Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen.

Cloning and Partial Characterization of an Endo-α-(1→6)-d-Mannanase Gene from Bacillus circulans.

Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1→6) [Manp-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria.

Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

New insights into the biosynthesis of mycobacterial lipomannan arising from deletion of a conserved gene.

Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.

Biosynthesis of mycobacterial lipoarabinomannan: role of a branching mannosyltransferase.

Lipoarabinomannan (LAM), one of the few known bacterial glycosylphosphoinositides (GPIs), occurs in various structural forms in Mycobacterium species. It has been implicated in key aspects of the physiology of Mycobacterium tuberculosis and the immunology and pathogenesis of tuberculosis. Yet, little is known of the biosynthesis of LAM. A bioinformatics approach identified putative integral membrane proteins, MSMEG4250 in Mycobacterium smegmatis and Rv2181 in M. tuberculosis, with 10 predicted transmembrane domains and a glycosyltransferase (GT) motif (DID), features that are common to eukaryotic mannosyltransferases (ManTs) of the GT-C superfamily that rely on polyprenyl-linked rather than nucleotide-linked sugar donors. Inactivation of M. smegmatis MSMEG4250 by allelic exchange resulted in altered growth and inability to synthesize lipomannan (LM) but accumulation of a previously uncharacterized, truncated LAM. MALDI-TOF/MS and NMR indicated a structure lower in molecular weight than the native molecule, a preponderance of 6-linked Manp residues, and the absence of 2,6-linked and terminal Manp. Complementation of the mutant with the corresponding ortholog of M. tuberculosis H37Rv restored normal LM/LAM synthesis. The data suggest that MSMEG4250 and Rv2181 are ManTs that are responsible for the addition of alpha(1-->2) branches to the mannan core of LM/LAM and that arrest of this branching in the mutant deters formation of native LAM. The results allow for the presentation of a unique model of LM and LAM biosynthesis. The generation of mutants defective in the synthesis of LM/LAM will help define the role of these GPIs in the immunology and pathogenesis of mycobacterial infections and physiology of the organism.

The carboxy terminus of EmbC from Mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan.

D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

A major cell wall lipopeptide of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-L-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phospho-{alpha}-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis.

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.