Putting the Leishmania Genome to Work: Functional Genomics by Transposon Trapping and Expression Profiling

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Jan, 2002  |  Pubmed ID: 11839181

Leishmania are important protozoan pathogens of humans in temperate and tropical regions. The study of gene expression during the infectious cycle, in mutants or after environmental or chemical stimuli, is a powerful approach towards understanding parasite virulence and the development of control measures. Like other trypanosomatids, Leishmania gene expression is mediated by a polycistronic transcriptional process that places increased emphasis on post-transcriptional regulatory mechanisms including RNA processing and protein translation. With the impending completion of the Leishmania genome, global approaches surveying mRNA and protein expression are now feasible. Our laboratory has developed the Drosophila transposon mariner as a tool for trapping Leishmania genes and studying their regulation in the form of protein fusions; a classic approach in other microbes that can be termed 'proteogenomics'. Similarly, we have developed reagents and approaches for the creation of DNA microarrays, which permit the measurement of RNA abundance across the parasite genome. Progress in these areas promises to greatly increase our understanding of global mechanisms of gene regulation at both mRNA and protein levels, and to lead to the identification of many candidate genes involved in virulence.

Non-pathogenic Trypanosomatid Protozoa As a Platform for Protein Research and Production

Protein Expression and Purification. Jul, 2002  |  Pubmed ID: 12135552

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30 mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogeneous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans.

Characterization of Quinonoid-dihydropteridine Reductase (QDPR) from the Lower Eukaryote Leishmania Major

The Journal of Biological Chemistry. Oct, 2002  |  Pubmed ID: 12151409

Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H(4)biopterin pools. LmQDPR shows good homology to metazoan quinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8-9 copies containing LmQDPR plus two other genes. QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H(4)biopterin throughout this family of human parasites.

Leishmania LPG3 Encodes a GRP94 Homolog Required for Phosphoglycan Synthesis Implicated in Parasite Virulence but Not Viability

The EMBO Journal. Sep, 2002  |  Pubmed ID: 12198148

Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide-phosphate repeat unit Galbeta1,4Manalpha1-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 Manalpha1-PO4 residue of the first repeat unit identified LPG3, the Leishmania 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.

Protozomics: Trypanosomatid Parasite Genetics Comes of Age

Nature Reviews. Genetics. Jan, 2003  |  Pubmed ID: 12509749

Trypanosomatid protozoans cause important diseases of humans and their domestic livestock. Various molecular genetic tools are now allowing rapid progress in understanding many of the unique aspects of the molecular and cell biology of these organisms. Diploidy and the lack or difficulty of sexual crossing has been a challenge for forward genetics, but powerful selections and functional complementation have helped to overcome it in Leishmania. RNA interference has been adapted for forward genetics in trypanosomes, in which it is also a powerful tool for reverse genetics. Interestingly, the efficacy of different genetic tools has steered research into different aspects of the biology of these parasites.

Transposon Mutagenesis of Mycobacterium Marinum Identifies a Locus Linking Pigmentation and Intracellular Survival

Infection and Immunity. Feb, 2003  |  Pubmed ID: 12540574

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.

Functional Identification of Galactosyltransferases (SCGs) Required for Species-specific Modifications of the Lipophosphoglycan Adhesin Controlling Leishmania Major-sand Fly Interactions

The Journal of Biological Chemistry. May, 2003  |  Pubmed ID: 12604613

Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Galbeta1,4Manalpha1-PO(4)) repeating units. In L. major these are extensively substituted with Gal(beta1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain beta1,3-galactosyltransferases (betaGalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain betaGalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression.

Improvements in Transfection Efficiency and Tests of RNA Interference (RNAi) Approaches in the Protozoan Parasite Leishmania

Molecular and Biochemical Parasitology. May, 2003  |  Pubmed ID: 12742588

Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania. RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei. For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively. This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids. However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L. donovani. Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect. Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct. These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae. The implications to parasite biology, gene amplification, and genetic analysis are discussed.

Identification of Genes Encoding Arabinosyltransferases (SCA) Mediating Developmental Modifications of Lipophosphoglycan Required for Sand Fly Transmission of Leishmania Major

The Journal of Biological Chemistry. Aug, 2003  |  Pubmed ID: 12750366

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 scbetaGal 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 beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs 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 betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania.

The Role(s) of Lipophosphoglycan (LPG) in the Establishment of Leishmania Major Infections in Mammalian Hosts

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2003  |  Pubmed ID: 12869694

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- mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1- 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- 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.

Ether Phospholipids and Glycosylinositolphospholipids Are Not Required for Amastigote Virulence or for Inhibition of Macrophage Activation by Leishmania Major

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12944391

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 GPI-anchored 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- knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1- thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1- grew well and maintained lipid rafts (detergent-resistant 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.

Functional Genetic Identification of PRP1, an ABC Transporter Superfamily Member Conferring Pentamidine Resistance in Leishmania Major

Molecular and Biochemical Parasitology. Aug, 2003  |  Pubmed ID: 12946844

Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.

Persistence Without Pathology in Phosphoglycan-deficient Leishmania Major

Science (New York, N.Y.). Aug, 2003  |  Pubmed ID: 12947201

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-) 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 lpg2- thus provides a platform for probing parasite factors implicated in persistence and its role in disease and immunity.

The H Region HTBF Gene Mediates Terbinafine Resistance in Leishmania Major

Molecular and Biochemical Parasitology. Sep, 2003  |  Pubmed ID: 12967714

An in Vitro System for Developmental and Genetic Studies of Leishmania Donovani Phosphoglycans

Molecular and Biochemical Parasitology. Aug, 2003  |  Pubmed ID: 14550894

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.

Sphingolipids Are Essential for Differentiation but Not Growth in Leishmania

The EMBO Journal. Nov, 2003  |  Pubmed ID: 14609948

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.

