Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis.

BACKGROUND: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. RESULTS: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 ß-glucose or 1 to 3 ß-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. CONCLUSIONS: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

Genetic metabolic complementation establishes a requirement for GDP-fucose in Leishmania.

To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Arap) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related L. major genes (FKP40 and AFKP80) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Arap into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Arap from exogenous d-Arap, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δafkp80- null mutants ablated d-Arap modifications of LPG as predicted, whereas Δfkp40- null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major, but unexpectedly, we were unable to generate fkp40-/afkp80- double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed "genetic metabolite complementation." First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δfkp40-/Δafkp80- double mutant was now readily obtained. As expected, the Δfkp40-/Δafkp80-/+TbGMD-GMER line lacked the capacity to generate GDP-Arap, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s).

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

BACKGROUND: Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. CONCLUSIONS/SIGNIFICANCE: These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

Characterization of a ricin-resistant mutant of Leishmania donovani that expresses lipophosphoglycan.

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one  Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(ß1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(ß1,4)[Glc(ß1,2)]Man and tetrasaccharide Gal(ß1,4)[Glc(ß1,2)Man(α1,2)]Man instead of the usual Gal(ß1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a ß-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms.

Leishmania promastigotes induce cytokine secretion in macrophages through the degradation of synaptotagmin XI.

Synaptotagmins (Syts) are type-I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. We recently discovered that Syt XI is a recycling endosome- and lysosome-associated protein that negatively regulates the secretion of TNF and IL-6. In this study, we show that Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. To demonstrate that cytokine release was dependent on GP63-mediated degradation of Syt XI, small interfering RNA-mediated knockdown of Syt XI before infection revealed that the effects of small interfering RNA knockdown and GP63 degradation were not cumulative. In mice, i.p. injection of GP63-expressing parasites led to an increase in TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the inoculation site. Both of these cell types have been shown to be infection targets and aid in the establishment of infection. In sum, our data revealed that GP63 induces proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. This study provides new insight on how Leishmania exploits the immune response to establish infection.

Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi.

The lipophosphoglycan (LPG) of Leishmania major has a major role in the attachment to Phlebotomus papatasi midgut. Here, we investigated the comparative structural features of LPG of L. turanica, another species transmitted by P. papatasi. The mAb WIC 79.3, specific for terminal Gal(ß1,3) side-chains, strongly reacted with L. turanica LPG. In contrast, L. turanica LPG was not recognized by arabinose-specific mAb 3F12. In conclusion, LPGs from L. major and L. turanica are similar, with the latter being less arabinosylated than L. major's. The high galactose content in L. turanica LPG is consistent with its predicted recognition by P. papatasi lectin PpGalec.

Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts.

BACKGROUND: Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review. SCOPE OF THE REVIEW: The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species. MAJOR CONCLUSIONS: LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection. GENERAL SIGNIFICANCE: Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics.

Exclusion of synaptotagmin V at the phagocytic cup by Leishmania donovani lipophosphoglycan results in decreased promastigote internalization.

Regulators of membrane fusion play an important role in phagocytosis, as they regulate the focal delivery of endomembrane that is required for optimal internalization of large particles. During internalization of Leishmania promastigotes, the surface glycolipid lipophosphoglycan (LPG) is transferred to the macrophage membrane and modifies its fusogenic properties. In this study, we investigated the impact of LPG on the recruitment of the exocytosis regulator synaptotagmin V (Syt V) at the area of internalization and on the early steps of phagocytosis. Using Leishmania donovani LPG-defective mutants and LPG-coated particles, we established that LPG reduces the phagocytic capacity of macrophages and showed that it causes exclusion of Syt V from the nascent phagosome. Silencing of Syt V inhibited phagocytosis to the same extent as LPG, and these effects were not cumulative, consistent with a Syt V-dependent mechanism for the inhibition of phagocytosis by LPG. Previous work has revealed that LPG-mediated exclusion of Syt V from phagosomes prevents the recruitment of the vacuolar ATPase and acidification. Thus, whereas exclusion of Syt V from phagosomes in the process of formation may be beneficial for the creation of a hospitable intracellular niche, it reduces the phagocytic capacity of macrophages. We propose that the cost associated with a reduced internalization rate may be compensated by increased survival, and could lead to a greater overall parasite fitness.

Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern.

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded ß1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.

