AftD, a novel essential arabinofuranosyltransferase from mycobacteria.
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
Skovierová
Henrieta
H
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
Larrouy-Maumus
Gérald
G
Zhang
Jian
J
Kaur
Devinder
D
Barilone
Nathalie
N
Korduláková
Jana
J
Gilleron
Martine
M
Guadagnini
Stéphanie
S
Belanová
Martina
M
Prevost
Marie-Christine
MC
Gicquel
Brigitte
B
Puzo
Germain
G
Chatterjee
Delphi
D
Brennan
Patrick J
PJ
Nigou
Jérôme
J
Jackson
Mary
M
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005685:Galactans / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D005685:Galactans; D008070:Lipopolysaccharides; C050016:lipoarabinomannan; D016695:Glycosyltransferases; C005653:arabinogalactan