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1.  The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite 
Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana complex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.
PMCID: PMC4182287  PMID: 25336895
Leishmania amazonensis; comparative genomics; phylogenomics
2.  Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ 
The FASEB Journal  2014;28(7):3050-3063.
In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ.
PMCID: PMC4210457  PMID: 24732131
ecotin; Toll; interferon; ISP2
3.  The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants 
PLoS Genetics  2014;10(2):e1004007.
Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.
Author Summary
Some plant trypanosomes, single-celled organisms living in phloem sap, are responsible for important palm diseases, inducing frequent expensive and toxic insecticide treatments against their insect vectors. Other trypanosomes multiply in latex tubes without detriment to their host. Despite the wide range of behaviors and impacts, these trypanosomes have been rather unceremoniously lumped into a single genus: Phytomonas. A battery of molecular probes has been used for their characterization but no clear phylogeny or classification has been established. We have sequenced the genomes of a pathogenic phloem-specific Phytomonas from a diseased South American coconut palm and a latex-specific isolate collected from an apparently healthy wild euphorb in the south of France. Upon comparison with each other and with human pathogenic trypanosomes, both Phytomonas revealed distinctive compact genomes, consisting essentially of single-copy genes, with the vast majority of genes shared by both isolates irrespective of their effect on the host. A strong cohort of enzymes in the sugar metabolism pathways was consistent with the nutritional environments found in plants. The genetic nuances may reveal the basis for the behavioral differences between these two unique plant parasites, and indicate the direction of our future studies in search of effective treatment of the crop disease parasites.
PMCID: PMC3916237  PMID: 24516393
4.  Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen 
PLoS Pathogens  2014;10(1):e1003886.
The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.
Author Summary
The African trypanosome, which is transmitted by the tsetse fly, causes the usually fatal disease Sleeping Sickness in humans and a wasting disease, called Nagana, in livestock in sub-Saharan Africa. There are no vaccines available against the diseases, and various problems are associated with current drug treatments (including toxicity to the patient and parasite drug resistance). Thus, it is important to identify essential parasite proteins that could be targeted by novel drugs. Protein kinases (PKs) are important cell signalling molecules, and are generally considered to have potential as drug targets. Here we report the construction of a library of trypanosome cell lines that allows us to specifically deplete each of the trypanosome's 190 PKs individually and analyse their function. Using this library, we show that ≥42 PKs are essential for proliferation of the mammalian-infective bloodstream form of the parasite (and thus have potential as drug targets), and demonstrate that 24 of these play important roles in coordinating cell division. We also shed light on how the parasite develops during its life cycle as it passes from the mammalian bloodstream form to the tsetse fly gut by identifying the first two PKs that regulate this life cycle developmental step.
PMCID: PMC3894213  PMID: 24453978
5.  Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing “Red-Shifted” Luciferase 
Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.
Methodology/Principal findings
Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5′-variant surface glycoprotein untranslated region (UTR) and a downstream 3′-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.
The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.
Author Summary
Parasites of the Trypanosoma brucei species complex are the causative agents of human African trypanosomiasis. There is an urgent need for new drugs to treat this debilitating and potentially fatal infection, especially in its late stage, when parasites have entered the central nervous system. Factors which hamper drug development include the limitations of the current murine models for stage II disease. In vivo bioluminescence imaging is a non-invasive technique that can be used to monitor infections in real time and is a powerful new approach for studying drug effectiveness. However, application of this imaging technology to trypanosome infections has been restricted because of lack of sensitivity. In this paper, we have taken a major step to resolve this problem. The enhanced sensitivity in infected mice is based on the high level expression in trypanosomes of a “red-shifted” luciferase variant that greatly improves bioluminescence detection in deep tissue. The system which we have developed should be a widely applicable tool for providing new insights into the infection biology of T. brucei.
PMCID: PMC3836995  PMID: 24278497
6.  Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB 
PLoS ONE  2013;8(10):e77460.
Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.
PMCID: PMC3797739  PMID: 24146999
7.  Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2 
The FEBS journal  2013;280(11):2608-2621.
Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca2+ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has is an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most likely a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P2 and P3 positions and for small residues at the prime-side of a peptide substrate. Studies into the effects of Ca2+ on the enzyme revealed the presence of two calcium-binding sites and a reversible structural modification of the enzyme upon Ca2+-binding. In addition, the concentration of Ca2+ used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P2 were examined, suggesting that Ca2+ activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity, and Ca2+ activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes, and make attractive drug targets due to their absence in humans.
PMCID: PMC3779824  PMID: 23506317
Metacaspase; substrate specificity; structural modification; kinetic parameters; calcium binding
8.  In Vivo Imaging of Trypanosome-Brain Interactions and Development of a Rapid Screening Test for Drugs against CNS Stage Trypanosomiasis 
Human African trypanosomiasis (HAT) manifests in two stages of disease: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 post-infection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain.
