Crystal structure of the Leishmania major MIX protein: a scaffold protein that mediates protein-protein interactions.

Infection by Leishmania and Trypanosoma causes severe disease and can be fatal. The reduced effectiveness of current treatments is largely due to drug resistance, hence the urgent need to develop new drugs, preferably against novel targets. We have recently identified a mitochondrial membrane-anchored protein, designated MIX, which occurs exclusively in these parasites and is essential for virulence. We have determined the crystal structure of Leishmania major MIX to a resolution of 2.4 Å. MIX forms an all α-helical fold comprising seven α-helices that fold into a single domain. The distribution of helices is similar to a number of scaffold proteins, namely HEAT repeats, 14-3-3, and tetratricopeptide repeat proteins, suggesting that MIX mediates protein-protein interactions. Accordingly, using copurification and mass spectroscopy we were able to identify several proteins that may interact with MIX in vivo. Being parasite specific, MIX is a promising new drug target and, thus, the structure and potential interacting partners provide a basis for structure-guided drug discovery.

The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation.

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.

Fine mapping of Leishmania major susceptibility Locus lmr2 and evidence of a role for Fli1 in disease and wound healing.

Genetic linkage studies of the host response to Leishmania major, the causative agent of cutaneous leishmaniasis, have identified significant genetic complexity in humans and mice. In the mouse model, multiple loci have been implicated in susceptibility to infection, but to date, the genes underlying these loci have not been identified. We now describe the contribution of a novel candidate gene, Fli1, to both L. major resistance and enhanced wound healing. We have previously mapped the L. major response locus, lmr2, to proximal chromosome 9 in a genetic cross between the resistant C57BL/6 strain and the susceptible BALB/c strain. We now show that the presence of the resistant C57BL/6 lmr2 allele in susceptible BALB/c mice confers an enhanced L. major resistance and wound healing phenotype. Fine mapping of the lmr2 locus permitted the localization of the lmr2 quantitative trait locus to a 5-Mb interval comprising 21 genes, of which microarray analysis was able to identify differential expression in 1 gene-Fli1. Analysis of Fli1 expression in wounded and L. major-infected skin and naïve and infected lymph nodes validated the importance of Fli1 in lesion resolution and wound healing and identified 3 polymorphisms in the Fli1 promoter, among which a GA repeat element may be the important contributor.

Inhibitors of Leishmania GDP-mannose pyrophosphorylase identified by high-throughput screening of small-molecule chemical library.

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.

The role of host genetics in leishmaniasis.

Leishmaniasis is one of the world's important infectious diseases. It is prevalent in tropical and subtropical regions of the world and endemic in 88 countries, with two million new cases of leishmaniasis reported annually. As a complex disease, the pathology of leishmaniasis varies and is determined by factors such as the environment, the insect vector, and parasite and host genetics. The contributing host genetics involve multiple genes; thus, the mouse model of leishmaniasis has been exploited extensively in an attempt to identify and dissect the contribution of disease modifier genes to pathogenesis. This review summarizes recent advances in the identification of genetic loci involved in the host response to Leishmania spp. in the mouse model and in the human situation.

Leishmania major CorA-like magnesium transporters play a critical role in parasite development and virulence.

Establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium. We show that two Leishmania major proteins designated MGT1 and MGT2, which play a critical role in these processes, belong to the two-transmembrane domain (2-TM-GxN) cation transporter family and share homology with the major bacterial magnesium transporter CorA. Although both are present in the endoplasmic reticulum throughout the life cycle of the parasite, MGT1 is more highly expressed in the infectious metacyclic parasites, while MGT2 is enriched in the immature procyclic stages. The two proteins, although predicted to be structurally similar, have features that suggest different regulatory or gating mechanisms. The two proteins may also be functionally distinct, since only MGT1 complements an Escherichia coli DeltaCorA mutant. In addition, deletion of one mgt1 allele from L. major led to increased virulence, while deletion of one allele of mgt2 resulted in slower growth and total loss of virulence in vitro and in vivo. This loss of virulence may be due to an impaired transformation of the parasites into amastigotes. Deletion of both mgt1 alleles in the hemizygous MGT2 knockdown parasites reversed the growth defect and partially restored virulence. Our data indicate that the MGTs play a critical role in parasite growth, development and virulence.

Decreased IL-10 and IL-13 production and increased CD44hi T cell recruitment contribute to Leishmania major immunity induced by non-persistent parasites.

Leishmaniasis is currently classified as category 1 disease, i.e. emerging and uncontrolled. Since the importance of persistent infection for maintaining an effective long-lasting protective response is controversial, the present study asks whether immunisation with non-persistent parasites leads to protection against Leishmania infection and to the recruitment of T cells of a specific phenotype. Our study shows that vaccination of susceptible BALB/c mice with live Leishmania major phosphomannomutase-deficient parasites, which are avirulent and non-persistent in vivo, leads to protection against infection. Immunisation with phosphomannomutase-deficient parasites neither leads to differences in IFN-gamma, IL-12, IL-4 production nor alters the expression of effector and memory markers, including CD62L, IL-7Ralpha and IL-2Ralpha, when compared with unvaccinated controls. Observed protection is due to the ability of vaccinated animals to suppress early IL-10 and IL-13 production and to recruit a higher number of antigen-experienced CD44hiCD4+ and CD44hiCD8+ T cells into draining LN following infection. Thus, expansion of T-cell numbers and their rapid recruitment to LN upon infection as well as the restriction of IL-13 and IL-10 production leading to high IFN-gamma/IL-10 ratio play an important role in protection against Leishmania affecting the outcome of the disease in favour of the host.

