Galactofuranose in mycobacteria and nematodes

Abstract

All cells are covered in a coat of carbohydrates. These sugars participate in many important processes and are often essential for viability. Polysaccharides assembled by both prokaryotic and eukaryotic pathogens contain monosaccharide building blocks not used by mammals. For example, galactofuranose (Galf), the 5-membered ring conformation of galactose, is distributed broadly across many types of bacteria, lower eukaryotes, and invertebrates. However, mammals do not use Galf in their glycans. Since many human pathogens require Galf for viability, enzymes necessary for Galf biosynthesis and incorporation into polysaccharides represent worthy drug targets. In this thesis, I examine incorporation of Galf by glycosyltransferases and the importance of this sugar to mycobacterial and nematode physiology. In Chapter 1, I review cell envelope assembly for members of the Corynebacterineae suborder.

Many organisms within this suborder represent major public health threats and all require the presence of the galactan, a polysaccharide composed primarily by Galfresidues. In Chapter 2, 1 evaluate the consequences of Galfinhibition within the free-living nematode, C. elegans. Results from these experiments validate Galf biosynthesis as a worthy drug target for parasitic nematodes. In Chapter 3, I report the biochemical characterization of the glycosyltransferase G1fT1. This enzyme initiates polymerization of the galactan, and we find that G1fT 1 contains remarkable substrate selectivity. We propose that Nature has evolved two galactofuranosyl transferases so that G1fT1 can effectively discriminate between substrates that will later be elongated by the promiscuous enzyme, G1ff2. In Chapter 4, I explore structure-function relationships between galactan chain length and mycobacterial physiology.

Results from these experiments demonstrate that galactan chain length creates an important determinant of cell envelope mechanical integrity and periplasm size. In Chapter 5, I purify and test activity of uncharacterized G1fT2 orthologs. These proteins were thought to install unique linkages that are not created by mycobacterial G1fT2s. However, our results demonstrate that these proteins also create the same linkages as previously characterized enzymes, implying that G1fT2 regioselectivity is conserved.