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29 pages, 6023 KiB

Open AccessArticle

De Novo Transcriptome Meta-Assembly of the Mixotrophic Freshwater Microalga Euglena gracilis

by Javier Cordoba, Emilie Perez, Mick Van Vlierberghe, Amandine R. Bertrand, Valérian Lupo, Pierre Cardol and Denis Baurain

Genes 2021, 12(6), 842; https://doi.org/10.3390/genes12060842 - 29 May 2021

Cited by 9 | Viewed by 3832

Abstract

Euglena gracilis is a well-known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular belonging to the same eukaryotic phylum as the parasitic trypanosomatids. As its nuclear genome has proven difficult to sequence, reliable [...] Read more.

Euglena gracilis is a well-known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular belonging to the same eukaryotic phylum as the parasitic trypanosomatids. As its nuclear genome has proven difficult to sequence, reliable transcriptomes are important for functional studies. In this work, we assembled a new consensus transcriptome by combining sequencing reads from five independent studies. Based on a detailed comparison with two previously released transcriptomes, our consensus transcriptome appears to be the most complete so far. Remapping the reads on it allowed us to compare the expression of the transcripts across multiple culture conditions at once and to infer a functionally annotated network of co-expressed genes. Although the emergence of meaningful gene clusters indicates that some biological signal lies in gene expression levels, our analyses confirm that gene regulation in euglenozoans is not primarily controlled at the transcriptional level. Regarding the origin of E. gracilis, we observe a heavily mixed gene ancestry, as previously reported, and rule out sequence contamination as a possible explanation for these observations. Instead, they indicate that this complex alga has evolved through a convoluted process involving much more than two partners. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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25 pages, 4307 KiB

Open AccessArticle

Genomic and Transcriptomic Analysis for Identification of Genes and Interlinked Pathways Mediating Artemisinin Resistance in Leishmania donovani

by Sushmita Ghosh, Aditya Verma, Vinay Kumar, Dibyabhaba Pradhan, Angamuthu Selvapandiyan, Poonam Salotra and Ruchi Singh

Genes 2020, 11(11), 1362; https://doi.org/10.3390/genes11111362 - 17 Nov 2020

Cited by 5 | Viewed by 3349

Abstract

Current therapy for visceral leishmaniasis (VL), compromised by drug resistance, toxicity, and high cost, demands for more effective, safer, and low-cost drugs. Artemisinin has been found to be an effectual drug alternative in experimental models of leishmaniasis. Comparative genome and transcriptome analysis of [...] Read more.

Current therapy for visceral leishmaniasis (VL), compromised by drug resistance, toxicity, and high cost, demands for more effective, safer, and low-cost drugs. Artemisinin has been found to be an effectual drug alternative in experimental models of leishmaniasis. Comparative genome and transcriptome analysis of in vitro-adapted artesunate-resistant (K133AS-R) and -sensitive wild-type (K133WT) Leishmania donovani parasites was carried out using next-generation sequencing and single-color DNA microarray technology, respectively, to identify genes and interlinked pathways contributing to drug resistance. Whole-genome sequence analysis of K133WT vs. K133AS-R parasites revealed substantial variation among the two and identified 240 single nucleotide polymorphisms (SNPs), 237 insertion deletions (InDels), 616 copy number variations (CNVs) (377 deletions and 239 duplications), and trisomy of chromosome 12 in K133AS-R parasites. Transcriptome analysis revealed differential expression of 208 genes (fold change ≥ 2) in K133AS-R parasites. Functional categorization and analysis of modulated genes of interlinked pathways pointed out plausible adaptations in K133AS-R parasites, such as (i) a dependency on lipid and amino acid metabolism for generating energy, (ii) reduced DNA and protein synthesis leading to parasites in the quiescence state, and (iii) active drug efflux. The upregulated expression of cathepsin-L like protease, amastin-like surface protein, and amino acid transporter and downregulated expression of the gene encoding ABCG2, pteridine receptor, adenylatecyclase-type receptor, phosphoaceylglucosamine mutase, and certain hypothetical proteins are concordant with genomic alterations suggesting their potential role in drug resistance. The study provided an understanding of the molecular basis linked to artemisinin resistance in Leishmania parasites, which may be advantageous for safeguarding this drug for future use. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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20 pages, 3798 KiB