Author Summary
Trypanosoma brucei, a parasite transmitted by the bite of tsetse fly, is responsible for the disease human African trypanosomiasis (HAT). In advanced stages of HAT, trypanosomes invade the central nervous system (CNS), resulting in an array of neurological symptoms, and eventually death. Existing drugs for treatment of HAT are highly unsatisfactory and new safe drugs are urgently needed. Currently, potential drugs for HAT are screened in a mouse model that relies on the emergence of trypanosomes from tissues and their detection in blood. This can take up to 200 days, making selection and further development of new drugs slow and costly. Here, we employ in vivo imaging and genetically modified trypanosomes to monitor parasite distribution throughout the body in live infected mice. Our bioluminescence imaging approach provides sensitive detection of trypanosomes at sites of infection, allowing more rapid and more effective in vivo screening of candidate HAT drugs. Higher resolution intra-vital microscopy was used to investigate trypanosome dynamics in the brain and their accessibility to drugs during infection. These approaches allow more sensitive real time tracking of trypanosomes during chronic infections and will provide new insights about trypanosome pathogenesis in future experiments.
PMCID: PMC3749981  PMID: 23991236
9.  Trypanosoma brucei brucei: Endocytic recycling is important for mouse infectivity 
Experimental Parasitology  2011;127(4-4):777-783.
Graphical abstract
► Trypanosomes evade the immune response by antigenic variation. ► Trypanosomes also remove immune effectors from the surface by endocytosis. ► Partially defective endocytic uptake does not compromise mouse infectivity. ► Recycling pathway defects do compromise mouse infectivity.
Endocytosis in the African trypanosome, Trypanosoma brucei, is intimately involved in maintaining homeostasis of the cell surface proteome, morphology of the flagellar pocket and has recently been demonstrated as a bona fide drug target. RNAi-mediated knockdown of many factors required for endocytic transport, including several small GTPases, the major coat protein clathrin and a clathrin-associated receptor, epsinR, results in rapid cell death in vitro. Rapid loss of viability in vitro precludes meaningful investigation by RNAi of the roles of trypanosome endocytosis in vivo. Here we have sought to address this issue using strategies designed to produce milder effects on the endocytic system than complete functional ablation. We created a trypanosome clathrin heavy chain hemizygote and several lines expressing mutant forms of Rab5 and Rab11, described previously. All are viable in in vitro culture, with negligible impact to proliferative rates or cell cycle. Clathrin hemizygotes express clathrin heavy chain at ∼50% of wild type levels, but despite this demonstrate no defect to growth in mice, while none of the Rab5 mutants affected proliferation in vivo, despite clear evidence for effects on endocytosis. By contrast we find that expressing a dominantly active Rab11 mutant led to compromised growth in mice. These data indicate that trypanosomes likely tolerate the effects of partly decreased clathrin expression and alterations in early endocytosis, but are more sensitive to alterations in the recycling arm of the pathway.
PMCID: PMC3080601  PMID: 21256128
Trypanosome; Protein transport; Vesicle trafficking; Endocytosis; Recycling
10.  Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes 
Cardiovascular Research  2013;100(2):325-335.
African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response.
Methods and results
Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events.
These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function.
PMCID: PMC3797627  PMID: 23892734
Sarcoplasmic reticulum; Cardiomyocyte; Calcium; Trypanosomiasis; Trypanosome
11.  Imaging of the host/parasite interplay in cutaneous leishmaniasis 
Experimental Parasitology  2010;126(3):310-317.
An understanding of host–parasite interplay is essential for the development of therapeutics and vaccines. Immunoparasitologists have learned a great deal from ‘conventional’ in vitro and in vivo approaches, but recent developments in imaging technologies have provided us (immunologists and parasitologists) with the ability to ask new and exciting questions about the dynamic nature of the parasite–immune system interface. These studies are providing us with new insights into the mechanisms involved in the initiation of a Leishmania infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations can be further developed to understand the immunology of cutaneous Leishmania infection in vivo.
PMCID: PMC3427850  PMID: 20501336
Leishmania; Immunology; Microscopy
12.  Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei 
PLoS ONE  2013;8(6):e67327.
The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.
PMCID: PMC3689728  PMID: 23805309
13.  Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase 
PLoS ONE  2012;7(12):e51300.
The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.
PMCID: PMC3519623  PMID: 23251490
14.  The role of conserved residues of chagasin in the inhibition of cysteine peptidases 
Febs Letters  2008;582(4-3):485-490.
We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10–100-fold increases in the Ki for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.
PMCID: PMC2607524  PMID: 18201565
Z-Phe-Arg-MCA, carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin; PBS, phosphate buffered saline; cruzain, recombinant cruzipain truncated at the C-terminal extension; DTT, dithiothreitol; EDTA, ethylenidiaminetetracetic acid disodium salt 2-hydrate; E-64, l-trans-epoxysuccinylleucylamido-(4-guanidino) butane; IPTG, isopropyl-β-d-thiogalactopyranoside; Chagasin; Cysteine peptidase; Inhibitor; Mutant; Trypanosoma
15.  Distinct Roles in Autophagy and Importance in Infectivity of the Two ATG4 Cysteine Peptidases of Leishmania major* 
The Journal of Biological Chemistry  2012;288(5):3678-3690.