Vaccination with Phosphoglycan-deficient Leishmania Major Protects Highly Susceptible Mice from Virulent Challenge Without Inducing a Strong Th1 Response

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2004  |  Pubmed ID: 15004184

Long-term immunity to Leishmania may require the continued presence of parasites, but previous attempts to create attenuated parasites that persist without causing disease have had limited success. Since Leishmania major mutants that lack lipophosphoglycan and other secreted phosphoglycans, termed lpg2-, persist indefinitely in infected mice without inducing any disease, we tested their ability to provide protection to virulent L. major challenge. In response to leishmanial Ag stimulation, cells from lpg2--infected mice produced minimal levels of IL-4 and IL-10, as well as very low levels of IFN-gamma. Nevertheless, when BALB/c mice infected with lpg2- parasites were challenged with virulent L. major they were protected from disease. Thus, these findings report on attenuated parasites that may be used to induce long-term protection against leishmaniasis and indicate that the immunity induced can be maintained in the absence of a strong Th1 response.

The LPG1 Gene Family of Leishmania Major

Molecular and Biochemical Parasitology. Jul, 2004  |  Pubmed ID: 15138063

In Leishmania major, the core of the abundant surface lipophosphoglycan (LPG) is structurally related to that of the smaller glycosylinositolphospholipids (GIPLs) in containing galactosylfuranose (Gal(f)) residues in a Gal(f) (beta1, 3)Man motif. However, deletion of the putative Gal(f)-transferase (Gal(f)T) LPG1 affected Gal(f) incorporation in LPG but not GIPLs. We hypothesized that the presumptive GIPL Gal(f)-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 Gal(f) -containing GIPLs. In contrast, overexpression of these three led to elevated galactose incorporation in glycoproteins. Gal(f)-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 Gal(f)T.

Expression Profiling Using Random Genomic DNA Microarrays Identifies Differentially Expressed Genes Associated with Three Major Developmental Stages of the Protozoan Parasite Leishmania Major

Molecular and Biochemical Parasitology. Jul, 2004  |  Pubmed ID: 15138069

To complete its life cycle, protozoan parasites of the genus Leishmania undergo at least three major developmental transitions. However, previous efforts to identify genes showing stage regulated changes in transcript abundance have yielded relatively few. Here we used expression profiling to assess changes in transcript abundance in three stages: replicating promastigotes and infective non-replicating metacyclics, which occur in the sand fly vector, and in the amastigote stage residing with macrophage phagolysosomes in mammals. Microarrays were developed containing 11,484 PCR products that included a number of known genes and 10,464 random 1 kb genomic DNA fragments. Arrays were hybridized in triplicate and genes showing two-fold or greater changes in 2/3 experiments were scored as differentially expressed. Remarkably, only about one percent of the DNAs expression varied by this criteria, in either stage comparison. Northern blot analysis confirmed the predicted change in mRNA abundance for most of these (68%). This set of genes included most of those previously identified in the literature as differentially regulated as well as a number of novel genes. Notably, Leishmania maxicircle transcripts showed strong up-regulation in metacyclic and amastigote parasites, probably associated with changes in parasite energy metabolism. However, current data suggest that expression profiling using shotgun DNA libraries significantly underestimates the extent of regulated transcripts.

In Vitro Shuttle Mutagenesis Using Engineered Mariner Transposons

Methods in Molecular Biology (Clifton, N.J.). 2004  |  Pubmed ID: 15153636

Advances in our understanding of the protozoan parasite Leishmania have been facilitated by the development of molecular and genetic tools. One powerful approach for gene identification and analysis is transposon mutagenesis. This can be performed directly in vivo, but often it is more convenient to generate transpositions in vitro for subsequent analysis in vivo, in a process termed "shuttle mutagenesis." The Drosophila element mariner is well suited for application by either route. Minimal mariner elements containing cis-acting elements required for transposition have been generated, which can be further modified to suit the needs of the experimenter. Additional genetic markers and/or reporters can be introduced, which are useful for procedures such as insertional mutagenesis, shotgun sequencing, or the generation of protein and transcriptional fusions for subsequent analysis. Active transposase can readily be generated following expression in Escherichia coli, and efficiencies of 10-3/target can be obtained, allow-ing the generation of large transposon insertion libraries suitable for subsequent screening in vivo. This chapter explains the steps necessary to purify active Mos1 transposase and conduct an in vitro transposition reaction. We also discuss some of the considerations relevant to the design and application of functional mariner elements (donor plasmids) relevant to studies in Leishmania and other organisms.

Identification of a Compensatory Mutant (lpg2-REV) of Leishmania Major Able to Survive As Amastigotes Within Macrophages Without LPG2-dependent Glycoconjugates and Its Significance to Virulence and Immunization Strategies

Infection and Immunity. Jun, 2004  |  Pubmed ID: 15155672

Different Leishmania species rely to different extents on abundant glycoconjugates, such as lipophosphoglycan (LPG) and related molecules, in mammalian infections. Previously, we showed that Leishmania major deletion mutants lacking the Golgi GDP-mannose transporter LPG2, which is required for assembly of the dominant phosphoglycan (PG) repeats of LPG, were unable to survive in macrophages. These lpg2- mutants, however, retained the ability to generate asymptomatic, persistent infections in mice. In contrast, Ilg and colleagues showed that Leishmania mexicana LPG2 mutants retained virulence for mice. Here we identified a partial revertant population of the L. major lpg2- mutants (designated lpg2(-)REV) that had regained the ability to replicate in macrophages and induce disease pathology through a compensatory change. Like the lpg2 parent, the lpg2(-)REV revertant was unable to synthesize LPG2-dependent PGs in the promastigote stage and thus remained highly attenuated in the ability to induce infection. However, after considerable delay lpg2(-)REV revertant-infected mice exhibited lesions, and amastigotes isolated from these lesions were able to replicate within macrophages despite the fact that they were unable to synthesize PGs. Thus, in some respects, the lpg2(-)REV amastigotes resemble L. mexicana amastigotes. Future studies of the gene(s) responsible may shed light on the mechanisms employed by L. major to survive in the absence of LPG2-dependent glycoconjugates and may also improve the potential of the lpg2- L. major line to serve as a live parasite vaccine by overcoming its tendency to revert toward virulence.

