Eukaryotic UDP-Galactopyranose Mutase ( GLF Gene) in Microbial and Metazoal Pathogens

Abstract

Galactofuranose (Gal f ) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal f is synthesized as the nucleotide sugar UDP-Gal f by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal f synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal f synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal f synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal f plays critical roles in cellular viability and virulence.

Unusual Galactofuranose Modification of a Capsule Polysaccharide in the Pathogenic Yeast Cryptococcus neoformans

“…We next engineered a strain lacking Galf by deleting the gene encoding UDP-galactopyranose mutase, the enzyme that catalyzes the formation of the Galf donor molecule, UDP-Galf. The eukaryotic gene encoding UDP-galactopyranose mutase, GLF, was discovered by bioinformatics analysis in Leishmania, and an ortholog was identified in Cryptococcus; the activity corresponding to both sequences was demonstrated by heterologous expression in prokaryotes (43). Here we show that as expected the GXMGal from glf⌬ mutants was devoid of Galf.…”

“…neoformans JEC21 (43). In this study, we replaced the genomic coding sequence of GLF with a nourseothricin resistance marker (NAT) by homologous recombination.…”

Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

“…After inoculation of the parasite into the mammalian host by the sand fly bite, LPG plays important roles in the establishment of the infection by conferring resistance to lysis by complement, protection from oxidative damage, and remodeling of the initial phagolysome, including the induction of a transient delay in phagolysosomal fusion (13)(14)(15). The roles of LPG deduced by a variety of biochemical and cellular studies have been supported by studies of mutant parasites lacking genes of the LPG biosynthetic pathway (13,(16)(17)(18)(19).…”

“…A key linkage within the LPG core involves galactosylfuranose (Galf ), an unusual parasite sugar not made by the mammalian host (21,22). The precursor for Galf is UDP-Galf, which arises from the action of cytosolic UDP-galactopyranose mutase (UGM, encoded by the gene GLF) (16,23), and glf Ϫ knockouts confirm the requirement for this gene in Leishmania LPG biosynthesis (18). Although Galf occurs on other Leishmania glycoconjugates, including the abundant glycosylinositolphosphatidylinositols (GIPLs), previous studies establish that GIPLs play little role in L. major infectivity (24) and, accordingly, glf Ϫ knockouts show phenotypes identical to lpg1 Ϫ parasites lacking LPG alone (18,19,25).…”

Leishmania UDP-sugar Pyrophosphorylase

“…This glycocalyx is particularly rich in galactose occurring either in the pyranosic form (Gal) or the more unusual furanosic form (Galf). 4 Its biosynthesis thus depends on the availability of the nucleotide-activated sugar UDP-galactopyranose (UDP-Gal), which can be interconverted into UDP-galactofuranose (UDP-Galf) by the specific enzyme UDP-galactopyranose mutase (2,3). Consequently, mutants deficient in the formation of UDP-Galf or in the transport of UDP-Gal into the secretory pathway organelles present an altered glycocalyx associated with parasite attenuation (4 -7).…”