Leishmaniasis is a group of vector-borne neglected tropical diseases caused by the protozoan parasites of the genus Leishmania. The disease is transmitted to humans by infected sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World (Serafim et al., 2021). Leishmaniasis is a significant public health, social, and economic problem affecting the marginalized segments of the population in tropical and subtropical countries, with almost one billion people at risk of infection (Alvar et al., 2006). Although the reporting rate is low in some endemic countries, 205,986 cutaneous and 12,842 visceral leishmaniasis cases were reported to the WHO in 2022 (Ruiz-Postigo et al., 2023). Multiple factors hinder the achievement of the Sustainable Development Goals to control leishmaniasis, including the absence of effective treatment and vaccine (Bamorovat et al., 2024).

The clinical manifestations of the disease vary according to the parasite species, geographical distribution, and the patient's immune response. Leishmania infantum is the causative agent of fatal visceral leishmaniasis. It is also reported to cause cutaneous leishmaniasis in Brazil and Central American countries (Murray et al., 2005, Sasidharan and Saudagar, 2021). The parasite species, geographical regions, patient type, and poor patient adherence affect the efficacy of currently available treatments. In addition, actual medications are complicated by severe side effects, high cost, low accessibility, painful administration, and the requirement of trained healthcare professionals (Ghorbani and Farhoudi, 2017, Sundar et al., 2012). Therefore, developing new therapies to treat the disease and finding parasite-specific targets are urgent.

The linear chromosome ends of eukaryotic organisms are prone to two biological problems: the end-replication and end-protection problems, which are solved by telomerase and telomere-binding proteins. Telomeres are tracts of double-stranded (dsDNA) G-rich repetitive DNA that end with a G-rich single-stranded (ssDNA) 3′ overhang protrusion, whose length varies among species. Telomeres and associated proteins help maintain eukaryotes' genome stability by distinguishing normal chromosome ends from DNA double-strand breaks (DSB) (Blackburn et al., 2006, Zakian, 2012). The telomere repeats of most eukaryotic organisms, including Leishmania spp., are composed of the repetitive TTAGGG sequence that presents some variations in other eukaryotes, such as the ciliates (TTGGGG), and is elongated by telomerase (Assis et al., 2021, Cano, 2001, Moyzis et al., 1988, Zakian, 2012). Telomerase is a ribonucleoprotein (RNP) complex that comprises the catalytic unit known as telomerase reverse transcriptase (TERT), the integral RNA called telomerase RNA (TER), and associated proteins. The TERT adds the telomeric repeats to the 3′ G-rich overhang using the template sequence provided by the telomerase RNA with subsequent C-strand filling by DNA polymerase (Autexier and Lue, 2006, Blackburn and Collins, 2011, Feng et al., 2017, MacNeil et al., 2016).

Besides the elongation of telomeres by telomerase, several telomere binding proteins, some of which are species-specific, ensure telomere protection by regulating telomerase activity, enhancing the higher ordered secondary structure of telomeres (t-loop and G-quadruplex formation), and inhibiting a local DNA damage response (Arnoult and Karlseder, 2015, Hockemeyer and Collins, 2015). In humans, a group of six telomere-specific proteins form a complex called the shelterin complex that caps and maintains telomere homeostasis. The telomere repeat binding factors (TRF1, TRF2) bind to the telomeric dsDNA. TRF2 recruits the repressor activator protein 1 (RAP1), and this association represses homologous recombination in telomeres. The TRF1-interacting nuclear factor 2 (TIN2) forms the bridge between telomeric ssDNA and dsDNA binding proteins. In contrast, protection of telomere 1 (POT1) interacts with TPP1 (adrenocortical dysplasia protein, ACD, homolog) and binds to the telomeric ssDNA. (De Lange, 2018, Tesmer et al., 2023). POT1 is the protein that interacts with and protects the G-rich 3′ overhang, preventing a local response to DNA damage. The TPP1/POT1 subcomplex recruits telomerase for telomere elongation and is also involved in the enzyme's processivity (Cai et al., 2024, Li et al., 2023, Tesmer et al., 2023). The interaction of these proteins with the telomerase RNP is critical for enzyme recruitment, processivity, and telomere elongation during the S-phase of the cell cycle (Frank et al., 2015, Zhong et al., 2012).

In T. brucei, although no canonical shelterin is present, several functional analogs have been identified, including TbTRF, TbRAP1, TbTIF2, and, more recently, TelAP1, TelAP2, and Polε. TbTRF, TbRAP1, and TbTIF2 are essential for telomere integrity and subtelomeric VSG gene regulation, and TelAP1 and TelAP2 associate with the same complex composed of TRF, TIF2, and RAP1 and may perform some function in telomeres. While Polε, a translesion DNA polymerase, suppresses telomerase-mediated G-strand elongation and helps ensure proper telomere C-strand synthesis (Li, 2023). Apart from the telomerase components (TERT and TER), which preserve some structural and functional conservation (De Oliveira et al., 2024, Shiburah et al., 2025), with a few exceptions, Leishmania has a different set of telomere-associated proteins. Some ssDNA binding proteins, such as RPA-1, RBP38, and CalA1, were pulled down together with DNA polymerase alpha in telomerase-positive extracts (Fernández et al., 2004). All of them showed to interact in vitro and in vivo with telomeric DNA, and RPA-1 shares with human POT1 a canonical OBfold DNA-binding domain and other functional features, such as protecting the telomeric ssDNA from 3′ to 5′ exonuclease digestion and unfolding telomeric G-quadruplex (Fernandes et al., 2019, Morea et al., 2017, Neto et al., 2007, Fernandes et al., 2020). Also, two Myb-containing DNA-binding proteins were identified and partially characterized. One is a structural ortholog of the vertebrate TRF2 and TbTRF, and the other, TBP1, remains to be functionally characterized (Da Silva et al., 2010, Lira et al., 2007).