The Application of Gene Expression Microarray Technology to Kinetoplastid Research

Current Molecular Medicine. Sep, 2004  |  Pubmed ID: 15357212

Protozoan parasites in the order Kinetoplastida cause severe disease primarily in tropical and subtropical areas. Vaccines to control these diseases have shown some promise, but none are in active clinical use. Drug treatments are available for all of the acute infections, but the emergence of resistance and an unresponsive chronic phase are current problems. Rapid advances in genomic technology open the possibility of discovering new genes that can contribute to vaccine initiatives or serve as targets for development of new drugs. The DNA microarray is a genomic technology, which is being applied to new gene discovery in kinetoplastid parasites. Both cDNA and genomic microarrays for Leishmania major have identified a number of new genes that are expressed in a stage-specific fashion and preliminary results from a L. donovani genomic microarray also demonstrated new gene discovery. A microarray of Trypanosoma brucei genomic fragments identified new genes whose expression differs between the insect borne stage and the human infectious stage of the parasite. The next few years, building on this foundational work, should witness the most exciting stage as microarrays are applied to questions such as the basis of drug resistance, post kala azar dermal leishmaniasis, the regulation of differentiation to infectious stages, linking coordinately regulated pathways of genes and development of genetically defined parasites that may have potential as live attenuated vaccines.

Central Memory T Cells Mediate Long-term Immunity to Leishmania Major in the Absence of Persistent Parasites

Nature Medicine. Oct, 2004  |  Pubmed ID: 15448686

Infection with Leishmania major induces a protective immune response and long-term resistance to reinfection, which is thought to depend upon persistent parasites. Here we demonstrate that although effector CD4(+) T cells are lost in the absence of parasites, central memory CD4(+) T cells are maintained. Upon secondary infection, these central memory T cells become tissue-homing effector T cells and mediate protection. Thus, immunity to L. major is mediated by at least two distinct populations of CD4(+) T cells: short-lived pathogen-dependent effector cells and long-lived pathogen-independent central memory cells. These data suggest that central memory T cells should be the targets for nonlive vaccines against infectious diseases requiring cell-mediated immunity.

Flypaper for Parasites

Cell. Oct, 2004  |  Pubmed ID: 15507202

In this issue, Kamhawi et al. (2004) describe the identification of an insect galectin as the receptor for the stage-specific Leishmania adhesin lipophosphoglycan (LPG). This interaction is critical for parasite survival in the midgut of its sand fly vector. The results open new avenues for studies of insect immunity, transmission binding vaccines, and host-parasite coevolution.

Characterization of a Defensin from the Sand Fly Phlebotomus Duboscqi Induced by Challenge with Bacteria or the Protozoan Parasite Leishmania Major

Infection and Immunity. Dec, 2004  |  Pubmed ID: 15557638

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Leishmania Salvage and Remodelling of Host Sphingolipids in Amastigote Survival and Acidocalcisome Biogenesis

Molecular Microbiology. Mar, 2005  |  Pubmed ID: 15720561

Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2-) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2- amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out 'headgroup' remodelling. Additionally, we describe a new defect of the spt2- promastigotes involving 'empty' acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2- amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.

Eukaryotic UDP-galactopyranose Mutase (GLF Gene) in Microbial and Metazoal Pathogens

Eukaryotic Cell. Jun, 2005  |  Pubmed ID: 15947206

Galactofuranose (Gal(f)) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal(f) is synthesized as the nucleotide sugar UDP-Gal(f) by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal(f) synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal(f) synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal(f) synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal(f) plays critical roles in cellular viability and virulence.

Identification of a DNA Fragment That Increases Mitotic Stability of Episomal Linear DNAs in Leishmania Major

International Journal for Parasitology. Aug, 2005  |  Pubmed ID: 15996670

The centromere is a specialized region of eukaryotic chromosomes, the site of kinetochore formation, spindle attachment and regulation of chromosome segregation during mitotic and meiotic cell divisions. To identify sequences which increase mitotic stability and/or act as potential centromeres in Leishmania major, we first generated libraries of Leishmania linear artificial chromosomes (LACs) bearing 30 kb inserts of randomly selected genomic DNAs. These were introduced into parasites, and then their stability was assessed following a period of 10 passages of growth in the absence of selective pressure. Approximately 80% of the 108 transfectants tested lost their LACs promptly and only 20% of the recombinants were retained; of these six showed strong but partial stability (maintained in 30-46% of cells). Mapping and sequencing of one clone (cSC10), which confers the highest degree of maintenance, revealed the presence of a sequence that was found within another stable episome, and which is dispersed in the genome of L. major. The implications of these data to the possible mechanisms of chromosomal maintenance are discussed.

The Genome of the Kinetoplastid Parasite, Leishmania Major

Science (New York, N.Y.). Jul, 2005  |  Pubmed ID: 16020728

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

Structures of Leishmania Major Pteridine Reductase Complexes Reveal the Active Site Features Important for Ligand Binding and to Guide Inhibitor Design

Journal of Molecular Biology. Sep, 2005  |  Pubmed ID: 16055151

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value.

Reconstitution of GDP-mannose Transport Activity with Purified Leishmania LPG2 Protein in Liposomes

The Journal of Biological Chemistry. Jan, 2005  |  Pubmed ID: 15542612

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-containing liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-Fuc). 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.

