The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. the model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. the analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, EPM UNIFESP, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilLNBio CNPEM, Lab Nacl Biociencias, Campinas, SP, BrazilLGE UNICAMP, Lab Genom & Expressao, Campinas, SP, BrazilInst Agron Campinas, Ctr Pesquisa & Desenvolvimento Recursos Geneti Ve, Campinas, SP, BrazilUniv Calif San Diego, Sch Med, Dept Pediat, San Diego, CA 92103 USAUniversidade Federal de São Paulo, UNIFESP, Dept Ciencia & Tecnol, Sao Jose Dos Campos, BrazilUniv N Carolina, Sch Med, Dept Genet, Chapel Hill, NC USAUniv Fed Minas Gerais, ICB UFMG, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, EPM UNIFESP, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Ciencia & Tecnol, Sao Jose Dos Campos, BrazilFAPESP: 07/50551-2FAPESP: 10/19335-4Web of Scienc

Ultrastructural studies on parasitic flagellates

Summary available: p.4

Thianthrene is a novel inhibitor of Leishmania donovani pteridine reductase 1 (PTR1)

Pteridine reductase 1 (PTR1) from Leishmania donovani is a short chain reductase that catalyses the NADPH-dependent reduction of folates and pterins. It has gained attention as a therapeutic target because it acts as a metabolic bypass for dihydrofolate reductase (DHFR) targeting drugs and is thought to be responsible for the failure of conventional therapies against the trypanosomatids. In the present study, we report the identification of thianthrene as a potent inhibitor of L. donovani PTR1 (LdPTR1) based on both structure-based virtual screening and experimental verification. Thianthrene displayed uncompetitive mixed type inhibition in a recombinant enzyme inhibition assay. In addition, cell based assays and flow cytometry showed that the intracellular amastigotes were inhibited by thianthrene in vitro. The results of our study could be considered for the development of novel therapeutics based on PTR1 inhibition

The Genome Sequence Of Leishmania (leishmania) Amazonensis: Functional Annotation And Extended Analysis Of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment. © The Author 2013.206567581(2010) Control of the Leishmaniasis WHOTechnical Report Series, , WHO. WHO Press: GenevaLainson, R., Shaw, J.J., (1987) The leishmaniases in biology and medicine. Evolution, classification and geographical distributionBates, P.A., Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies (2007) Int. J. 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Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

Polyomic characterisation of polyene drug resistance in Leishmania spp.

Amphotericin B is the compound of choice for the treatment of leishmaniasis, however a definitive mode of action and full knowledge of causes of resistance to this polyene are still poor. The aim of this project is to use a polyomic approach to characterise laboratory generated mutant lines of Leishmania spp., selected for resistance against the polyene antifungals, amphotericin B and nystatin. While previous work has characterised multiple lines of L. mexicana resistant to amphotericin B, this is the first report of resistant lines selected against nystatin in Leishmania spp. Ergosta-7,22-dien-3-ol and cholesta-5,7,22-trienol, were the two main sterol intermediates replacing ergosterol in all eight polyeneresistant lines of Leishmania spp. The former sterol intermediate was associated with five novel mutations in sterol C5-desaturase, in two and six AmBR- and NysR-mutants and the latter resulted from changes in C24-sterol methyl transferase, along with deletion of the miltefosine transporter and its neighbouring gene downstream in two AmBR lines. Interestingly, switching from ergosterol to these two sterol intermediates was associated with an increased and an attenuated inflammatory response in vivo, respectively. In all cases, viable parasites were recovered post-infection and the retention of resistance in vivo was confirmed. In addition, response to treatment with amphotericin B was observed only in wild type parasites. Untargeted metabolomics provided hints towards modes of action in addition to the binding to ergosterol. Upregulation of the pentose phosphate pathway plays a central role as a key provider of NADPH suggesting an immediate pulse of oxidative stress associated with addition of the drug. Amphotericin B treatment rapidly altered lipid metabolism, decreasing the abundance of Acetyl-CoA, NADPH, leucine and mevalonate. In all mutants, the total or partial loss of the key membrane sterol ergosterol lead to amphotericin B resistance. All polyene resistant mutants were more susceptible to pentamidine and paromomycin. Conversely, miltefosine resistance was found in all mutants, with this increase being more pronounced in two lines showing a deletion of the miltefosine transporter. The grounds of cross-resistance to a new library of sterol inhibitors, 1,2,3-triazolylsterols, was also assessed. The most active hits showed a micromolar potency, albeit a mode of action independent of the inhibition of sterols is suggested. Considering the increase of resistance against the antileishmanials and the limited therapies available, this thesis provides valuable information on the MoA and resistance of polyenes in Leishmania, should the resistance against AmB, the drug of choice for leishmaniasis, increases in clinical settings, and to improve the discovery of potential new drug targets

