Abstract
The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.
Highlights
The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development
The cell wall core, which is essential for viability, consists of peptidoglycan (PG)5 in covalent attachment via phosphoryl-Nacetylglucosaminosyl-rhamnosyl linkage units (P-GlcNAc-Rha) with the heteropolysaccharide arabinogalactan (AG); AG is in turn esterified at its non-reducing ends to long-chain (C70–C90) mycolic acids (Fig. 1)
The galactan is biosynthesized in the cytoplasm by two bifunctional galactosyltransferases, with the first two Galf residues added to the linker unit by the galactosyltransferase GlfT1 (Rv3782) and the remaining alternating 5- and 6-linked Galf residues added by GlfT2 (Rv3808c)
Summary
Nyl phosphate (Dec-P) carrier lipid with formation of the linker unit followed by the addition of Galf and Araf residues (10 –12). A WecA-like transferase encoded by Rv1302 in the genome of M. tuberculosis H37Rv transfers GlcNAc 1-phosphate to Dec-P to form Dec-P-P-GlcNAc (GL-1) This step is followed by the attachment of a rhamnosyl residue from dTDPRha to the 3-position of GlcNAc in a reaction catalyzed by WbbL1 to form GL-2 (Dec-P-P-GlcNAc-Rha), the “linker unit” (Fig. 1). LCPlike proteins have further been involved in the transfer of capsular polysaccharides to PG and the glycosylation of cell wall-associated proteins in a variety of other Gram-positive organisms [22,23,24,25,26,27,28,29] These reports led us to investigate the possible involvement and therapeutic potential of the three LCP-like proteins encoded by the M. tuberculosis genome in the ligation of AG to PG. Our results highlight the participation of two of these proteins in the phosphotransferase reaction leading to the ligation of AG and PG, a crucial step in the assembly of the entire complex cell wall of M. tuberculosis
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