Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci.

BACKGROUND: Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. RESULTS: Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. CONCLUSION: Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

Effect of the endoplasmic reticulum stressor tunicamycin in Angomonas deanei heat-shock protein expression and on the association with the endosymbiotic bacterium.

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.

Tubastatin A, a deacetylase inhibitor, as a tool to study the division, cell cycle and microtubule cytoskeleton of trypanosomatids.

Trypanosoma cruzi is a protozoan of great medical interest since it is the causative agent of Chagas disease, an endemic condition in Latin America. This parasite undergoes epigenetic events, such as phosphorylation, methylation and acetylation, which play a role in several cellular processes including replication, transcription and gene expression. Histone deacetylases (HDAC) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. Tubastatin A (TST) is a specific HDAC6 inhibitor that affects cell growth and promotes structural modifications in cancer cells and parasites. In the present study, we demonstrated that T. cruzi epimastigote cell proliferation and viability are reduced after 72 h of TST treatment. The results obtained through different microscopy methodologies suggest that this inhibitor impairs the polymerization dynamics of cytoskeleton microtubules, generating protozoa displaying atypical morphology and cellular patterns that include polynucleated parasites. Furthermore, the microtubules of treated protozoa were more intensely acetylated, especially at the anterior portion of the cell body. A cell cycle analysis demonstrated an increase in the number of trypanosomatids in the G2/M phase. Together, our results suggest that TST should be explored as a tool to study trypanosomatid cell biology, including microtubule cytoskeleton dynamics, and as an antiparasitic drug.

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship.

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.

Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

Chromosomal assembly of the nuclear genome of the endosymbiont-bearing trypanosomatid Angomonas deanei.

Angomonas deanei is an endosymbiont-bearing trypanosomatid with several highly fragmented genome assemblies and unknown chromosome number. We present an assembly of the A. deanei nuclear genome based on Oxford Nanopore sequence that resolves into 29 complete or close-to-complete chromosomes. The assembly has several previously unknown special features; it has a supernumerary chromosome, a chromosome with a 340-kb inversion, and there is a translocation between two chromosomes. We also present an updated annotation of the chromosomal genome with 10,365 protein-coding genes, 59 transfer RNAs, 26 ribosomal RNAs, and 62 noncoding RNAs.

Antileishmanial activity of the essential oils of Myrcia ovata Cambess. and Eremanthus erythropappus (DC) McLeisch leads to parasite mitochondrial damage.

Leishmania amazonensis is a species causative of cutaneous and anergic diffuse cutaneous leishmaniasis, treatment-resistant form, in the New World. Plants essential oils exhibit great potential as microbicide agents. We described the composition of the essential oils of two plants native from Brazil, Myrcia ovata, with geranial and neral as major constituents, and Eremanthus erythropappus, with α-bisabolol. In vitro effects of these essential oils on L. amazonensis promastigotes growth and ultrastructure were analysed as well as their cytotoxicity to murine macrophages. Both oils were highly active with IC50/96 h of 8.69 and 9.53 µg/mL for M. ovata and E. erythropappus against promastigotes and caused ultrastructural alterations including mitochondrial enlargement. Cytotoxicity for murine macrophages varied with the oil concentrations. The IC50 low values of both M. ovata and E. erythropappus oils against L. amazonensis and their relative low cytotoxicity to mammal host cells support their potential use against cutaneous leishmaniasis.

The Importance of Glycerophospholipid Production to the Mutualist Symbiosis of Trypanosomatids.

The symbiosis in trypanosomatids is a mutualistic relationship characterized by extensive metabolic exchanges between the bacterium and the protozoan. The symbiotic bacterium can complete host essential metabolic pathways, such as those for heme, amino acid, and vitamin production. Experimental assays indicate that the symbiont acquires phospholipids from the host trypanosomatid, especially phosphatidylcholine, which is often present in bacteria that have a close association with eukaryotic cells. In this work, an in-silico study was performed to find genes involved in the glycerophospholipid (GPL) production of Symbiont Harboring Trypanosomatids (SHTs) and their respective bacteria, also extending the search for trypanosomatids that naturally do not have symbionts. Results showed that most genes for GPL synthesis are only present in the SHT. The bacterium has an exclusive sequence related to phosphatidylglycerol production and contains genes for phosphatidic acid production, which may enhance SHT phosphatidic acid production. Phylogenetic data did not indicate gene transfers from the bacterium to the SHT nucleus, proposing that enzymes participating in GPL route have eukaryotic characteristics. Taken together, our data indicate that, differently from other metabolic pathways described so far, the symbiont contributes little to the production of GPLs and acquires most of these molecules from the SHT.

The effect of the biflavonoid 2″,3″-dihydroochnaflavone on Trypanosoma cruzi Y strain.

Trypanosoma cruzi is the causative agent of Chagas disease which affects 8 million people in Latin America. The parasite possesses high capacity to evade host immune system and the available drugs to treat Chagas disease present low efficacy combined to serious side effects to patients. Therefore, the identification of alternative therapeutics is essential. Brazilian flora exhibits an immense diversity of metabolites with great potential to be developed into new drugs. We investigated the action of 2″,3″-dihydroochnaflavone a biflavonoid extracted from Luxemburgia nobilis Eichler ex Engl. (Ochnaceae) against T. cruzi (Y strain). Our experiments showed that this compound is effective against parasite epimastigote forms, presenting IC50 value of (2.5 ± 0.1) µM after 96 h of treatment. Ultrastructure alterations were also detected in treated epimastigotes especially mitochondrial enlargement at the kinetoplast region. At the concentration of 30 µM, the compound killed (61.6 ± 3.37)% of the parasite in its amastigote form. In addition, at the same concentration, the compound killed all trypamastigotes growing within murine macrophages after 7-9 days of infection. Nonetheless, the biflavonoid concentrations were harmless to murine enriched population of lymphocytes and peritoneal macrophages. These results indicate that 2″,3″- dihydroochnaflavone presents activity against T. cruzi.

Electron Microscopy Techniques Applied to Symbiont-Harboring Trypanosomatids: The Association of the Bacterium with Host Organelles.

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.

Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of glycosomes is their harboring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle's membrane-homologs of the ABC and MCF transporter families-and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium.

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.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity.

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

Trichostatin A induces Trypanosoma cruzi histone and tubulin acetylation: effects on cell division and microtubule cytoskeleton remodelling.

Trypanosoma cruzi, the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.

Trichostatin A induces Trypanosoma cruzi histone and tubulin acetylation: effects on cell division and microtubule cytoskeleton remodelling

Trypanosoma cruzi, the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

Trichostatin A induces Trypanosoma cruzi histone and tubulin acetylation: effects on cell division and microtubule cytoskeleton remodelling

Trypanosoma cruzi, the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.