Molecular analysis of MAP kinase kinase signaling in Leishmania

Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated.Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated

Molecular and functional characterization of the quality control Valosin-containing protein VCP/p97 and of its co-factors in Leishmania

Leishmania est un parasite protozoaire eucaryote unicellulaire qui infecte plus de 1.6 millions de personnes chaque année dans plus de 98 pays. Aucun vaccin humain est actuellement disponible et peu de traitements efficaces sont utillisés pour lutter contre le large spectre de pathologies causées par Leishmania. Récemment, l’étude du contrôle de la qualité des protéines chez Leishmania infantum a révélé que DDX3, une DEAD-box hélicase à ARN dotée de multiples fonctions dans le métabolisme de l’ARN et la signalisation cellulaire, joue un rôle central dans le contrôle de qualité des protéines dans la mitochondrie. Une étude plus approfondie de ce mécanisme a révélé des interactions potentielles de DDX3 avec des composantes clés de la réponse cellulaire au stress, en particulier avec une protéine de la famille des AAA + ATPases, VCP/p97/Cdc48. Comme VCP/p97/Cdc48 participe à de multiples étapes dans le contrôle de qualité des protéines en utilisant son hydrolyse de l’ATP pour séparer les protéines ubiquitinées de leurs partenaires et les acheminer au protéasome 26S pour dégradation, nous avons émis l’hypothèse que l’homologue très conservé chez Leishmania, LiVCP, pourrait agir de la même façon. Cette étude a permis la caractérisation fonctionnelle de l’homologue VCP chez Leishmania, son rôle dans la réponse du parasite au stress et sa survie dans les macrophages, ses interactions potentielles avec d’autres partenaires dont des cofacteurs clés, ainsi que la modélisation 3D des interactions LiVCP-cofacteurs. En utilisant des mutants génétiquement générés ayant moins de copies du gène LiVCP ou des mutants dominants négatifs avec une activité VCP altérée, nous avons démontré que LiVCP est un gène essentiel et que les mutants VCP sont incapables de survivre sous le shock de la chaleur et présentent un déficit de croissance très marqué chez les amastigotes. De plus, nous avons montré une forte accumulation de protéines polyubiquitinées et une sensibilité accrue au stress protéotoxique chez ces mutants, soutenant la fonction de chaperone sélective de l'ubiquitine de LiVCP. Grâce à des analyses in silico et à la «protéomique en réseau» en utilisant des études de co-immunoprécipitation et de spectrométrie de masse (LC-MS / MS), nous avons établi le premier réseau protéique de VCP chez les parasites protozoaires et déterminé que p47, FAF2, UFD1, PUB1 et l’hétérodimère NPL4-UFD1 étaient les principaux cofacteurs de LiVCP. Enfin, nos travaux nous ont permis de faire progresser nos connaissances générales sur la protéine essentielle VCP et le contrôle de la qualité des protéines chez Leishmania et d’indiquer quelques perspectives intéressantes pour approfondir notre compréhension sur ces mécanismes importants non seulement chez Leishmania mais aussi chez d’autres trypanosomatides.Leishmania is a unicellular eukaryotic protozoan parasite that infects over 1.6 million people each year in more than 98 countries. No human vaccine is currently available and few effective treatments are used to combat the broad spectrum of diseases caused by Leishmania. Recently, studies on Protein Quality Control in Leishmania infantum revealed that the multifunctional DEAD-box RNA helicase DDX3 involved among others in RNA metabolism and cell signaling plays a central role in mitochondrial protein quality control. Further studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly with the conserved AAA+ ATPase VCP/ p97/Cdc48. As VCP is associated with many ubiquitin-dependent cellular pathways that are central to protein quality control in other eukaryotic systems using its ATP hydrolysis to separate ubiquitinated proteins from their partners and bring them to the 26S proteasome for degradation, we hypothesized that the Leishmania highly conserved counterpart, LiVCP, might act in similar way. This study enabled the functional characterization of the Leishmania VCP homolog, its role in the parasite's response to stress and survival inside macrophages, its potential interactions with other partners including key VCP cofactors, and the homology 3D modeling of LiVCP-cofactor interactions. Using genetically engineered mutants with fewer copies of the LiVCP gene or dominant negative mutants with altered VCP activity, we demonstrated that LiVCP is an essential gene and that VCP mutants are unable to survive under heat stress and exhibit a very marked growth defect in amastigotes. In addition, we showed a high accumulation of polyubiquitinated proteins and increased susceptibility to proteotoxic stress in these mutants, supporting that LiVCP has an ubiquitin selective chaperone function. Using "network proteomics" analyses by co-immunoprecipitation and mass spectrometry (LC-MS/MS) studies, we established the first VCP protein network in protozoan parasites and determined p47, FAF2, UFD1, PUB1 and the NPL4-UFD1 heterodimer as the major cofactors of LiVCP. Overall, our work allowed us to advance general knowledge of the essential role of VCP in Leishmania protein quality control and to propose some interesting perspectives to deepen our understanding of these important pathways not only in Leishmania but also in other trypanosomatids

