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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 Ipg- 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(β1,4)Man(α1)-PO4 typical of wild-type L donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4)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 β-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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The most abundant surface glycoconjugate of the Leishmania promastigotes is lipophosphoglycan, a glycosylphosphatidyl-inositol-anchored polymer of the repeating disaccharide-phosphate Gal(β1,4)Manα1-PO4 unit. This complex molecule possesses properties that contribute to the ability of Leishmania to modulate macrophage signaling pathways during the initiation of infection. [Copyright &y& Elsevier]

The dominant glycoconjugate on the cell surface of all Leishmania promastigotes is an unusual glycoconjugate named lipophosphoglycan (LPG). Its relative abundance, unique structure, and cellular location have implicated LPG as an essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of protein kinase C (PKC) of host macrophages. This article summarizes the evidence that LPG is inhibitory toward PKC activation in macrophages and discusses the implication of such inhibition on intramacrophage survival of the parasite. [ABSTRACT FROM AUTHOR]

Investigates the mechanism by which lipophosphoglycan (LPG) protein from Leishmania donovani inhibits protein kinase C (PKC) isolated from rat brain. PKC binding to sucrose-loaded vesicles; Ineffectiveness of an LPG fragment containing only one repeating disaccharide unit; Purification of rat brain PKC; Trypsin cleavage of PKCalpha.

Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania parasites, was recently shown to be a potent inhibitor of viral infection. The mechanism by which this natural membrane amphiphile compound inhibits membrane fusion was investigated in this study using a simple model membrane system and a synthetic peptide corresponding to the fusion peptide of simian immunodeficiency virus (SIV). At low concentration (< 10 μM), LPG inhibits SIV-induced lipid mixing of large unilamellar vesicles composed of an equimolar mixture of egg phosphatidylcholine and egg phosphatidylethanolamine. Importantly, this inhibition was observed regardless of which LPG was inserted in the inner monolayer, the outer monolayer or both sides of the membrane, suggesting that the inner monolayer plays a determining role in membrane fusion. Fourier transform infrared spectroscopy revealed that LPG induced a conformational change of SIV fusion peptide without affecting its capacity to interact with the lipid membrane. This structural change was shown not to depend on the LPG localization and was observed even when LPG was exclusively associated to the inner lipid membrane. [ABSTRACT FROM AUTHOR]

Presents a study that developed a quantitative essay for the attachment of living promastigotes to whole midguts. Methods; Results; Discussion.

Reviews the structural aspects of the lipophosphoglycan (LPG) of Leishmania parasites. LPG of promastigotes and amastigotes; Developmental modification during promastigote metacyclogenesis; Glycosylphosphoinositides; Extracellular LPG-like glycoconjugates; LPG functions in sandfly-Leishmania interactions and in the bloodstream; Intracellular functions; Biosynthesis and immunological aspects of LPG.

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. [ABSTRACT FROM AUTHOR]

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 Galβ1,4Manα1-PO4 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. [ABSTRACT FROM AUTHOR]

In Leishmania major, the core of the abundant surface lipophosphoglycan (LPG) is structurally related to that of the smaller glycosylinositolphospholipids (GIPLs) in containing galactosylfuranose (Galf) residues in a Galf (β1, 3)Man motif. However, deletion of the putative Galf-transferase (GalfT) LPG1 affected Galf incorporation in LPG but not GIPLs. We hypothesized that the presumptive GIPL Galf-transferases could be homologous to LPG1, and identified three related genes in the L. major genome. These were termed LPG1L, LPG1R, and LPG1G, the latter of which was found in three identical copies located at the telomeres of chromosomes 5, 19, and 32 based on Leishmania genome project data. Neither LPG1 nor its homologues LPG1L and LPG1R were involved in the biosynthesis of GIPLs, as an lpg1-/lpg1l-/lpg1r- triple knockout (the first such in Leishmania) grew normally and made wild-type levels of Galf -containing GIPLs. In contrast, overexpression of these three led to elevated galactose incorporation in glycoproteins. Galf-containing glycoproteins had not been described in Leishmania but occur at high levels in other closely related trypanosomatids including Trypanosoma cruzi, Crithidia, Leptomonas, and Endotrypanum, and LPG1L and LPG1R homologs were detected in these species. These data suggest that the glyco-synthetic capabilities of Leishmania and perhaps other trypanosomatids may be larger than previously thought, with some activities being ‘cryptic’ in different lineages and potentially serving as reservoirs for glycoconjugate variation during evolution. Future tests will address whether the LPG1G family encodes the hypothesized GIPL-specific GalfT. [Copyright &y& Elsevier]