Open AccessArticle

Identification of Novel Interspersed DNA Repetitive Elements in the Trypanosoma cruzi Genome Associated with the 3′UTRs of Surface Multigenic Families

by Simone Guedes Calderano, Milton Yutaka Nishiyama Junior, Marjorie Marini, Nathan de Oliveira Nunes, Marcelo da Silva Reis, José Salvatore Leister Patané, José Franco da Silveira, Julia Pinheiro Chagas da Cunha and Maria Carolina Elias

Genes 2020, 11(10), 1235; https://doi.org/10.3390/genes11101235 - 21 Oct 2020

Cited by 2 | Viewed by 2508

Abstract

Trypanosoma cruzi is the etiological agent of Chagas disease, which affects millions of people in Latin America. No transcriptional control of gene expression has been demonstrated in this organism, and 50% of its genome consists of repetitive elements and members of multigenic families. [...] Read more.

Trypanosoma cruzi is the etiological agent of Chagas disease, which affects millions of people in Latin America. No transcriptional control of gene expression has been demonstrated in this organism, and 50% of its genome consists of repetitive elements and members of multigenic families. In this study, we applied a novel bioinformatics approach to predict new repetitive elements in the genome sequence of T. cruzi. A new repetitive sequence measuring 241 nt was identified and found to be interspersed along the genome sequence from strains of different DTUs. This new repeat was mostly on intergenic regions, and upstream and downstream regions of the 241 nt repeat were enriched in surface protein genes. RNAseq analysis revealed that the repeat was part of processed mRNAs and was predominantly found in the 3′ untranslated regions (UTRs) of genes of multigenic families encoding surface proteins. Moreover, we detected a correlation between the presence of the repeat in the 3′UTR of multigenic family genes and the level of differential expression of these genes when comparing epimastigote and trypomastigote transcriptomes. These data suggest that this sequence plays a role in the posttranscriptional regulation of the expression of multigenic families. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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16 pages, 3262 KiB

Open AccessArticle

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability

by Annika Bea, Constanze Kröber-Boncardo, Manpreet Sandhu, Christine Brinker and Joachim Clos

Genes 2020, 11(10), 1198; https://doi.org/10.3390/genes11101198 - 14 Oct 2020

Cited by 3 | Viewed by 1947

Abstract

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small [...] Read more.

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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24 pages, 2577 KiB

Open AccessArticle

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

by Vanessa Adaui, Constanze Kröber-Boncardo, Christine Brinker, Henner Zirpel, Julie Sellau, Jorge Arévalo, Jean-Claude Dujardin and Joachim Clos

Genes 2020, 11(10), 1159; https://doi.org/10.3390/genes11101159 - 30 Sep 2020

Cited by 9 | Viewed by 4130

Abstract

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis [...] Read more.

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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19 pages, 3760 KiB

Open AccessArticle

Genomic Organization and Generation of Genetic Variability in the RHS (Retrotransposon Hot Spot) Protein Multigene Family in Trypanosoma cruzi

by Werica P. Bernardo, Renata T. Souza, André G. Costa-Martins, Eden R. Ferreira, Renato A. Mortara, Marta M. G. Teixeira, José Luis Ramirez and José F. Da Silveira

Genes 2020, 11(9), 1085; https://doi.org/10.3390/genes11091085 - 17 Sep 2020

Cited by 7 | Viewed by 3150

Abstract

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS [...] Read more.

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS protein family in T. cruzi was demonstrated by phylogenetic and recombination analyses. Transcribed sequences carrying the RHS domain were classified into ten distinct groups of monophyletic origin. We identified numerous recombination events among the RHS and traced the origins of the donors and target sequences. The transcribed RHS genes have a mosaic structure that may contain fragments of different RHS inserted in the target sequence. About 30% of RHS sequences are located in the subtelomere, a region very susceptible to recombination. The evolution of the RHS family has been marked by many events, including gene duplication by unequal mitotic crossing-over, homologous, as well as ectopic recombination, and gene conversion. The expression of RHS was analyzed by immunofluorescence and immunoblotting using anti-RHS antibodies. RHS proteins are evenly distributed in the nuclear region of T. cruzi replicative forms (amastigote and epimastigote), suggesting that they could be involved in the control of the chromatin structure and gene expression, as has been proposed for T. brucei. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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20 pages, 2107 KiB

Open AccessArticle

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations

by África Sanchiz, Esperanza Morato, Alberto Rastrojo, Esther Camacho, Sandra González-de la Fuente, Anabel Marina, Begoña Aguado and Jose M. Requena

Genes 2020, 11(9), 1036; https://doi.org/10.3390/genes11091036 - 02 Sep 2020

Cited by 9 | Viewed by 3040

Abstract

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and [...] Read more.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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15 pages, 1465 KiB

Open AccessArticle

Comparative Analysis of the Minimum Number of Replication Origins in Trypanosomatids and Yeasts

by Marcelo S. da Silva, Marcela O. Vitarelli, Bruno F. Souza and Maria Carolina Elias

Genes 2020, 11(5), 523; https://doi.org/10.3390/genes11050523 - 08 May 2020

Cited by 6 | Viewed by 3238

Abstract

Single-celled eukaryote genomes predominantly replicate through multiple origins. Although origin usage during the S-phase has been elucidated in some of these organisms, few studies have comparatively approached this dynamic. Here, we developed a user-friendly website able to calculate the length of the cell [...] Read more.

Single-celled eukaryote genomes predominantly replicate through multiple origins. Although origin usage during the S-phase has been elucidated in some of these organisms, few studies have comparatively approached this dynamic. Here, we developed a user-friendly website able to calculate the length of the cell cycle phases for any organism. Next, using a formula developed by our group, we showed a comparative analysis among the minimum number of replication origins (MO) required to duplicate an entire chromosome within the S-phase duration in trypanosomatids (Trypanosoma cruzi, Leishmania major, and Trypanosoma brucei) and yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe). Using the data obtained by our analysis, it was possible to predict the MO required in a situation of replication stress. Also, our findings allow establishing a threshold for the number of origins, which serves as a parameter for genome approaches that map origins. Moreover, our data suggest that when compared to yeasts, trypanosomatids use much more origins than the minimum needed. This is the first time a comparative analysis of the minimum number of origins has been successfully applied. These data may provide new insight into the understanding of the replication mechanism and a new methodological framework for studying single-celled eukaryote genomes. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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Review

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15 pages, 1040 KiB

Open AccessReview

The Maze Pathway of Coevolution: A Critical Review over the Leishmania and Its Endosymbiotic History

by Lilian Motta Cantanhêde, Carlos Mata-Somarribas, Khaled Chourabi, Gabriela Pereira da Silva, Bruna Dias das Chagas, Luiza de Oliveira R. Pereira, Mariana Côrtes Boité and Elisa Cupolillo

Genes 2021, 12(5), 657; https://doi.org/10.3390/genes12050657 - 27 Apr 2021

Cited by 17 | Viewed by 2955

Abstract

The description of the genus Leishmania as the causative agent of leishmaniasis occurred in the modern age. However, evolutionary studies suggest that the origin of Leishmania can be traced back to the Mesozoic era. Subsequently, during its evolutionary process, it achieved worldwide dispersion [...] Read more.

