Identification of Compounds Preventing A. fumigatus Biofilm Formation by Inhibition of the Galactosaminogalactan Deacetylase Agd3.

The opportunistic fungus Aspergillus fumigatus causes a set of diseases ranging from allergy to lethal invasive mycosis. Within the human airways, A. fumigatus is embedded in a biofilm that forms not only a barrier against the host immune defense system, but also creates a physical barrier protecting the fungi from chemicals such as antifungal drugs. Novel therapeutic strategies aim at combining drugs that inhibit biofilm synthesis or disrupt existing biofilm with classical antimicrobials. One of the major constituents of A. fumigatus biofilm is the polysaccharide galactosaminogalactan (GAG) composed of α1,4-linked N-acetylgalactosamine, galactosamine, and galactose residues. GAG is synthesized on the cytosolic face of the plasma membrane and is extruded in the extracellular space, where it is partially deacetylated. The deacetylase Agd3 that mediates this last step is essential for the biofilm formation and full virulence of the fungus. In this work, a previously described enzyme-linked lectin assay, based on the adhesion of deacetylated GAG to negatively charged plates and quantification with biotinylated soybean agglutinin was adapted to screen microbial natural compounds, as well as compounds identified in in silico screening of drug libraries. Actinomycin X2, actinomycin D, rifaximin, and imatinib were shown to inhibit Agd3 activity in vitro. At a concentration of 100 µM, actinomycin D and imatinib showed a clear reduction in the biofilm biomass without affecting the fungal growth. Finally, imatinib reduced the virulence of A. fumigatus in a Galleria mellonella infection model in an Agd3-dependent manner.

Characterization of a gene cluster involved in Aspergillus fumigatus zwitterionic glycosphingolipid synthesis.

The human pathogenic fungus Aspergillus fumigatus synthesizes the zwitterionic glycolipid Manα1,3Manα1,6GlcNα1,2IPC, named Af3c. Similar glycosphingolipids having a glucosamine (GlcN) linked in α1,2 to inositolphosphoceramide (IPC) as core structure have only been described in a few pathogenic fungi. Here, we describe an A. fumigatus cluster of 5 genes (AFUA_8G02040 to AFUA_8G02090) encoding proteins required for the glycan part of the glycosphingolipid Af3c. Besides the already characterized UDP-GlcNAc:IPC α1,2-N-acetylglucosaminyltransferase (GntA), the cluster encodes a putative UDP-GlcNAc transporter (NstA), a GlcNAc de-N-acetylase (GdaA), and 2 mannosyltransferases (OchC and ClpC). The function of these proteins was inferred from analysis of the glycolipids extracted from A. fumigatus strains deficient in one of the genes. Moreover, successive introduction of the genes encoding GntA, GdaA, OchC, and ClpC in the yeast Saccharomyces cerevisiae enabled the reconstitution of the Af3c biosynthetic pathway. Absence of Af3c slightly reduced the virulence of A. fumigatus in a Galleria mellonella infection model.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites.

Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the Δmic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum Δpofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Protein O- and C-Glycosylation pathways in Toxoplasma gondii and Plasmodium falciparum.

Apicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N- and O-glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N-glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O-glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O-fucosylation and C-mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.

Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening.

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-containing Adhesins of the TRAP Family.

In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites. Here, we demonstrate that also the micronemal protein MIC2 secreted by Toxoplasma gondii tachyzoites is C-hexosylated. When expressed in a mammalian cell line deficient in C-mannosylation, P. falciparum and T. gondii Dpy19 homologs were able to modify TSR domains of the micronemal adhesins TRAP/MIC2 family involved in parasite motility and invasion. In vitro, the apicomplexan enzymes can transfer mannose to a WXXWXXC peptide but, in contrast to C. elegans or mammalian C-mannosyltransferases, are inactive on a short WXXW peptide. Since TSR domains are commonly found in apicomplexan surface proteins, C-mannosylation may be a common modification in this phylum.

Aspergillus fumigatus Cap59-like protein A is involved in α1,3-mannosylation of GPI-anchors.

