RESUMO
Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.
Assuntos
Bactérias/metabolismo , Replicação do DNA , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Mitocôndrias/genética , Proteínas de Protozoários/genética , Trypanosomatina/genética , Divisão Celular , Núcleo Celular , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Protozoário/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Simbiose , Trypanosomatina/metabolismo , Trypanosomatina/microbiologiaRESUMO
In this chapter we describe different electron microscopy techniques such as freeze fracture, deep etching, and three-dimensional reconstruction, obtained by electron tomography or focused ion beam scanning electron microscopy (FIB-SEM), combined with quick-freezing methods in order to reveal aspects of the cell structure in trypanosomatids. For this purpose, we chose protists that evolve in a mutualistic way with a symbiotic bacterium. Such cells represent excellent models to study the positioning and distribution of organelles, since the symbiotic bacterium interacts with different organelles of the host trypanosomatid. We demonstrate that the employment of such techniques can show the proximity and even the interaction of the symbiotic bacterium with different structures of the protist host, such as the nucleus and the glycosomes. In addition, the quick-freezing approach can reveal new aspects of the gram-negative bacterial envelope, such as the presence of a greatly reduced cell wall between the two membrane units.
Assuntos
Bactérias/citologia , Microscopia Eletrônica de Varredura/métodos , Trypanosomatina/microbiologia , Núcleo Celular/microbiologia , Parede Celular , Microcorpos/microbiologia , Microscopia Eletrônica de Varredura/instrumentação , Simbiose , Trypanosomatina/citologiaRESUMO
Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.
Assuntos
Fenômenos Fisiológicos Bacterianos , Proteoma/genética , Simbiose/fisiologia , Trypanosomatina/microbiologia , Trypanosomatina/genéticaRESUMO
Trypanosomatids of the genera Angomonas and Strigomonas (subfamily Strigomonadinae) have long been known to contain intracellular beta-proteobacteria, which provide them with many important nutrients such as haem, essential amino acids and vitamins. Recently, Kentomonas sorsogonicus, a divergent member of Strigomonadinae, has been described. Herein, we characterize the genome of its endosymbiont, Candidatus Kinetoplastibacterium sorsogonicusi. This genome is completely syntenic with those of other known Ca. Kinetoplastibacterium spp., but more reduced in size (~742 kb, compared with 810-833 kb, respectively). Gene losses are not concentrated in any hot-spots but are instead distributed throughout the genome. The most conspicuous loss is that of the haem-synthesis pathway. For long, removing haemin from the culture medium has been a standard procedure in cultivating trypanosomatids isolated from insects; continued growth was considered as an evidence of endosymbiont presence. However, we demonstrate that, despite bearing the endosymbiont, K. sorsogonicus cannot grow in culture without haem. Thus, the traditional test cannot be taken as a reliable criterion for the absence or presence of endosymbionts in trypanosomatid flagellates. It remains unclear why the ability to synthesize such an essential compound was lost in Ca. K. sorsogonicusi, whereas all other known bacterial endosymbionts of trypanosomatids retain them.
Assuntos
Betaproteobacteria/genética , Genoma Bacteriano , Heme/metabolismo , Simbiose , Trypanosomatina/microbiologia , Betaproteobacteria/efeitos dos fármacos , Betaproteobacteria/crescimento & desenvolvimento , Vias Biossintéticas , Heme/farmacologia , Filogenia , Análise de Sequência de DNARESUMO
Reactive oxygen species (ROS) are toxic molecules involved in several biological processes such as cellular signaling, proliferation, differentiation and cell death. Adaptations to oxidative environments are crucial for the success of the colonization of insects by protozoa. Strigomonas culicis is a monoxenic trypanosomatid found in the midgut of mosquitoes and presenting a life cycle restricted to the epimastigote form. Among S. culicis peculiarities, there is an endosymbiotic bacterium in the cytoplasm, which completes essential biosynthetic routes of the host cell and may represent an intermediary evolutive step in organelle origin, thus constituting an interesting model for evolutive researches. In this work, we induced ROS resistance in wild type S. culicis epimastigotes by the incubation with increasing concentrations of hydrogen peroxide (H2O2), and compared the oxidative and energetic metabolisms among wild type, wild type-H2O2 resistant and aposymbiotic strains. Resistant protozoa were less sensitive to the oxidative challenge and more dependent on oxidative phosphorylation, which was demonstrated by higher oxygen consumption and mitochondrial membrane potential, increased activity of complexes II-III and IV, increased complex II gene expression and higher ATP production. Furthermore, the wild type-H2O2 resistant strain produced reduced ROS levels and showed lower lipid peroxidation, as well as an increase in gene expression of antioxidant enzymes and thiol-dependent peroxidase activity. On the other hand, the aposymbiotic strain showed impaired mitochondrial function, higher H2O2 production and deficient antioxidant response. The induction of H2O2 resistance also led to a remarkable increase in Aedes aegypti midgut binding in vitro and colonization in vivo, indicating that both the pro-oxidant environment in the mosquito gut and the oxidative stress susceptibility regulate S. culicis population in invertebrates.