The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of the Leishmania infectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPG in vivo. Here we used an Leishmania major lpg1[sup -] mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1[sup -] was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion for Leishmania macrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanism in vitro and in vivo, unlike human hosts. In contrast, lpg1[sup -] parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG. [ABSTRACT FROM AUTHOR]

Examines the effects of lipophosphoglycan (LPG) of Leishmania donovani on the fusion of influenza virus. Integration of influenza hemagglutinin protein to planar lipid bilayers; Importance of influenza hemagglutinin protein in membrane fusion; Positive curvature-promoting properties of LPG.

Explores a specialized pathway affecting virulence glycoconjugates of Leishmania. Functional complementation identifying the gene LPG2, which encodes an integral Golgi membrane protein implicated in intracellular compartmentalization of lipophosphoglycan; Data suggesting that LPG2 participates in a specialized virulence pathway.

Focuses on a major step in the infectious cycle of protozoan parasite Leishmania major. Differentiation of parasites within the sand fly vector to the highly infective metacyclic promastigote stage; Tetrahydrobiopterin levels; Major factor controlling the extent of metacyclogenesis.

Background: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its speciesspecific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)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 β-glucose or 1 to 3 β-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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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(β1,4)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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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(β1,3) 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] 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. [ABSTRACT FROM AUTHOR]

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 β-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 fromthe 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 ~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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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-PO4) 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 LPGSB functions overlap but are not equivalent, since the lpg5A- mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the Ipg5B- 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. [ABSTRACT FROM AUTHOR]

Abstract: 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-γ 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-γ production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design. [Copyright &y& Elsevier]

Abstract: 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 scβ-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. [Copyright &y& Elsevier]

Clearance of pathogens by phagocytosis and their killing in phagolysosomes is a key aspect of our innate ability to fight infectious agents. Leishmania parasites have evolved ways to survive and replicate in macrophages by inhibiting phagosome maturation and avoiding the harsh environment of phagolysosomes. We describe here that during this process Leishmania donovani uses a novel strategy involving its surface lipophosphoglycan (LPG), a virulence factor impeding many host functions, to prevent the formation or disrupt lipid microdomains on the phagosome membrane. LPG acts locally on the membrane and requires its repetitive carbohydrate moieties to alter the organization of microdomains. Targeting and disruption of functional foci, where proteins involved in key aspects of phagolysosome biogenesis assemble, is likely to confer a survival advantage to the parasite. [ABSTRACT FROM AUTHOR]

Abstract: During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1–2 (β1–3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis. [Copyright &y& Elsevier]

Activated nucleotide sugars required for the synthesis of glycoconjugates within the secretory pathway of eukaryotes are provided by the action of nucleotide sugar transporters (NSTs). Typically, NSTs are studied in microsomal preparations from wild-type or mutant lines; however, in this setting it can be difficult to assess NST properties because of the presence of glycosyltransferases and other interfering activities. Here we have engineered Leishmania donovani to express high levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal polyhistidine tag. The functional LPG2-HIS was solubilized, purified by metal affinity chromatography, and reconstituted into phosphatidylcholine-conraining liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent Km of 6.6 µM. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDPFuc). In contrast to the activity in crude microsomes, transport was not dependent on the presence of divalent cations. Thus, reconstitution of transport activity using purified LPG2 protein in liposomes provides firm experimental evidence that a single polypeptide is solely required for NST activity and is able to mediate the uptake of multiple substrates. These studies are relevant to the study of NST structure and function in both protozoan parasites as well as their higher eukaryotic hosts. [ABSTRACT FROM AUTHOR]