Glycosylphosphatidylinositol (GPI) attaches a variety of eukaryotic proteins to the outer leaflet of the plasma membrane. In fungi, these proteins may also be transferred to the cell wall, to which they are covalently linked via a remnant of the GPI-anchor. They play crucial physiological roles in cell-cell interactions, adhesion or cell wall biogenesis. The biosynthesis of GPI-anchors in the endoplasmic reticulum, their transfer to proteins, early remodelling and transport to the Golgi apparatus has been fairly well described. In contrast, almost nothing is known about the genes and enzymes involved in adding glycan side chains to GPI after protein attachment. In this study, we characterized an α1,3-mannosyltransferase involved in maturation of GPI-anchors from the pathogenic fungus Aspergillus fumigatus. This enzyme shows homology to Cryptococcus neoformans Cap59p, a putative glycosyltransferase involved in capsule formation and virulence, and was thus named Cap59-like protein A (ClpA). Targeted deletion of the clpA gene in A. fumigatus led to absence of α1,3-mannose from mature GPI-anchors. The enzyme was further located to the Golgi-like apparatus of A. fumigatus and was shown to be active in the yeast Saccharomyces cerevisiae.

Characterization of an N-acetylglucosaminyltransferase involved in Aspergillus fumigatus zwitterionic glycoinositolphosphoceramide biosynthesis.

Glycoinositolphosphoceramides (GIPCs) are complex sphingolipids present at the plasma membrane of various eukaryotes with the important exception of mammals. In fungi, these glycosphingolipids commonly contain an α-mannose residue (Man) linked at position 2 of the inositol. However, several pathogenic fungi additionally synthesize zwitterionic GIPCs carrying an α-glucosamine residue (GlcN) at this position. In the human pathogen Aspergillus fumigatus, the GlcNα1,2IPC core (where IPC is inositolphosphoceramide) is elongated to Manα1,3Manα1,6GlcNα1,2IPC, which is the most abundant GIPC synthesized by this fungus. In this study, we identified an A. fumigatus N-acetylglucosaminyltransferase, named GntA, and demonstrate its involvement in the initiation of zwitterionic GIPC biosynthesis. Targeted deletion of the gene encoding GntA in A. fumigatus resulted in complete absence of zwitterionic GIPC; a phenotype that could be reverted by episomal expression of GntA in the mutant. The N-acetylhexosaminyltransferase activity of GntA was substantiated by production of N-acetylhexosamine-IPC in the yeast Saccharomyces cerevisiae upon GntA expression. Using an in vitro assay, GntA was furthermore shown to use UDP-N-acetylglucosamine as donor substrate to generate a glycolipid product resistant to saponification and to digestion by phosphatidylinositol-phospholipase C as expected for GlcNAcα1,2IPC. Finally, as the enzymes involved in mannosylation of IPC, GntA was localized to the Golgi apparatus, the site of IPC synthesis.

Biosynthesis of the fungal cell wall polysaccharide galactomannan requires intraluminal GDP-mannose.

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy.

Structural insight into the unique substrate binding mechanism and flavin redox state of UDP-galactopyranose mutase from Aspergillus fumigatus.

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and reduced flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg-182 and Arg-327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A, and R327A. These results support our hypothesis that Arg-182 and Arg-327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis.

CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function.

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed ß-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact.

Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.

Towards the crystal structure elucidation of eukaryotic UDP-galactopyranose mutase.

UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Eukaryotic UGMs from Aspergillus fumigatus and Leishmania major have been purified to homogeneity by means of Ni(2+)-affinity chromatography and crystallized. Eukaryotic UGM structure elucidation was not straightforward owing to high pseudo-symmetry, twinning and very low anomalous signal. Phasing to 2.8 Å resolution using SAD was successful for L. major UGM. However, the maps could only be improved by iterative density modification and manual model building. High pseudo-symmetry and twinning prevented correct space-group assignment and the completion of structure refinement. The structure of A. fumigatus UGM to 2.52 Å resolution was determined by molecular replacement using the incomplete 2.8 Å resolution L. major UGM model.

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Farnesol misplaces tip-localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus.

Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Deltamnt1 andDeltaglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.

A single UDP-galactofuranose transporter is required for galactofuranosylation in Aspergillus fumigatus.

Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translocates into the organelles of the secretory pathway. Here we report the first identification of a Golgi-localized nucleotide sugar transporter, named GlfB, with specificity for a UDP-Galf. In vitro transport assays established binding of UDP-Galf to GlfB and excluded transport of several other nucleotide sugars. Furthermore, the implication of glfB in the galactofuranosylation of A. fumigatus glycoconjugates and galactomannan was demonstrated by a targeted gene deletion approach. Our data reveal a direct connection between galactomannan and the organelles of the secretory pathway that strongly suggests that the cell wall-bound polysaccharide originates from its glycosylphosphatidylinositol-anchored form.

Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major.

The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.