A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

Leishmania Encodes a Bacterium-like 2,4-Dienoyl-Coenzyme A Reductase That Is Required for Fatty Acid β-Oxidation and Intracellular Parasite Survival

Leishmaniaspp. are protozoan parasites that cause a spectrum of im-portant diseases in humans. These parasites develop as extracellular promastigotesin the digestive tract of their insect vectors and as obligate intracellular amastigotesthat infect macrophages and other phagocytic cells in their vertebrate hosts.Promastigote-to-amastigote differentiation is associated with marked changes in me-tabolism, including the upregulation of enzymes involved in fatty acid -oxidation,which may reflect adaptation to the intracellular niche. Here, we have investigatedthe function of one of these enzymes, a putative 2,4-dienoyl-coenzyme A (CoA) re-ductase (DECR), which is specifically required for the -oxidation of polyunsaturatedfatty acids. TheLeishmaniaDECR shows close homology to bacterial DECR proteins,suggesting that it was acquired by lateral gene transfer. It is present in othertrypanosomatids that have obligate intracellular stages (i.e.,Trypanosoma cruziandAngomonas) but is absent from dixenous parasites with an exclusively extracellularlifestyle (i.e.,Trypanosoma brucei). A DECR-green fluorescent protein (GFP) fusionprotein was localized to the mitochondrion in both promastigote and amastigotestages, and the levels of expression increased in the latter stages. ALeishmania ma-jorΔdecrnull mutant was unable to catabolize unsaturated fatty acids and accumu-lated the intermediate 2,4-decadienoyl-CoA, confirming DECR’s role in -oxidation.Strikingly, theL. majorΔdecrmutant was unable to survive in macrophages and wasavirulent in BALB/c mice. These findings suggest that -oxidation of polyunsaturatedfatty acids is essential for intracellular parasite survival and that the bacterial originof key enzymes in this pathway could be exploited in developing new therapies.Peer Reviewe

Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy

The CD20 antigen is an attractive target for specific treatment of B- cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human β-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 15-amino acid linker and fused to human β-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human β-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single-chain anti-CD2O antibody and human β- glucuronidase for future use in the activation of anticancer prodrugs in B- cell lymphoma

Golgi-Located NTPDase1 of <i>Leishmania major</i> Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

<div><p>Parasitic protozoa, such as <i>Leishmania</i> species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The <i>Leishmania major</i> genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An <i>L. major</i> LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of <i>L. major</i> strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the <i>L. major</i> NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the <i>Leishmania major</i> NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.</p></div

Subcutaneous infection of BALB/c mice with either amastigote (A, D) or promastigote (B, C, E) <i>L. major</i>.

<p>A. Mice were infected with either 10⁵ wild type <i>L. major</i> (squares) or 10⁵<i>L. major</i> NTPD1 null mutant (triangles) amastigotes and lesion scores monitored weekly. Error bars represent S.E.M. (<i>n</i> = 5). No significant difference in lesion size was observed at any time point (<i>P</i>>0.05, unpaired t-test). B. Mice were infected with either 10⁶ wild type <i>L. major</i> (squares) or 10⁶<i>L. major</i> NTPD1 null mutant (triangles) parasites and lesion scores monitored weekly. Error bars represent S.E.M. (<i>n</i> = 10). Significant differences in lesion size were observed at all time points from week 6 inclusive (<i>P</i><0.05, unpaired t-test). C. Mice were infected with either 10⁶ wild type <i>L. major</i> + pIR1SAT (squares), 10⁶<i>L. major</i> NTPD1 null mutant + pIR1SAT (closed triangles) or 10⁶<i>L. major</i> NTPD1 null mutant + pIR1SAT-<i>ntpd1</i> (open triangles). Error bars represent S.E.M. (<i>n</i> = 5). D and E. Mice were infected with either 10⁵ wild type <i>L. major</i> (squares) or 10⁵<i>L. major</i> NTPD2 null mutant (circles) parasites and lesion scores monitored weekly. Error bars represent S.E.M. (<i>n</i> = 5). No significant difference in lesion size was observed between strains at any individual time point (<i>P</i>>0.05, two-way ANOVA).</p

Proposed model for the role of NTPD1 in Golgi nucleotide-sugar transport and LPG synthesis.

<p>UDP-galactose and GDP-mannose/GDP-arabinose are transported into the Golgi via transporters LPG5A/LPG5B <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003402#pntd.0003402-Capul1" target="_blank">[37]</a> and LPG2 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003402#pntd.0003402-Ma1" target="_blank">[65]</a> respectively. Galactose and mannose-phosphate are cleaved for use in phosphoglycan synthesis. Following cleavage, GMP is exchanged for GDP-mannose transport into the lumen. In the case of UDP, hydrolysis to UMP is catalyzed by NTPD1, allowing efficient ongoing transport of UDP-galactose into the Golgi lumen.</p

Analysis of purified LPG.

<p>A. LPG extracted from <i>L. major</i> wild type (WT) and <i>L. major Δntpd1</i> after SDS-PAGE and silver staining, demonstrating a clear difference in apparent molecular weight. Numbers indicate approximate molecular weight markers (kDa). B. Elution profile during octyl-Sepharose chromatography of LPG extracted from wild type <i>L. major</i> (squares) and the NTPD1 null mutant (triangles). LPG content was determined by orcinol staining [3:5-dihydroxy-toluene, BDH; 0.2%(w/v) in 10% H₂SO₄ and 50% ethanol], followed by colour development at 100°C and comparison to a known standard. The 1-propanol gradient concentration (open circles) was measured refractometrically. C. Fractionation of the dephosphorylated repeat units of LPG from wild-type and NTPD1 null mutant promastigotes. LPG was purified by octyl-Sepharose chromatography, depolymerised with 40 mM trifluoroacetic acid (8 min, 100°C) and dephosphorylated with calf intestinal alkaline phosphatase. The repeat units were desalted by passage over a mixed bed ion exchange column and chromatographed by HPAEC. The numbers at the top of the profile represent the elution positions of dextran oligomers (number of glucose units).</p

Subcellular localization of LmNTPDase-GFP fusion proteins.

<p>A. Western blot using anti-GFP antibody demonstrating production of GFP-fusion proteins of the correct sizes by <i>L. major</i> parasites transfected with either pXG-NTPD1-GFP or pXG-NTPD2-GFP, and secretion of NTPD2-GFP into the culture supernatant. Lane 1: <i>L. major</i> + pXG-NTPD1-GFP (whole cell lysate, C), Lane 2: <i>L. major</i> + pXG-NTPD1-GFP culture supernatant (SN), Lane 3: <i>L. major</i> + pXG-NTPD2-GFP C, Lane 4, <i>L. major</i> + pXG-NTPD2-GFP SN. Samples were developed simultaneously on one membrane, with the vertical line representing removal of unrelated intervening lanes. B. Localization of NTPD1-GFP to the Golgi apparatus. Top panel: <i>L. major</i> co-transfected with pXG-NTPD1-GFP and pXG-LPG1-mCherry; middle panel: <i>L. major</i> co-transfected with pXG-NTPD1-GFP and pXG-SAT-mCherry; bottom panel: <i>L. major</i> co-transfected with pXG-/GFP+ and pXG-LPG1-mCherry. Arrow indicates co-localisation of NTPD1-GFP and LPG1-mCherry in the Golgi apparatus. Hoechst staining highlights the parasite nucleus (diffuse staining) and kinetoplast (dense staining), with the Golgi apparatus (top and bottom panel, mCherry) in the region adjacent to the kinetoplast (as expected).</p