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International audience; Glycoside hydrolases (GHs) are enzymes that catalyze the hydrolysis of glycosidic bonds in glycoconjugates, oligo-and polysaccharides. A classification of these enzymes based on conserved sequence and structure motifs supported by the Carbohydrate Active Enzyme (CAZy) database has proven useful in the systematic groupings of similar enzymes into families. The human pathogen Mycobacterium tuberculosis employs 30 GHs to perform a variety of different functions, which can be divided into four broad categories: alpha-glucan metabolism, peptidoglycan remodeling, a-glycan hydrolysis and alpha-demannosylation. The review presented here shows how the GHs that have been characterized play a role in each category. Expanding the genomic analysis of GH presence to other Mycobacterium species has highlighted the importance of certain families-most notably GH13 and GH23-in the general genomic make-up of mycobacteria. Since many GHs are still uncharacterized and considered as ``conserved hypothetical'' proteins, the grouping of them into respective families provides a strong prediction on their putative biological functions.

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the

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.

Mycobacterium abscessus is an emerging pathogenic mycobacterium involved in pulmonary and mucocutaneous infections, presenting a serious threat for patients with cystic fibrosis (CF). The lack of an efficient treatment regimen and the emergence of multidrug resistance in clinical isolates require the development of new therapeutic strategies against this pathogen. Reverse genetics has revealed genes that are present in M. abscessus but absent from saprophytic mycobacteria and that are potentially involved in pathogenicity. Among them, MAB_3593 encodes MgtC, a known virulence factor involved in intramacrophage survival and adaptation to Mg 2+ deprivation in several major bacterial pathogens. Here, we demonstrated a strong induction of M. abscessus MgtC at both the transcriptional and translational levels when bacteria reside inside macrophages or upon Mg 2+ deprivation. Moreover, we showed that M. abscessus MgtC was recognized by sera from M. abscessus -infected CF patients. The intramacrophage growth (J774 or THP1 cells) of a M. abscessus knockout mgtC mutant was, however, not significantly impeded. Importantly, our results indicated that inhibition of MgtC in vivo through immunization with M. abscessus mgtC DNA, formulated with a tetrafunctional amphiphilic block copolymer, exerted a protective effect against an aerosolized M. abscessus challenge in CF (ΔF508 FVB) mice. The formulated DNA immunization was likely associated with the production of specific MgtC antibodies, which may stimulate a protective effect by counteracting MgtC activity during M. abscessus infection. These results emphasize the importance of M. abscessus MgtC in vivo and provide a basis for the development of novel therapeutic tools against pulmonary M. abscessus infections in CF patients.

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 μg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 μg/ml and 1 μg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 μg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10. Fil: Velappan, Anand Babu. Sastra University; India Fil: Charan Raja, Mamilla R.. Sastra University; India Fil: Datta, Dhrubajyoti. Indian Institute of Science Education and Research Pune; India Fil: Tsai, Yi Ting. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Halloum, Iman. Université de Montpellier; Francia. Centre National de la Recherche Scientifique; Francia Fil: Wan, Baojie. University of Illinois; Estados Unidos Fil: Kremer, Laurent. Université de Montpellier; Francia. Inserm; Francia. Centre National de la Recherche Scientifique; Francia Fil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Franzblau, Scott G.. University of Illinois; Estados Unidos Fil: Kar Mahapatra, Santanu. Sastra University; India Fil: Debnath, Joy. Sastra University; India

