Diverse telomeres in trypanosomatids

Abstract

Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the ‘end replication and protection’ problems, associated with linearity. At the nucleotide level, telomeres typically represent stretches of tandemly arranged telomeric repeats, which vary in length and sequence among different groups of organisms. Recently, a composition of the telomere-associated protein complex has been scrutinized in Trypanosoma brucei. In this work, we subjected proteins from that list to a more detailed bioinformatic analysis and delineated a core set of 20 conserved proteins putatively associated with telomeres in trypanosomatids. Out of these, two proteins (Ku70 and Ku80) are conspicuously missing in representatives of the genus Blastocrithidia, yet telomeres in these species do not appear to be affected. In this work, based on the analysis of a large set of trypanosomatids widely different in their phylogenetic position and life strategies, we demonstrated that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. Our analysis showed that the expression of two proteins predicted to be associated with telomeres (those encoding telomerase and telomere-associated hypothetical protein orthologous to Tb927.6.4330) may directly affect and account for the differences in telomere length within the species of the Leishmania mexicana complex.

Introduction

Trypanosomatidae is a family of protozoan parasites possessing a single large mitochondrion, which encompasses a network of catenated circular DNA molecules, the so-called kinetoplast or kDNA (Maslov et al., 2019). These species have been attracting research attention because of numerous unique or rare biochemical and molecular traits, such as trans-splicing and polycistronic transcription (Clayton, 2019; Michaeli, 2011), mitochondrial RNA editing (Aphasizheva et al., 2020), presence of modified nucleotides (van Luenen et al., 2012) and unusual organelles (Szöör et al., 2014; Docampo, 2016), or a bizarre variation of the nuclear genetic code (Záhonová et al., 2016). Most of these flagellates are monoxenous (with one host in their life cycle) parasites restricted to invertebrates (Maslov et al., 2013), while members of the genera Endotrypanum, Leishmania, Phytomonas, Porcisia and Trypanosoma have switched to dixeny (two-host life cycle) and infect vertebrates or plants in addition to invertebrates (Lukeš et al., 2018). It is established beyond a reasonable doubt that the dixenous species have evolved from the monoxenous ancestor(s) independently several times (Lukeš et al., 2014). Notably, several Leishmania and Trypanosoma spp. are of medical importance, as they cause severe human diseases, and are fairly well-studied (Stuart et al., 2008; Nussbaum et al., 2010).

Telomeres typically represent repetitive physical ends of linear eukaryotic chromosomes, variable in length and sequence in different groups of organisms (Fulnečková et al., 2013). Their main role is to protect chromosome ends from being recognized and processed as DNA double-strand breaks by the cellular repair machinery in order to prevent chromosomal end-to-end fusions (Pfeiffer and Lingner, 2013). Such shielding is provided by the telomere-associated protein complexes (Lewis and Wuttke, 2012) or specific complementary DNA structures, such as telomere loops (t-loops) facilitating the protection of chromosome ends (Tomáška et al., 2019). It is generally assumed that telomeres undergo gradual shortening with each round of cell division because of incomplete lagging strand synthesis of linear DNA templates by DNA polymerases, known as the ‘end replication problem’ (Olovnikov, 1973; Greider, 1990; Hackett and Greider, 2002). In order to overcome this problem and, thus, prevent telomere shortening, cells engage a dedicated enzyme called telomerase (Greider and Blackburn, 1985).

