22 research outputs found
Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis
Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The main structural elements of the cell wall consist of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are covalently attached to a complex polysaccharide, arabinogalactan (AG), via a unique α-l-rhamnopyranoseâ(1â3)-α-d-GlcNAc-(1âP) linker unit. While the molecular genetics associated with PG and AG biosynthetic pathways have been largely delineated, the mechanism by which these two major pathways converge has remained elusive. In Gram-positive organisms, the LytR-CpsA-Psr (LCP) family of proteins are responsible for ligating cell wall teichoic acids to peptidoglycan, through a linker unit that bears a striking resemblance to that found in mycobacterial arabinogalactan. In this study, we have identified Rv3267 as a mycobacterial LCP homolog gene that encodes a phosphotransferase which we have named Lcp1. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is capable of ligating AG to PG in a cell-free radiolabeling assay
Synthesis of a homologous series of galactofuranose-containing mycobacterial arabinogalactan fragments
Mycobacteria, including the human pathogen Mycobacterium tuberculosis the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the mycobacterial cell wall is a glycoconjugate called the mycolic acidâarabinogalactanâpeptidoglycan (mAGP) complex. Inhibition of mAGP biosynthesis is a proven strategy for developing anti-mycobacterial drugs and thus understanding the pathways and enzymes involved in the assembly of this molecule is of interest. In this paper we describe the chemical synthesis of a panel nine oligosaccharides fragments (4â12) of the galactan domain of the mAGP complex designed as biosynthetic probes. These structures, ranging in size from a hexasaccharide to a tetradecasaccharide, are potential substrates for two biosynthetic enzymes, GlfT2 and AftA, and represent the largest mycobacterial galactan fragments synthesized to date. The route developed was iterative and provided multi-milligram quantities of the target molecules 4â12 in good overall yield.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author
Synthesis of the Docosanasaccharide Arabinan Domain of Mycobacterial Arabinogalactan and a Proposed Octadecasaccharide Biosynthetic Precursor
Biosynthesis of the Methylthioxylose Capping Motif of Lipoarabinomannan in Mycobacterium tuberculosis
International audienc
The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan
International audienceDespite the impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and /composition in response to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes in its structure, but no reports exist supportings this assumption. Herein, using [MS2] recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we identified found that the mycobacterial protein GlfH1 (Rv3096), which protein exhibits an exo-ÎČ-D-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal ÎČ-(1,5) and ÎČ-(1,6)-Galf linkages. The characterization of this galactosidase represents the a first step towards understanding the remodeling of mycobacterial AG
The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the α(1âŻââŻ5) arabinan backbone in the early stages of cell wall arabinan biosynthesis
The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ÎaftAÎemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumÎaftA, C. glutamicumÎemb, C. glutamicumÎaftAÎemb and C. glutamicumÎaftBÎaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) âprimedâ galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1âŻââŻ5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum
Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis.
International audienceUDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis
Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides
Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria
Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine
© 2017 Prados-Rosales et al.Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice rea
Biosynthesis of the Methylthioxylose Capping Motif of Lipoarabinomannan in <i>Mycobacterium tuberculosis</i>
Lipoarabinomannan
(LAM) is a lipoglycan found in abundant quantities
in the cell envelope of all mycobacteria. The nonreducing arabinan
termini of LAM display species-specific structural microheterogeneity
that impacts the biological activity of the entire molecule. <i>Mycobacterium tuberculosis</i>, for instance, produces mannoside
caps made of one to three α-(1 â 2)-Man<i>p</i>-linked residues that may be further substituted with an α-(1
â 4)-linked methylthio-d-xylose (MTX) residue. While
the biological functions and catalytic steps leading to the formation
of the mannoside caps of <i>M. tuberculosis</i> LAM have
been well established, the biosynthetic origin and biological relevance
of the MTX motif remain elusive. We here report on the discovery of
a five-gene cluster dedicated to the biosynthesis of the MTX capping
motif of <i>M. tuberculosis</i> LAM, and on the functional
characterization of two glycosyltransferases, MtxS and MtxT, responsible,
respectively, for the production of decaprenyl-phospho-MTX (DP-MTX)
and the transfer of MTX from DP-MTX to the mannoside caps of LAM.
Collectively, our NMR spectroscopic and mass spectrometric analyses
of <i>mtxS</i> and <i>mtxT</i> overexpressors
and knockout mutants support a biosynthetic model wherein the conversion
of 5âČ-methylthioadenosine, which is a ubiquitous byproduct
of spermidine biosynthesis, into 5âČ-methylthioribose-1-phosphate
precedes the formation of a 5âČ-methylthioribose nucleotide
sugar, followed by the epimerization at C-3 of the ribose residue,
and the transfer of MTX from the nucleotide sugar to decaprenyl-phosphate
yielding the substrate for transfer onto LAM. The conservation of
the MTX biosynthetic genes in a number of Actinomycetes suggests that
this discrete glycosyl substituent may be more widespread in prokaryotes
than originally thought