Gérald Larrouy-Maumus

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium–host interaction

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC‐SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose‐dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC‐SIGN‐transfected Raji cells, but no differences in IL‐10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose‐capped LAM does not dominate the Mycobacterium–host interaction.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

The immunomodulatory lipoglycans, lipoarabinomannan and lipomannan, are exposed at the mycobacterial cell surface

By labeling surface carbohydrates, we found that a pool of lipoglycans, cell wall associated, is exposed at the cell surface of mycobacteria and thus, most probably, inserted in the outer leaflet of the outer membrane. In contrast, plasma membrane anchored lipoglycans are not accessible to surface labeling. This result supports the role of lipoglycans as key immunomodulatory molecules but raises the question of their transport from the plasma membrane, where they are synthesized, to the outermost layers of the envelope, where they can act as modulins. The data are discussed in terms of consequences for cell envelope organization.

Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate.

Here we identify the amino acid transporter AnsP1 as the unique aspartate importer in the human pathogen Mycobacterium tuberculosis. Metabolomic analysis of a mutant inactivated in AnsP1 revealed the transporter is essential for M. tuberculosis to assimilate nitrogen from aspartate. Virulence of the AnsP1 mutant is impaired in vivo, revealing aspartate is a primary nitrogen source required for host colonization by the tuberculosis bacillus.

Metabolomic strategies for the identification of new enzyme functions and metabolic pathways

Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity‐based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non‐physiological functions. This review discusses state‐of‐the‐art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources.

AftD, a novel essential arabinofuranosyltransferase from mycobacteria

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis

Background: The biogenesis of 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) found in the outer membrane of Mycobacterium tuberculosis is ill defined. Results: DAT synthesis is cytosolic. Chp2-mediated transesterification reactions between DAT substrates yield PAT on the periplasmic face of the membrane. Conclusion: DAT and PAT biosynthesis is topologically split across the membrane. Significance: DAT and PAT biosynthesis and transport are coupled and dependent on the MmpL10 transporter. A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activity-based metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region. Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.

Biosynthetic origin of the galactosamine substituent of Arabinogalactan in Mycobacterium tuberculosis.

The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenyl-phospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40–50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane. The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.