The Leish niche: the secretome of Leishmania and its role in parasite virulence

Leishmaniasis is a neglected tropical parasitic disease that causes several debilitating manifestations. No commercially available vaccine exists against this disease, and treatment strategies are far from ideal with the emergence of resistance, coupled with toxic side effects of many of the drugs available. Rational drug design relies on knowledge of the cell biology of the parasite and the interplay between the parasite and its hosts. Production of secreted proteins, the secretome, has become a known strategy for parasite invasion and persistence in host cells, however, host-parasite interaction is still not well defined. Virulence factors secreted by the parasite mediate the host-parasite interaction and create a niche permissive for parasite proliferation. They therefore represent potential therapeutic targets and vaccine candidates. Here, the use of secretomics was implemented to investigate these virulence factors. Parasite conditioned culture supernatant, containing the secretome, was characterised by morphological, immunochemical and proteomic analyses. Here, we optimised and extended current methods and applied them to the medically relevant amastigote stage. Method development and validation was implemented to extract a reproducible secretome in vitro. Induction of cell stress was managed and cell viability maintained to minimise interference of intracellular proteins. A total of 256 proteins were reproducibly identified in the secretome of promastigotes and 36 proteins were reproducibly identified in the secretome of amastigotes. Analysis of their protein abundance index (emPAI) allowed comparison of the relative abundance of proteins and functions of the secretome throughout the parasite life cycle. Differences in the putative functions of nutrient salvage, protease production and antioxidant activity were observed. Analyses reveal that many proteins lack a signal peptide and as such are thought to be released by nonclassical secretion mechanisms. Several exosome-associated proteins and membrane proteins were also detected in the secretome, suggesting the occurrence of secretion by exosomes or microvesicles. Extended comparative analyses between the secretome of parasites with differing phenotypes allowed us to infer functionality of the secretome in the parasite’s survival but also variations within the same species which result in differing disease outcomes. Dysregulation in the secretion of various proteins in attenuated parasites implicates these proteins in the virulence of the parasite. An increase in the secretion of pro-inflammatory mediators and destructive proteases by parasites isolated from patients with chronic cutaneous leishmaniasis compared to those from patients with self-healing lesions, indicates the role of the parasite in the chronicity of cutaneous leishmaniasis. Here, we demonstrate an applicable method for the study of the Leishmania mexicana promastigote and amastigote secretome. Results suggest that the secretome plays a role in disease progression and virulence. Proteomic analyses of the secretome, like this study presented here, provide crucial information on the host:parasite interaction for the identification of therapeutic targets and potential vaccine candidates for the provision of safer treatments and new vaccines for eradication of this disease

High Throughput Screening to Identify, Develop and Analyse Inositol Phosphorylceramide Synthase Inhibitors as Novel Antileishmanials

Leishmaniasis and Human African trypanosomiasis are tropical diseases caused by kinetoplastid parasites that together affect over 12 million people, with an estimated 400 million at risk worldwide. Both are potentially fatal, yet the current treatments available are expensive and many have toxic side effects. Emerging resistance to many current drugs is also a concern; novel therapeutic agents are therefore urgently required. One novel target for drug discovery previously identified in the group is sphingolipid synthesis. Sphingolipids are ubiquitous biomolecules found in nature and are both structural membrane components and signalling molecules. Inositol phosphorylceramide synthase (IPCS) is an essential enzyme involved in kinetoplastid sphingolipid synthesis that has no mammalian equivalent, making it an attractive drug target. Whilst specific inhibitors of the fungal IPCS are known, they are unsuitable as pharmaceuticals. The overall aim of this project was to identify novel inhibitors of this enzyme that could be further investigated as potential antikinetoplastid drugs. The first stage involved the construction of Saccharomyces cerevisiae strains as expression systems of the kinetoplastid IPCS enzymes. The strain complemented with the Leishmania major enzyme was subsequently used in the development and optimisation of a robust high throughput screening (HTS)-compatible assay. This was used to screen the 1.8 million compound library stored at the GlaxoSmithKline research site in Tres Cantos in what is believed to be the largest screening project undertaken by an academic group to date. 500 compounds were identified as selective inhibitors of the L. major IPCS enzyme, and 216 of these were selected for additional investigation. Further compound triage was achieved by means of a screening process involving multiple in cellulo assays against both Leishmania parasites and mammalian cells. Six compounds demonstrating both high potency and selectivity were identified. Following additional biochemical testing, the two most potent compounds were found to share a common benzazepane chemical structure. Investigation of analogues of these compounds permitted the identification of preliminary structure-activity relationship data, which identified several possible avenues for further investigation