Maria Carolina Elias

Transcription rate modulation through the Trypanosoma cruzi life cycle occurs in parallel with changes in nuclear organisation.

In trypanosomes transcription occurs as large polycistronic units, with trans-splicing and polyadenylation generating each individual mRNA. There are no defined RNA polymerase II promoters and mRNA stabilisation is most likely the process controlling levels of differentially expressed mRNAs, since no selective modulation of gene activity has even been reported at the transcriptional level. Here, we show a large decrease in the transcription rates by RNA polymerases I and II when proliferative forms of Trypanosoma cruzi (epimastigotes and amastigotes) transform into non-proliferative and infective forms (trypomastigotes). We also show that these changes in transcription occur in parallel with modifications in the nuclear structure. While nuclei of proliferative forms are round, contain small amounts of peripheral heterochromatin and a large nucleolus, nuclei of trypomastigotes are elongated, the nucleolus disappears and the heterochromatin occupies most of the nuclear compartment. The decrease in the transcription parallels the nucleolus disassembly, as seen by the dispersion of nucleolar antigens. As T. cruzi cycles continuously through proliferative and infective forms, the molecular mechanisms involved in the control of nuclear organisation and chromatin remodelling can be revealed by this system.

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

Predicting the proteins of angomonas deanei, strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus.

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

Histone H1 is phosphorylated in non-replicating and infective forms of Trypanosoma cruzi.

The nuclear structure changes during the differentiation from growing to infective stages of Trypanosoma cruzi. As histone modifications have been correlated with structural and functional changes of chromatin, we investigated whether histones in T. cruzi are modified during the life cycle of this protozoan parasite. We found that histone H1 isolated from proliferating forms (epimastigotes) and from differentiated/infective forms (trypomastigotes) have a distinct migrating pattern in Triton-acetic acid-urea gel electrophoresis. While epimastigotes contain predominantly a fast migrating form, a slow migrating band is prominent in trypomastigotes. By metabolically labeling the cells with radioactive phosphate, we demonstrated that the slow migrating histone H1 band is phosphorylated, and that after alkaline phosphatase treatment, it migrates as the fast form. Parasites arrested at the onset of the S phase of the cell cycle with hydroxyurea (HU) also predominantly have the phosphorylated form of histone H1, suggesting that phosphorylation occurs in non-replicating stages of T. cruzi. We also found that the phosphorylated histone H1 is more weakly associated with the chromatin, being preferentially released at 150 mM NaCl. Therefore, histone H1 phosphorylation varies during the life cycle of T. cruzi, and might be related to changes in the chromatin structure.

Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

Lipid profile and cardiovascular risk in two Amazonian populations

OBJECTIVE To compare the lipid profiles and coronary heart disease risks of 2 Brazilian Amazonian populations as follows: a riverside population (village of Vigia) and an urban population (city of Bel m in the state of Par ). METHODS Fifty individuals controlled for age and sex were assessed in each region, and the major risk factors for coronary heart disease were analyzed. RESULTS According to the National Cholesterol Education Program (NCEP III) and using the Framingham score, both populations had the same absolute risk of events (Vigia = 5.4 +/- 1 vs Bel m = 5.7 +/- 1), although the population of Vigia had a lower consumption of saturated fat (P<0.0001), a greater consumption of mono- and polyunsaturated fat (P<0.03), in addition to lower values for body mass index (25.4 +/- 0.6 vs 27.6 +/- 0.7 kg/m , P<0.02), of biceps skin fold (18.6 1.1 vs 27.5 +/- 1.3 mm, P<0.0001), of triceps skin fold (28.7 +/- 1.2 vs 37.3 +/- 1.7 mm, P<0.002), and of total cholesterol (205 +/- 5 vs 223 +/- 6 mg/dL, P< 0.03) and triglycerides (119 +/- 9 vs 177 +/- 18 mg/dL, P<0.005). Both populations did not differ in regard to HDL-C (46 +/- 1 vs 46 +/- 1 mg/dL), LDL-C (135 +/- 4 vs 144 +/- 5 mg/dL) and blood pressure (SBP 124 +/- 3 vs 128 +/- 3 mmHg; DBP 80 +/- 2 vs 82 +/- 2 mmHg). CONCLUSION The riverside and urban populations of Amazonia had similar cardiovascular risks. However, the marked difference in the variables studied suggests that different strategies of prevention should be applied.

Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number.

Single molecule analysis of Trypanosoma brucei DNA replication dynamics

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5′ extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.