Between Armour and Weapons — Cell Death Mechanisms in Trypanosomatid Parasites

Among the pathogenic protozoa, trypanosomatids stand out due to their medical and economic impact, especially for low-income populations in tropical countries. Together, sleeping sickness, Chagas disease and leishmaniasis affect millions of humans and animals worldwide, yet are neglected by the pharmaceutical industry. The current drugs for trypanosomatid infections are limited and unsatisfactory, with severe side effects leading to reduced quality of life and, in several instances, to the abandonment of treatment. An intense search for alternative compounds has been performed, aiming at specific parasite targets by cellular, molecular and biochemical approaches. One interesting strategy could be interference with the protozoan cell death pathways. However, these pathways are poorly understood in unicellular eukaryotes, with the controversial existence and uncertain biological relevance of programmed cell death (PCD). This chapter will discuss apoptosis-like and autophagic cell death and necrosis in Trypanosoma brucei, Trypanosoma cruzi and Leishmania sp. and the possible implications of these pathways for the parasite life cycle and infection persistence. It will also revisit the genomic and proteomic metadata of these trypanosomatids in the literature to rebuild the map of cell death proteins expressed under different conditions. The interaction of leading candidates with parasite-specific molecules, especially with enzymes that regulate key steps in the cell death process, is a rational and attractive alternative for drug development for these neglected diseases

Ultrastructural and Cytotoxic Effects of Metarhizium robertsii Infection on Rhipicephalus microplus Hemocytes

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL−1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control

Biological effects of trans, trans-farnesol in Leishmania amazonensis

IntroductionFarnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite.MethodsThe study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome.ResultsExogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production.DiscussionThe findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections

Estudo do mecanismo de ação de derivados naftoimodazólicos de β-lapachona sobre Trypanosoma cruzi

Submitted by Repositório Arca ([email protected]) on 2019-07-02T18:34:38Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) rubem_barreto_ioc_mest_2006.pdf: 2980156 bytes, checksum: e9dfd8e4d2ea48e74b5a4bb19d9f7b4e (MD5)Approved for entry into archive by Raquel Dinelis ([email protected]) on 2019-09-25T18:42:04Z (GMT) No. of bitstreams: 2 rubem_barreto_ioc_mest_2006.pdf: 2980156 bytes, checksum: e9dfd8e4d2ea48e74b5a4bb19d9f7b4e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-09-25T18:42:04Z (GMT). No. of bitstreams: 2 rubem_barreto_ioc_mest_2006.pdf: 2980156 bytes, checksum: e9dfd8e4d2ea48e74b5a4bb19d9f7b4e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Foram sintetizados e ensaiados sobre formas tripomastigotas sangüíneas de Trypanosoma cruzi 60 derivados de naftoquinonas pertencentes aos grupos dos imidazóis, oxazóis, fenoxazóis e das fenazinas. Os compostos mais ativos foram três naftoimidazóis derivados de β-lapachona com grupamento fenila (N1), 3-indolila (N2) ou p-metil fenila (N3) ligado ao anel imidazólico. Estudos com N1, N2 e N3 demonstraram atividade sobre tripomastigotas (IC50/1 d oscilando na faixa de 12 a 37 ?M na ausência de sangue, e entre 62 e 68 ?M na presença de sangue), amastigotas (IC50/1 d variando entre 9 e 14 ?M) e epimastigotas (IC50/1 d entre 30 e 83 ?M). Tratamento de macrófagos com os três naftomidazóis por 4 dias inibiu a infecção em até 91,2% (N1), 93,9% (N2) e 94,1% (N3). A infecção foi também diminuída em cardiomiócitos tratados com N1 (31-47%). Danos nos macrófagos ocorreram apenas em concentrações pelo menos 10x maiores que as utilizadas durante os ensaios. Estes resultados nos estimulam a realizar estudos in vivo. Tratamento de epimastigotas com N1, N2 ou N3 induziu alterações morfológicas como inchaço mitocondrial, ruptura da \201CTrans Golgi Network\201D e desorganização de reservosomos Apesar do dano observado nessas organelas, os compostos não interferiram com a marcação de transferrina-Au em compartimentos endocíticos iniciais, sugerindo que o efeito sobre a via endocítica ocorra apenas na porção terminal da via (reservosomos). Foram ainda observados em epimastigotas tratados perfis de retículo endoplasmático envolvendo diferentes organelas. Tripomastigotas tratados apresentaram inchaço mitocondrial, alterações na rede de kDNA, formação de \201Cblebs\201D na membrana plasmática e condensação anormal de cromatina, além de uma perda da eletrondensidade de acidocalcisomos (tratamento com N1). Os três naftoimidazóis inibiram a atividade de succinato citocromo c redutase em epimastigotas, bem como o processo de metaciclogênese. Resultados obtidos pela análise ultra-estrutural e por citometria de fluxo apontam a mitocôndria e compartimentos acídicos de epimastigotas e tripomastigotas de T. cruzi como alvos iniciais de N1, N2 e N3. A inibição da succinato citocromo c redutase reforça a hipótese do comprometimento mitocondrial em conseqüência do tratamento. O efeito dos três compostos sobre o DNA de T. cruzi foi semelhante ao efeito de ?-lapachona, previamente descrito pelo grupo do Dr. R. Docampo. É possível que tal efeito seja decorrente da estrutura planar dos naftoimidazóis e desta naftoquinona.Sixty naphthoquinone derivatives – classified either as oxazoles, imidazoles, phenoxazoles or phenazines - were synthesized and assayed against T. cruzi bloodstream trypomastigotes. The most active compounds were naphthoimidazole derived from β-lapachone and containing a phenyl (N1), a 3-indolyl (N2) or a pmethyl phenyl (N3) moiety linked to the imidazole ring. Assays with N1, N2 and N3 demonstrated activity on trypomastigotes (IC50/1 d ranging between 12 to 37 µM in the absence of blood, and between 62 to 68 µM in the presence of blood), amastigotes (IC50/1 d ranging between 9 and 14 µM), and epimastigotes (IC50/1 d ranging between 30 and 83 µM). Treatment of macrophages with the naphthoimidazoles for 4 days inhibited the infection up to 91.2% (N1), 93.9% (N2) and 94.1% (N3). Infection was also inhibited in N1-treated cardiomyocytes (31- 47%). Damage to macrophages occurred only in concentrations 10x higher than that used during the assays. These results stimulate us to perform further in vivo studies. Treatment of epimastigotes with N1, N2 or N3 induced morphological alterations such as mitochondrial swelling, Trans Golgi Network disruption and reservosomes disorganization. Despite the observed damage to these organelles, the compounds did not interfere with transferrin-Au labeling in early endocytic compartments, suggesting that the effect on the endocytic pathway occurs only at the pre-lysosomal level (reservosomes). Endoplasmic reticulum profiles surrounding organelles were also observed in epimastigotes. Trypomastigotes treated with N1, N2 or N3 presented mitochondrial swelling, alterations in the kDNA network, bleb formation at the plasma membrane, abnormal chromatin condensation, and loss of electron-density in acidocalcisomes (only in N1-treated parasites). All three derivatives inhibited the activity of succinate cytochrome c reductase, as well as the metacyclogenesis process. The ultrastructural data together with flow cytometry results point to the mitochondrion and acidic compartments of T. cruzi epimastigotes and trypomastigotes as initial targets of N1, N2 and N3. The inhibition of succinate cytochrome c reductase reinforces the hypothesis of mitochondrial susceptibility due to the treatment. The effect of all three compounds on T. cruzi DNA was similar to that previously described for β-lapachone by the group of Dr. R. Docampo. It is possible that such effect is caused by the planar structure of the naphthoimidazoles and of this naphthoquinone

The Double-Edged Sword in Pathogenic Trypanosomatids: The Pivotal Role of Mitochondria in Oxidative Stress and Bioenergetics

Made available in DSpace on 2015-04-17T17:04:28Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) solange_castro2etal_IOC_2014.pdf: 2655199 bytes, checksum: 043e207b171c3027dba1db2ddf47f731 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilThe pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement ofmitochondrialDNA(kinetoplastDNA).Due tomitochondrial differences betweenmammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids’ life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatidtargeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation

Submitted by sandra infurna ([email protected]) on 2016-04-28T17:24:16Z No. of bitstreams: 1 laura_farnesi_etal_IOC_2015.pdf: 1801229 bytes, checksum: 0e5e68098ccc098cd106a0cc133754a2 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-28T17:38:41Z (GMT) No. of bitstreams: 1 laura_farnesi_etal_IOC_2015.pdf: 1801229 bytes, checksum: 0e5e68098ccc098cd106a0cc133754a2 (MD5)Made available in DSpace on 2016-04-28T17:38:41Z (GMT). No. of bitstreams: 1 laura_farnesi_etal_IOC_2015.pdf: 1801229 bytes, checksum: 0e5e68098ccc098cd106a0cc133754a2 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bilogia Molecular de Insetos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Fisiologia e Controle de Artrópodes Vetores. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Fisiologia e Controle de Artrópodes Vetores. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Estadual do Norte Fluminense Darcy Ribeiro. Centro de Biociências e Biotecnologia. Laboratório de Química e Função de Proteínas e Peptídeos. Campos de Goytacazes, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus

TrypanocidalActivity of Natural Sesquiterpenoids Involves Mitochondrial Dysfunction, ROS Production and Autophagic Phenotype in <i>Trypanosoma cruzi</i>

Chagas disease is a neglected tropical disease that is caused by the protozoan Trypanosoma cruzi and represents a serious health problem, especially in Latin America. The clinical treatment of Chagas disease is based on two nitroderivatives that present severe side effects and important limitations. In folk medicine, natural products, including sesquiterpenoids, have been employed for the treatment of different parasitic diseases. In this study, the trypanocidal activity of compounds isolated from the Chilean plants Drimys winteri, Podanthus mitiqui and Maytenus boaria on three T. cruzi evolutive forms (epimastigote, trypomastigote and amastigote) was evaluated. Total extracts and seven isolated sesquiterpenoids were assayed on trypomastigotes and epimastigotes. Polygodial (Pgd) from D. winteri, total extract from P. mitiqui (PmTE) and the germacrane erioflorin (Efr) from P. mitiqui were the most bioactive substances. Pgd, Efr and PmTE also presented strong effects on intracellular amastigotes and low host toxicity. Many ultrastructural effects of these substances, including reservosome disruption, cytosolic vacuolization, autophagic phenotype and mitochondrial swelling (in the case of Pgd), were observed. Flow cytometric analysis demonstrated a reduction in mitochondrial membrane potential in treated epimastigotes and an increase in ROS production and high plasma membrane permeability after treatment with Pgd. The promising trypanocidal activity of these natural sesquiterpenoids may be a good starting point for the development of alternative treatmentsforChagas disease