Telomeres of kinetoplastids share many traits with those of other eukaryotes. They have the canonical sequence (5′-ttaggg-3′) found in vertebrates, end with a t-loop, are associated with capping protein complexes and maintained by telomerases (Muñoz-Jordán et al., 2001; Conte and Cano, 2005; Fulnečková et al., 2013). Similar to the situation in other eukaryotic pathogens, genes encoding trypanosomatid virulence factors are often located in the sub-telomeric regions and their expression may be co-regulated with telomeres (Chiurillo et al., 1999; Dobson et al., 2006; Hovel-Miner et al., 2012). Moreover, transposable elements are often found in association with telomeres (Pardue et al., 1997; Rahnama et al., 2020). In agreement with this, a sub-telomeric region of Leptomonas pyrrhocoris chromosome contains an integrated copy of an RNA-dependent RNA polymerase putatively originating from an RNA virus of the family Tombusviridae infecting this flagellate (Grybchuk et al., 2018), and possibly contributing to the retrotransposon translocation within the trypanosomatid genome. Telomeric regions of kinetoplastid chromosomes also possess several features distinguishing them from their counterpart in most of the other eukaryotes. For example, the telomeres of Trypanosoma brucei increase in length (by approximately 10 bp per generation) until they reach an equilibrium (Bernards et al., 1983; Pays et al., 1983; Horn et al., 2000). In trypanosomatids, a modified nucleobase, base J (β-D-gluco-pyranosyl-oxy-methyl-uracil) is involved in RNA polymerase II transcription termination and is preferentially localized to telomeres (Borst and van Leeuwen, 1997; Genest et al., 2007; van Luenen et al., 2012).

To the best of our knowledge, there has been very little systematic effort to analyse telomeres in trypanosomatids outside the medically relevant Trypanosoma and Leishmania spp. (Fu et al., 1998; Fu and Barker, 1998a, 1998b; Chiurillo et al., 1999, 2002; Muñoz-Jordán et al., 2001; Janzen et al., 2004; Conte and Cano, 2005; Genest and Borst, 2007). Therefore, we decided to do that for a wide range of trypanosomatids with a special emphasis on largely neglected parasites of insects, which are not pathogenic to humans. We selected more than 20 proteins from a set of recently defined putative trypanosomatid telomere-associated proteins (Reis et al., 2018) for more detailed in silico analyses. For most of these proteins, some functional information is available [Table 1; the TriTrypDB (Aslett et al., 2010) gene IDs are used throughout the text]. The predicted telomere-associated complex appears to be a cohort of proteins with widely variable functions, from ribosome and proteasome subunits to telomerase and even DNA repair proteins (Boulton and Jackson, 1998; Paugam et al., 2003; Riha and Shippen, 2003; Janzen et al., 2004; Dreesen et al., 2005; Ruvinsky and Meyuhas, 2006; Chico et al., 2011; Sandhu et al., 2013; Nenarokova et al., 2019). In this work, we analysed the evolutionary history of telomere-associated proteins in Kinetoplastea, performed a systematic analysis of telomere length variation among trypanosomatids on the dataset, which incorporates a wide range of understudied monoxenous members of the family Trypanosomatidae, and established a correlation between the level of transcription for several analysed telomere-associated proteins and the telomere length.

Table 1. Predicted telomere-associated protein complex composition in T. brucei

Materials and methods

In silico analyses

A putative set of telomere-associated proteins of T. brucei brucei TREU927 (Reis et al., 2018) were used as queries for BLAST searches (Altschul et al., 1990) against a dataset of annotated proteins of 64 trypanosomatids and the eubodonid Bodo saltans. First, BLASTp searches were performed with an E-value set to 1 and all the hits with an E-value not exceeding 10⁻¹⁵ were retained. If the respective sequence was not identified among annotated proteins, the searches were repeated with the tBLASTn algorithm against a database of genome sequences. In case no protein was identified in the genome, HMMER v.3.3 (Eddy, 2009), a more sensitive method for the identification of divergent homologues based on hidden Markov models was employed. Annotated proteins and assembled genome sequences were downloaded from the NCBI Genome (Sayers et al., 2019) and TriTrypDB v. 45/46 (Aslett et al., 2010) databases. The validity of the hits was confirmed using reciprocal BLAST searches against T. brucei proteins and alignments including the query and all identified proteins, if necessary. The resulting gene presence/absence table and a cladogram manually written in a Newick format based on recent publications (Butenko et al., 2019; Kostygov et al., 2014, 2020; Kostygov and Yurchenko, 2017; Lukeš et al., 2018; Frolov et al., 2019; Kato et al., 2019) were used for Dollo parsimony analysis in the Count software (Csűrös, 2010) and results were visualized in a graphical editor.