From our recent survey of non-pigmented rapidly growing mycobacteria in the Parisian water system, three groups of isolates (taxons 1-3) corresponding to possible novel species were selected for taxonomic study. The three taxa each formed creamy white, rough colonies, had an optimal growth temperature of 30 °C, hydrolyzed Tween 80, were catalase-positive at 22 °C and expressed arylsulfatase activity. All three were susceptible to amikacin, ciprofloxacin and tigecycline. The three taxa produced specific sets of mycolic acids, including one family that has never previously been described, as determined by thin layer chromatography and nuclear magnetic resonance. The partial rpoB sequences (723 bp) showed 4-6 % divergence from each other and more than 5 % differences from the most similar species. Partial 16S rRNA gene sequences showed 99 % identity within each species. The most similar sequences for 16S rRNA genes (98-99 % identity over 1444-1461 bp) were found in the Mycobacterium fortuitum group, Mycobacterium septicum and Mycobacterium farcinogenes. The three taxa formed a new clade (bootstrap value, 99 %) on trees reconstructed from concatenated partial 16S rRNA, hsp65 and rpoB sequences. The above results led us to propose three novel species for the three groups of isolates, namely Mycobacterium lutetiense sp. nov. [type strain 071T=ParisRGMnew_1T (CIP 110656T=DSM 46713T)], Mycobacterium montmartrense sp. nov. [type strain 196T=ParisRGMnew_2T (CIP 110655T=DSM 46714T)] and Mycobacteriu marcueilense sp. nov. [type strain of 269T=ParisRGMnew_3T (CIP 110654T=DSM 46715T)].

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.

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.

Animal models are fundamental to advance knowledge of disease pathogenesis and to test/develop new therapeutic strategies. Most of the current knowledge about the pathogenic mechanisms underpinning autoimmune demyelination processes implicating autoantigens has been obtained using the Experimental Autoimmune Neuritis (EAN) animal model. The most widely used EAN model is obtained by active immunization of Lewis rats using a peptide, P0 (180–199), issuing from the major peripheral nervous system myelin protein. But this model mimics only the classical monophasic acute form of demyelinating polyradiculoneuropathy, i.e. Guillain-Barre syndrome (GBS). We developed a new model by immunizing Lewis rats using the same immunodominant neuritogenic peptide P0 (180–199) but this time with its S-palmitoyl derivative, S-palm P0 (180–199). All of the animals immunized with the S-palm P0 (180–199) peptide developed a chronic relapsing-remitting form of the disease corresponding to the electrophysiological criteria of demyelination (slow sensory nerve conduction velocity, prolonged motor nerve latency, partial motor nerve conduction blocks) with axon degeneration. These findings were confirmed by immunohistopathology study and thus, appear to mimic human chronic inflammatory demyelinating polyradiculopathy (CIDP). This new model opens up new avenues of research for testing new anti-inflammatory and neuroprotective therapeutic strategies.

The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.

Although it was identified in the cell wall of several pathogenic mycobacteria, the biological properties of dimycolyl-diarabino-glycerol have not been documented yet. In this study an apolar glycolipid, presumably corresponding to dimycolyl-diarabino-glycerol, was purified from Mycobacterium marinum and subsequently identified as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1')-glycerol (designated Mma_DMAG) using a combination of nuclear magnetic resonance spectroscopy and mass spectrometry analyses. Lipid composition analysis revealed that mycolic acids were dominated by oxygenated mycolates over α-mycolates and devoid of trans-cyclopropane functions. Highly purified Mma_DMAG was used to demonstrate its immunomodulatory activity. Mma_DMAG was found to induce the secretion of proinflammatory cytokines (TNF-α, IL-8, IL-1β) in human macrophage THP-1 cells and to trigger the expression of ICAM-1 and CD40 cell surface antigens. This activation mechanism was dependent on TLR2, but not on TLR4, as demonstrated by (i) the use of neutralizing anti-TLR2 and -TLR4 antibodies and by (ii) the detection of secreted alkaline phosphatase in HEK293 cells co-transfected with the human TLR2 and secreted embryonic alkaline phosphatase reporter genes. In addition, transcriptomic analyses indicated that various genes encoding proinflammatory factors were up-regulated after exposure of THP-1 cells to Mma_DMAG. Importantly, a wealth of other regulated genes related to immune and inflammatory responses, including chemokines/cytokines and their respective receptors, adhesion molecules, and metalloproteinases, were found to be modulated by Mma_DMAG. Overall, this study suggests that DMAG may be an active cell wall glycoconjugate driving host-pathogen interactions and participating in the immunopathogenesis of mycobacterial infections.