Insect galectins are associated with embryonic development or immunity against pathogens. Here, we show that they can be exploited by parasites for survival in their insect hosts. PpGalec, a tandem repeat galectin expressed in the midgut of the sand fly Phlebotomus papatasi, is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event crucial for parasite survival, and accounts for species-specific vector competence. PpGalec is thus identified as a key molecule controlling vector competence for the most widely distributed form of cutaneous leishmaniasis in the Old World. In addition, these studies demonstrate the feasibility of using midgut receptors for parasite ligands as target antigens for transmission-blocking vaccines. [Copyright &y& Elsevier]

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes which has been shown to be critical for parasite-sand fly vector interactions. To provide additional evidence for its importance in transmission, the LPGs from three Leishmania tropica strains that differ in their capability to infect sand flies, were biochemically characterized. One of these strains, ISER/IL/98/LRC-L747, was isolated from a Phlebotomus sergenti female collected in the Judean Desert close to Jerusalem. The other strains originated from a different focus in the Galilee region of northern Israel. One was isolated from a patient (MHOM/IL/02/Ofri-LRC-L863) and the other from a naturally infected Phlebotomus arabicus female (IARA/IL/00/Amnunfly1-LRC-L810). The LPG structures of the isolates from the Galilee (L863 and L810) were similar to each other, but differed in the LPG repeat units from the Judean Desert isolate (L747). The terminal sugar in the side chains of the repeat units of LPG purified from L863 and L810 was β-galactose and was not capped with glucose, unlike L747 and a previously characterized L. tropica strain from Iraq (L36). Since L810 was isolated from P. arabicus and L747 from P. sergenti, variations in the structure of their LPGs may explain their capacity to infect different sand fly species.vsp sp="1">Index Descriptors and Abbreviations: LPG, lipophosphoglycan; PBS, phosphate-buffered saline; FACE, fluorophore-assisted carbohydrate electrophoresis; CE, capillary electrophoresis, ANTS; 8-aminonaphthalene-1,3,6-trisulfate; APTS, 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt [Copyright &y& Elsevier]

Ether phospholipids are major components of the membranes of humans and Leishmania. In protozoan parasites they occur separately or as part of the glycosylphosphatidylinositol (GPI) anchor of molecules implicated in virulence, such as lipophosphoglycan (LPG), smaller glycosylinositolphospholipids (GIPLs), and GPIanchored proteins. We generated null mutants of the Leishmania major alkyldihydroxyacetonephosphate synthase (ADS), the first committed step of ether lipid synthesis. Enzymatic analysis and comprehensive mass spectrometric analysis showed that ads1[sup -] knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1[sup -] thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1[sup -] grew well and maintained lipid rafts (detergentresistant membranes). In virulence tests it closely resembled LPG-deficient L. major, including sensitivity to complement and an inability to survive the initial phase of macrophage infection. Likewise it retained the ability to inhibit host cell signaling and to form infectious amastigotes from the few parasites surviving the establishment defect. These findings counter current proposals that GIPLs are required for amastigote survival in the mammalian host or that parasite lyso-alkyl or alkylacyl-GPI anchors are solely responsible for inhibition of macrophage activation. [ABSTRACT FROM AUTHOR]

