Identification of a Novel Arabinofuranosyltransferase (AftA) Involved in Cell Wall Arabinan Biosynthesis in Mycobacterium tuberculosis

Abstract

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor ␤-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from ␤-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.The Corynebacterianeae represent a distinct group within Grampositive bacteria, with prominent members being the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae, and Corynebacterium diphtheriae (1). In addition, nonpathogenic bacteria belonging to this taxon, such as Corynebacterium glutamicum and Corynebacterium efficiens, are used in the industrial production of amino acids (2). A common feature of the Corynebacterianeae is that they possess an unusual cell wall architecture (3-5). The cell wall is dominated by an essential heteropolysaccharide, arabinogalactan (AG), 5 linked to both peptidoglycan and mycolic acids, forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex (3-6). The biosynthesis of the arabinan domain of AG, which is made up of ␣135-, ␣133-, and ␤132-glycosyl linkages, results from the sequential addition of arabinofuranose (Araf ) residues from the sugar donor ␤-D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) (7-9), by a set of unique arabinofuranosyltransferases termed the Emb proteins, of which three paralogues exist in Mycobacterium avium (10) and M. tuberculosis (11). The anti-tuberculosis drug ethambutol (EMB) specifically inhibits AG biosynthesis (12), and the molecular target of EMB occupies the embCAB locus in M. tuberculosis (11). Upon individual disruption of embC, embA, and embB in My...

Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

“…Moreover, initial Araf residues at branching sites could require specialized arabinofuranosyltransferases as already observed for AftA (19), and it has to be considered that even further specialized arabinofuranosyltransferases might exist to incorporate Araf into lipoarabinomannan. Clearly additional arabinofuranosyltransferases still remain to be identified in Corynebacterianeae.…”

“…Arabinofuranosyltransferase Activity with Membrane Preparations of C. glutamicum, C. glutamicum⌬aftB, and C. glutamicum⌬aftB pMSX-Mt-aftB-Membranes were prepared as described previously (19,24) and resuspended in 50 mM MOPS (pH 7.9) containing 5 mM ␤-mercaptoethanol and 10 mM MgCl 2 (buffer A) to a final concentration of 15-10 mg/ml.…”

“…Indeed, Liu and Mushegian (29) identified 15 members of the GT-C superfamily, representing candidates involved in the biosynthesis of cell wall related glycans and lipoglycans in M. tuberculosis. We have continued our earlier studies (9,16,19) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex in Corynebacterianeae by studying mutants of C. glutamicum and the orthologous genes and enzymes of M. tuberculosis. Herein we present Rv3805c as a new arabinofuranosyltransferase of the GT-C superfamily that is responsible for the transfer of Araf residues from DPA to the arabinan domain to form terminal ␤(1 3 2)-linked Araf residues, which marks the "end point" for AG arabinan biosynthesis before decoration with mycolic acids.…”

“…MALDI-TOF mass spectrometry (MS) analyzes of per-Omethylated AG of C. glutamicum deleted of its single arabinofuranosyltransferase, Cg-emb, revealed that the 8th, 10th, and 12th Galf residue possessed singular Araf residues (9). These specific Araf residues were recently shown to be transferred by a specialized arabinofuranosyltransferase AftA, whose gene in all Corynebacterianeae analyzed to date is adjacent to the emb cluster (19). These initial Araf residues "prime" the galactan backbone for further attachment of ␣(1 3 5)-linked Araf residues.…”

“…with respect to the deletion of Cg-emb (9) and Cg-aftA (19), which are otherwise essential in M. tuberculosis. 5 The structural basis of AG is now well defined (4,5,8); conversely, aspects of its biogenesis remained poorly resolved.…”