Eosinophils have recently been shown to participate in innate immune responses against mycobacteria. We have investigated whether Mycobacterium bovis BCG regulate the human eosinophil immune response. A negative correlation between mycobacteria internalization and eosinophil activation was observed. In addition, mannose-capped lipoarabinomannan from M. bovis BCG (ManLAM) failed to induce a significant release of eosinophil peroxidase and TNF-α. Noteworthy, ManLAM exhibited a potent inhibitory effect on eosinophil peroxidase release by TLR2-activated eosinophils involving the complement receptor-3 molecule and the phosphatidylinositol-3 kinase pathway. ManLAM, generally present in pathogenic mycobacteria, plays an important role in modulating eosinophil-dependent immune response.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 μg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the α-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, α-mycolic acid cyclopropanation was inducible in cultures grown at 25 °C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 °C compared with 37 °C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of α-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a ΔpcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.

International audience; Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg-1529. Disruption of Msmeg-1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.

Introduction Les anticorps monoclonaux anti-PD1 (programmed cell death protein 1) comme le nivolumab et le pembrolizumab sont utilises comme anticancereux. Ils peuvent provoquer parfois des effets secondaires musculaires potentiellement fatals. Observation Un patient de 78 ans avait pour antecedent un melanome cutane traite il y a 4 ans par chirurgie. Suite a une recidive ganglionnaire inoperable, un traitement par pembrolizumab a ete debute. Trois semaines apres la premiere perfusion, un ptosis bilateral non fluctuant est apparu, suivi quelques jours plus tard d’une diplopie binoculaire, d’une dysphagie et d‘une dysphonie. L’IRM cerebrale et l’analyse du liquide cephalo-rachidien etaient normales et la recherche d’anticorps anti-neurones, anti-recepteur de l’acetylcholine et d’anticorps associes aux myosites etait negative. Les CPK etaient elevees a 11 500 UI/L (n Discussion Plusieurs cas de myopathie inflammatoire aigue fatale apres traitement par anticorps anti-PD1 ont deja ete rapportes. Ces myopathies avaient un debut quelques semaines apres la premiere cure et une predominance oculo-bulbaire et respiratoire. L’histologie musculaire retrouvait le plus souvent une myosite necrosante. L’absence de necrose dans notre cas etait peut-etre liee au fait que la biopsie a ete realisee precocement. Conclusion Le traitement par pembrolizumab peut se compliquer d’une myopathie inflammatoire aigue severe, a predominance oculo-bulbaire et respiratoire, potentiellement fatale.

International audience; SUMMARY Mycobacterium tuberculosis (M. tb) has a complex life style in different environments and involving several developmental stages. The success of M. tb results from its remarkable capacity to survive within the infected host, where it can persist in a non-replicating state for several decades. The survival strategies developed by M. tb are linked to the presence of an unusual cell envelope. However, little is known regarding its capacity to modulate and adapt production of cell wall components in response to environmental conditions or to changes in cell shape and cell division. Signal sensing leading to cellular responses must be tightly regulated to allow survival under variable conditions. Although prokaryotes generally control their signal transduction processes through two-component systems, signalling through Ser/Thr phosphorylation has recently emerged as a critical regulatory mechanism in bacteria. The genome of M. tb possesses a large family of eukaryotic-like Ser/Thr Protein Kinases (STPKs). The physiological roles of several mycobacterial STPK substrates are connected to cell shape/division and cell envelope biosynthesis. Although these regulatory mechanisms have mostly been studied in Mycobacterium, Ser/Thr phosphorylation appears also to regulate cell division and peptidoglycan synthesis in Corynebacterium and Streptomyces. This review focuses on the proteins which have been identified as STPK substrates and involved in the synthesis of major cell envelope components and cell shape/division in actinomycetes. It is also intended to describe how phosphorylation affects the activity of peptidoglycan biosynthetic enzymes or cell division proteins.

