Targeted Gene Deletion of Leishmania major UDP-galactopyranose Mutase Leads to Attenuated Virulence

Abstract

Considering the high incidence of galactofuranose (Gal f ) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal f in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal f metabolism by providing UDP-Gal f to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal f metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal f is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf ؊ mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.

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).…”

“…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). In this work, we used the dd fusion approach to regulate LPG expression through control of UGM levels, yielding parasites that when placed in the ''on'' and ''off'' states closely resemble WT and LPG-deficient mutants.…”

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

“…Interestingly, both mutants continued to express LPG fragments and an abundant class of glycophosphatidylinositol lipids (GIPLs), which is expressed in both the promastigote and amastigote stages (48). The lpg2 Ϫ/Ϫ mutant expresses a Gal-Gal-Galf-Man-ManGlcN-Inos-P-lyso-alkyl glycerol (24) and normal levels of GIPLs, whereas the GLF mutant should continue to express the LPG fragment Man-Man-GlcN-Inos-P-lyso-alkyl glycerol as well as truncated GIPLs, which differ from the LPG fragment in bearing an acyl-alkyl glycerol anchor (51). However, none of these molecules (GIPL, truncated LPG, or truncated GIPLs) seemed to be the target of the VIP analog.…”

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

“…Kinetoplastids incorporate Galf into a protective glycocalyx that enhances establishment of disease and promotes survival of these parasites within their insect vectors (8). In Leishmania, loss of Galf synthesis or of the key galactofuranosyltransferase required to synthesize surface lipophosphoglycan results in severe defects in survival both in the insect and in the early stages of mammalian infection (15)(16)(17)(18). In the pathogenic fungus Aspergillus fumigatus, the presence of Galf in secreted glycoproteins, glycolipids, and cell wall polysaccharides (7,8) improves cell integrity (19 -21) and reduces adhe-sion to mammalian epithelial and endothelial surfaces by shielding underlying mannan structures (20).…”