Demonstration by Heterologous Expression That the Leishmania SCA1 Gene Encodes an Arabinopyranosyltransferase

Glycobiology. Mar, 2006  |  Pubmed ID: 16272216

In part of the life cycle within their sand fly vector, Leishmania major parasites first attach to the fly's midgut through their main surface adhesin lipophosphoglycan (LPG) and later resynthesize a structurally distinct LPG that results in detachment and eventual transmission. One of these structural modifications requires the addition of alpha1,2-D-arabinopyranose caps to beta1,3-galactose side chains in the phosphoglycan repeat unit domain of LPG. We had previously identified two side chain arabinose genes (SCA1/2) that were involved in the alpha1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha1,2-D-ArapT activity, resulting in arabinopyranosylation of beta1,3-Gal side chains in LPG (hereafter called side chain D-arabinopyranosyltransferase [sc-D-ArapT]). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-D-ArapT. SCA1 expression constructs introduced into both mammalian COS-7 cells and the baculovirus-sf9 cell system exhibited considerable expression of the protein. However, functional sc-D-ArapT activity was observed only in the latter. In in vitro assays incubated with guanidine 59-diphosphate (GDP)-D-[3H]Arap as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-D-ArapT activity was observed when microsomes from the baculovirus-sf9 cells were incubated in presence of the LPG acceptor. No activity was observed in the absence of LPG. These results demonstrate that SCA1 encodes a sc-D-ArapT and provide the first example of heterologous expression of a D-ArapT gene.

Immunization with Persistent Attenuated Delta Lpg2 Leishmania Major Parasites Requires Adjuvant to Provide Protective Immunity in C57BL/6 Mice

Infection and Immunity. Jan, 2006  |  Pubmed ID: 16369039

Leishmania major parasites lacking the GDP-mannose transporter, termed Deltalpg2 parasites, fail to induce disease in mice but persist long-term. We previously found that Deltalpg2 organisms protect BALB/c mice from virulent L. major challenge. In contrast, we report here that Deltalpg2 parasites induce protective immunity in C57BL/6 mice only when administered with CpG-containing oligodeoxynucleotides, indicating that parasite persistence alone is not sufficient to maintain protective immunity to L. major.

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

Molecular and Biochemical Parasitology. Apr, 2006  |  Pubmed ID: 16464509

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.

Biochemical and Genetic Analysis of Methylenetetrahydrofolate Reductase in Leishmania Metabolism and Virulence

The Journal of Biological Chemistry. Dec, 2006  |  Pubmed ID: 17032644

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr(-)) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr(-) mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr(-) mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr(-) mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.

Redirection of Sphingolipid Metabolism Toward De Novo Synthesis of Ethanolamine in Leishmania

The EMBO Journal. Feb, 2007  |  Pubmed ID: 17290222

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.

A Lipophosphoglycan-independent Development of Leishmania in Permissive Sand Flies

Microbes and Infection / Institut Pasteur. Mar, 2007  |  Pubmed ID: 17307009

Leishmaniases are serious parasitic diseases the etiological organisms of which are transmitted by insect vectors, phlebotominae sand flies. Two sand fly species, Phlebotomus papatasi and P. sergenti, display remarkable specificity for Leishmania parasites they transmit in nature, but many others are broadly permissive to the development of different Leishmania species. Previous studies have suggested that in 'specific' vectors the successful parasite development is mediated by parasite surface glycoconjugates and sand fly lectins, however we show here that interactions involving 'permissive' sand flies utilize another molecules. We did find that the abundant surface glycoconjugate lipophosphoglycan, essential for attachment of Leishmania major in the specific vector P. papatasi, was not required for parasite adherence or survival in the permissive vectors P. arabicus and Lutzomyia longipalpis. Attachment in several permissive sand fly species instead correlated with the presence of midgut glycoproteins bearing terminal N-acetyl-galactosamine and with the occurrence of a lectin-like activity on Leishmania surface. This new binding modality has important implications for parasite transmission and evolution. It may contribute to the successful spreading of Leishmania due to their adaptation into new vectors, namely transmission of L. infantum by Lutzomyia longipalpis; this event led to the establishment of L. infantum/chagasi in Latin America.

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

The Journal of Biological Chemistry. May, 2007  |  Pubmed ID: 17347153

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.

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

Infection and Immunity. Sep, 2007  |  Pubmed ID: 17606605

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.

Characterization of Inositol Phosphorylceramides from Leishmania Major by Tandem Mass Spectrometry with Electrospray Ionization

Journal of the American Society for Mass Spectrometry. Sep, 2007  |  Pubmed ID: 17627842

We describe tandem mass spectrometric approaches, including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species seen as [M - H](-) and [M - 2H + Li](-) ions in the negative-ion mode as well as [M + H](+), [M + Li](+), and [M - H + 2Li](+) ions in the positive-ion mode. Following CAD in an ion-trap or a triple-stage quadrupole instrument, the [M - H](-) ions of IPC yielded fragment ions reflecting only the inositol and the fatty acyl substituent of the molecule. In contrast, the mass spectra from MS(3) of [M - H - Inositol](-) ions contained abundant ions that are readily applicable for assignment of the fatty acid and long-chain base (LCB) moieties. Both the product-ion spectra from MS(2) and MS(3) of the [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) ions also contained rich fragment ions informative for unambiguous assignment of the fatty acyl substituent and the LCB. However, the sensitivity of the ions observed in the forms of [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) (Alk = Li, Na) is nearly 10 times less than that observed in the [M - H](-) form. In addition to the major fragmentation pathways leading to elimination of the inositol or inositol monophosphate moiety, several structurally informative ions resulting from rearrangement processes were observed. The fragmentation processes are similar to those previously reported for ceramides. While the tandem mass spectrometric approach using MS(n) (n = 2, 3) permits the structures of the Leishmania major IPCs consisting of two isomeric structures to be unveiled in detail, tandem mass spectra from constant neutral loss scans may provide a simple method for detecting IPC in mixtures.

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania Major

The Journal of Biological Chemistry. Jan, 2008  |  Pubmed ID: 17981801

For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH(2)-THF). 5,10-CH(2)-THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP(-) mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-(14)C]glycine into DNA. The gcvP(-) mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP(-) promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH(2)-THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH(2)-THF synthesis.

