Re-emergence of Gamma-like-II and emergence of Gamma-S:E661D SARS-CoV-2 lineages in the south of Brazil after the 2021 outbreak.

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.

Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration.

The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration

The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

Fluorescence-based assay for accurate measurement of transcriptional activity in trypanosomatid parasites.

Trypanosomatid parasites are causative agents of neglected human diseases. Their lineage diverged early from the common eukaryotic ancestor, and they evolved singular mechanisms of gene expression that are crucial for their survival. Studies on unusual and essential molecular pathways lead to new drug targets. In this respect, assays to analyze transcriptional activity will provide useful information to identify essential and specific factors. However, the current methods are laborious and do not provide global and accurate measures. For this purpose, a previously reported radiolabeling in vitro nascent mRNA methodology was used to establish an alternative fluorescent-based assay that is able to precisely quantify nascent mRNA using both flow cytometry and a high-content image system. The method allowed accurate and global measurements in Trypanosoma brucei, a representative species of trypanosomatid parasites. We obtained data demonstrating that approximately 70% of parasites from a population under normal growth conditions displayed mRNA transcriptional activity, whilst the treatment with α-amanitin (75 µg/ml) inhibited the polymerase II activity. The adaptation of the method also allowed the analyses of the transcriptional activity during the cell cycle. Therefore, the methodology described herein contributes to obtaining precise measurements of transcriptional rates using multiparametric analysis. This alternative method can facilitate investigations of genetic and biochemical processes in trypanosome parasites and consequently provide additional information related to new treatment or prophylaxis strategies involving these important human parasites.

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii.

Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans.

Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: proof of concept.

The scarcity of molecular tools for genetic manipulation is a critical obstacle for functional genomics studies on Trypanosoma cruzi. The current study adapted an inducible site-specific recombination system based on Dimerizable CRE recombinase (DiCRE). Two vectors for stable transfection were created, a first one to express inactive portions of DiCRE recombinase, and a second plasmid containing the loxP sites to test DiCRE activity. After integrating both constructs into the T. cruzi genome, it was shown that DiCRE recombinase can be efficiently used to manipulate its genome by allowing the removal of selectable markers thus generating homogeneous populations. The DiCRE recombinase success allows conditional knockout and the removal of selectable markers without prior parasite modification, which also facilitate the transferring of DiCRE recombinase to different T. cruzi strains.

Comparative genomics of proteins involved in RNA nucleocytoplasmic export.

BACKGROUND: The establishment of the nuclear membrane resulted in the physical separation of transcription and translation, and presented early eukaryotes with a formidable challenge: how to shuttle RNA from the nucleus to the locus of protein synthesis. In prokaryotes, mRNA is translated as it is being synthesized, whereas in eukaryotes mRNA is synthesized and processed in the nucleus, and it is then exported to the cytoplasm. In metazoa and fungi, the different RNA species are exported from the nucleus by specialized pathways. For example, tRNA is exported by exportin-t in a RanGTP-dependent fashion. By contrast, mRNAs are associated to ribonucleoproteins (RNPs) and exported by an essential shuttling complex (TAP-p15 in human, Mex67-mtr2 in yeast) that transports them through the nuclear pore. The different RNA export pathways appear to be well conserved among members of Opisthokonta, the eukaryotic supergroup that includes Fungi and Metazoa. However, it is not known whether RNA export in the other eukaryotic supergroups follows the same export routes as in opisthokonts. METHODS: Our objective was to reconstruct the evolutionary history of the different RNA export pathways across eukaryotes. To do so, we screened an array of eukaryotic genomes for the presence of homologs of the proteins involved in RNA export in Metazoa and Fungi, using human and yeast proteins as queries. RESULTS: Our genomic comparisons indicate that the basic components of the RanGTP-dependent RNA pathways are conserved across eukaryotes, and thus we infer that these are traceable to the last eukaryotic common ancestor (LECA). On the other hand, several of the proteins involved in RanGTP-independent mRNA export pathways are less conserved, which would suggest that they represent innovations that appeared later in the evolution of eukaryotes. CONCLUSIONS: Our analyses suggest that the LECA possessed the basic components of the different RNA export mechanisms found today in opisthokonts, and that these mechanisms became more specialized throughout eukaryotic evolution.

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens.

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.

Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi.

Sera of Chagasic patients react with antigens from the tomato parasite Phytomonas serpens

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These fagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identifed, resulting in 22 different putative proteins. The identifed proteins were classifed into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi.

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185bp long and encodes a 44.3kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.

Deletion of copies of the gene encoding old yellow enzyme (TcOYE), a NAD(P)H flavin oxidoreductase, associates with in vitro-induced benznidazole resistance in Trypanosoma cruzi.

Old yellow enzyme (OYE) is a NAD(P)H flavin oxidoreductase that in Trypanosoma cruzi (TcOYE) catalyzes prostaglandin PGF2alpha synthesis and reduction of some trypanocidal drugs. We performed DNA microarray analysis and it revealed that the levels of transcription of the TcOYE gene were six-fold lower in a T. cruzi population with in vitro-induced resistance to benznidazole (BZ) (17LER) than in the wild-type (17WTS). Further we investigated the TcOYE levels in 15 T. cruzi strains and clones that were either susceptible or naturally resistant to BZ and nifurtimox, or had in vivo-selected resistance to BZ. Northern blot and real-time RT-PCR analyses confirmed our finding that TcOYE transcription levels were lower in 17LER than in 17WTS. In contrast, we detected no differences in TcOYE transcription levels between other T. cruzi samples. All T. cruzi strains contained four copies of TcOYE gene, except 17LER that contained only one. A 42kDa TcOYE protein was detected in all T. cruzi strains tested. The expression of this protein was similar for all samples, with the exception of 17LER for which the protein was nearly seven-fold less expressed. The chromosomal location of the TcOYE gene and the polymorphisms detected in TcOYE nucleotide and amino acid sequences of the T. cruzi strains are associated with the zymodeme but not with drug-resistance phenotype. Our data show that one of the mechanisms conferring in vitro-induced BZ resistance to T. cruzi correlates with deletion of copies of the TcOYE gene. In contrast, the in vivo and natural resistance to BZ are mediated by different mechanisms.