The first descriptions of the Leishmania telomeres organization were from L. braziliensis, L. major, L. lainsoni, and L. donovani (Fu and Barker, 1998, Chiurillo et al., 2000). All Leishmania sp. chromosome ends contain repetitive DNA at the telomeric region, which in L. major, L. amazonensis, and L. donovani are tandem repeats of the conserved hexameric sequence TTAGGG (Fu and Barker, 1998, Chiurillo et al., 2000, Chiurillo and Ramírez, 2002, Conte and Cano, 2005), but in L. braziliensis, are represented by repetitions of CCCTAACCCGTGGA sequences (Fu and Barker, 1998). The cloning of L. major and L. donovani chromosome ends revealed that their 3′ G-overhang comprises nine nucleotides (5′-GGTTAGGGT-OH 3′) (Chiurillo et al., 2000, Chiurillo and Ramírez, 2002), whereas in L. amazonensis, it is 12 nt long (5′-GTTAGGGTTAGG-3′) (Conte and Cano, 2005) and in L. donovani, the telomere terminates with 5′-GGTTAGGGT-3′ sequences (Chiurillo et al., 2000). Unusual telomeric repeats, such as single-nucleotide mutations in the canonical telomeric repeats, were also reported in other Leishmania species (Fu and Melville, 2002).

At the subtelomeric position are the Leishmania Conserved Telomere Associated Sequences (LCTAS), subdivided into two conserved sequence blocks (CSB1 and CSB2) (Fu and Barker, 1998, Conte and Cano, 2005). In L. braziliensis, LCTAS are in a single copy per chromosome end (Fu et al., 1998, Fu and Barker, 1998), whereas they are tandemly repeated in other Leishmania species (Fu and Barker, 1998, Conte and Cano, 2005). Besides LCTAS, L. braziliensis and L. donovani also contain other subtelomeric elements. A 1.6 kb and a 247 bp tandemly repeated sequence, for example, are specific to the L. braziliensis complex (Fu and Barker, 1998), and octamers and a 62 bp repeat are found at L. donovani subtelomeres (Chiurillo et al., 2000). The organization of LCTAS showed the polymorphic nature of the Leishmania chromosome ends (Fu and Melville, 2002), which is more concentrated at the subtelomeric regions.

In other Kinetoplastids, such as Trypanosoma brucei, all chromosome ends contain the canonical telomeric repeats (TTAGGG) and a short 30 bp long 3′ G-rich overhang terminating in 5′-GGGTTAGGG′-3′ (Cano, 2001, Dreesen et al., 2007, Ersfeld, 2011). A large subtelomeric region containing active or silent bloodstream or metacyclic VSG (Variant Surface Glycoprotein) genes is accompanied by a 70 bp repeat. Also, arrays of a telomere-derived 29-bp repeat separate telomeres from the subtelomeric AT-rich sequences in megabase and medium chromosomes (Weiden et al., 1991). The subtelomeric region of minichromosomes, in contrast, contains a 177 bp satellite DNA and the AT-rich sequences (Cano, 2001). Trypanosoma cruzi telomeres also contain the canonical hexameric repeats and share with T. brucei the same 3′ G-overhang sequence. Upstream to the telomeric repeat is a 198 bp telomere junction that separates telomeres from the subtelomeric region, which generally contains pseudogenes of the gp85 and rsh multigene families or pseudogenes from the dispersed gene family-1 (dgf-1) and retrotransposable elements (Chiurillo et al., 1999; Kim et al., 2005).

Despite telomeres being characterized in different species of Leishmania and Trypanosoma, there is no information about the L. infantum chromosome ends and telomere organization, although Leishmania infantum is considered a model organism for visceral leishmaniasis. Here, we uncovered the structure and complexity of the parasite's telomeric and subtelomeric regions using gold standard methods (i.e., Southern blot) and long-read Oxford nanopore sequencing (ONT). We showed that the chromosome ends of L. infantum share some similarities with L. donovani but present many species-specific peculiarities, such as a highly diverse and complex subtelomeric organization containing frequent octameric repeats intercalated by interstitial telomeric hexamers and a 62 bp Leishmania conserved telomere-associated sequence containing the CSB2 and other elements. Also, L. infantum telomeres show a different distribution dependent on the cell cycle phase. Moreover, a low percentage of telomeric variant repeats (TVRs) is found interspersed among the conserved TTAGGG hexameric repeats. This finding has some importance since TVRs can be implicated in alterations in the parasite telomere structure and function, and in protein binding sites at the telomeres. All these findings add valuable information to complete the L. infantum genomic map and can be used to explore telomere dynamics, which may ultimately help the drug design efforts aimed at disrupting the specific mechanisms that parasites use to maintain their genome stability.