Phylogenomic and Functional Analysis of Pterin-4a-carbinolamine Dehydratase Family (COG2154) Proteins in Plants and Microorganisms

Plant Physiology. Apr, 2008  |  Pubmed ID: 18245455

Pterin-4a-carbinolamine dehydratases (PCDs) recycle oxidized pterin cofactors generated by aromatic amino acid hydroxylases (AAHs). PCDs are known biochemically only from animals and one bacterium, but PCD-like proteins (COG2154 in the Clusters of Orthologous Groups [COGs] database) are encoded by many plant and microbial genomes. Because these genomes often encode no AAH homologs, the annotation of their COG2154 proteins as PCDs is questionable. Moreover, some COG2154 proteins lack canonical residues that are catalytically important in mammalian PCDs. Diverse COG2154 proteins of plant, fungal, protistan, and prokaryotic origin were therefore tested for PCD activity by functional complementation in Escherichia coli, and the plant proteins were localized using green fluorescent protein fusions. Higher and lower plants proved to have two COG2154 proteins, a mitochondrial one with PCD activity and a noncanonical, plastidial one without. Phylogenetic analysis indicated that the latter is unique to plants and arose from the former early in the plant lineage. All 10 microbial COG2154 proteins tested had PCD activity; six of these came from genomes with no AAH, and six were noncanonical. The results suggested the motif [EDKH]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWF]-x(9)-[HW]-x(8,15)-D as a signature for PCD activity. Organisms having a functional PCD but no AAH partner include angiosperms, yeast, and various prokaryotes. In these cases, PCD presumably has another function. An ancillary role in molybdopterin cofactor metabolism, hypothesized from phylogenomic evidence, was supported by demonstrating significantly lowered activities of two molybdoenzymes in Arabidopsis thaliana PCD knockout mutants. Besides this role, we propose that partnerless PCDs support the function of as yet unrecognized pterin-dependent enzymes.

Leishmania Major Intracellular Survival is Not Altered in SHP-1 Deficient Mev or CD45-/- Mice

Experimental Parasitology. Nov, 2008  |  Pubmed ID: 18682252

Protozoan parasites of the genus Leishmania escape from the immune response by interfering with signal transduction pathways of its host cell, the macrophage, thereby establishing permissive conditions for intracellular survival. Inhibition of macrophage activation after Leishmania infection has been suggested to require activation of the host cell phosphatase SHP-1. However, by utilizing infections of SHP-1 deficient (me(v)) and CD45 null mutant mice or macrophages, we provide evidence that intracellular survival of Leishmania major is not generally dependent on these cellular phosphatases.

Developmentally Regulated Sphingolipid Synthesis in African Trypanosomes

Molecular Microbiology. Oct, 2008  |  Pubmed ID: 18699867

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.

Migratory Dermal Dendritic Cells Act As Rapid Sensors of Protozoan Parasites

PLoS Pathogens. Nov, 2008  |  Pubmed ID: 19043558

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i) protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.

Methylene Tetrahydrofolate Dehydrogenase/cyclohydrolase and the Synthesis of 10-CHO-THF Are Essential in Leishmania Major

Molecular Microbiology. Mar, 2009  |  Pubmed ID: 19183277

10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP(+)-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF (K(i) 105 nM), as was Leishmania growth (EC(50) 1.1 microM). Previous studies showed null ftl(-) mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1(-) null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of approximately 1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1(-) null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.

Leishmania Donovani Lacking the Golgi GDP-Man Transporter LPG2 Exhibit Attenuated Virulence in Mammalian Hosts

Experimental Parasitology. Jul, 2009  |  Pubmed ID: 19328787

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.

Demonstration of Genetic Exchange During Cyclical Development of Leishmania in the Sand Fly Vector

Science (New York, N.Y.). Apr, 2009  |  Pubmed ID: 19359589

Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore full genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fly vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

A Novel Role for Stat1 in Phagosome Acidification and Natural Host Resistance to Intracellular Infection by Leishmania Major

PLoS Pathogens. Apr, 2009  |  Pubmed ID: 19381261

Intracellular parasites of the genus Leishmania generate severe diseases in humans, which are associated with a failure of the infected host to induce a protective interferon gamma (IFNgamma)-mediated immune response. We tested the role of the JAK/STAT1 signaling pathway in Leishmania pathogenesis by utilizing knockout mice lacking the signal transducer and activator of transcription 1 (Stat1) and derived macrophages. Unexpectedly, infection of Stat1-deficient macrophages in vitro with promastigotes from Leishmania major and attenuated LPG1 knockout mutants (lpg(-)) specifically lacking lipophosphoglycan (LPG) resulted in a twofold increased intracellular growth, which was independent of IFNgamma and associated with a substantial increase in phagosomal pH. Phagosomes in Stat1-/- macrophages showed normal maturation as judged by the accumulation of the lysosomal marker protein rab7, and provided normal vATPase activity, but were defective in the anion conductive pathway required for full vesicular acidification. Our results suggest a role of acidic pH in the control of intracellular Leishmania growth early during infection and identify for the first time an unexpected role of Stat1 in natural anti-microbial resistance independent from its function as IFNgamma-induced signal transducer. This novel Stat1 function may have important implications to studies of other pathogens, as the acidic phagolysosomal pH plays an important role in antigen processing and the uncoating process of many viruses.

Regulated Expression of the Leishmania Major Surface Virulence Factor Lipophosphoglycan Using Conditionally Destabilized Fusion Proteins

Proceedings of the National Academy of Sciences of the United States of America. May, 2009  |  Pubmed ID: 19383793

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

Leishmania Major Lacking Arginase (ARG) Are Auxotrophic for Polyamines but Retain Infectivity to Susceptible BALB/c Mice

Molecular and Biochemical Parasitology. May, 2009  |  Pubmed ID: 19393161

Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from L-ornithine has been well studied in protozoa, the metabolic origin(s) of L-ornithine have received less attention. Arginase (EC 3.5.3.1) catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg(?) knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or L-ornithine for growth. A complemented line (arg(?)/+ARG) expressing arginase from a multi-copy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors ?-difluoromethylornithine (DFMO) and N(?)-hydroxy-l-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg(?) parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of l-arginine metabolism in both pro- and anti-parasitic responses involving host nitric oxide synthase, which requires L-arginine to generate NO.

