Specificity of the interrelations of Leishmania and host cells in vitro

Experiments on cross infection of peritoneal macrophages of mice with Leishmania of reptiles L. gymnodactyli and free cells of abdominal cavity of caucasian Agama (some part of which is composed by fibroblasts) with Leishmania of mammals L. major and L. donovani have shown the possibility of reproduction of the above species both in reptiles and mammals. The persistence of L. gymnodactyli and L. major in macrophages of mice was traced up to 10 days, the abundance of L. gymnodactyli during the whole period of observations being lower than that of L. major. The abundance of the above Leishmania in these cells happened to be higher than in the cells of reptiles. In the cells of reptiles the infection with these three species of Leishmania was eliminated by 5-6 days. More activity internalization of Leishmania of reptiles into cells of reptiles as compared to Leishmania of mammals was revealed that, apparently, reflects a definite degree of their adaptation to existence in reptiles in vivo.     

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.     

The existence of lipophosphoglycanlike molecules in Trichomonads

The trichomonods, Trichomonas vaginalis and Tritrichomonas foetus, appear to express novel lipophosphoglycon (LPG) like glycoconjugates on their cell surface, which are structurally distinct from Leishmania LPGs'. In this article, Bibhuti Singh discusses the structural and cellular aspects o f these molecules, and speculates on their biological significance.     

Myristoyl-CoA:protein N-myristoyltransferase,an essential enzyme and potential drug target in kinetoplastid parasites

Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.     

Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.     

Intermediary metabolism of Trypanosoma cruzi

In this article, Julio Urbino discusses the characteristics o f the intermediary metabolism of Trypanosoma cruzi (the causative agent of Chagas disease), which are responsible for the unusual capacity of this parasite to use carbohydrates or amino acids as carbon and energy sources without drastic changes in its catabolic enzyme levels(1-3). Many, but not all, o f the metabolic capabilities of this organism are shared with Leishmania and the procyclic form o f the African trypanosomes, and the reviewer presents a metabolic model which is also consistent with the information available on these other parasites(2,4).     

SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.     

Molecular and cellular characterization of an AT-hook protein from Leishmania

AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.     

Effect of polymorphisms on ligand binding by mouse major urinary proteins

Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx-binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed.     

Dendritic cells in Leishmania infection

Dendritic cells (DCs) are key elements of the immune system, which function as sentinel in the periphery and alert T lymphocytes about the type of invading antigen and address their polarisation, in order to mount an efficacious immune response. Leishmania spp. are parasitic protozoa which may cause severe disease in humans and domestic animals. In this work, the main studies concerning the role of DCs in Leishmania infection are reviewed, in both the murine and human models. In particular, the importance of the genetic status of the hosts and of the different Leishmania species in modulating DC-mediated immune response is examined. In addition, different approaches of DC-based vaccination against experimental leishmaniasis, which could have important future applications, are summarised.     

Leishmania major induces deactivation of extracellular signal regulated kinases 2 in human U937 macrophage like cells

The protozoan parasite of the genus Leishmania has developed strategies to evade host defence mechanisms. Leishmania (L.) parasites interfere with several signalling pathways to inhibit phagocyte functions. In the present study, we analysed possible alteration of MAPK activation during infection of human U937 cell line with Leishmania major parasites. Analysis of whole cell lysates by anti-phosphotyrosine immunoblotting, showed that the pattern of tyrosine phosphorylated proteins were different for undifferentiated, PMA differentiated and Leishmania major infected cells. Cell infection induces a decrease in tyrosine phosphorylation of several host cell proteins, including PMA-induced tyrosine phosphorylated proteins. Leishmania major also caused a time dependent inhibition of ERK2 phosphorylation which correlates with the inhibition of ERK activity. This Leishmania induced effect was blocked when the cells were treated with a PTP inhibitor, prior to infection. These results suggest that Leishmania major may interfere with MAPK mediated signal transduction of the host cell through the inhibition of ERK2 activation and that this effect may be mediated by induction of protein tyrosine phosphatases activities.     

Leishmania proteins derived from recombinant DNA: current status and next steps

The parasite Leishmania is a major cause of disease worldwide. In the past 15 years, many groups have analysed DNA-derived proteins from Leishmania. Large amounts of data obtained by these groups can be collated to direct future research into Leishmania and to find novel immunological mechanisms and information about its metabolism. International coordination will increase both the basic knowledge about Leishmania and the capacity to apply this knowledge to combat leishmaniases.     

Alkylacylglycerolipid domain of GPI molecules of Leishmania is responsible for inhibition of PKC-mediated c-fos expression

Glycosylphosphatidylinositols (GPIs) are the most abundant molecules present in the membranes of the parasitic protozoa Leishmania responsible for multiple forms of leishmaniasis. Among the prominent biological activities displayed by the major Leishmania GPIs [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] is the inhibition of macrophage functions such as the protein kinase C (PKC)-dependent signaling pathway. The bioactivity of Leishmania GPIs is in contrast to Trypanosoma brucei and Plasmodium falciparum GPIs, which activate the macrophage functions. To address the question as to which structural domain of Leishmania GPIs is responsible for dramatic down-regulation of PKC-dependent transient c-fos expression, the chemically synthesized defined alkylacylglycerolipids domain of corresponding GPIs, and LPG and GIPLs isolated from Leishmania donovani, were evaluated for inhibition of PKC and c-fos expression in macrophages. The results presented here demonstrate that the unusual lipid domain of Leishmania GPIs is primarily responsible for inhibition of PKC-dependent transient c-fos expression.     

Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host

Leishmania are intracellular protozoan parasites that reside primarily in host mononuclear phagocytes. Infection of host macrophages is initiated by infective promastigote stages and perpetuated by an obligate intracellular amastigote stage. Studies undertaken over the last decade have shown that the composition of the complex surface glycocalyx of these stages (comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans and free GPI glycolipids) changes dramatically as promastigotes differentiate into amastigotes. Marked stage-specific changes also occur in the expression of other plasma membrane components, including type-1, polytopic and peripheral membrane proteins, reflecting the distinct microbicidal responses and nutritional environments encountered by these stages. More recently, a number of Leishmania mutants lacking single or multiple surface components have been generated. While some of these mutants are less virulent than wild type parasites, many of these mutants exhibit only mild or no loss of virulence. These studies suggest that, 1) the major surface glycocalyx components of the promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or minor role in macrophage invasion, 2) that there is considerable functional redundancy in the surface glycocalyx and/or loss of some components can be compensated for by the acquisition of equivalent host glycolipids, 3) the expression of specific nutrient transporters is essential for life in the macrophage and 4) the role(s) of some surface components differ markedly in different Leishmania species. These mutants will be useful for identifying other surface or intracellular components that are required for virulence in macrophages.     

Pathogenomics analysis of Leishmania spp.: flagellar gene families of putative virulence factors

The trypanosomatid flagellar apparatus contains conventional and unique features, whose roles in infectivity are still enigmatic. Although the flagellum and the flagellar pocket are critical organelles responsible for all vesicular trafficking between the cytoplasm and cell surface, still very little is known about their roles in pathogenesis and how molecules get to and from the flagellar pocket. The ongoing analysis of the genome sequences and proteome profiles of Leishmania major and L infantum, Trypanosoma cruzi, T. brucei, and T. gambiensi ( www.genedb.org ), coupled with our own work on L. chagasi (as part of the Brazilian Northeast Genome Program- www.progene.ufpe.br ), prompted us to scrutinize flagellar genes and proteins of Leishmania spp. promastigotes that could be virulence factors in leishmaniasis. We have identified some overlooked parasite factors such as the MNUDC-1 (a protein involved in nuclear development and genomic fusion) and SQS (an enzyme of sterol biosynthesis), among the described flagellar gene families. A database concerning the results of this work, as well as of other studies of Leishmania and its organelles, is available at http://nugen.lcc.uece.br/LPGate . It will serve as a convenient bioinformatics resource on genomics and pathology of the etiological agents of leishmaniasis.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.     

Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope

Lipophosphoglycan is a major surface molecule of Leishmania, protozoa parasites, which are the causative agents of leishmaniasis, a disease that annually afflicts millions of people worldwide. The oligosaccharide structures of lipophosphoglycan varies among species, and epitopes of these species-specific oligosaccharides are suggested to be implicated in the interaction of Leishmania with macrophages as well as species-specific tissue tropism observed in leishmaniasis. The recognition of the species-specific variation of oligosaccharides is likely to be mediated by host carbohydrate-binding proteins, lectins, but the identities of the lectins remain elusive. Galectin-3 is a mammalian soluble beta-galactoside-binding lectin and is expressed in macrophages, dendritic cells, and keratinocytes, as well as fibroblasts, all of which are present in the site of Leishmania infection. In this paper, we found that galectin-3 binds to lipophosphoglycan of Leishmania major but not to those of Leishmania donovani through L. major-specific polygalactose epitopes. Association of galectin-3 with L. major led to the cleavage of galectin-3, resulting in truncated galectin-3 containing the C-terminal lectin domain but lacking the N-terminal domain implicated in lectin oligomerization. This cleavage was inhibited by the galectin-3 antagonist lactose, as well as 1,10-ortho-phenanthroline, suggesting that galectin-3 is cleaved by zinc metalloproteases after its binding to lipophosphoglycans. The modulation of various innate immunity reactions by galectin-3 is affected by its oligomerization; therefore, we propose the L. major-specific truncation of galectin-3 may contribute to the species-specific immune responses induced by Leishmania.     

Temperature-induced expression of proteins in Leishmania mexicana amazonensis. A 22-kDa protein is possibly localized in the mitochondrion

Temperature increase is an integral part of Leishmania life cycle, and plays a major role in stage transformation. Analysis of the temperature-dependent pattern of protein synthesis on two-dimensional gel electrophoresis shows that, in addition to the conserved heat-shock type of response in which expression of the major 70-kDa and 83-kDa heat-shock proteins is observed, a group of low-molecular-mass (17-40 kDa) proteins is induced in promastigotes of Leishmania mexicana amazonensis at elevated temperatures. Immuno-gold labelling with antibodies raised against the heat-induced 22-kDa proteins was localized mainly in the mitochondrion of Leishmania parasites, though labelling was observed also in the nucleus. The correlation of this finding with various reports on induction of mitochondrial enzymes in response to temperature stress in other organisms is discussed.