We demonstrate thatMycobacterium tuberculosisGroEL1 is phosphorylated by PknF at two positions, Thr25 and Thr54. Unexpectedly,Mycobacterium smegmatisGroEL1 is not a substrate of its cognate PknF. This study shows that the phosphorylation profiles of conserved proteins are species dependent and provide insights that may explain the numerous biological functions of these important proteins.

International audience; The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections., Author Summary The incidence of non-tuberculous mycobacterial infections has recently increased and has even surpassed tuberculosis as a public health concern in many developed countries. These infections require long treatment regimens that are often unsuccessful. Among these, Mycobacterium abscessus has emerged as perhaps the most difficult-to-manage pathogen, especially in cystic fibrosis patients. Unfortunately, very little is known regarding the contributions of the pro-inflammatory and innate immune responses during M. abscessus infection. Here, we exploited the transparency of zebrafish embryos to study, at high resolution, the interactions of M. abscessus with macrophages and neutrophils, and found that both cell types are required to control the infection. We also describe the dramatic consequences of impaired TNF/IL8 immunity on the outcome of the infection. Most importantly, by tracking the dynamics of neutrophil mobilization, we demonstrated the crucial role of these cells in the formation and integrity of protective granulomas. Together, our data provide a significant advance in deciphering the immunopathology of M. abscessus infection, which is particularly relevant for understanding the exquisite vulnerability of cystic fibrosis patients to this bacterium.

Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.

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.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

The zebrafish offers many advantages that motivated and validated its use to study the virulence of numerous human pathogens, including viruses, bacteria and fungi. Its immune system is homologous to the one of mammals. The optical transparency of zebrafish embryos allows non-invasive and real-time monitoring of the infection processes through the use of imaging techniques. The zebrafish is therefore a useful and powerful model to study host-pathogen interactions at a cellular level. It may be used to describe pathophysiological events and subversion mechanisms that are specific to each pathogen. In addition to increasing our understanding of the host immune defense, this model is of high potential for medical application, being particularly amenable to high-throughput screening for the discovery of new anti-infective molecules.

SummaryMycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential—presumably providing the housekeeping chaperone functions—while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates—long-chain fatty acid components of the mycobacterial cell wall—specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56–C68) fatty acids, and strains with altered mycolate profiles—including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA—are defective in biofilm formation.

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These α-alkyl, β-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C24-C26 fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The β-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg46 revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg161 → Ala substitution. Our structural studies suggested that His258, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys122.

Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non-pathogenic mycobacterial species. TLC analysis of [(14)C]-acetate- or [(14)C]-glycerol-labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol-related lipid. Subsequent purification, followed by structural determination using (1)H-nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl-diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well-known type II fatty acid synthase (FAS-II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [(14)C]-acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH-susceptible and -resistant Mycobacterium tuberculosis strains. Therefore, THL and other beta-lactones may be promising drugs for the development of new antitubercular therapy.

The Mycobacterium tuberculosis cell envelope differs substantially from the canonical cell wall structures of both gram-negative and gram-positive bacteria. This chapter reviews the current understanding of the major cell wall waxes found in M. tuberculosis, along with their functions and biosynthesis. Mycolic acids have unique characteristics essential for maintaining the cell wall structure, and their physiological role may possibly be correlated to individual mycolates. The major biological functions associated with mycolic acid either bound to arabinogalactan (AG) or associated with trehalose are summarized. The phthiocerol dimycoserosates (PDIMs) are major waxes of the tubercle bacillus. Two types of fatty acid-synthesizing systems, fatty acid synthase I (FAS-I) and fatty acid synthase II (FAS-II), achieve the enzymatic cycles. Through genetic and biochemical approaches, two genes of the FAS-II system, inhA and kasA, have been postulated to encode the primary target of isoniazid (INH). Therefore, due to conflicting reports, the mode of action of INH has recently been reexamined. First, studies performed in vivo demonstrated that overexpression of InhA, but not KasA, in M. smegmatis, M. bovis BCG, and M. tuberculosis conferred increased resistance to INH. Second, in vitro assays using purified KasA or InhA demonstrated that KatG-activated INH inhibited InhA activity but not KasA activity. A recent review of mycolic acid biosynthesis discusses the possibility that mycobacteria may use more than one pathway for the biosynthesis of mycolates.