PTR1-dependent Synthesis of Tetrahydrobiopterin Contributes to Oxidant Susceptibility in the Trypanosomatid Protozoan Parasite Leishmania Major

Current Genetics. Jun, 2009  |  Pubmed ID: 19396443

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 (-) null mutants were 18-fold more sensitive to H(2)O(2) than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H(2)O(2) susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H(4)B metabolism specifically. Neither H(2)O(2) consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.

The Enzymes of the 10-formyl-tetrahydrofolate Synthetic Pathway Are Found Exclusively in the Cytosol of the Trypanosomatid Parasite Leishmania Major

Molecular and Biochemical Parasitology. Aug, 2009  |  Pubmed ID: 19450731

In most organisms 10-formyl-tetrahydrofolate (10-CHO-THF) participates in the synthesis of purines in the cytosol and formylation of mitochondrial initiator methionyl-tRNA(Met). Here we studied 10-CHO-THF biosynthesis in the protozoan parasite Leishmania major, a purine auxotroph. Two distinct synthetic enzymes are known, a bifunctional methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (DHCH) or formyl-tetrahydrofolate ligase (FTL), and phylogenomic profiling revealed considerable diversity for these in trypanosomatids. All species surveyed contain a DHCH1, which was shown recently to be essential in L. major. A second DHCH2 occurred only in L. infantum, L. mexicana and T. cruzi, and as a pseudogene in L. major. DHCH2s bear N-terminal extensions and we showed a LiDHCH2-GFP fusion was targeted to the mitochondrion. FTLs were found in all species except Trypanosoma brucei. L. major ftl(-) null mutants were phenotypically normal in growth, differentiation, animal infectivity and sensitivity to a panel of pteridine analogs, but grew more slowly when starved for serine or glycine, as expected for amino acids that are substrates in C1-folate metabolism. Cell fractionation and western blotting showed that both L. major DHCH1 and FTL were localized to the cytosol and not the mitochondrion. These localization data predict that in L. major cytosolic 10-formyl-tetrahydrofolate must be transported into the mitochondrion to support methionyl-tRNA(Met) formylation. The retention in all the trypanosomatids of at least one enzyme involved in 10-formyl-tetrahydrofolate biosynthesis, and the essentiality of this metabolite in L. major, suggests that this pathway represents a promising new area for chemotherapeutic attack in these parasites.

Leishmania Major Phosphoglycans Influence the Host Early Immune Response by Modulating Dendritic Cell Functions

Infection and Immunity. Aug, 2009  |  Pubmed ID: 19487470

The precise role of Leishmania glycoconjugate molecules including phosphoglycans (PGs) and lipophosphoglycan (LPG) on host cellular responses is still poorly defined. Here, we investigated the interaction of Leishmania major LPG2 null mutant (lpg2(-)), which lacks both PGs and LPG, with dendritic cells (DCs) and the subsequent early immune response in infected mice. Surprisingly, the absence of phosphoglycans did not influence expression pattern of major histocompatibility complex class II (MHC II), CD40, CD80, and CD86 on DCs in vitro and in vivo. However, lpg2(-) L. major induced significantly higher production of interleukin-12p40 (IL-12p40) by infected bone marrow-derived DCs (BMDCs) than wild-type (WT) parasites in vitro. Furthermore, the production of IL-12p40 by draining lymph node cells from lpg2(-) mutant-infected mice was higher than those from WT L. major-infected mice. In model antigen presentation experiments, DCs from lpg2(-) mutant-infected mice induced more gamma interferon (IFN-gamma) and IL-2 production by Leishmania-specific T cells than those from WT-infected mice. Lymphocytes isolated from mice infected for 3 days with lpg2(-) parasites produce similar levels of IFN-gamma, but significantly less IL-4 and IL-10 than WT controls. Decreased IL-4 production was also seen in another general PG-deficient mutant lacking the Golgi UDP-galactose transporters (lpg5A(-) lpg5B(-)), but not with the lpg1(-) mutant lacking only LPG, thereby implicating PGs generally in the reduction of IL-4 production. Thus, Leishmania PGs influence host early immune response by modulating DC functions in a way that inhibits antigen presentation and promotes early IL-4 response, and their absence may impact the balance between Th1 and Th2 responses.

Inoculation of Killed Leishmania Major into Immune Mice Rapidly Disrupts Immunity to a Secondary Challenge Via IL-10-mediated Process

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2009  |  Pubmed ID: 19666482

Recovery from natural or experimental Leishmania major infection, the causative agent of cutaneous leishmaniasis, results in development of durable immunity in mice and humans that is manifested as rapid control of parasite replication and resolution of cutaneous lesion after secondary challenge. This form of "infection-induced" immunity is thought to occur naturally in endemic areas and is generally considered the gold standard for any effective vaccine against cutaneous leishmaniasis. To determine factors that might heighten or abrogate infection-induced immunity, we investigated the impact of inoculating dead antigen in the form of killed Leishmania parasites to healed mice. We show that inoculation of killed parasites into mice that resolved their primary virulent L. major infection results in rapid and relatively sustained loss of infection-induced immunity. This loss of immunity was not due to the inability of killed parasites to induce inflammatory responses (such as delayed type hypersensitivity), but it was related to their failure to induce robust IFN-gamma response. Furthermore, inoculation of killed Leishmania parasites into healed mice led to rapid expansion of IL-10-producing CD4(+)CD25(+)Foxp3(+) T cells in lymph nodes draining the primary infection site. Treatment with anti-CD25 or anti-IL-10R mAb abolished killed parasite-induced loss of immunity. Our study suggests that vaccination with killed parasites could predispose naturally immune individuals to become susceptible to new infections and/or disease reactivation. This may account for the lack of efficacy of such vaccines in field trials in endemic regions. These findings have important implications for vaccine design and vaccination strategies against human cutaneous leishmaniasis.

Degradation of Host Sphingomyelin is Essential for Leishmania Virulence

PLoS Pathogens. Dec, 2009  |  Pubmed ID: 20011126

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl(-)) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl(-) mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl(-) was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology.