Assuntos
Aedes/parasitologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético/genética , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/farmacologia , Proteínas de Protozoários/genética , Trypanosomatina/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Antioxidantes/metabolismo , Betaproteobacteria/metabolismo , Evolução Biológica , Resistência a Medicamentos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Trato Gastrointestinal/parasitologia , Regulação da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Simbiose/fisiologia , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/genética , Trypanosomatina/microbiologiaRESUMO
The mutualistic relationship between trypanosomatids and their respective endosymbiotic bacteria represents an excellent model for studying metabolic co-evolution since the symbiont completes essential biosynthetic routes of the host cell. In this work, we investigated the influence of the endosymbiont on the energy metabolism of Strigomonas culicis by comparing the wild strain with aposymbiotic protists. The bacterium maintains a frequent and close association with glycosomes, which are distributed around the prokaryote. Furthermore, 3D reconstructions revealed that the shape and distribution of glycosomes are different in symbiont-bearing protists compared to symbiont-free cells. Results of bioenergetic assays showed that the presence of the symbiont enhances the O2 consumption of the host cell. When the quantity of intracellular or released glycerol was evaluated, the aposymbiotic strain presented higher values when compared to symbiont-containing cells. Furthermore, inhibition of oxidative phosphorylation by potassium cyanide increased the rate of glycerol release and slightly diminished the ATP content in cells without the symbiont, indicating that the host trypanosomatid enhances its fermentative activity when the bacterium is lost.
Assuntos
Fenômenos Fisiológicos Bacterianos , Simbiose , Trypanosomatina/microbiologia , Metabolismo EnergéticoRESUMO
In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a nonpathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here, we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α-tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont-containing trypanosomatids and to clarify important aspects of symbiosis and cell evolution.
Assuntos
Bactérias/citologia , Proteínas de Protozoários/genética , Simbiose , Trypanosomatina/microbiologia , Bactérias/genética , Divisão Celular , Proteínas de Protozoários/metabolismo , Interferência de RNA , Trypanosomatina/genética , Trypanosomatina/metabolismo , Trypanosomatina/ultraestrutura , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
UNLABELLED: We describe a novel symbiotic association between a kinetoplastid protist, Novymonas esmeraldas gen. nov., sp. nov., and an intracytoplasmic bacterium, "Candidatus Pandoraea novymonadis" sp. nov., discovered as a result of a broad-scale survey of insect trypanosomatid biodiversity in Ecuador. We characterize this association by describing the morphology of both organisms, as well as their interactions, and by establishing their phylogenetic affinities. Importantly, neither partner is closely related to other known organisms previously implicated in eukaryote-bacterial symbiosis. This symbiotic association seems to be relatively recent, as the host does not exert a stringent control over the number of bacteria harbored in its cytoplasm. We argue that this unique relationship may represent a suitable model for studying the initial stages of establishment of endosymbiosis between a single-cellular eukaryote and a prokaryote. Based on phylogenetic analyses, Novymonas could be considered a proxy for the insect-only ancestor of the dixenous genus Leishmania and shed light on the origin of the two-host life cycle within the subfamily Leishmaniinae. IMPORTANCE: The parasitic trypanosomatid protist Novymonas esmeraldas gen. nov., sp. nov. entered into endosymbiosis with the bacterium "Ca. Pandoraea novymonadis" sp. nov. This novel and rather unstable interaction shows several signs of relatively recent establishment, qualifying it as a potentially unique transient stage in the increasingly complex range of eukaryotic-prokaryotic relationships.
Assuntos
Burkholderiaceae/fisiologia , Simbiose , Trypanosomatina/microbiologia , Burkholderiaceae/classificação , Burkholderiaceae/citologia , Burkholderiaceae/isolamento & purificação , Equador , Filogenia , Trypanosomatina/classificação , Trypanosomatina/citologia , Trypanosomatina/genéticaRESUMO
BACKGROUND: Angomonas deanei is a trypanosomatid parasite of insects that has a bacterial endosymbiont, which supplies amino acids and other nutrients to its host. Bacterium loss induced by antibiotic treatment of the protozoan leads to an aposymbiotic strain with increased need for amino acids and results in increased production of extracellular peptidases. In this work, a more detailed examination of A. deanei was conducted to determine the effects of endosymbiont loss on the host calpain-like proteins (CALPs), followed by testing of different calpain inhibitors on parasite proliferation. RESULTS: Western blotting showed the presence of different protein bands reactive to antibodies against calpain from Drosophila melanogaster (anti-Dm-calpain), lobster calpain (anti-CDPIIb) and cytoskeleton-associated calpain from Trypanosoma brucei (anti-CAP5.5), suggesting a possible modulation of CALPs influenced by the endosymbiont. In the cell-free culture supernatant of A. deanei wild type and aposymbiotic strains, a protein of 80 kDa cross-reacted with the anti-Dm-calpain antibody; however, no cross-reactivity was found with anti-CAP5.5 and anti-CDPIIb antibodies. A search in A. deanei genome for homologues of D. melanogaster calpain, T. brucei CAP5.5 and lobster CDPIIb calpain revealed the presence of hits with at least one calpain conserved domain and also with theoretical molecular mass consistent with the recognition by each antibody. No significant hit was observed in the endosymbiont genome, indicating that calpain molecules might be absent from the symbiont. Flow cytometry analysis of cells treated with the anti-calpain antibodies showed that a larger amount of reactive epitopes was located intracellularly. The reversible calpain inhibitor MDL28170 displayed a much higher efficacy in diminishing the growth of both strains compared to the non-competitive calpain inhibitor PD150606, while the irreversible calpain inhibitor V only marginally diminished the proliferation. CONCLUSIONS: Altogether, these results indicate that distinct calpain-like molecules are expressed by A. deanei, with a possible modulation in the expression influenced by the endosymbiont. In addition, treatment with MDL28170 affects the growth rate of both strains, as previously determined in the human pathogenic species Leishmania amazonensis and Trypanosoma cruzi, with whom A. deanei shares immunological and biochemical relationships.
Assuntos
Bactérias/crescimento & desenvolvimento , Calpaína/antagonistas & inibidores , Calpaína/biossíntese , Glicoproteínas/metabolismo , Simbiose , Trypanosomatina/crescimento & desenvolvimento , Trypanosomatina/microbiologia , Calpaína/genética , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/genéticaRESUMO
Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.