The description of the genus Leishmania as the causative agent of leishmaniasis occurred in the modern age. However, evolutionary studies suggest that the origin of Leishmania can be traced back to the Mesozoic era. Subsequently, during its evolutionary process, it achieved worldwide dispersion predating the breakup of the Gondwana supercontinent. It is assumed that this parasite evolved from monoxenic Trypanosomatidae. Phylogenetic studies locate dixenous Leishmania in a well-supported clade, in the recently named subfamily Leishmaniinae, which also includes monoxenous trypanosomatids. Virus-like particles have been reported in many species of this family. To date, several Leishmania species have been reported to be infected by Leishmania RNA virus (LRV) and Leishbunyavirus (LBV). Since the first descriptions of LRVs decades ago, differences in their genomic structures have been highlighted, leading to the designation of LRV1 in L. (Viannia) species and LRV2 in L. (Leishmania) species. There are strong indications that viruses that infect Leishmania spp. have the ability to enhance parasitic survival in humans as well as in experimental infections, through highly complex and specialized mechanisms. Phylogenetic analyses of these viruses have shown that their genomic differences correlate with the parasite species infected, suggesting a coevolutionary process. Herein, we will explore what has been described in the literature regarding the relationship between Leishmania and endosymbiotic Leishmania viruses and what is known about this association that could contribute to discussions about the worldwide dispersion of Leishmania. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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26 pages, 2916 KiB

Open AccessReview

Trypanosoma Cruzi Genome: Organization, Multi-Gene Families, Transcription, and Biological Implications

by Alfonso Herreros-Cabello, Francisco Callejas-Hernández, Núria Gironès and Manuel Fresno

Genes 2020, 11(10), 1196; https://doi.org/10.3390/genes11101196 - 14 Oct 2020

Cited by 26 | Viewed by 5131

Abstract

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people. Although its first genome dates from 2005, its complexity hindered a complete assembly and annotation. However, the new sequencing methods have improved genome annotation of some strains elucidating the broad genetic [...] Read more.

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people. Although its first genome dates from 2005, its complexity hindered a complete assembly and annotation. However, the new sequencing methods have improved genome annotation of some strains elucidating the broad genetic diversity and complexity of this parasite. Here, we reviewed the genomic structure and regulation, the genetic diversity, and the analysis of the principal multi-gene families of the recent genomes for several strains. The telomeric and sub-telomeric regions are sites with high recombination events, the genome displays two different compartments, the core and the disruptive, and the genome plasticity seems to play a key role in the survival and the infection process. Trypanosoma cruzi (T. cruzi) genome is composed mainly of multi-gene families as the trans-sialidases, mucins, and mucin-associated surface proteins. Trans-sialidases are the most abundant genes in the genome and show an important role in the effectiveness of the infection and the parasite survival. Mucins and MASPs are also important glycosylated proteins of the surface of the parasite that play a major biological role in both insect and mammal-dwelling stages. Altogether, these studies confirm the complexity of T. cruzi genome revealing relevant concepts to better understand Chagas disease. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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24 pages, 1406 KiB

Open AccessReview

Of Drugs and Trypanosomatids: New Tools and Knowledge to Reduce Bottlenecks in Drug Discovery

by Arijit Bhattacharya, Audrey Corbeil, Rubens L. do Monte-Neto and Christopher Fernandez-Prada

Genes 2020, 11(7), 722; https://doi.org/10.3390/genes11070722 - 29 Jun 2020

Cited by 32 | Viewed by 5236

Abstract

Leishmaniasis (Leishmania species), sleeping sickness (Trypanosoma brucei), and Chagas disease (Trypanosoma cruzi) are devastating and globally spread diseases caused by trypanosomatid parasites. At present, drugs for treating trypanosomatid diseases are far from ideal due to host toxicity, elevated [...] Read more.

Leishmaniasis (Leishmania species), sleeping sickness (Trypanosoma brucei), and Chagas disease (Trypanosoma cruzi) are devastating and globally spread diseases caused by trypanosomatid parasites. At present, drugs for treating trypanosomatid diseases are far from ideal due to host toxicity, elevated cost, limited access, and increasing rates of drug resistance. Technological advances in parasitology, chemistry, and genomics have unlocked new possibilities for novel drug concepts and compound screening technologies that were previously inaccessible. In this perspective, we discuss current models used in drug-discovery cascades targeting trypanosomatids (from in vitro to in vivo approaches), their use and limitations in a biological context, as well as different examples of recently discovered lead compounds. Full article

(This article belongs to the Special Issue Kinetoplastid Genomics and Beyond)

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