Amplification of an Alternate Transporter Gene Suppresses the Avirulent Phenotype of Glucose Transporter Null Mutants in Leishmania Mexicana

Molecular Microbiology. Jan, 2009  |  Pubmed ID: 19017272

A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana, in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate beta-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle.

Phospholipid and Sphingolipid Metabolism in Leishmania

Molecular and Biochemical Parasitology. Apr, 2010  |  Pubmed ID: 20026359

In many eukaryotes, phospholipids (PLs) and sphingolipids (SLs) are abundant membrane components and reservoirs for important signaling molecules. In Leishmania, the composition, metabolism, and function of PLs and SLs differ significantly from those in mammalian cells. Although only a handful of enzymes have been experimentally characterized, available data suggest many steps of PL/SL metabolism are critical for Leishmania viability and/or virulence, and could be a source for new drug targets. Further studies of genes involved in the synthesis (de novo and salvage) and degradation of PLs and SLs will reveal their diverse effects on Leishmania pathogenesis.

Leishmania Major Glycosylation Mutants Require Phosphoglycans (lpg2-) but Not Lipophosphoglycan (lpg1-) for Survival in Permissive Sand Fly Vectors

PLoS Neglected Tropical Diseases. 2010  |  Pubmed ID: 20084096

Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1(-)), and the phosphoglycan (PG)-deficient mutant lpg2(-). The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.

Proteophosphoglycan Confers Resistance of Leishmania Major to Midgut Digestive Enzymes Induced by Blood Feeding in Vector Sand Flies

Cellular Microbiology. Jul, 2010  |  Pubmed ID: 20088949

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.

Deletion of UDP-glucose Pyrophosphorylase Reveals a UDP-glucose Independent UDP-galactose Salvage Pathway in Leishmania Major

Glycobiology. Jul, 2010  |  Pubmed ID: 20335578

The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.

Monitoring the Efficacy of Antimicrobial Photodynamic Therapy in a Murine Model of Cutaneous Leishmaniasis Using L. Major Expressing GFP

Journal of Biophotonics. Jun, 2010  |  Pubmed ID: 20376860

A murine model of cutaneous leishmaniasis with green fluorescent protein positive (GFP+) L. major enables the monitoring of parasitic load via measurements of GFP fluorescence intensity, allowing for a faster and more efficient way of monitoring the clinical outcome of photodynamic therapy (PDT). This model may provide new insights on the phototoxic aspects in PDT. Although PDT regimens may be somewhat different in humans, it is expected that the developed model will facilitate the optimization and clinical translation of PDT as a therapy for cutaneous leishmaniasis and the eventual development of topical PDT treatments for other granulomatous infections.

Phosphoproteome Dynamics Reveal Heat-shock Protein Complexes Specific to the Leishmania Donovani Infectious Stage

Proceedings of the National Academy of Sciences of the United States of America. May, 2010  |  Pubmed ID: 20404152

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

A Role for Tetrahydrofolates in the Metabolism of Iron-sulfur Clusters in All Domains of Life

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20489182

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Expansion of the Target of Rapamycin (TOR) Kinase Family and Function in Leishmania Shows That TOR3 is Required for Acidocalcisome Biogenesis and Animal Infectivity

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20551225

Target of rapamycin (TOR) kinases are key regulators of cell growth, proliferation, and structure in eukaryotes, processes that are highly coordinated during the infectious cycle of eukaryotic pathogens. Database mining revealed three TOR kinases in the trypanosomatid parasite Leishmania major, as defined by homology to the phosphoinositide 3-kinase-related kinase (PIKK) family and a signature conserved FKBP12/rapamycin-binding domain. Consistent with the essential roles of TOR complexes in other organisms, we were unable to generate null TOR1 or TOR2 mutants in cultured L. major promastigotes. In contrast, tor3(-) null mutants were readily obtained; while exhibiting somewhat slower growth, tor3(-) maintained normal morphology, rapamycin sensitivity, and differentiation into the animal-infective metacyclic stage. Significantly, tor3(-) mutants were unable to survive or replicate in macrophages in vitro, or to induce pathology or establish infections in mice in vivo. The loss of virulence was associated with a defect in acidocalcisome formation, as this unique organelle was grossly altered in tor3- mutants and no longer accumulated polyphosphates. Correspondingly, tor3- mutants showed defects in osmoregulation and were sensitive to starvation for glucose but not amino acids, glucose being a limiting nutrient in the parasitophorous vacuole. Thus, in Leishmania, the TOR kinase family has expanded to encompass a unique role in AC function and biology, one that is essential for parasite survival in the mammalian infective stage. Given their important roles in cell survival and virulence, inhibition of TOR kinase function in trypanosomatids offers an attractive target for chemotherapy.

A Transposon Toolkit for Gene Transfer and Mutagenesis in Protozoan Parasites

Genetica. Mar, 2010  |  Pubmed ID: 19763844

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood. The availability of the genome sequence of these protozoa sets the stage for using more comprehensive, genome-wide strategies to study gene function. Transposons are effective tools for the systematic introduction of genetic alterations and different transposition systems have been adapted to study gene function in these human pathogens. A mariner transposon toolkit for use in vivo or in vitro in Leishmania parasites has been developed and can be used in a variety of applications. These modified mariner elements not only permit the inactivation of genes, but also mediate the rescue of translational gene fusions, bringing a major contribution to the investigation of Leishmania gene function. The piggyBac and Tn5 transposons have also been shown to mobilize across Plasmodium spp. genomes circumventing the current limitations in the genetic manipulation of these organisms.