Structural and functional association of Trypanosoma brucei MIX protein with cytochrome c oxidase complex.

A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei.

Fishing for anti-leishmania drugs: principles and problems.

To date, there are no vaccines against any of the major parasitic diseases including leishmaniasis, and chemotherapy is the main weapon in our arsenal. Current drugs are toxic and expensive, and are losing their effectiveness due to parasite resistance. The availability of the genome sequence of two species of Leishmania, Leishmania major and Leishmania infantum, as well as that of Trypanosoma brucei and Trypanosoma cruzi should provide a cornucopia of potential new drug targets. Their exploitation will require a multi-disciplinary approach that includes protein structure and function and high throughput screening of random and directed chemical libraries, followed by in vivo testing in animals and humans. We outline the opportunities that are made possible by recent technologies, and potential problems that need to be overcome.

WITHDRAWN: The molecular basis of Leishmania pathogenesis.

The Publisher regrets that this article was an accidental duplication of an article that has already been published in PARA 37/10 (2007) 1047-1051, doi:10.1016/S0020-7519(07)00216-0. The duplicate article has therefore been withdrawn.

A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania.

The single mitochondrion of kinetoplastids divides in synchrony with the nucleus and plays a crucial role in cell division. However, despite its importance and potential as a drug target, the mechanism of mitochondrial division and segregation and the molecules involved are only partly understood. In our quest to identify novel mitochondrial proteins in Leishmania, we constructed a hidden Markov model from the targeting motifs of known mitochondrial proteins as a tool to search the Leishmania major genome. We show here that one of the 17 proteins of unknown function that we identified, designated mitochondrial protein X (MIX), is an oligomeric protein probably located in the inner membrane and expressed throughout the Leishmania life cycle. The MIX gene appears to be essential. Moreover, even deletion of one allele from L. major led to abnormalities in cell morphology, mitochondrial segregation and, importantly, to loss of virulence. MIX is unique to kinetoplastids but its heterologous expression in Saccharomyces cerevisiae produced defects in mitochondrial morphology. Our data show that a number of mitochondrial proteins are unique to kinetoplastids and some, like MIX, play a central role in mitochondrial segregation and cell division, as well as virulence.

Structure of Leishmania mexicana phosphomannomutase highlights similarities with human isoforms.

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

Membrane transporters in the relict plastid of malaria parasites.

Malaria parasites contain a nonphotosynthetic plastid homologous to chloroplasts of plants. The parasite plastid synthesizes fatty acids, heme, iron sulfur clusters and isoprenoid precursors and is indispensable, making it an attractive target for antiparasite drugs. How parasite plastid biosynthetic pathways are fuelled in the absence of photosynthetic capture of energy and carbon was not clear. Here, we describe a pair of parasite transporter proteins, PfiTPT and PfoTPT, that are homologues of plant chloroplast innermost membrane transporters responsible for moving phosphorylated C3, C5, and C6 compounds across the plant chloroplast envelope. PfiTPT is shown to be localized in the innermost membrane of the parasite plastid courtesy of a cleavable N-terminal targeting sequence. PfoTPT lacks such a targeting sequence, but is shown to localize in the outermost parasite plastid membrane with its termini projecting into the cytosol. We have identified these membrane proteins in the parasite plastid and determined membrane orientation for PfoTPT. PfiTPT and PfoTPT are proposed to act in tandem to transport phosphorylated C3 compounds from the parasite cytosol into the plastid. Thus, the transporters could shunt glycolytic derivatives of glucose scavenged from the host into the plastid providing carbon, reducing equivalents and ATP to power the organelle.

Functional comparison of mouse CIRE/mouse DC-SIGN and human DC-SIGN.

CIRE/mDC-SIGN is a C-type lectin we originally identified as a molecule differentially expressed by mouse dendritic cell (DC) populations. Immunostaining with a CIRE/mDC-SIGN-specific mAb revealed that CIRE/mDC-SIGN is indeed on the surface of some CD4+, CD4- 8- DCs and plasmacytoid pre-DCs, but not on CD8+ DCs. It has been proposed that CIRE/mDC-SIGN is the functional orthologue of human DC-SIGN (hDC-SIGN), a molecule that both enhances T cell responses and facilitates antigen uptake. We assessed if CIRE/mDC-SIGN and hDC-SIGN exhibit functional similarities. CIRE/mDC-SIGN is down-regulated upon activation, but unlike hDC-SIGN, incubation with IL-4 and IL-13 did not enhance CIRE/mDC-SIGN expression, indicating differences in gene regulation. Like hDC-SIGN, CIRE/mDC-SIGN bound mannosylated residues. However, we could detect no role for CIRE/mDC-SIGN in T cell-DC interactions and the protein did not bind to pathogens known to interact with hDC-SIGN, including Leishmania mexicana, cytomegalovirus, HIV and lentiviral particles bearing the Ebolavirus glycoprotein. The binding of CIRE/mDC-SIGN to hDC-SIGN ligands was not rescued when CIRE/mDC-SIGN was engineered to express the stalk region of hDC-SIGN. We conclude that there are significant differences in the fine specificity of the C-type lectin domains of hDC-SIGN and CIRE/mDC-SIGN and that these two molecules may not be functional orthologues.

Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase.

Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macrophages. To define the biochemical composition of the phagosome and carbon source requirements of intracellular stages of L. major, we investigated the role and requirement for the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP). L. major FBP was constitutively expressed in both extracellular and intracellular stages and was primarily targeted to glycosomes, modified peroxisomes that also contain glycolytic enzymes. A L. major FBP-null mutant was unable to grow in the absence of hexose, and suspension in glycerol-containing medium resulted in rapid depletion of internal carbohydrate reserves. L. major Deltafbp promastigotes were internalized by macrophages and differentiated into amastigotes but were unable to replicate in the macrophage phagolysosome. Similarly, the mutant persisted in mice but failed to generate normal lesions. The data suggest that Leishmania amastigotes reside in a glucose-poor phagosome and depend heavily on nonglucose carbon sources. Feeding experiments with [(13)C]fatty acids showed that fatty acids are poor gluconeogenic substrates, indicating that amino acids are the major carbon source in vivo. The need for amino acids may have forced Leishmania spp. to adapt to life in the mature phagolysosome.

The wound repair response controls outcome to cutaneous leishmaniasis.

Chronic microbial infections are associated with fibrotic and inflammatory reactions known as granulomas showing similarities to wound-healing and tissue repair processes. We have previously mapped three leishmaniasis susceptibility loci, designated lmr1, -2, and -3, which exert their effect independently of T cell immune responses. Here, we show that the wound repair response is critically important for the rapid cure in murine cutaneous leishmaniasis caused by Leishmania major. Mice congenic for leishmaniasis resistance loci, which cured their lesions more rapidly than their susceptible parents, also expressed differentially genes involved in tissue repair, laid down more ordered collagen fibers, and healed punch biopsy wounds more rapidly. Fibroblast monolayers from these mice repaired in vitro wounds faster, and this process was accelerated by supernatants from infected macrophages. Because these effects are independent of T cell-mediated immunity, we conclude that the rate of wound healing is likely to be an important component of innate immunity involved in resistance to cutaneous leishmaniasis.

Genes and susceptibility to leishmaniasis.

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly where they grow as motile, flagellated promastigotes in the gut, and the mammalian macrophage where they grow intracellularly as non-flagellated amastigotes. Leishmaniasis is the outcome of an evolutionary 'arms race' between the host's immune system and the parasite's evasion mechanisms which ensure survival and transmission in the population. The spectrum of disease manifestations and severity reflects the interaction between the genome of the host and that of the parasite, and the pathology is caused by a combination of host and parasite molecules. This chapter examines the genetic basis of host susceptibility to disease in humans and animal models. It describes the genetic tools used to map and identify susceptibility genes, and the lessons learned from murine and human cutaneous leishmaniasis.

Insight into the self-association of key enzymes from pathogenic species.

Self-association of protein monomers to higher-order oligomers plays an important role in a plethora of biological phenomena. The classical biophysical technique of analytical ultracentrifugation is a key method used to measure protein oligomerisation. Recent advances in sedimentation data analysis have enabled the effects of diffusion to be deconvoluted from sample heterogeneity, permitting the direct identification of oligomeric species in self-associating systems. Two such systems are described and reviewed in this study. First, we examine the enzyme dihydrodipicolinate synthase (DHDPS), which crystallises as a tetramer. Wild-type DHDPS plays a critical role in lysine biosynthesis in microbes and is therefore an important antibiotic target. To confirm the state of association of DHDPS in solution, we employed sedimentation velocity and sedimentation equilibrium studies in an analytical ultracentrifuge to show that DHDPS exists in a slow dimer-tetramer equilibrium with a dissociation constant of 76 nM. Second, we review works describing the hexamerisation of GDP-mannose pyrophosphorylase (GDP-MP), an enzyme that plays a critical role in mannose metabolism in Leishmania species. Although the structure of the GDP-MP hexamer has not yet been determined, we describe a three-dimensional model of the hexamer based largely on homology with the uridyltransferase enzyme, Glmu. GDP-MP is a novel drug target for the treatment of leishmaniasis, a devastating parasitic disease that infects more than 12 million people worldwide. Given that both GDP-MP and DHDPS are only active in their oligomeric states, we propose that inhibition of the self-association of critical enzymes in disease is an emerging paradigm for therapeutic intervention.