The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.

Introduction Les APL (ligands peptidiques modifies) sont des analogues des peptides antigeniques natifs capables de supprimer la reponse des lymphocytes T pathogenes en laissant le reste du systeme immunitaire intact. Objectifs Recherche d’un APL efficace dans le traitement du modele animal de polyradiculonevrite chronique induit chez les rats Lewis avec le peptide P0(180-199) S-palmitoyle « S- palmP0(180-199) ». Pour cela, trois APL ont ete synthetises : APL1, APL2, APL3. Patients et methodes Etude de la capacite des APL a induire une reponse immunitaire in vitro (proliferation lymphocytaire ; cytokines a j10) sur les cellules des ganglions lymphatiques de rats immunises avec le S-palmP0(180-199). Etude de l’activite protectrice in vivo des APL (evaluation clinique quotidienne ; proliferation lymphocytaire ; cytokines a j14) par co-immunisation des rats avec le S-palmP0(180-199) plus un des APL. Etude de la myeline et de l’inflammation du nerf sciatique et de la queue de cheval par immuno-histochimie. Resultats Les APL induisent une reponse proliferative in vitro similaire au peptide P0(180-199) mais seul le traitement avec l’APL3 permet une amelioration des signes cliniques et une reduction significative de l’inflammation. En effet, il existe une reduction de la production de cytokines pro-inflammatoires (in vitro et in vivo), une diminution des macrophages et des cellules secretrices d’IL-17 sur le nerf sciatique et la queue de cheval ainsi qu’une preservation des fibres myelinisees. Discussion La suite du projet sera d’etudier l’activite therapeutique de l’APL3 en l’injectant a des rats Lewis 2 jours apres le debut des symptomes (j12) puis dans un second temps d’etudier l’effet de l’APL3 palmitoyle. Nous savons que la palmitoylation du peptide P0(180-199) augmente son effet neuritogene, nous chercherons donc a savoir si la palmitoylation de l’APL augmente son effet protecteur. Conclusion L’APL3 semble etre le plus efficace pour reduire la reaction immunitaire et favoriser la recuperation clinique dans la polyradiculonevrite chronique. Il offre une piste serieuse a explorer en vue de developper une therapie antigenique dans cette pathologie. Informations complementaires Bourse JNLF pour annee recherche 2014–2015.

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) α1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.

NTRODUCTION: The aim of this study was to investigate the association between visual hallucinations in dementia with Lewy bodies (DLB) and brain perfusion using single-photon emission computed tomography. METHODS: We retrospectively included 66 patients with DLB, 36 of whom were having visual hallucinations (DLB-hallu) and 30 of whom were not (DLB-c). We assessed visual hallucination severity on a 3-point scale of increasing severity: illusions, simple visual hallucinations and complex visual hallucinations. We performed voxel-level comparisons between the two groups and assessed correlations between perfusion and visual hallucinations severity. RESULTS: We found a significant decrease in perfusion in the left anterior cingulate cortex, the left orbitofrontal cortex and the left cuneus in the DLB-hallu group compared with the DLB-c group. We also found a significant correlation between decreased bilateral anterior cingulate cortex, left orbitofrontal cortex, right parahippocampal gyrus, right inferior temporal cortex and left cuneus perfusion with the severity of hallucinations. CONCLUSIONS: Visual hallucinations seem to be associated with the impairment of anterior and posterior regions (secondary visual areas, orbitofrontal cortex and anterior cingulate cortex) involved in a top-down and bottom-up mechanism, respectively. Furthermore, involvement of the bilateral anterior cingulate cortex and right parahippocampal gyrus seems to lead to more complex hallucinations. journal article 2015 2015 02 17 imported

The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.

Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins inMycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel α1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present inMycobacterium smegmatis and other fast growing species. Tumor necrosis factor-α and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokine-inducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules.

Tuberculosis (TB), which kills more people than any other infectious disease, was declared a global emergency by the World Health Organization in 1993. The emergence of new Mycobacterium tuberculosis strains that are resistant to some or all current antitubercular drugs seriously hampers the control of the disease. Up to 50 million people may be infected with drug-resistant TB, with resistance being caused by inconsistent or partial treatment when patients do not comply with long-term chemotherapy. Resistance is often a corollary to HIV infection. Besides being more fatal, drug-resistant TB is more difficult and more expensive to treat. In addition to this human cost, TB also represents a significant economic burden for developing countries. Therefore, new approaches to the treatment of TB are needed. During the last few years, important efforts have been made in order to elucidate the molecular mechanism of action of antitubercular drugs and understand the genetic basis of acquired drug resistance in M. tuberculosis. The identification of novel targets requires the characterisation of biochemical pathways specific to mycobacteria. Many unique metabolic processes occur during the biosynthesis of cell wall components, including arabinogalactan and mycolic acids. In this review, the mode of action of first- and second-line agents, as well as the potentiality of some promising drugs that are still at an early stage of development will be described. Finally, some of the attractive targets offered by the mycobacterial cell wall for the rational design of new antitubercular drugs for a future and more effective control of the disease will be examined.

Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.

Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an α-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625–31631], which catalysed the formation of triacyl (Ac3)-PIM2 (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[14C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac3PIM3 (i.e. the trimannoside). The results indicate that pimC encodes an α-mannosyltransferase involved in Ac3PIM3 biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette—Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.

Tuberculosis (TB) was declared a global emergency by the WHO in 1993. Approximately one third of the world's population is infected with Mycobacterium tuberculosis. Despite the availability of effective chemotherapy, 3.5 million TB deaths occur each year. In addition to this human cost, TB also represents a significant economic burden for developing countries. The worldwide spread of TB is mainly caused by the emergence of multidrug-resistant (MDR) M. tuberculosis strains, thus underlining the need for novel antimycobacterial drugs with improved efficacy. Research in TB chemotherapy, as well as in vaccine development, has entered an intense phase in an attempt to combat this disease.

Introduction L’epitheliopathie en plaques (EEP) est une vascularite retinienne rare avec des cephalees et parfois des infarctus cerebraux (IC) que l’atteinte ophtalmologique peut preceder. Nous rapportons le cas d’un patient avec une EEP compliquee d’un infarctus cerebral. Observation Un homme de 24 ans fut adresse pour des scotomes centraux aigus associe a des cephalees en coup de tonnerre. L’examen ophtalmologique montrait des tâches blanches de la retine au fond d’œil, des anomalies de la perfusion retienne en plages a l’angiographie a la fluoresceine sans atteinte du segment anterieur en lien avec une epitheliopathie en plaques. Un mois apres, sont apparus un syndrome de Weber et des troubles de la conscience (NIH a 20). L’IRM cerebrale montrait de multiples infarctus cerebraux recents dans les territoires posterieur et anterieur, une occlusion de l’artere cerebrale posterieure droite, un aspect grele et irregulier des autres arteres intra-crâniennes et une leptomeningite. Le bilan infectieux, auto-immun et cardiovasculaire etait normal hormis une meningite lymphocytaire et la presence d’un anticoagulant circulant. Le traitement par corticotherapie et cyclophosphamide a permis une regression partielle des symptomes (NIH a 9). Discussion Il s’agit d’un cas d’EEP associee a une vascularite cerebrale avec IC bilateraux. Les etudes histologiques montrent un infiltrat inflammatoire granulomateux des arteres cerebrales de moyen calibre. Le bilan doit rechercher une cause infectieuse ou inflammatoire. Une meningite ou des cephalees sont des facteurs predictifs de complications cerebrovasculaires et justifie une surveillance neurologique, un traitement agressif precoce. Conclusion Le pronostic de l’epitheliopathie en plaques peut etre grave si une vascularite cerebrale y est associee. La presence d’une meningite et de cephalees doivent faire discuter d’emblee un traitement agressif.