Trypanosomatid isolates and cultivation

Cultures of Crithidia expoeki (BJ08.175), C. fasciculata (Cf-Cl), C. fasciculata (ATCC20358), C. termophilla (ATCC30817), L. pyrrhocoris (H10), L. seymouri (ATCC30220), Novymonas esmeraldas (E262AT), Strigomonas oncopelti (TCC290E) and Zelonia costaricensis (15EC) were grown in BHI medium (Oxoid/Thermo Fisher Scientific, Basingstoke, UK) supplemented with 2 μg mL⁻¹ Hemin (Sigma-Aldrich, St. Louis, USA) and 50 units mL⁻¹ of Penicillin/Streptomycin (BioSera, Nuaillé, France) at 23°C. Cultures of Endotrypanum monterogeii (ATCC30507), Herpetomonas samuelpessoai, Leishmania (Leishmania) major (LV39), L. (L.) amazonensis (Josefa, LV78, LV79 and PH8), L. (L.) mexicana (M379), L. (Mundinia) martiniquensis (LEM2494), L. (M.) orientalis (PCM2), L. (Sauroleishmania) tarentolae (ParrotTarII), L. (Viannia) braziliensis (human2017), L. (V.) guyanensis (M4147), Phytomonas sp. (EM1), Porcisia deanei (TCC258) and P. hertigi (TCC260) were grown in M199 medium supplemented with 2 μg mL⁻¹ Biopterin, 2 μg mL⁻¹ Hemin (all Sigma-Aldrich), 25 mm HEPES (Lonza, Basel, Switzerland), 50 units mL⁻¹ of Penicillin/Streptomycin (BioSera) and 10% fetal bovine serum (BioWest, Nuaillé, France) at 23°C. Cultures of Angomonas deanei (CT-IOC 044), A. desouzai (CT-IOC 109), Blastocrithidia sp. (p57), Blastocrithidia triatomae, Blechomonas ayalai (B08-376), Blechomonas pulexsimulantis (ATCC50186), Herpetomonas muscarum (MMO-01), Jaenimonas sp. (Finn-01.02), Kentomonas sorsogonicus (MF-08.02), Lafontella sp. (GMO-01), Vickermania ingenoplastis (CP21), Wallacemonas collosoma (ATCC30261) and W. rigidus (PL11) were maintained in SDM medium (BioWest) supplemented with 10% fetal bovine serum (BioWest) and 50 units mL⁻¹ of Penicillin/Streptomycin (BioSera) at 23°C. In the cases of Lafontella and Endotrypanum, cultures were grown in a bi-phasic medium, overlaying blood agar. All species were validated by amplifying and sequencing the 18S rRNA gene as described previously (Kostygov et al., 2014).

Quantification of transcription level of genes encoding telomeric proteins using RT-qPCR

RNA was isolated and transcript levels of the telomeric proteins were assessed by RT-qPCR as described previously (Záhonová et al., 2014; Kraeva et al., 2019). Sequences of the specific primers for L. mexicana/amazonensis orthologues of T. brucei genes are listed in Supplementary Table 1. Expression values were normalized to those of 18S rRNA.

Southern blotting

The previously established terminal restriction fragment analysis of telomere lengths protocol was followed (Janzen et al., 2004). In brief, total genomic DNA from the log-phase grown cells was isolated and digested with AluI, HinfI and RsaI overnight. Restriction fragments were separated in 0.75% agarose gel, transferred to a ZetaProbe blotting membrane (Bio-Rad, Hercules, USA), probed with the DIG-labelled telomeric probe [CCCTAA]_x25 in the DIG Easy Hyb buffer (Roche Diagnostics, Indianapolis, USA), and visualized with the DIG Luminescent Detection Kit (Roche Diagnostics). The probe was labelled by the Dioxigenin NT Labeling Kit (Jena Bioscience GmbH, Jena, Germany). Statistics of the telomere lengths were obtained with an online tool WALTER (Web-based Analyser of the Length of TElomeRes) (Lyčka et al., 2021). For the loading and integrity control in the L. mexicana complex analysis, DNAs were processed as above, and the membrane was probed against a fragment of a gene encoding telomerase (LmxM.36.3930) (Supplementary Table 1).