Glycoconjugates have been shown to play important roles in Leishmania development. However, the ability to study these molecules and other processes would benefit greatly from improved methods for genetic manipulation and analysis of the amastigote stage. This is especially challenging for L. donovani, the agent of the most severe form of leishmaniasis, which can rapidly lose virulence during in vitro culture. Here we report on a clonal subline of an L. donovani 1S2D (LdBob or LdB), which differentiates readily from promastigotes to amastigotes in axenic culture, and maintains this ability during extended parasite cultivation in vitro. This derivative can be plated and transfected efficiently while grown as promastigotes or amastigotes. Importantly, LdB maintains the ability to differentiate while undergoing genetic alterations required for creation of gene knockouts and complemented lines. Like virulent L. donovani, LdB exhibits down-regulation of lipophosphoglycan (LPG) synthesis and up-regulation of A2 protein synthesis in amastigotes. We showed that knockouts of LPG2, encoding a Golgi GDP-mannose transporter, eliminated phosphoglycan synthesis in LdB axenic amastigotes. These and other data suggest that LdB axenic amastigotes will be generally useful as a differentiation model in studies of gene expression, virulence, glycoconjugate function and drug susceptibility in L. donovani. [Copyright &y& Elsevier]

At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scβGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain β1,2-arabinosyltransferases (scβAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scβAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scβAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scβAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic βAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania. [ABSTRACT FROM AUTHOR]

Discusses the use of evolutionary polymorphisms in lipophosphoglycan (LPG) structure and cross-species transfections to recover genes encoding the LPG side chain beta1-3-galactosyltransferases (GalT). Recovery of a dispersed family of six SCG genes; Exhibition of characteristics of eukaryotic GalT; Reflection of differences in the expression of strain-specific LPG galactosylation patterns.

Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide-phosphate repeat unit Galβ1,4Manα1-PO4, shared by other developmentally regulated molecules implicated in parasite virulence. Functional complementation of a Leishmania donovani LPG-defective mutant (OB1) accumulating a truncated LPG containing only the Manα1-PO4 residue of the first repeat unit identified LPG3, the Leishniania homolog of the mammalian endoplasmic reticulum (ER) chaperone GRP94. LPG3 resembles GRP94, as it localizes to the parasite ER, and lpg3- mutants show defects including down-regulation of surface GPI-anchored proteins and mild effects on other glycoconjugates. LPG3 binds cellular proteins and its Leishmania infantum GRP94 ortholog is highly immunogenic, suggesting a potential role in directing the immune response. However, null lpg3- mutants grow normally, are completely defective in the synthesis of phosphoglycans, and the LPG3 mRNA is regulated developmentally but not by stress or heat. Thus the role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes. Like the other glycoconjugate synthetic pathways in this parasite, its activity is focused on molecules implicated in virulence rather than viability. [ABSTRACT FROM AUTHOR]

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi. The distinguishing feature of LPG of procyclic L. chagasi consisted of β1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite–sand fly interactions. [Copyright &y& Elsevier]

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide?sugar transporter that transfers UDP?glucuronic acid, UDP?N-acetylglucosamine and possibly UDP?xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue. [ABSTRACT FROM AUTHOR]

Provides advice concerning issues in clinical medicine. Guidelines for the safe use of over-the-counter hydrocortisone products; Procedures for chest tube irrigation; Recommendations on using filters for hyperalimentation therapy; Definition of lateral decubitus position.

A multicenter study of the overall costs and impact on medication administration of sterile cartridge-needle units and conventional disposable syringes with multiple- and single-dose vials was conducted in six hospitals in the Voluntary Hospitals of America system. In this era of increasing limits on resources in health care, personnel strategies, hospital purchases, work patterns, and department relationships, virtually all systems within hospitals are under ongoing examination for efficiency and cost-effectiveness.

Leishmania infections involve an acute phase of replication within macrophages, typically associated with pathology. After recovery parasites persist for long periods, which can lead to severe disease upon reactivation. Unlike the role of host factors, parasite factors affecting persistence are poorly understood. Leishmania major lacking phosphoglycans (lpg2[sup-]) were unable to survive in sand flies and macrophages, but retained the ability to persist indefinitely in the mammalian host without inducing disease. The L. major Ipg2[sup-] thus provides a platform for probing parasite factors implicated in persistence and its role in disease and immunity. [ABSTRACT FROM AUTHOR]