Introduction Le bilan etiologique de premiere intention d’une myelite transverse subaigue est bien codifiee. Dans les formes dont l’etiologie demeure indeterminee, la place de la biopsie medullaire meriterait d’etre discutee. Observation Nous rapportons les cas de trois patients, ayant presente des tableaux cliniques de myelite subaigue, a la phase initiale des symptomes. Une IRM medullaire avait permis de mettre en evidence des hypersignaux T2 etendus de la moelle, associes selon les cas, a une prise de contraste apres injection de gadolinium. L’etude du LCR retrouvait une hyperproteinorachie majeure, souvent isolee. Le bilan infectiologique n’etait jamais concluant. Selon le contexte de chaque patient, differentes causes de myelite inflammatoire avaient ete evoquees, sans confirmation paraclinique : tuberculose medullaire pour le cas numero 1, myelite aigue post-vaccinale pour le cas numero 2 et myelite d’origine inflammatoire pour le cas numero 3. Ils ont alors beneficie d’un traitement etiologique ainsi que d’une corticotherapie au long cours, permettant dans un premier temps une amelioration des symptomes et une regression de la myelite sur l’IRM. Par la suite, devant la recidive des symptomes a plusieurs reprises et l’absence d’arguments formels pour une etiologie, une biopsie medullaire a finalement ete realisee. Celle-ci a permis de mettre en evidence une tumeur gliale de haut grade. Discussion Le recours a une biopsie medullaire merite d’etre discute lors d’un tableau de myelite subaigue d’etiologie indeterminee, apres bilan etiologique de premiere intention, et lors de la recidive des symptomes, malgre une prise en charge therapeutique supposee adaptee. Il n’existe cependant pas de reel consensus dans la litterature quant a son indication a des phases precoces. Conclusion Les glioblastomes medullaires primaires sont une entite rare. Leur presentation peut s’apparenter a une myelite transverse subaigue. La realisation precoce d’une biopsie medullaire permettrait d’optimiser la prise en charge de nos patients.

Introduction Un sous-type severe de polyradiculonevrite inflammatoire demyelinisante chronique (PIDC) associe a la presence d’anticorps anti-contactine 1, proteine des noeuds de Ranvier, a recemment ete decrit. Nous decrivons le cas de 2 patients. Observation Patient 1 : homme de 78 ans ayant presente un episode de polyradiculonevrite aigue en janvier 2016 avec atteinte multiples des paires crâniennes, tetra-paresie et ataxie proprioceptive severe des 4 membres. L’evolution est initialement favorable apres traitement par immunogloblines polyvalentes intraveineuses (IGIV). En mars, on note une degradation progressive des symptomes avec une ataxie et un tremblement proprioceptif majeur associes a une dysphagie et une dysphonie. Les IGIV et les corticoides sont inefficaces. Patient 2 : homme de 49 ans qui presente depuis 2000 des troubles sensitifs d’aggravation progressive suivi de troubles de la marche. La clinique se degrade rapidement en 2015 avec l’apparition d’une ataxie proprioceptive avec tremblement majeur. Les traitements par IGIV, corticoides, metothrexate et cyclophosphamide sont inefficaces. Dans les 2 cas, l’electromyogramme rempli les criteres EFNS de CIDP avec une nette predominance sensitive. La ponction lombaire retrouve une hyperproteinorachie majeure. Des anticorps anti-contactine sont mis en evidence et un traitement par Rituximab a debute. Discussion Les CIDP associees aux anticorps anti-contactine 1 presentent un debut souvent subaigu, une atteinte sensitive severe avec ataxie proprioceptive et tremblement intentionnel. La reponse aux IGIV et corticoides est mauvaise. Les anticorps anti-contactine sont de type IgG4 expliquant une bonne efficacite du Rituximab. Ces patients doivent etre depistes le plus tot possible car un traitement par Rituximab debute precocement serait plus efficace. Conclusion Il faut connaitre est depister les patients avec CIDP a anticorps anti-contactine car l’evolution peut etre severe et la reponse therapeutique est mauvaise. Un traitement par Rituximab doit etre envisage rapidement chez ces patients.