Identification of Transport-critical Residues in a Folate Transporter from the Folate-biopterin Transporter (FBT) Family

The Journal of Biological Chemistry. Jan, 2010  |  Pubmed ID: 19923217

The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT) family within the major facilitator superfamily. Both proteins transport folates when expressed in Escherichia coli. Because the structural requirements for transport activity are not known for any FBT protein, we applied mutational analysis to identify residues that are critical to transport and interpreted the results using a comparative structural model based on E. coli lactose permease. Folate transport was assessed via the growth of an E. coli pabA abgT strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate. In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions abolished folate uptake without affecting Slr0642 expression in membranes, whereas other mutations had no effect. Residues important for function mostly line the predicted central cavity and are concentrated in the core alpha-helices H1, H4, H7, and H10. The essential residue locations are consistent with a folate-binding site lying roughly equidistant from both faces of the transporter. Arabidopsis has eight FBT proteins besides At2g32040, often lacking conserved critical residues. When six of these proteins were expressed in E. coli or in Leishmania folate or pterin transporter mutants, none showed evidence of folate or pterin transport activity, and only At2g32040 was isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such negative data could reflect roles in transport of other substrates. These studies provide the first insights into the native structure and catalytic mechanism of FBT family carriers.

Sphingolipids in Parasitic Protozoa

Advances in Experimental Medicine and Biology. 2010  |  Pubmed ID: 20919659

The surface of most protozoan parasites relies heavily upon lipid-anchored molecules, to form protective barriers and play critical functions required for infectivity. Sphingolipids (SLs) play important roles through their abundance and involvement in membrane microdomain formation, as well as serving as the lipid anchor for many of these molecules and in some but possibly not all species, as important signaling molecules. Interactions of parasite sphingolipid metabolism with that of the host may potentially contribute to parasite survival and/or host defense. In this chapter we summarize current knowledge of SL structure, synthesis and function in several of the major parasitic protozoan groups.

Retention and Loss of RNA Interference Pathways in Trypanosomatid Protozoans

PLoS Pathogens. 2010  |  Pubmed ID: 21060810

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.

Leishmania Major Survival in Selective Phlebotomus Papatasi Sand Fly Vector Requires a Specific SCG-encoded Lipophosphoglycan Galactosylation Pattern

PLoS Pathogens. 2010  |  Pubmed ID: 21085609

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 β1,3-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.

Differential Microbicidal Effects of Human Histone Proteins H2A and H2B on Leishmania Promastigotes and Amastigotes

Infection and Immunity. Mar, 2011  |  Pubmed ID: 21189319

Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1(0)) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Mφs) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites.

Leishmania RNA Virus Controls the Severity of Mucocutaneous Leishmaniasis

Science (New York, N.Y.). Feb, 2011  |  Pubmed ID: 21311023

Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

The Susceptibility of Trypanosomatid Pathogens to PI3/mTOR Kinase Inhibitors Affords a New Opportunity for Drug Repurposing

PLoS Neglected Tropical Diseases. Aug, 2011  |  Pubmed ID: 21886855

Target repurposing utilizes knowledge of "druggable" targets obtained in one organism and exploits this information to pursue new potential drug targets in other organisms. Here we describe such studies to evaluate whether inhibitors targeting the kinase domain of the mammalian Target of Rapamycin (mTOR) and human phosphoinositide-3-kinases (PI3Ks) show promise against the kinetoplastid parasites Trypanosoma brucei, T. cruzi, Leishmania major, and L. donovani. The genomes of trypanosomatids encode at least 12 proteins belonging to the PI3K protein superfamily, some of which are unique to parasites. Moreover, the shared PI3Ks differ greatly in sequence from those of the human host, thereby providing opportunities for selective inhibition.

Muco-cutaneous Leishmaniasis in the New World: the Ultimate Subversion

Virulence. Nov-Dec, 2011  |  Pubmed ID: 21971185

Infection by the human protozoan parasite Leishmania can lead, depending primarily on the parasite species, to either cutaneous or mucocutaneous lesions, or fatal generalized visceral infection. In the New World, Leishmania (Viannia) species can cause mucocutaneous leishmaniasis (MCL). Clinical MCL involves a strong hyper-inflammatory response and parasitic dissemination (metastasis) from a primary lesion to distant sites, leading to destructive metastatic secondary lesions especially in the nasopharyngal areas. Recently, we reported that metastasizing, but not non-metastatic strains of Leishmania (Viannia) guyanensis, have high burden of a non-segmented dsRNA virus, Leishmania RNA Virus (LRV). Viral dsRNA is sensed by the host Toll-like Receptor 3 (TLR3) thereby inducing a pro-inflammatory response and exacerbating the disease. The presence of LRV in Leishmania opens new perspectives not only in basic understanding of the intimate relation between the parasite and LRV, but also in understanding the importance of the inflammatory response in MCL patients.

Folate Metabolic Pathways in Leishmania

Essays in Biochemistry. 2011  |  Pubmed ID: 22023442

Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

[Mucocutaneous Leishmaniasis and an Undesired Passenger]

Médecine Sciences : M/S. Nov, 2011  |  Pubmed ID: 22130014

Remodeling of Protein and MRNA Expression in Leishmania Mexicana Induced by Deletion of Glucose Transporter Genes

Molecular and Biochemical Parasitology. Jan, 2011  |  Pubmed ID: 20869991

Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.

Phenylalanine Hydroxylase (PAH) from the Lower Eukaryote Leishmania Major

Molecular and Biochemical Parasitology. Jan, 2011  |  Pubmed ID: 20887755

Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H(4)B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah(-) null mutant and an episomal complemented overexpressing derivative (pah-/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah(-) parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah(-) showed an increased sensitivity to Tyr deprivation, while the pah(-)/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah(-) showed no alterations in H(4)B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

Killed but Metabolically Active Leishmania As a Novel Whole-cell Vaccine for Visceral Leishmaniasis

Clinical and Vaccine Immunology : CVI. Feb, 2012  |  Pubmed ID: 22323556

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of ultraviolet A radiation. This treatment generates permanent, covalent DNA crosslinks within parasites, and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA-L. major and KBMA-L. infantum chagasi (KBMA-Lic). Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA-Lic parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after six months, KBMA-Lic were undetectable in the organs of mice at this time point. In vitro, KBMA-Lic retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA-Lic correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA-Lic displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.