Leishmania parasites alternate between hosts, facing environmental changes that demand rapid gene expression adaptation. Lacking canonical RNA polymerase II promoters, transcription in these eukaryotes is polycistronic, with gene regulation occurring post-transcriptionally. Although non-coding RNAs (ncRNAs) have been identified in Leishmania transcriptomes, their functions remain unclear. Recognizing RNA structure's importance, we performed a genome-wide alignment of L. braziliensis and related species, identifying conserved RNA structures, 38 of which overlap with known ncRNAs. One such ncRNA, lncRNA45, was functionally characterized. Using a knockout cell line, we demonstrated that lncRNA45 is crucial for parasite fitness. Reintroducing the wild type lncRNA45 restored fitness, while a version with a single nucleotide substitution in the structured region did not. This mutation also altered RNA-protein interactions. These findings suggest that lncRNA45's regulatory role and protein interactions rely on its secondary structure. This study highlights the significance of structured lncRNAs in Leishmania biology and their potential as therapeutic targets. Further research into these ncRNAs could uncover new parasite regulation mechanisms and inspire novel treatment strategies.

Leishmania parasites alternate between hosts, facing environmental changes that demand rapid gene expression adaptation. Lacking canonical RNA polymerase II promoters, transcription in these eukaryotes is polycistronic, with gene regulation occurring post-transcriptionally. Although non-coding RNAs (ncRNAs) have been identified in Leishmania transcriptomes, their functions remain unclear. Recognizing RNA structure's importance, we performed a genome-wide alignment of L. braziliensis and related species, identifying conserved RNA structures, 38 of which overlap with known ncRNAs. One such ncRNA, lncRNA45, was functionally characterized. Using a knockout cell line, we demonstrated that lncRNA45 is crucial for parasite fitness. Reintroducing the wild-type lncRNA45 restored fitness, while a version with a single nucleotide substitution in the structured region did not. This mutation also altered RNA-protein interactions. These findings suggest that lncRNA45's regulatory role and protein interactions rely on its secondary structure. This study highlights the significance of structured lncRNAs in Leishmania biology and their potential as therapeutic targets. Further research into these ncRNAs could uncover new parasite regulation mechanisms and inspire novel treatment strategies. ### Competing Interest Statement The authors have declared no competing interest.

Trypanosomatids are early-divergent eukaryotes that have adapted to parasitism. During their life cycles, these parasites switch between a mammalian and an invertebrate host, and the ability to adapt their metabolism to different nutritional sources is instrumental for their success. In the invertebrate host, these protists have access to high amounts of amino acids and efficiently utilise it for energy production. Proline is a particularly efficient energy source for trypanosomes. Glutamate is also efficiently used by Trypanosoma cruzi and can be converted into proline as part of the glutamate-proline pathway prior to its intramitochondrial catabolism. By employing a series of genetic modifications and functional analysis, we show here that Leishmania parasites, the causative agents of leishmaniases, can utilise proline, glutamate and glutamine as energy sources, and although these parasites possess all the genes necessary for the biosynthesis of proline from glutamate, this pathway has, at best, limited function, with at least one of its components (pyrroline-5-carboxylate reductase) assuming divergent functions in different life cycle stages of the parasite. In fact, we show that the catabolism of glutamate is independent of proline biosynthesis and the former is most likely directly imported into the mitochondrion and catabolised to recover the cellular redox metabolism and increase mitochondrial membrane potential. Moreover, our data suggest a relevant role for glutamate dehydrogenase in nutritional stress response in Leishmania. These findings highlight relevant differences in amino acid metabolism between Trypanosoma and Leishmania and suggest a diversification in amino acid metabolic pathways within Trypanosomatidae.

The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5’ end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that the lack of ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3’UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3’UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Leishmania are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, Leishmania regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, Leishmania are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations. Typically, such events indicate genome instability in other eukaryotes, coinciding with genetic abnormalities, but for Leishmania, exploiting these products of genome instability can provide selectable substrates to catalyse necessary gene expression changes by modifying gene copy number. Unorthodox DNA replication, DNA repair, replication stress factors and DNA repeats are recognised in Leishmania as contributors to this intrinsic instability, but how Leishmania regulate genome plasticity to enhance fitness whilst limiting toxic under- or over-expression of co-amplified and co-transcribed genes is unclear. Herein, we focus on fresh, and detailed insights that improve our understanding of genome plasticity in Leishmania. Furthermore, we discuss emerging models and factors that potentially circumvent regulatory issues arising from polycistronic transcription. Lastly, we highlight key gaps in our understanding of Leishmania genome plasticity and discuss future studies to define, in higher resolution, these complex regulatory interactions.

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary among Leishmania species and that target protein methylation changes across different L. braziliensis life cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of the L. braziliensis PRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro , and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L . major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7 . It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L . major . Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of Lmj PRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi . Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies.

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a "regulatory code". Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Abstract RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

Background Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F 2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S ( Lbr PGF2S) expression and pathogenicity in mice. Methods Lbr PGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate Lbr PGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry- Lbr PGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. Results Leishmania braziliensis promastigotes synthesize prostaglandin F 2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. Lbr PGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of Lbr PGF2S leads to an increase of infectivity in vitro . The data suggest that Lbr PGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. Conclusions Lbr PGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of Lbr PGF2S in L. braziliensis . Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.

Through whole-genome sequencing analysis, we identified non-Leishmania parasites isolated from a man with a fatal visceral leishmaniasis–like illness in Brazil. The parasites infected mice and reproduced the patient’s clinical manifestations. Molecular epidemiologic studies are needed to ascertain whether a new infectious disease is emerging that can be confused with leishmaniasis.

In the last 30 years, significant advances in genetic manipulation tools along with complete genome and transcriptome sequencing have advanced our understanding of the biology of Leishmania parasites and their interplay with the sand fly and mammalian hosts. High-throughput sequencing in association with CRISPR/Cas9 have prepared the ground for significant advances. Given the richness of the progress made over the last decade, in this article, we focused on the most recent contributions of genome-wide and transcriptome analyses of Leishmania spp., which permit the comparison of life cycle stages, the evaluation of different strains and species in their natural niches and in the field and the simultaneously comparison of the gene expression profiles of parasites and hosts.