Transcriptome and proteome changes triggered by overexpression of the transcriptional regulator Maf1 in the human pathogen Leishmania major.

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites.

RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.

Synanthropic triatomines in Hidalgo state, Mexico: Spatial-temporal distribution, domestic transmission cycle, and natural infection with Trypanosoma cruzi.

Triatomine vectors are responsible for the main route of transmission of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This illness is potentially life-threatening and highly disabling and represents a major public health concern in the endemic countries in Latin America. The analysis of the spatial and temporal occurrence of triatomine insects is critical, since control strategies strongly depend on the vector species found within each area. Such knowledge is non-existent in Hidalgo state, an endemic region of Chagas disease in Mexico. A Geographic Information System (GIS) was used to analyze broad-scale spatial and temporal patterns of synanthropic triatomines collected in Hidalgo. Data was taken from the Institute of Epidemiological Diagnosis and Reference (InDRE) of Mexico and the state program of Vector Control of the Secretary of Health, covering the period of 1997-2019. Our analyses demonstrate a differential distribution of Triatoma dimidiata, T. mexicana, T. gerstaeckeri and T. barberi, which are the four predominant species, and that climate, temperature, and precipitation are some of the drivers of their distribution pattern. Notably, we report the presence of T. nitida, T. pallidipennis and T. phyllosoma for the first time in the state. In addition, we found seasonal variations of the populations of T. mexicana and T. gerstaeckeri, but not for T. dimidiata, whose population remains constant throughout the year. The insects were found mainly intradomicile (81.79%), followed by peridomicile (17.56%) and non-domestic areas (0.65%), with an average T. cruzi infection of 16.4%. Based on this evidence, priority sites for vector control intervention were identified. Our findings are very valuable for understanding the epidemiology of Chagas disease, the generation of future potential risk maps and for the development and implementation of effective and targeted vector control programs in Hidalgo state.

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major.

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

Gene expression in trypanosomatid parasites.

The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

Motility patterns of Trypanosoma cruzi trypomastigotes correlate with the efficiency of parasite invasion in vitro.

Numerous works have demonstrated that trypanosomatid motility is relevant for parasite replication and sensitivity. Nonetheless, although some findings indirectly suggest that motility also plays an important role during infection, this has not been extensively investigated. This work is aimed at partially filling this void for the case of Trypanosoma cruzi. After recording swimming T. cruzi trypomastigotes (CL Brener strain) and recovering their individual trajectories, we statistically analyzed parasite motility patterns. We did this with parasites that swim alone or above monolayer cultures of different cell lines. Our results indicate that T. cruzi trypomastigotes change their motility patterns when they are in the presence of mammalian cells, in a cell-line dependent manner. We further performed infection experiments in which each of the mammalian cell cultures were incubated for 2 h together with trypomastigotes, and measured the corresponding invasion efficiency. Not only this parameter varied from cell line to cell line, but it resulted to be positively correlated with the corresponding intensity of the motility pattern changes. Together, these results suggest that T. cruzi trypomastigotes are capable of sensing the presence of mammalian cells and of changing their motility patterns accordingly, and that this might increase their invasion efficiency.

Dynamics of Mammalian Cell Infection by Trypanosoma cruzi trypomastigotes.

In a recent work we demonstrated that Trypanosoma cruzi trypomastigotes change their motility patterns in the presence of mammalian cells, that the extent of the changes depends on the cell line, and that this extent is positively correlated with the efficiency with which parasites invade the different cell lines. These results open the question of what cellular characteristics are relevant for parasite identification and invasion. In the present work, we tackled such question. We performed infection-kinetics experiments on various cell lines, and developed a mathematical model to simulate the experimental outcomes. An analysis of the cell-parasite mechanisms included in the model, together with the parameter values that allowed it to replicate the experimental results, suggests that a process related to the cell replication rate may strongly influence the parasite invasion efficiency, and the infection dynamics in general.

Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites.

BACKGROUND: The protozoan pathogens Leishmania major, Trypanosoma brucei and Trypanosoma cruzi (the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an in silico analysis of the Tritryps genome sequences. RESULTS: Our analysis indicated the presence of 83, 66 and 120 genes in L. major, T. brucei and T. cruzi, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the L. major genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between T. cruzi and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps. CONCLUSION: A low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major.

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene.

BACKGROUND: Leishmania and other trypanosomatid parasites possess atypical mechanisms of gene expression, including the maturation of mRNAs by trans-splicing and the involvement of RNA Polymerase III in transcription of all snRNA molecules. Since snRNAs are essential for trans-splicing, we are interested in the study of the sequences that direct their expression. Here we report the characterization of L. major U2 snRNA promoter region. RESULTS: All species of Leishmania possess a single U2 snRNA gene that contains a divergently-oriented tRNA-Ala gene in the upstream region. Between these two genes we found a tRNA-like sequence that possesses conserved boxes A and B. Primer extension and RT-qPCR analyses with RNA from transiently-transfected cells showed that transcription of L. major U2 snRNA is almost abolished when boxes A and B from the tRNA-like are deleted or mutated. The levels of the U2 snRNA were also highly affected when base substitutions were introduced into box B from the tRNA-Ala gene and the first nucleotides of the U2 snRNA gene itself. We also demonstrate that the tRNA-like is transcribed, generating a main transcript of around 109 bases. As pseudouridines in snRNAs are required for splicing in other organisms, we searched for this modified nucleotide in the L. major U2 snRNA. Our results show the presence of six pseudouridines in the U2 snRNA, including one in the Sm site that has not been reported in other organisms. CONCLUSIONS: Four different regions control the transcription of the U2 snRNA gene in L. major: boxes A and B from the neighbor tRNA-like, box B from the upstream tRNA-Ala gene and the first nucleotides of the U2 snRNA. Thus, the promoter region of L. major U2 snRNA is different from any other promoter reported for snRNAs. Pseudouridines could play important roles in L. major U2 snRNA, since they were found in functionally important regions, including the branch point recognition region and the Sm binding site.

Experimental and Mathematical-Modeling Characterization of Trypanosoma cruzi Epimastigote Motility.

The present work is aimed at characterizing the motility of parasite T. cruzi in its epimastigote form. To that end, we recorded the trajectories of two strains of this parasite (a wild-type strain and a stable transfected strain, which contains an ectopic copy of LYT1 gene and whose motility is known to be affected). We further extracted parasite trajectories from the recorded videos, and statistically analysed the following trajectory-step features: step length, angular change of direction, longitudinal and transverse displacements with respect to the previous step, and mean square displacement. Based on the resulting observations, we developed a mathematical model to simulate parasite trajectories. The fact that the model predictions closely match most of the experimentally observed parasite-trajectory characteristics, allows us to conclude that the model is an accurate description of T. cruzi motility.