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Aetiologic agents of diseases such as malaria and toxoplasmosis are found in representatives of the phylum Apicomplexa. Therefore, apicomplexan parasites are known to have a significant impact on public health. Epigenetic factors such as histone acetylation/deacetylation are among the main mechanisms of gene regulation in these parasites. Histone deacetylases (HDACs) have aroused a great deal of interest over the past 20 years for being promising targets in the development of drugs for treating several diseases such as cancer. In addition, they have also been shown to be effective for parasitic diseases. However, little is known about the structure of these proteins, as well as their interactions with specific ligands. In this paper, we modelled 14 HDACs from different apicomplexan parasites and performed molecular docking with 12 ligands analogous to the HDAC inhibitors FR235222 and apicidin, which had previously been tested against Toxoplasma gondii and Plasmodium falciparum. In this in silico study, we were able to gather relevant structural data regarding these proteins as well as insights into protein–ligand interactions for testing and developing drugs for these diseases.

Background We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil. Methods The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil. Results Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. Conclusions Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná.

The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication.

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite., Author summary Trypanosoma cruzi is the causative agent of Chagas disease, a tropical neglected illness that affects 6 million people worldwide, mostly in Latin America. Our research group focuses on the elucidation of DNA repair and metabolism of T. cruzi, and on the possible implications of those mechanisms in both parasite genetic diversity and Chagas disease development. In this work, we investigated the involvement of homologous recombination in the oxidation-induced damage response, and in DNA repair, in T. cruzi cells after gamma radiation exposure. We also examined whether TcRad51 –a key protein for homologous recombination–could be an important factor for T. cruzi’s infectivity. We generated genetically-modified T. cruzi cells for RAD51 and studied the in vitro and in vivo infectivity of these mutant cells. Our results indicate that TcRad51 is a key protein involved in the repair of oxidative and DNA double-strand breaks lesions in two crucial developmental forms of T. cruzi.

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response.

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.

A total of 100 years have passed since the discovery of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas’ disease. Since its discovery, the molecular and cellular biology of this early divergent eukaryote, as well as its interactions with the mammalian and insect hosts, has progressed substantially. It is now clear that this parasite presents unique mechanisms controlling gene expression, DNA replication, cell cycle and differentiation, generating several morphological forms that are adapted to survive in different hosts. In recent years, the relationship between the chromatin structure and nuclear organization with the unusual transcription, splicing, DNA replication and DNA repair mechanisms have been investigated in T. cruzi. This article reviews the relevant aspects of these mechanisms in relation to chromatin and nuclear organization.

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for "histone code" reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi . One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.

Epigenetics is the study of heritable changes in gene expression that occur independent of the DNA sequence. Due to their intimacy with DNA, histones have a central role in chromatin structure and epigenetic regulation. Their tails are subject to posttranslational modifications (PTMs) that together with chromatin-remodeling proteins control the access of different proteins to DNA and allow a precise response to different environmental conditions. The first part of this chapter is dedicated to histone enrichment methods that allow the study of histones using techniques such as immunoblot or mass spectrometry for the mapping of the histone PTM network. Next we describe chromatin immunoprecipitation-based techniques (ChIP) for study of the epigenome. ChIP followed by microarray or next-generation sequencing enables the precise genomic localization of protein-DNA interactions. These techniques for genome-wide profiling of chromatin provide powerful and efficient tools to study the epigenome.

Pin1-type peptidyl-prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)-Pro residues, regulating various cellular events. Previously, we reported a Pin1-type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1-like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi-mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two-hybrid assay, we identified a MORN domain-containing protein as putative Pin1-binding partners. These data suggest that Pin1-mediated signaling mechanism plays a different role in protozoan parasites.