Results and discussion

The core set of proteins putatively involved in telomere maintenance in kinetoplastids is conserved

To study the phylogenetic distribution of proteins predicted to be involved in telomere maintenance (Reis et al., 2018), we analysed the presence/absence of the corresponding 24 genes in the available genomes of trypanosomatids and their close eubodonid relative, B. saltans (Table 2). Most of the studied proteins (20 of 24) are well conserved and we consider them as a core set putatively involved in telomere maintenance in kinetoplastids. It is worth noting that the telomere association and function in telomere maintenance has already been confirmed for some of these proteins, while some others have not been functionally characterized yet. Thus, the composition of the core set of proteins involved in telomere maintenance, as defined previously (Reis et al., 2018) and discussed herein, should be taken with caution. Despite the fact that most of the respective genes are conserved across Kinetoplastea (Fig. 1, Table 2) and, thus appear to be present in the kinetoplastid common ancestor, we came across several interesting exceptions that are discussed in detail below.

Fig. 1. Gains and losses of genes encoding putative telomere-associated proteins in kinetoplastids.

Table 2. Presence of genes putatively involved in telomere maintenance in kinetoplastids

Species analysed by Southern blotting are shaded.

+, identified; empty, not identified; *, identified in strain UA301.

A set of three proteins (orthologues of T. brucei Tb927.3.1560, Tb927.9.5020 and Tb927.11.370) was acquired by the common ancestor of trypanosomatids upon the separation from bodonids (Fig. 1). One of them, Tb927.3.1560 [TIF-2, an orthologue of mammalian TINF2 (Jehi et al., 2014a; 2014b)] was suggested to be essential, as it is involved in shelterin (a protein complex implicated in telomere protection) assembly and telomerase-mediated telomere length maintenance in other organisms (Walne et al., 2008; Frank et al., 2015). Yet, it is not present in bodonids and was secondarily lost in all other trypanosomatids outside of the genera Paratrypanosoma, Trypanosoma and Blechomonas (Fig. 1), raising a question of how do they cope with its absence or whether they replaced it with a functional analogue? Tb927.2.6100 is Trypanosoma-specific, confirming previous report (Beck et al., 2013). Surprisingly, this protein was shown to be specifically associated with kDNA, so its role in telomere maintenance, if any, remains to be elucidated by functional genetics approaches. Two proteins (orthologues of T. brucei Tb927.9.3930 and Tb927.9.4000) are present only in four species of the T. brucei group and may determine specific traits of these parasites.

An orthologue of Tb927.11.9870 (TelAP-1) is present in most species, but it is conspicuously absent from the representatives of two monoxenous groups (Blastocrithidia and Vickermania spp.) and most Phytomonas spp., plant pathogens with streamlined genomes (Porcel et al., 2014). While we cannot rule out a possibility that the protein is divergent beyond recognition by available bioinformatics tools, there may exist another component fulfilling the role of TelAP-1 in these species.

Of special attention is the absence of Tb927.3.5030 (Ku70) and Tb927.3.5030 (Ku80) orthologues in Blastocrithidia sp., which has a non-canonical nuclear genetic code with all three stop codons reassigned to encode amino acids (Záhonová et al., 2016). It has been recently proposed that such an absence may lead to the accumulation of numerous insertions in many protein-coding genes of these organisms (Nenarokova et al., 2019).

Trypanosomatid telomeres are variable in length

We performed a systematic screen of the telomere length across Trypanosomatidae by Southern blotting (Fig. 2, Table 3). Our analysis revealed that monoxenous Leishmaniinae (Kostygov and Yurchenko, 2017) of the genera Leptomonas, Novymonas and Zelonia have fairly short telomeres (weighted medians 900–1200 bp; hereafter only rounded weighted median data are compared in the text, see Table 3 for minimum and maximum values), while telomeres in analysed Crithidia spp. ranged from 400 bp in C. fasciculata Cf-C1 to 4300 bp in C. expoeki. These numbers correlate well with previous reports on telomere length in the selected representatives of the genera Crithidia, Leishmania and Trypanosoma (Genest et al., 2007). Of note, the repertoire of genes implicated in telomere maintenance is identical in these flagellates (Table 2), so these differences can be explained by either the presence of other proteins involved in this process, or (more likely) differences in gene expression. Telomeres of Blechomonas, Herpetomonas, Jaenimonas and Wallacemonas spp. are 1300–2100 bp long. The endosymbiont-containing Strigomonadinae [Angomonas, Kentomonas and Strigomonas spp. (Votýpka et al., 2014)] differ in telomere length, with S. oncopelti bearing the shortest chromosome ends of ~400 bp.

Fig. 2. Southern blotting analysis of telomere repeats in selected species of Trypanosomatidae. Marker sizes are indicated on the left. The vertical lines denote a composite image from the same blot. DNA integrity controls are presented in Supplementary Fig. 1 (left and middle panels).

Table 3. Telomere lengths (weighted median, minimum–maximum) in selected species of Trypanosomatidae

Representatives of three genera (Blastocrithidia, Leishmania and Trypanosoma) deserved special attention. Uniquely among trypanosomatids, Blastocrithidia spp. lack Ku proteins (Nenarokova et al., 2019), yet their telomeres are of similar length to telomeres of other trypanosomatids (600 and 1500 bp in B. triatomae and Blastocrithidia sp., respectively), arguing that either Ku proteins are dispensable for the telomere length maintenance in these species, or their loss can be compensated by other factors. Telomere sizes vary in different Trypanosoma spp. represented by short telomeres in T. mega (400 bp) and substantially longer telomeres in T. brucei Lister 427 (3100–3400 bp). In contrast to the previous report (Dreesen and Cross, 2008), we did not document differences in telomeres' length between the procyclic and bloodstream stages of T. brucei. However, both strains in our analysis have the same origin (Lister 427), while the abovementioned study compared Lister 427 and TREU927 strains. Similar to the cases discussed above, despite possessing the same repertoire of telomere-bound proteins, the distribution of telomere sizes in the Leishmania–Porcisia–Endotrypanum clade (Espinosa et al., 2018) is wide, exemplified by two extreme cases of P. hertigi (5000 bp) and L. mexicana (400 bp, Fig. 2). Variable telomere length in Leishmania spp. (and possibly other Leishmaniinae) may be explained by the presence of a stress-sensitive telomere-proximal replication activity outside S phase of the cell cycle in these species (Damasceno et al., 2020, 2021).

RNA level of telomerase and several telomere-associated proteins correlates with telomere length in the species of L. mexicana complex

We analysed telomere length and expression of the core set of proteins putatively involved in telomere maintenance in closely related species forming the L. mexicana complex (Eresh et al., 1994). Similar to the cases discussed above, telomeres in L. mexicana and four isolates of L. amazonensis greatly differed in length from ~400 bp in L. mexicana M379 to ~3400 bp in L. amazonensis LV79 (Fig. 3, Table 3, Supplementary Fig. 1). Such a wide range of telomere lengths correlated well with the expression of the Leishmania spp. telomerase (orthologue of Tb927.11.10190) and a telomere-associated hypothetical protein (orthologue of Tb927.6.4330). The higher expression of these proteins correlated with longer telomeres. The specific roles of these and other proteins remain to be further elucidated by functional studies.

Fig. 3. Transcript levels of telomere-associated proteins and telomere lengths in the species of L. mexicana complex. (A) Quantitative RT-PCR analysis of the core set of proteins implicated in telomere maintenance. Gene expression is presented as normalized means and standard deviations of three replicates. Data are presented in two graphs to account for differences in expression values. (B, C) Southern blotting analysis of telomere repeats (B) and telomerase-encoding gene (C, used as an additional DNA integrity control) in L. amazonensis LV78, LV79, PH8, Josefa and L. mexicana M379. Marker sizes are indicated on the left. DNA integrity controls are presented in Supplementary Fig. 1 (right panel).

Conclusions

The genome analysis has allowed us to identify a core set of 20 conserved proteins predicted to be responsible for telomere maintenance in trypanosomatids. Several proteins, previously identified in T. brucei pull-downs, are trypanosome-specific. Out of 20 proteins conserved in Trypanosomatidae, two (Ku70 and Ku80) are conspicuously missing in Blastocrithidia spp., yet telomeres in these species do not appear to be affected by their loss. We documented that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. One such group is a complex of species, related to L. mexicana. Our analysis demonstrated that the expression of several telomere-associated proteins correlates with the documented differences in telomere length within species of the L. mexicana complex, which is indicative of a potential role these proteins may play in the telomere length maintenance.