Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrateenzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an -(16)-mannosyltransferase (ManT) and a -(15,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating -(15) and -(16)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.

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Cell wall mannans of Candida albicans mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling. Graus et al. find that N-mannan structural features regulated by Candida mannosyltransfersases control glucan exposure. Loss of mannan increased the frequency and size of glucan exposures and changed multivalent receptor engagement. Changes to mannan structure in a bloodstream isolate are associated with elevated glucan exposure at the nanoscale.