The presence of nucleus in living organisms characterizes the Eukaryote domain. The nucleus compartmentalizes the genetic material surrounded by a double membrane called nuclear envelope. The nucleus has been observed since the advent of the light microscope, and sub-compartments such as nucleoli, diverse nuclear bodies and condensed chromosomes have been later recognized, being part of highly organized and dynamic structure. The significance and function of such organization has increased with the understanding of transcription, replication, DNA repair, recombination processes. It is now recognized as consequence of adding complexity and regulation in more complex eukaryotic cells. Here we provide a description of the actual stage of knowledge of the nuclear structure of Trypanosoma cruzi. As an early divergent eukaryote, it presents unique and/or reduced events of DNA replication, transcription and repair as well as RNA processing and transport to the cytosol. Nevertheless, it shows peculiar structure changes accordingly to the cell cycle and stage of differentiation. T. cruzi proliferates only as epimastigote and amastigote stages, and when these forms differentiate in trypomastigote forms, their cell cycle is arrested. This arrested stage is capable of invading mammalian cells and of surviving harsh conditions, such as the gut of the insect vector and mammalian macrophages. Transcription and replication decrease during transformation in trypomastigotes implicating large alterations in the nuclear structure. Recent evidences also suggest that T. cruzi nucleus respond to oxidative and nutritional stresses. Due to the phylogenetic proximity with other well-known trypanosomes, such as Trypanosoma brucei and Leishmania major, they are expected to have similar nuclear organization, although differences are noticed due to distinct life cycles, cellular organizations and the specific adaptations for surviving in different host environments. Therefore, the general features of T. cruzi nuclear structure regarding unique characteristics of this protozoan parasite will be described.

National Institutes of Health Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ellison Medical Foundation to the Center for Tropical and Emerging Global Diseases American Heart Association Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G(2) phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei. Univ Georgia, Ctr Trop & Emerging Global Dis, Athens, GA 30602 USA Univ Georgia, Dept Cellular Biol, Athens, GA 30602 USA Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Univ Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21949900 Rio de Janeiro, Brazil Inst Nacl Metrol Normalizacao & Qualidade Ind, Diretoria Programas, BR-25250020 Rio de Janeiro, Brazil Consejo Super Invest Cient, Inst Parasitol & Biomed Lopez Neyra, Granada 18100, Spain Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil National Institutes of Health: AI-077538 Web of Science

Background Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages. Results By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure. Conclusions The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

Histones of trypanosomes are quite divergent when compared to histones of most eukaryotes. Nevertheless, the histone H4 of Trypanosoma cruzi, the protozoan that causes Chagas' disease, is acetylated in the N terminus at lysines 4, 10, and 14. Here, we investigated the cellular distribution of histone H4 containing each one of these posttranslational modifications by using specific antibodies. Histone H4 acetylated at lysine 4 (H4-K4ac) is found in the entire nuclear space preferentially at dense chromatin regions, excluding the nucleolus of replicating epimastigote forms of the parasite. In contrast, histone H4 acetylated either at K10 or K14 is found at dispersed foci all over the nuclei and at the interface between dense and nondense chromatin areas as observed by ultrastructural immunocytochemistry. The level of acetylation at K4 decreases in nonreplicating forms of the parasites when compared to K10 and K14 acetylations. Antibodies recognizing the K14 acetylation strongly labeled cells at G2 and M stages of the cell cycle. Besides that, hydroxyurea synchronized parasites show an increased acetylation at K4, K10, and K14 after S phase. Moreover, we do not observed specific colocalization of K4 modifications with the major sites of RNA polymerase II. Upon gamma-irradiation that stops parasite replication until the DNA is repaired, dense chromatin disappears and K4 acetylation decreases, while K10 and K14 acetylation increase. These results indicate that each lysine acetylation has a different role in T. cruzi. While K4 acetylation occurs preferentially in proliferating situations and accumulates in packed chromatin, K10 and K14 acetylations have a particular distribution probably at the boundaries between packed and unpacked chromatin.

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: kcat/KM = 97.1 /M/s), like that found for human Par14. In contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell.