Françoise Laval

Surface-exposed glycopeptidolipids of mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.

MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis.

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram‐negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single‐chain antibodies against the surface‐exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C‐terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.

Mycobacterium tuberculosis proteins involved in mycolic acid synthesis and transport localize dynamically to the old growing pole and septum.

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles heel of the bacillus at all its growing stages.

Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria

ABSTRACT Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

Functional characterisation of three o-methyltransferases involved in the biosynthesis of phenolglycolipids in Mycobacterium tuberculosis.

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

The changes in mycolic acid structures caused by hadC mutation have a dramatic effect on the virulence of Mycobacterium tuberculosis.

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra‐long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4‐log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.

The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs.

The cyclic di‐GMP phosphodiesterase gene Rv1357c/BCG1419c affects BCG Pellicle production and In Vivo maintenance

Bacteria living in a surface‐attached community that contains a heterogeneous population, coated with an extracellular matrix, and showing drug tolerance (biofilms) are often linked to chronic infections. In mycobacteria, the pellicle mode of growth has been equated to an in vitro biofilm and meets several of the criteria mentioned above, while tuberculosis infection presents a chronic (latent) phase of infection. As mycobacteria lack most genes required to control biofilm production by other microorganisms, we deleted or expressed from the hsp60 strong promoter the only known c‐di‐GMP phosphodiesterase (PDE) gene in Mycobacterium bovis BCG. We found changes in pellicle production, cellular protein profiles, lipid production, resistance to nitrosative stress and maintenance in lungs and spleens of immunocompetent BALB/mice. Our results show that pellicle production and capacity to remain within the host are linked in BCG.

Structural elucidation and genomic scrutiny of the C60-C100 mycolic acids of Segniliparus rotundus.

Mycolic acids, very long-chain α-alkyl, β-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.

A novel mycolic acid species defines two novel genera of the Actinobacteria, Hoyosella and Amycolicicoccus.

Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (α-alkyl, β-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, (1)H-NMR spectroscopy and chemical degradations. The C(2)-C(3) cleavage of these C(30)-C(36) acids led to the formation of two fragments: saturated C(9)-C(11) acids, and saturated and unsaturated C(20)-C(25) aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C(24) aldehyde fragment was located at position ω16, whereas that found in the ethylenic fatty acids of the bacteria was at ω9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella.