Analysis and modelling of Trypanosomatidae family in vitro and ex vivo cultures. Chagas disease and leishmaniasis

Chagas disease and leishmaniasis cause thousands of deaths every year in the South countries. Those diseases are caused by the protozoa Trypanosoma cruzi and Leishmania spp., respectively. Both protozoa belong to the family Trypanosomatidae and have a digenetic life cycle. That is to say that they live between a vertebrate host and an invertebrate vector through which they are transmitted (sand flies (Phlebotominae) in Leishmania spp. and bloodsucking (Triatomine) in Trypanosoma cruzi). The main objective of this bachelor thesis is allowing the better understanding of Trypanosoma cruzi behaviour in in vitro and ex vivo cultures. As we could demonstrate that the behaviour of in vitro cultures of Leishmania spp. is pretty similar as Trypanosoma cruzi the understanding done with second will be useful for the first. In order to understand them, we have developed two mathematic models: a continuous model of the in vitro growth and an Individual-based Model of the ex vivo growth. The experimental work of in vivo cultures of the two protozoa has been done in the parasitology lab of the Department of Health Microbiology and Parasitology of the Universitat de Barcelona. An experimental method has been designed in order to know the growing curve of the epimastigotes during 20-30 days (no infective, reproductive, extracellular form, from Trypanosoma cruzi and Leishmania spp.) obtaining results that show a bi-lineal growth in the growing phase of the curve. The ex vivo experimental work with Trypanosoma cruzi has been done in the School of Pharmacy of the Ouro Preto Federal University, Brazil. Information from an experimental method that measures the number of infected eukaryotic cells out of the number of counted cells of the culture and the number of amastigotes (no infective, reproductive, intracellular form) in the infected cells for 72 hours. In last this case, the number of infected cells at 72 hours is maxim and the amastigotes number is maxim at 48 hours. From the in vitro results we have developed a continuous model where we can mathematically demonstrate the bi-lineal growth of the cultures and that there is a growing limitation due to the diffusion, or the lack of diffusion, of oxygen in the medium. Likewise, we think about a molecule secreted to the medium that inhibits the growing of the protozoa population in order to control it and facilitate the co-existence between the guest and the parasite. From the ex vivo results we have developed an Individual-based Model that allows us to hypothesize two facts. Firstly, when the tripomastigotes (infective, no reproductive, extracellular form) of Trypanosoma cruzi infect the eukaryotic cells, there may be a change in the membrane composition that does not allow the other trypomastigotes penetrate. Secondly, the complete growing cycle of the amastigotes may be larger than 72h, if not we ought to observe oscillations in their 72 hours behaviour. In order to keep progressing in the understanding of those diseases, is necessary to keep working with both models, as well as start working with in vivo models, since they are further similar at the protozoa's behaviour in humans.La malaltia del Chagas i la leishmaniasis causen milers de morts anualment en països del Sud. Aquestes malalties estan causades pels protozous Trypanosoma cruzi i Leishmania spp., respectivament. Ambdós protozous pertanyen a la família Trypanosomatidae i el seu un cicle de vida digenètic, és a dir, viuen entre un hoste vertebrat i un vector invertebrat mitjançant el qual es transmeten (flebòtoms (Phlebotominae) en el cas de Leishmania spp. i hemípters predadors (Triatomine) en el cas de Trypanosoma cruzi). L'objectiu principal d'aquest treball de final de grau és avançar en la comprensió del comportament de cultius in vitro i ex vivo del protozou Trypanosoma cruzi. Com que hem pogut demostrar que el seu comportament en cultius in vitro és molt semblant al de Leishmania spp., aquesta comprensió realitzada amb T. cruzi ens serà d'ús per a Leishmania spp. Per fer-ho, s'han desenvolupat dos models matemàtics: un model continu dels creixements in vitro i un model basat en l'individu dels creixements ex vivo. Els experiments in vitro amb els dos protozous s'han realitzat en el Departament de Microbiologia i Parasitologia Sanitàries de la Universitat de Barcelona. S'ha dissenyat un mètode experimental que mesura el perfil de creixement dels epimastigots durant 20-30 dies (forma no infectiva, reproductiva i extracel·lular tant de Trypanosoma cruzi com de Leishmania spp.) obtenint resultats que ens indiquen un clar comportament bilineal en la fase de creixement. Els experiments de creixement ex vivo amb Trypanosoma cruzi s'han realitzat a l'Escola de Farmàcia de la Universitat Federal de Ouro Preto (Brasil). S'ha utilitzat la informació d'un mètode experimental que mesura el nombre de cèl·lules eucariotes infectades en funció del nombre de cèl·lules contades del cultiu i el nombre d'amastigots (forma no infectiva, reproductiva, intracel·lular) presents en cada cèl·lula infectada durant 72 hores. En aquests cas s'ha observat que el nombre de cèl·lules infectades és màxim a les 72 hores i el nombre d'amastigots és màxim a les 48 hores. A partir dels resultats in vitro hem construït un model continu on hem pogut demostrar matemàticament el creixement bilineal dels cultius i on la limitació de creixement es deu a la difusió, o manca d'ella, d'oxigen en el medi. També, podem suposar que hi ha algun metabòlit secretat per els protozous que inhibeix el seu propi creixement controlant el creixement poblacional i facilitant la coexistència amb el hoste. A partir dels resultats ex vivo hem construït un model basat en l'individu que ens ha permès hipotetitzar dos fets. Primerament, quan els tripomastigots (forma infectiva, no reproductiva, extracel·lular) de Trypanosoma cruzi infecten a les cèl·lules eucariotes, aquestes canvien la seva composició de membrana perquè no puguin entrar més protozous. La segona hipòtesi és que el cicle de creixement complet dels amastigots dura més de 72h perquè sinó observaríem oscil·lacions en les gràfiques que representen el seu nombre absolut per cèl·lula infectada. Per seguir avançant en la comprensió d'aquestes malalties, cal seguir treballant amb ambdós models així com començar a treballar amb models in vivo i així aproximar-nos més al comportament en humans.La enfermedad de Chagas i la leishmaniasis provocan miles de muertos anualmente en los países del Sud. Estas enfermedades son causadas por los protozoos Trypanosoma cruzi y Leishmania spp., respectivamente. Ambos protozoos pertenecen a la familia Trypanosomatidae y su ciclo de vida es digenético, es decir, viven entre un hospedador vertebrado y un vector invertebrado con el que se transmiten (flebotómidos (Phlebotominae) para Leishmania spp. y hemípteros predadores (Triatomine) para Trypanosoma cruzi). El objetivo principal de este trabajo de final de grado es avanzar en la comprensión del comportamiento de cultivos in vitro y ex vivo del protozoo Trypanosoma cruzi. Como hemos podido demostrar que su comportamiento en cultivos in vitro es muy similar al de Leishmania spp., esta comprensión realizada con Trypanosoma cruzi nos será de uso para Leishmania spp. Para hacerlo, hemos desarrollado dos modelos matemáticos: un modelo continuo de los crecimientos in vitro y un modelo basado en el individuo de los crecimientos ex vivo. Los experimentos in vitro de los dos protozoos se han realizado en el Departamento de Microbiología y Parasitología Sanitarias de la Universitat de Barcelona. Se ha diseñado un método experimental que mide el perfil de crecimiento de los epimastigotes durante 20-30 días (forma no infectiva, reproductiva y extracelular de T. cruzi y Leishmania spp.) obteniendo resultados que nos indican un claro comportamiento bilineal en la fase de crecimiento. Los experimentos de crecimiento ex vivo con Trypanosoma cruzi se han realizado en la Escuela de Farmacia de la Universidad Federal de Ouro Preto (Brasil). Se ha utilizado la información de un método experimental que mide el número de células eucariotas infectadas en función del número de células contadas del cultivo y el número de amastigotes (forma no infectiva, reproductiva, intracelular) presentes en cada célula infectada durante 72 horas. En este caso se ha observado que el número de células infectadas es máximo a las 72 horas y el número de amastigotes es máximo a las 48 horas. A partir de los resultados in vitro hemos construido un modelo continuo donde hemos podido demostrar matemáticamente el crecimiento bilineal de los cultivos y donde la limitación de crecimiento se debe a la difusión, o falta de ella, de oxígeno en el medio. También, podemos suponer que hay algún metabolito secretado por los protozoos que inhibe su propio crecimiento controlando así el crecimiento poblacional y facilitando la coexistencia con el hospedador. A partir de los resultados ex vivo hemos construido un modelo basado en el individuo que nos ha permitido hipotetitar dos hechos. Primeramente, cuando los tripomastigotes (forma infectiva, no reproductiva, extracelular) de Trypanosoma cruzi infectan a las células eucariotas, estas cambian su composición de membrada para que no puedan entrar más protozoos. La segunda hipótesis es que el ciclo de crecimiento completo de los amastigotes dura más de 72 horas porque si no observaríamos oscilaciones en las gráficas que representan su número absoluto por célula infectada. Para seguir avanzando en la comprensión de estas enfermedades, es necesario seguir trabajando con ambos modelos, así como empezar a trabajar con modelos in vitro y así aproximarnos más al comportamiento en humanos

The Effects of N

Leishmaniasis is a disease that affects millions of people worldwide. The drugs that are available for the treatment of this infection exhibit high toxicity and various side effects. Several studies have focused on the development of new chemotherapeutic agents that are less toxic and more effective against trypanosomatids. We investigated the effects of N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide (C4) and its possible targets against L. amazonensis. The results showed morphological and ultrastructural alterations, depolarization of the mitochondrial membrane, the loss of cell membrane integrity, and an increase in the formation of mitochondrial superoxide anions in L. amazonensis treated with C4. Our results indicate that C4 is a selective antileishmanial agent, and its effects appear to be mediated by mitochondrial dysfunction

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

Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies

Genotypic and phenotypic diversity of trypanosomes infecting Australian marsupials and their association with the population decline of the brush-tailed bettong or woylie (Bettongia penicillata)

Trypanosomes are flagellated blood parasites that are capable of infecting virtually all classes of vertebrates. They range from non-pathogenic species to those that are highly pathogenic and are the causative agents of many diseases of medical and veterinary importance. While much is known of their impact on human health or economic development, a great deal less is known of those associated with wildlife. Within Australia, trypanosomes have been found naturally infecting a wide range of native marsupials, most of which are considered threatened or endangered. However, their research has largely been confined to the description of trypanosome morphology in blood, and a complete lack of information regarding their life cycle, virulence, and pathogenicity is evident. This study therefore, aimed to investigate the genotypic and phenotypic diversity of Trypanosoma spp. infecting Western Australia marsupials and to determine their potential pathogenicity with particular emphasis in the critically endangered marsupial, the woylie (Bettongia penicillata). The genotypic characterisation was achieved using a combination of sequencing and phylogenetic analysis of trypanosomes in the blood and tissues of nine different marsupial species, as well as the sequencing of partial fragments of the minicircles of the kinetoplast DNA of trypanosomes isolated in culture. The phenotypic characterisation involved a combination of histology, microscopy techniques, and in vitro experiments of cell infection and drug susceptibility. Results revealed that eight different genotypes belonging to three different Trypanosoma species: T. copemani, T. vegrandis, and T. sp H25 were found infecting woylies (Bettongia penicillata), quendas (Isoodon obesulus), quokkas (Setonix brachyurus), tammar wallabies (Macropus eugenii), banded hare wallabies (Lagostrophus fasciatus), boodies (Bettongia lesueur), Chuditches (Dasyurus geoffroii), common brush tailed possums (Trichosurus vulpecula), and western grey kangaroos (Macropus fuliginosus). However, the woylie was the only marsupial species where single individuals and single tissues were co-infected with genotypes belonging to the three different Trypanosoma species. Furthermore, T. copemani G2, the predominant trypanosome in the declining population of woylies, was shown to be able to infect tissue cells and generate a strong immune response characterised by tissue degeneration and necrosis in vital organs, suggesting an association between these infections and the decline of the woylie. Comparative analysis between T. copemani G2 and the pathogenic T. cruzi showed not only similarities in their capacity to infect tissue cells, but also in drug susceptibility and kinetoplast DNA organisation. In summary, this study not only contributes valuable information towards directing management decisions for endangered species where trypanosomes are known to be present at high prevalence levels, but also provides new knowledge about the evolutionary biology and relationships that Australian trypanosomes have with the exotic and pathogenic T. cruzi

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

Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long-term-infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. the extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro-irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non-irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Escola Paulista Medi, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Trop Med, Lab Soroepidemiol & Imunobiol, São Paulo, BrazilFdn Oswaldo Cruz FIOCRUZ, INCT DT, Salvador, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Prevent Med, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Medi, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Trop Med, Lab Soroepidemiol & Imunobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Prevent Med, São Paulo, BrazilFAPESP: 10/19335-4Web of Scienc