Antigen 85C inhibition restricts Mycobacterium tuberculosis growth through disruption of cord factor biosynthesis.

The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic ß-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

Functional expression of the PorAH channel from Corynebacterium glutamicum in cell-free expression systems: implications for the role of the naturally occurring mycolic acid modification.

PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. (15)N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.

O-mycoloylated proteins from Corynebacterium: an unprecedented post-translational modification in bacteria.

O-acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain alpha-alkyl and beta-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.

A deficiency in arabinogalactan biosynthesis affects Corynebacterium glutamicum mycolate outer membrane stability.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal beta(1 --> 2)-linked Araf residues. Here we show that Delta aftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a Delta aftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the Delta aftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum.

Identification of a stress-induced factor of Corynebacterineae that is involved in the regulation of the outer membrane lipid composition.

Corynebacterineae are gram-positive bacteria that possess a true outer membrane composed of mycolic acids and other lipids. Little is known concerning the modulation of mycolic acid composition and content in response to changes in the bacterial environment, especially temperature variations. To address this question, we investigated the function of the Rv3802c gene, a gene conserved in Corynebacterineae and located within a gene cluster involved in mycolic acid biosynthesis. We showed that the Rv3802 ortholog is essential in Mycobacterium smegmatis, while its Corynebacterium glutamicum ortholog, NCgl2775, is not. We provided evidence that the NCgl2775 gene is transcriptionally induced under heat stress conditions, and while the corresponding protein has no detectable activity under normal growth conditions, the increase in its expression triggers an increase in mycolic acid biosynthesis concomitant with a decrease in phospholipid content. We demonstrated that these lipid modifications are part of a larger outer membrane remodeling that occurs in response to exposure to a moderately elevated temperature (42 degrees C). In addition to showing an increase in the ratio of saturated corynomycolates to unsaturated corynomycolates, our results strongly suggested that the balance between mycolic acids and phospholipids is modified inside the outer membrane following a heat challenge. Furthermore, we showed that these lipid modifications help the bacteria to protect against heat damage. The NCgl2775 protein and its orthologs thus appear to be a protein family that plays a role in the regulation of the outer membrane lipid composition of Corynebacterineae under stress conditions. We therefore propose to name this protein family the envelope lipids regulation factor (ElrF) family.

Partial redundancy in the synthesis of the D-arabinose incorporated in the cell wall arabinan of Corynebacterineae.

The major cell wall carbohydrate of Corynebacterineae is arabinogalactan (AG), a branched polysaccharide that is essential for the physiology of these bacteria. Decaprenylphosphoryl-D-arabinose (DPA), the lipid donor of D-arabinofuranosyl residues of AG, is synthesized through a series of unique biosynthetic steps, the last one being the epimerization of decaprenylphosphoryl-beta-D-ribose (DPR) into DPA, which is believed to proceed via a sequential oxidation-reduction mechanism. Two proteins from Mycobacterium tuberculosis (Rv3790 and Rv3791) have been shown to catalyse this epimerization in an in vitro system. The present study addressed the exact function of these proteins through the inactivation of the corresponding orthologues in Corynebacterium glutamicum (NCgl0187 and NCgl0186, respectively) and the analysis of their in vivo effects on AG biosynthesis. We showed that NCgl0187 is essential, whereas NCgl0186 is not. Deletion of NCgl0186 led to a mutant possessing an AG that contained half the arabinose and rhamnose, and less corynomycolates linked to AG but more trehalose mycolates, compared with the parental strain. A candidate gene that may encode a protein functionally similar to NCgl0186 was identified in both C. glutamicum (NCgl1429) and M. tuberculosis (Rv2073c). While the deletion of NCgl1429 had no effect on AG biosynthesis of the mutant, the gene could complement the mycolate defect of the AG of the NCgl0186 mutant, strongly supporting the concept that the two proteins play a similar function in vivo. Consistent with this, the NCgl1429 gene appeared to be essential in the NCgl0186-inactivated mutant. A detailed bioinformatics analysis showed that NCgl1429, NCgl0186, Rv3791 and Rv2073c could constitute, with 52 other proteins belonging to the actinomycetales, a group of closely related short-chain reductases/dehydrogenases (SDRs) with atypical motifs. We propose that the epimerization of DPR to DPA involves three enzymes that catalyse two distinct steps, each being essential for the viability of the bacterial cells.

The key role of the mycolic acid content in the functionality of the cell wall permeability barrier in Corynebacterineae.

