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.

Implications of Flagellar Attachment Zone Proteins TcGP72 and TcFLA-1BP in Morphology, Proliferation, and Intracellular Dynamics in Trypanosoma cruzi.

The highly adaptable parasite Trypanosoma cruzi undergoes complex developmental stages to exploit host organisms effectively. Each stage involves the expression of specific proteins and precise intracellular structural organization. These morphological changes depend on key structures that control intracellular components' growth and redistribution. In trypanosomatids, the flagellar attachment zone (FAZ) connects the flagellum to the cell body and plays a pivotal role in cell expansion and structural rearrangement. While FAZ proteins are well-studied in other trypanosomatids, there is limited knowledge about specific components, organization, and function in T. cruzi. This study employed the CRISPR/Cas9 system to label endogenous genes and conduct deletions to characterize FAZ-specific proteins during epimastigote cell division and metacyclogenesis. In T. cruzi, these proteins exhibited distinct organization compared to their counterparts in T. brucei. TcGP72 is anchored to the flagellar membrane, while TcFLA-1BP is anchored to the membrane lining the cell body. We identified unique features in the organization and function of the FAZ in T. cruzi compared to other trypanosomatids. Deleting these proteins had varying effects on intracellular structures, cytokinesis, and metacyclogenesis. This study reveals specific variations that directly impact the success of cell division and differentiation of this parasite.

3D FIB-SEM structural insights into the architecture of sub-pellicular microtubules of Trypanosoma cruzi epimastigotes.

BACKGROUND INFORMATION: Trypanosomatidae, which includes eukaryotic species agents of diseases like leishmaniasis, sleeping sickness, and Chagas disease, have special structures and organelles not found in mammalian cells. They present a layer of microtubules, known as subpellicular microtubules (SPMT), located underneath the plasma membrane and responsible for preserving cell morphology, cell polarity, the position of single copy organelles, and morphological changes that occur throughout the protozoan life cycle. Even though a lot of knowledge about the SPMT is available, we still do not know exactly how each microtubule in the system is organized in three dimensions. Here, we use focused ion beam scanning electron microscopy (FIB-SEM) to analyze the tridimensional organization of epimastigotes SPMT. RESULTS: The high-resolution 3D analyses revealed that certain microtubules of the SPMT end more prematurely than the neighboring ones. CONCLUSIONS: These microtubules could (1) be shorter or (2) have the same length as the neighboring ones, assuming that those end up earlier at their other end, might be treadmilling/catastrophe events that have not yet been described in trypanosomatids.

Remarkable kinetoplast, cytostome-cytopharynx complex, and storage-related structures as dissected by three-dimensional reconstruction of Trypanosoma sp. 858 isolated from a toad (Amphibia: Anura).

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.

Alterations on growth and cell organization of Giardia intestinalis trophozoites after treatment with KH-TFMDI, a novel class III histone deacetylase inhibitor.

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.

Lysosome-like compartments of Trypanosoma cruzi trypomastigotes may originate directly from epimastigote reservosomes.

Trypanosoma cruzi epimastigote reservosomes store nutrients taken up during the intense endocytic activity exhibited by this developmental form. Reservosomes were classified as pre-lysosomal compartments. In contrast, trypomastigote forms are not able to take up nutrients from the medium. Interestingly, trypomastigotes also have acidic organelles with the same proteases contained in epimastigote reservosomes. Nevertheless, the origin and function of these organelles have not been disclosed so far. Given the similarities between the compartments of epimastigotes and trypomastigotes, the present study aimed to investigate the origin of metacyclic trypomastigote protease-containing organelles by tracking fluorospheres or colloidal gold particles previously stored in epimastigotes' reservosomes throughout metacyclogenesis. Using three-dimensional reconstruction of serial electron microscopy images, it was possible to find trypomastigote compartments containing the tracer. Our observations demonstrate that the protease-containing compartments from metacyclic trypomastigotes may originate directly from the reservosomes of epimastigotes.

The cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes disassembles during cell division.

The cytostome-cytopharynx complex is the main site for endocytosis in epimastigotes of Trypanosoma cruzi It consists of an opening at the plasma membrane surface - the cytostome - followed by a membrane invagination - the cytopharynx. In G1/S cells, this structure is associated with two specific sets of microtubules, a quartet and a triplet. Here, we used electron microscopy and electron tomography to build 3D models of the complex at different stages of the cell cycle. The cytostome-cytopharynx is absent in late G2 and M phase cells, whereas early G2 cells have either a short cytopharynx or no visible complex, with numerous vesicles aligned to the cytostome-cytopharynx microtubules. The microtubule quartet remains visible throughout cell division (albeit in a shorter form), and is duplicated during G2/M. In contrast, the microtubule triplet is absent during late G2/M. Cells in cytokinesis have an invagination of the flagellar pocket membrane likely to represent early stages in cytostome-cytopharynx assembly. Cells in late cytokinesis have two fully developed cytostome-cytopharynx complexes. Our data suggest that the microtubule quartet serves as a guide for new cytostome-cytopharynx assembly.

Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis.

Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the cytostome-cytopharynx complex - an anterior opening (cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis - the transformation of epimastigotes into human-infective metacyclic trypomastigotes - the cytostome-cytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (∼300nm in diameter, larger than that of the cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt cytostome-cytopharynx reassembly in amastigotes, upon host cell infection.

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions.

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.

The three-dimensional structure of the cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes.

The cytostome-cytopharynx complex is the main site of endocytosis of Trypanosoma cruzi epimastigotes. Little is known about the detailed morphology of this remarkable structure. We used serial electron tomography and focused-ion-beam scanning electron microscopy to reconstruct the entire complex, including the surrounding cytoskeleton and vesicles. Focusing on cells that had taken up gold-labeled tracers, we produced three-dimensional snapshots of the process of endocytosis. The cytostome cytoskeleton was composed of two microtubule sets--a triplet that started underneath the cytostome membrane, and a quartet that originated underneath the flagellar-pocket membrane and followed the preoral ridge before reaching the cytopharynx. The two sets accompanying the cytopharynx formed a 'gutter' and left a microtubule-free side, where vesicles were found to be associated. Cargo was unevenly distributed along the lumen of the cytopharynx, forming clusters. The cytopharynx was slightly longer during the G2 phase of the cell cycle, although it did not reach the postnuclear region owing to a bend in its path. Therefore, the cytopharynx is a dynamic structure, undergoing remodeling that is likely associated with endocytic activity and the preparation for cell division.

Tratamento da lombalgia através do dispositivo lombo abdominal e da reeducação postural global / Low Back Pain Treatment via Lumbar Abdominal Device and Global Postural Reeducation

O objetivo desse trabalho foi verificar se o dispositivo lombo abdomonal (DAL) associado ao tratamento de Reeducação Postural Global (RPG) proporciona alteração significativa da lombalgia mensurada através da Escala Visual Analógica da dor (EVA) Foram selecionados para o estudo 32 voluntários, que foram divididos em dois grupos de 16 individuos, G1 e G2. O G1, grupo controle, foi submetido ao tratamento fisioterapêutico, utilizando a técnica de RPG, e o G2, grupo caso, além de ser tratado pela RPG, foi submetido ao uso continuo do DAL. Ao início e término de cada sessão, o voluntário foi questionado em relação à presença e intensidade de dor por meio da EVA. Quando realizada a análise intra grupo, os valores da EVA obtidos após tratamento foram significativamente menores em ambos os grupos (p<0.05). Ao compararmos os grupos não houve diferença estatisticamente significante na Eva pós tratamento. Tanto a RPG utilizada isoladamente, quanto associada ao DAL, apresenta diminuição significativa da dor após o tratamento.

The purpose of these research was to find out if the back abdominal devece (DAL) associated to a treatment fo Global Postural Reeducation (RPG) provides significance alteration of back studies measure thought Analogic Visual Scale of pain (EVA). It was selected to the study 32 volunteers that were divided in two groups of 16 individuals, G1 e G2. The G1, the control group, was submitted to physiotherapy treatment, using the technique of RPG, and the G2, the case group, besides the treatment of RPG, was submitted to the continue use od DAL. From the beginning to the end each session, the volunteer was questioned in relation to the presence and intensity of pain trought the EVA. The EVA, after treatment, wasn't different between the groups. When realized the analysis inside groups, the values of EVA obtained after treatment were minor expressive in both groups (p<0,05). As much as the RPG put in use isolated, as much as the DAL, shows significant decrease of pain after treatment.

Efeito de diferentes doses de enxofre no consumo voluntário e nas populações de protozoários do rúmen de novilhas mestiças alimentadas com capim-elefante de baixa qualidade / Effect of different doses of sulfur on voluntary intake of low-quality elephant grass and estimates of ruminal protozoa populations in crossbred heifers

O experimento foi conduzido no Campo Experimental de Coronel Pacheco - MG da EMBRAPA Gado de Leite. O efeito de doses de enxofre (sulfato de amônio, 0,15; 0,31; 0,46 e 0,92 por cento de S na matéria verde / dia) na população de protozoários ruminais, foi avaliado utilizando-se quatro novilhas 7/8 Holandês X Zebu, arranjadas em um quadrado latino de 4 x 4. Forneceu-se diariamente capim-elefante de baixa qualidade (76,1 por cento FDN na MS), picado, com correção do teor de PB para 7 por cento com uréia, mais mistura mineral sem enxofre fornecida diretamente no rúmen. Foram feitas amostragens do conteúdo ruminal, uma hora após a alimentação. A estimativa das populações microbianas ruminais foi realizada por microscópica direta. Os resultados foram transformados para logaritmos decimais e avaliados estatisticamente. Não houve diferença significativa entre os tratamentos para microrganismos. O tratamento 0,92 por centoS apresentou o menor consumo de matéria seca e ainda, causou início de intoxicação em dois animais. De acordo com esses resultados, doses de 0,31 por cento de enxofre adicionadas a dieta promoveram o incremento das populações de microganismos ruminais e com isso um maior consumo voluntário.

The experiment was carried out in the Experimental Field of Coronel Pacheco belonging to EMBRAPA Dairy Cattle, Minas Gerais. The effects of the doses of sulfur (ammonium sulfate, 0.15 percent, 0.31 percent, 0.46 percent and 0.92 percentS fresh matter/day) on the ruminal protozoa population was evaluated by utilizing four 7/8 Holstein x Zebu, heifers arranged in 4 x 4 Latin square. Low quality elephant grass (76.1 percent NDF in DM) , chopped with correction of the CP content to 7 percent with urea plus a mineral mixture without sulfur given directly into the rumen. Samplings of the ruminal content were done, one hour after feeding. The estimate of the ruminal microbial populations was done by means of the direct microscopy. The results were transformed to decimal logarithm and evaluated statistically. There were no significant differences among the treatments for microorganisms. The 0.92 percentS treatment presented the least dry matter intake and, in addition, caused start of intoxication in two animals. According to those results, doses of 0.31 percent of sulfur added to the diet promoted the increase of the ruminal microorganism populations and hence a greater voluntary intake.