Retention and loss of RNA interference pathways in trypanosomatid protozoans.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies.

Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO(4)] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2(-) or lpg5A(-)/5B(-)) or LPG alone (lpg1(-)) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36-72 h after the infective feed, all parasites developed well except the lpg2(-) and lpg5A(-)/5B(-) mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2(-) in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2(-) promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.

Differential midgut attachment of Leishmania (Viannia) braziliensis in the sand flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia.

The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG). An upregulation of beta-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia) braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V.) braziliensis with its natural vectors Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. Using two in vitro binding techniques, phosphoglycans (PGs) derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was approximately 11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL) in Brazil is discussed.

The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V.

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galbeta1,4Manalpha1-PO(4) units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.

Leishmania donovani lacking the Golgi GDP-Man transporter LPG2 exhibit attenuated virulence in mammalian hosts.

Surface phosophoglycans such as lipophosphoglycan (LPG) or proteophosphoglycan (PPG) and glycosylinositol phospholipids (GIPLs) modulate essential interactions between Leishmania and mammalian macrophages. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by LPG2. LPG2-null (lpg2(-)) Leishmania major cannot establish macrophage infections or induce acute pathology, whereas lpg2(-)Leishmania mexicana retain virulence. lpg2(-)Leishmania donovani has been reported to survive poorly in cultured macrophages but in vivo survival has not been explored. Herein we discovered that, similar to lpg2(-)L. major, lpg2(-)L. donovani promastigotes exhibited diminished virulence in mice, but persisted at consistently low levels. lpg2(-)L. donovani promastigotes could not establish infection in macrophages and could not transiently inhibit phagolysosomal fusion. Furthermore, lpg2(-) promastigotes of L. major, L. donovani and L. mexicana were highly susceptible to complement-mediated lysis. We conclude that phosphoglycan assembly and expression mediated by L. donovani LPG2 are important for promastigote and amastigote virulence, unlike L. mexicana but similar to L. major.

Comparisons of mutants lacking the Golgi UDP-galactose or GDP-mannose transporters establish that phosphoglycans are important for promastigote but not amastigote virulence in Leishmania major.

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(beta1,4)Man(alpha1-PO(4))]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2(-) L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2(-), instead resembling the L. major lipophosphoglycan-deficient lpg1(-) mutant. Metacyclic lpg5A(-)/lpg5B(-) promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A(-)/lpg5B(-) mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2(-) Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major.

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO(4)) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A(-) and lpg5B(-) mutants produced PGs, an lpg5A(-)/5B(-) double mutant was completely deficient. PG synthesis was restored in the lpg5A(-)/5B(-) mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A(-) mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B(-) mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes.

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.

Genomic organization and expression of the expanded SCG/L/R gene family of Leishmania major: internal clusters and telomeric localization of SCGs mediating species-specific LPG modifications.

Stage-specific modifications to the abundant surface lipophosphoglycan (LPG) adhesin of Leishmania play critical roles in binding and release of the parasite during its infectious cycle in the sand fly, and control the ability of different fly species to transmit different parasite strains and species. In Leishmania major Friedlin V1, binding to a sand fly midgut lectin is mediated by side chain galactosyl (scGal) modifications of the LPG phosphoglycan (PG) repeats, while release occurs following arabinose-capping of scGals. Previously we identified a family of six SCG genes encoding PG scbeta-galactosyltransferases, and here we show that the extended SCG gene family (now termed SCG/L/R) encompasses 14 members in three subfamilies (SCG, SCGL and SCGR). Northern blot and RT-PCR analyses suggest that most of the SCG/L/R genes are expressed, with distinct patterns during the infectious cycle. The six SCGR subfamily genes are clustered and interspersed with the two SCA genes responsible for developmentally regulated arabinosylation of PG scGals; relationships amongst the SCGR revealed clear evidence of extensive gene conversion. In contrast, the seven SCG 'core' family members are localized adjacent to telomeres. These telomeres share varying amounts of sequence upstream and/or downstream of the SCG ORFs, again providing evidence of past gene conversions. Multiple SCG1-7 RNAs were expressed simultaneously within parasite populations. Potentially, telomeric localization of SCG genes may function primarily to facilitate gene conversion and the elaboration of functional evolutionary diversity in the degree of PG sc-galactosylation observed in other strains of L. major.

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis.

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.