Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

Capul, Althea A.; Barron, Tamara; Dobson, Deborah E.; Turco, Salvatore J.; Beverley, Stephen M.

doi:10.1074/jbc.m610869200

Cited by 56 publications

(63 citation statements)

References 71 publications

(81 reference statements)

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“…The activation of this monosaccharide was lately shown to be independent from UDP-Glc synthesis because an L. major UGP deletion mutant still expresses Gal-containing molecules. 5 Herein, we report the identification and characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate Gal-1-P into UDP-Gal constituting the Isselbacher pathway for UDP-Gal synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Leishmania Genome-Although enzymatic epimerization of UDP-glucose is clearly not the sole source of UDP-galactose in Leishmania major (15), 5 the genome of this parasite lacks an obvious UDP-glucose:␣-D-galactose-1-phosphate uridylyltransferase enzyme (EC 2.7.7.12). BLAST searches of the L. major genome revealed, however, the existence of a gene (LmjF17.1160) displaying ϳ32% identity with pea sprout USP (19) and 15% identity with L. major UGP (22,24).…”

Section: Identification Of a Putative Udp-sugar Pyrophosphorylase Inmentioning

confidence: 99%

“…Analysis of an L. major UDP-glucose pyrophosphorylase (UGP) deletion mutant 5 revealed the presence of Gal-containing molecules underlining the existence of a UDP-Gal biosynthetic pathway independent of UDP-Glc biosynthesis. Here, we report the identification, cloning, and characterization of an L. major UDP-sugar pyrophosphorylase (USP) (EC 2.7.7.64) with broad substrate specificity, including Gal-1-P and glucose 1-phosphate (Glc-1-P).…”

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confidence: 99%

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Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

Journal of Biological Chemistry

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The Leishmania parasite glycocalyx is rich in galactosecontaining 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.

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Section: Discussionmentioning

confidence: 99%

Section: Identification Of a Putative Udp-sugar Pyrophosphorylase Inmentioning

confidence: 99%

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confidence: 99%

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Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

Journal of Biological Chemistry

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“…Briefly, the gene encoding mCherry or firefly luciferase was cloned into the XmaI site of pIR1SAT, an integrating vector that was kindly provided to us by Stephen M. Beverley of Washington University, St. Louis, MO (8). After electroporation (12) and selection on semisolid medium, correct insertion was verified by Southern blotting (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

et al. 2011

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The vector-borne protozoan Leishmania infantum chagasi causes minimal inflammation after inoculation into skin but disseminates to cause fatal visceral leishmaniasis. To define the inflammatory response at the parasite inoculation site, we introduced metacyclic L. infantum chagasi promastigotes intradermally into BALB/c mouse ears and studied inflammatory cells over 7 days. Ly6G؉ neutrophils rapidly infiltrated the dermis, peaking after 6 to 24 h. Macrophages and NK cells next infiltrated the dermis, and NK followed by B cells expanded in draining lymph nodes. Parasite-containing phagocytes were tracked with fluorescent mCherry-labeled L. infantum chagasi. Ly6G؉ neutrophils contained the greatest proportion of intracellular parasites 6 to 24 h after inoculation, whereas dermal macrophages harbored the majority of intracellular parasites after 2 to 7 days. These observations were validated microscopically. Low doses of antibody transiently depleted mice of neutrophils, leaving other cells intact. Combined results of in vivo imaging, flow cytometry, and quantitative PCR showed that neutrophil depletion slowed the clearance of extracellular (luciferase-positive) promastigotes during the first 24 h after inoculation yet decreased the numbers of leukocytes containing intracellular (mCherry-positive) parasites. From 3 days onward, total L. infantum chagasi-containing dermal leukocytes and total L. infantum chagasi parasites in draining lymph nodes were similar in both groups. Nonetheless, a second wave of L. infantum chagasi-containing neutrophils occurred 7 days after parasite inoculation into neutrophildepleted mice, corresponding to the time of neutrophil recovery. Thus, neutrophils were recruited to the dermis even late after inoculation, and L. infantum chagasi trafficked through neutrophils in both neutrophil-depleted and control mice, albeit with different kinetics. Recruitment of neutrophils and transient parasite residence in neutrophils may play a role in nonulcerative forms of leishmaniasis.Parasites belonging to the genus Leishmania cause a spectrum of human diseases, the most deadly of which is visceral leishmaniasis. Leishmania infantum chagasi is one of the two most common etiologic agents of visceral leishmaniasis in humans. During natural infection, a bolus of metacyclic promastigotes is delivered into a hemorrhagic dermal lesion formed by a feeding female phlebotomine sand fly (5). Parasites quickly encounter soluble and cellular microbicidal immune elements. Rather than succumb, many are taken up by phagocytic host cells, where they transform to intracellular amastigotes, a form that can multiply and survive in phagolysosomes (9, 48).Although the majority of host cells harboring Leishmania sp. amastigotes are macrophages, intracellular amastigotes have been observed in other mammalian cell types as well, including dendritic cells (DCs), fibroblasts, and neutrophils (6,21,28,33). Recent studies suggest that neutrophils can promote the early establishment of intradermal infection with Leishmania major...

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“…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).…”

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confidence: 99%

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

Silva

Owens²,

Murta³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.inducible expression ͉ FK506 ͉ glycoconjugates ͉ pathogen ͉ trypanosomatid protozoa

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Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

Cited by 56 publications

References 71 publications

Leishmania UDP-sugar Pyrophosphorylase

Leishmania UDP-sugar Pyrophosphorylase

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

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

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