ces adaptations ont également été observées chez Mycobacterium tuberculosis, mycobactérie pathogène pulmonaire stricte de l’homme responsable de la tuberculose. Parmi les gènes induits par M. abscessus au sein des amibes figurent des gènes impliqués dans le transport de polyamines, la biosynthèse du MoCo (molybdopterin cofactor) ainsi que l’assemblage des centres fer-soufre (Fe-S). L’induction de tels gènes, décrits comme des facteurs de virulence chez certaines bactéries intracellulaires, contribuerait à la virulence de M. abscessus en permettant sa survie intramacrophagique. Quarante-cinq gènes ont été identifiés comme fortement induits en amibes chez M. abscessus. Mycobacterium chelonae, appartenant au même complexe génomique que M. abscessus et responsable exclusivement d’infections cutanées, ne présente pas de telles inductions après analyse de son transcriptome intracellulaire, ce qui pourrait expliquer son absence de survie en MФ. Cinq opérons recouvrant 10 gènes ont été délétés au sein du génome de M. abscessus par recombinaison homologue. Ces gènes sont requis pour la survie de M. abscessus en amibes et en MФ. La surexpression chez M. chelonae de deux de ces gènes, MAB_1517c et MAB_2649, codant respectivement une protéine TcmP (tetracenomycin polyketide synthesis O-methyltransferase) et une MmpS (mycobacterial membrane protein small), a conféré une survie intra-macrophagique à M. chelonae, suggérant que l’induction de ces gènes en amibes favorise la survie intracellulaire de M. abscessus en MФ. Enfin, l’analyse du transcriptome de M. abscessus en MФ révèle des adaptations propres à la vie intramacrophagique. Différents gènes particulièrement induits sont impliqués dans le métabolisme de la proline, la sécrétion de protéine par le système de sécrétion de type II ou appartiennent la voie MEP (méthylérythritol phosphate), des voies biologiques contribuant à la virulence de pathogènes. De plus, parmi les gènes induits, certains correspondent à des activités de N-acétylation, d’oxydoréduction, de liaison à l’oxygène ou de détoxification de l’oxyde nitrique par des dioxygénases qui sont enrichies. Comparé à 5 autres opérons délétés, sélectionnés selon leur niveau d’induction et leur activité biologique, le gène eis2 (MAB_4532c), codant une N-acétyltransférase, est essentiel à la survie de M. abscessus en MФ.L’utilisation d’une approche complémentaire, le criblage d’une banque de mutants par transposition chez M. abscessus en amibes, a révélé le rôle essentiel du gène mmpl8 (mycobacterial membrane protein large 8) parmi la famille de protéines MmpL, impliquées dans le transport et/ou la synthèse de lipides mycobactériens. Chez M. abscessus, l’absence de production de cette protéine est corrélée à un défaut d’export d’un nouveau glycolipide (GDND, glycosyl diacetylated nonadecyl diol) et à un phénotype délétère pour la bactérie en MФ.En conclusion, notre travail a montré le rôle fondamental de l’amibe dans l’apprêtement de M. abscessus à la survie intramacrophagique. Trois gènes ayant fait l’objet d’études approfondies, mmpL8, eis2 ainsi que le gène eccB4 (MAB_3759c) mis en évidence par le crible amibien de la banque de mutants de transposition, participent à ce phénotype, confirmant le caractère pathogène de M. abscessus.