Introduction La polyradiculonevrite chronique (CIDP) est une pathologie inflammatoire demyelinisante du systeme nerveux peripherique (SNP) d’origine auto-immune et dont les ressources therapeutiques sont actuellement limitees. Objectifs Etudier l’effet des immunoglobulines humaines (IVIg) et d’un fragment Fc recombinant (FcRec) sur un modele animal de CIDP (EAN-chronique) obtenu chez des rats Lewis par immunisation active avec le peptide S-palm P0 (180–99). Patients et methodes Des rats Lewis immunises avec le peptide S-palm P0 (180–199) ont ete traites avec les IVIg ou le FcRec par injection intra peritoneale journaliere et pendant 5 jours des le debut de l’apparition des signes cliniques. Une evaluation clinique quotidienne a ete faite pendant 60 jours. Nous avons etudie a 18 et 60 jours post-immunisation (jpi) le taux d’IL-17 dans le serum. Une analyse immunohistochimique du nerf sciatique et de la queue de cheval a 60 jpi a evalue la myeline, l’infiltration cellulaire et l’IL-17. Resultats Le traitement therapeutique par IVIg ou FcRec a non seulement diminue la severite de l’EAN-chronique mais egalement supprime la chronicite de la maladie. Une meilleure efficacite du traitement avec le FcRec par rapport a l’IVIg a ete demontree au niveau histologique avec les fibres myelinisees bien preservees et l’accumulation de macrophages et de cellules IL-17+ dans les nerfs sciatiques fortement reduite. Les traitements ont egalement reduit de maniere significative le taux serique d’IL-17. Discussion L’etude actuelle fournit pour la premiere fois des preuves directes que l’IVIg est efficace dans le traitement d’un nouveau modele animal mimant la CIDP humaine et suggere qu’un nouveau compose, le FcRec, serait plus efficace que l’IVIg. Nos resultats ont demontre que ces traitements peuvent supprimer la chronicite de la maladie, inhiber l’inflammation dans le SNP et diminuer dans le sang le taux d’IL-17. Conclusion Ces donnees suggerent un role therapeutique potentiel des FcRec sur la CIDP humaine et confirment l’interet de le tester dans cette pathologie.

Objectives Two β-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum β-lactamase, BlaMab, indicating that the combination of β-lactams with a BlaMab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of BlaMab production on the efficacy of β-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of β-lactams intracellularly and in an animal model. Methods We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-β-lactam β-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding BlaMab to assess the extent of BlaMab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection. Results We showed that avibactam efficiently inactivated BlaMab via the reversible formation of a covalent adduct. An inhibition of BlaMab by avibactam was observed in both infected macrophages and zebrafish. Conclusions Our data identify avibactam as the first efficient inhibitor of BlaMab and strongly suggest that β-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, including transporters and sensor kinases. The KdpF peptide, which is cotranscribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s). The mycobacterial KdpF can interact with the KdpD histidine kinase, and kdpF overexpression has been shown to reduce intramacrophage replication of Mycobacterium bovis BCG. In this study, we investigated whether KdpF displays similar behavior in another intracellular pathogen, Salmonella enterica serovar Typhimurium. We show that Salmonella KdpF can interact with KdpD in a bacterial two-hybrid assay. We have constructed a Salmonella strain overexpressing kdpF, and we have investigated expression of the kdp regulon, as well as intramacrophage survival. We show that kdpF overexpression reduces expression of kdpA and kdpD genes under potassium limitation. Moreover, kdpF overexpression increases intramacrophage multiplication of S. Typhimurium. Hence, our results indicate that KdpF can play a regulatory role in S. Typhimurium, modulating kdp gene expression and intramacrophage survival, but in a way that differs from the one reported for M. bovis BCG.