Background: ATG4 is a cysteine peptidase crucial for macroautophagy.
Results: Gene deletion mutants show that the two ATG4s of Leishmania perform distinct roles, although there is some redundancy.
Conclusion: ATG4s are not individually essential but macroautophagy, a process important in the virulence of the parasite, requires one.
Significance: Highlights the distinct roles of ATG4 isoforms and their importance for autophagy and parasite infectivity.
Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.
PMCID: PMC3561585  PMID: 23166325
Autophagy; Cysteine Protease; Leishmania; Parasite; Parasite Metabolism; Peptidases; Protease; ATG4
16.  Identification of Lead Compounds Targeting the Cathepsin B-Like Enzyme of Eimeria tenella 
Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.
PMCID: PMC3294901  PMID: 22143531
17.  High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a 
Microbiology  2012;158(Pt 9):2262-2271.
The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.
PMCID: PMC3542813  PMID: 22723289
18.  ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major 
PLoS Pathogens  2012;8(5):e1002695.
Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.
Author Summary
Leishmaniasis is a disease of humans that is of major significance throughout many parts of the world. It is caused by the protozoan parasite Leishmania and mammals are infected through the bite of a sand fly in which the parasite develops. Parasite remodelling crucial for generation of the human-infective forms is aided by the catabolic process known as autophagy in which cell material is packaged within organelles called autophagosomes and subsequently broken down in the digestive lysosomal compartment. Here we show that autophagy in Leishmania requires the coordinated actions of two pathways, one of which involves a protein called ATG5. We have generated parasite mutants lacking this protein and shown that ATG5 is required for both autophagosome formation and also maintenance of a fully functional mitochondrion. The mutants lacking ATG5 have increased mitochondrial mass and phospholipid content, high levels of oxidants and reduced membrane potential, all being hallmarks of a dysfunctional mitochondrion with impaired ability for energy generation. Our results have thus revealed that a functional autophagic pathway is crucial for phospholipid homeostasis and mitochondrial function in the parasite and important for the parasite's differentiation, infectivity and virulence to its mammalian host.
PMCID: PMC3355087  PMID: 22615560
19.  Correction: Cytokinesis in Bloodstream Stage Trypanosoma brucei Requires a Family of Katanins and Spastin 
PLoS ONE  2012;7(3):10.1371/annotation/aa6cd97c-cfb5-4afe-aa25-f5ecfed07980.
PMCID: PMC3297657
20.  Autophagy in protists 
Autophagy  2011;7(2):127-158.
Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.
PMCID: PMC3039767  PMID: 20962583
autophagy; ubiquitination; pexophagy; evolution; free-living protist; parasitic protist; life-cycle differentiation, Trypanosomatidae; Apicomplexa; drug discovery
21.  Cytokinesis in Bloodstream Stage Trypanosoma brucei Requires a Family of Katanins and Spastin 
PLoS ONE  2012;7(1):e30367.
Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.
PMCID: PMC3261199  PMID: 22279588
22.  Inhibition of Eimeria tenella CDK-related Kinase 2: From Target Identification to Lead Compounds 
ChemMedChem  2010;5(8):1259-1271.
Apicomplexan parasites encompass several human-pathogenic as well as animal-pathogenic protozoans like Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella is the causative agent of coccidiosis a disease of chickens, which causes tremendous economic losses to the world poultry industry. Considerable increase of drug resistance makes it necessary to develop and pursue new therapeutic strategies. Cyclin-dependent kinases (CDKs) are key molecules in the regulation of the cell cycle and are therefore prominent target proteins in parasitic diseases. Bioinformatic analysis revealed four potential CDK-like proteins of which one – E. tenella CDK-related kinase 2 (EtCRK2) – is already cloned, expressed and characterized.[1] Using the CDK specific inhibitor Flavopiridol in EtCRK2 enzyme assays and schizont maturation assays we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as template to built protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP-binding site between EtCRK2 and HsCDK2 as well as chicken CDK3 have been addressed for the optimization of selective ATP-competitive inhibitors. Virtual screening and “wet-bench” high throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized leading to a set of four promising lead compounds inhibiting EtCRK2.
PMCID: PMC3252702  PMID: 20575139
Coccidiosis; Virtual Screening; Transferases; Drug Discovery; Bioinformatics
23.  α-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB 
ChemMedChem  2010;5(10):1734-1748.
Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.
PMCID: PMC3245848  PMID: 20799311
cysteine proteases; inhibitors; ketoheterocycle; parasite CPB; Trypanosoma
Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes - highlighting that MIF is not a virulence factor in all species of Leishmania.
PMCID: PMC3242041  PMID: 19187777
25.  Morphological Events during the Cell Cycle of Leishmania major ▿ ‖ 
Eukaryotic Cell  2011;10(11):1429-1438.
The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of β-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in Leishmania.
PMCID: PMC3209043  PMID: 21926331

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