No abstract

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides -arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy.AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules.Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is noncovalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

Accurate identification of tuberculosis (TB), caused by Mycobacterium tuberculosis, is important for global disease management. Point-of-care serological tests may improve TB diagnosis; however, specificities of available serodiagnostics are sub-optimal. We employed genetically encoded fragment-based discovery (GE-FBD) to select ligands for antibodies directed against the mycobacterial cell wall component lipoarabinomannan (LAM), a potent antigen. GE-FBD employed a phage displayed library of 10 heptapeptides, chemically modified with an arabinofuranosyl hexasaccharide fragment of LAM (Ara), and the anti-LAM antibody CS-35 as a bait. The selection gave rise to glycopeptides with an enhanced affinity and selectivity for CS-35 but not for 906.4321 antibody, both of which bind to Ara with a comparable affinity. Multivalent assays incorporating the discovered ligands Ara-ANSSFAP, Ara-DAHATLR and Ara-TTYVVNP exhibited up to 19-fold discrimination between CS-35 and 906.4321. The use of the Ara antigen alone failed to distinguish these antibodies. Thus, GE-FBD gives rise to ligands that differentiate monoclonal antibodies with enhanced specificity. This technology could facilitate the development of effective point-of-care serological tests for mycobacterial and other infections.