Recently, it has been shown that trehalose and mycolic acids are essential for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, and important but not indispensable to the survival of Corynebacterium glutamicum. Therefore, to investigate the function of mycolic acids in both the permeability of the cell wall to small nutrients and antibiotics, and the excretion of amino acids by C. glutamicum, a trehalose-deficient mutant of the L-lysine producer ATCC 21527, designated LP Delta treS Delta otsA Delta treY, was constructed. By using different carbon sources in either the presence or the absence of external trehalose, a set of endogenously trehalose-free LP Delta treS Delta otsA Delta treY cells that exhibited various mycolate contents was generated. The results showed that the structure of the arabinogalactan of these different cell types of LP Delta treS Delta otsA Delta treY was not affected when the mycolic acid layer was either missing or impaired. Nevertheless, cells were more susceptible to antibiotics, and the permeability of their cell walls to glycerol was increased. Interestingly, a concomitant increase in the excretion of both L-lysine and L-glutamate was also observed, indicating that the mycolic acid content of the permeability barrier (and not only the peptidoglycan and/or the arabinogalactan) is implicated in the glutamate excretion process.

The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae.

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.

Structural elucidation of the predominant motifs of the major cell wall arabinogalactan antigens from the borderline species Tsukamurella paurometabolum and Mycobacterium fallax.

Tsukamurella paurometabolum and Mycobacterium fallax are members of the suprageneric actinomycete group Corynebacterineae that possesses a cell wall skeleton composed of a peptidoglycan to which an arabinogalactan is covalently attached. This polysaccharide is further modified by esterification with C60-C80 mycolic acid residues in mycobacteria and T. paurometabolum. However, M. fallax and T. paurometabolum produce polyenoic (up to six double bonds) mycolic acids whereas the most common type of mycobacterial mycolates, called alpha-mycolates, are mono- and di-enoic or -cyclopropanated mycolic acids. To determine whether this difference also applied to the structures of cell wall arabinogalactans, competitive inhibition experiments using antibodies raised against the cell wall from Mycobacterium bovis and the arabinogalactans from T. paurometabolum and M. fallax were performed. They demonstrated the structural identity between the polysaccharide of M. fallax and those of mycobacteria and showed a strong similarity between the latter polysaccharides and that of T. paurometabolum. Structural analyses of the per-O-alkylated alditol fragments derived from the polysaccharides by gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR) spectroscopy of the intact solubilized polysaccharides demonstrated that the polysaccharides from the two species analyzed contained all the major structural features previously characterized in mycobacterial arabinogalactans. These include (1) the homogalactan of alterning 5-linked galactofuranosyl (Galf) and 6-linked Galf residues, (2) a linear 5-linked arabino furanosyl (Araf), (3) a beta-Araf-(1-->2)-alpha-Araf disaccharide branched on both position 3 and position 5 of an alpha-Araf unit, and (4) a 5-linked-alpha-Araf unit branched on both position 3 and position 5 of an alpha-Araf residue. The polysaccharide from T. paurometabolum possesses additional structural domains composed of a terminal (t) Araf directly linked to either a 5-linked-alpha-Araf or to both position 3 and position 5 of a 3,5-linked alpha-Araf unit. Both the remarkable similarity of arabinogalactans from Corynebacterineae and their genus- and/or species-specificities are reflected in their 13C NMR spectra that may be used as a valuable help in the identification of members of the actinomycete group.

Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum.

Mycoloyltransferases (Myts) play an essential role in the biogenesis of the cell envelope of members of the Corynebacterineae, a group of bacteria that includes the mycobacteria and corynebacteria. While the existence of several functional myt genes has been demonstrated in both mycobacteria and corynebacteria (cmyt), the disruption of any of these genes has at best generated cell-wall-defective but always viable strains. To investigate the importance of Myts on the physiology of members of the Corynebacterineae, a double mutant of Corynebacterium glutamicum was constructed by deleting cmytA and cmytB, and the consequences of the deletion on the viability of the mutant, the transfer of corynomycoloyl residues onto its cell-wall arabinogalactan and trehalose derivatives, and on its cell envelope ultrastructure were determined. The double mutant strain failed to grow at 34 degrees C and exhibited a growth defect and formed segmentation-defective cells at 30 degrees C. Biochemical analyses showed that the double mutant elaborated 60 % less cell-wall-bound corynomycolates and produced less crystalline surface layer proteins associated with the cell surface than the parent and cmytA-inactivated mutant strains. Freeze-fracture electron microscopy showed that the DeltacmytA DeltacmytB double mutant, unlike the wild-type and cmytA-inactivated single mutant strains, frequently exhibited an additional fracture plane that propagated within the plasma membrane and rarely exposed the S-layer protein. Ultra-thin sectioning of the double mutant cells showed that they were totally devoid of the outermost layer. Complementation of the double mutant with the wild-type cmytA or cmytB gene restored completely or partially this phenotype. The data indicate that Myts are important for the physiology of C. glutamicum and reinforce the concept that these enzymes would represent good targets for the discovery of new drugs against the pathogenic members of the Corynebacterineae.

New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum.

Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope. We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C. The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt). In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C. glutamicum and three cmyt genes in C. diphtheriae. In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis. In C. glutamicum CGL2005 cmytE is a pseudogene. The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains. Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain. For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene. Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.

Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis.

Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis, the cell wall mycoloylation decreases by 40% when fbpC is knocked out. To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA- and fbpB-inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC-disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC-inactivated mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides.