Leishmaniases are a group of neglected tropical diseases caused by more than twenty different species of parasites of the genus Leishmania, presenting a variety of symptoms and degrees of severeness. These protozoans parasites are at the mainstream of biology/medical studies since understanding their biological particularities is crucial for the future development of antiparasitic therapies. Moreover, the comprehension of their cell cycle and its molecular mechanisms is of great value for the search of new possible treatments. In that sense, telomeres emerge as a relevant subject in Leishmania spp. molecular biology research. They are the physical ends of eukaryotic chromosomes, granting stability to the genome and continued cell proliferation. Telomere elongation/maintenance is accomplished by a ribonucleoproteic complex called telomerase. The enzyme synthesis and adds repetitive telomeric sequences to the chromosome end termini using the 3’overhangs as substrates. In mammals, their gradual loss after many cell duplications can determine the cell fate by inducing replicative senescence or apoptosis. Here, we cover the main aspects of telomerase activity in Leishmania spp.
Telomerase is the ribonucleoprotein responsible for telomere elongation. The enzyme adds short repetitive sequences (e.g. 5’-TTAGGG-3’) at the single-stranded 3’G overhangs of each chromosome end. Telomerase is essential to regulating telomere lengths and, consequently, for genomic stability. Telomerase comprises two main components, the protein Telomerase Reverse Transcriptase (TERT) and Telomerase RNA (TER), which are minimally required for the enzyme activity in vitro .
TERT is the catalytic component, and TER contains the template used by TERT to synthesize the telomeric repeats. In vivo, these two components form a complex with accessory proteins, which are necessary for biogenesis, enzyme activity, and nucleotide addition processivity . The first report of telomerase activity in Leishmania spp. was reported by Cano et al. (1999), and later, the enzyme purification and its biochemical characterization were described by Giardini et al., (2011).
Telomerase activity was detected in all three parasite life forms (amastigote, procyclic promastigote, and metacyclic promastigote), presenting the canonical properties of other telomerases. However, the catalytic activity was shown to be temperature and life-stage dependent. The gene encoding the Leishmania spp. TERT was characterized by Giardini et al., (2006), and the TER component was described later by Vasconcelos et al., 2014.
In trypanosomatids, as in most eukaryotes, the TERT component is structurally composed of four structural/functional domains: The Telomerase Essential N-terminal (TEN), Telomerase RNA-Binding Domain (TRBD), Reverse Transcriptase domain (RT), and the C-terminal extension (CTE). The TEN domain is involved in telomerase recruitment to the telomeres and enzyme processivity. The TRBD interacts with TER and is connected to the TEN domain by an unstructured linker creating the RNA-binding pocket that binds single-stranded and paired RNA. Both domains can also interact with proteins that stabilize the complex and help to recruit telomerase to telomeres and to regulate enzyme activity . The RT is the catalytic core of the enzyme, which interacts with TER through the pseudoknot region, and is involved in the interactions with the RNA–DNA hybrid. Finally, the CTE domain stabilizes the RNA–DNA duplex, and differently from the other three domains, it is less conserved among different species
Leishmania TERT preserves all the canonical domains found in other TERTs but shows some amino acid substitutions specific to the genus. The knockout of Leishmania TERT seems to be harmful to the parasite since it induces a gradual decrease in cell density in the culture, apparent G1/G0 cell cycle arrest, morphological alterations, and telomere shortening (unpublished data).
The RNA component TER varies in length and sequence, presenting a conserved secondary structure in most eukaryotes. Variations of the TER size and sequence are observed among different organisms, and they are more prominent than TERT, which is conserved even among different taxa. In Leishmania spp., TER (LeishTER) is about ~2100 nucleotides long, and the mature molecule modified by trans-splicing contains a 5’ cap, a spliced leader sequence (SL), and a 3’ polyA tail. It also presents a conserved TBE (Template Boundary Element) motif C[U/C]GUCA in helix II, responsible for interacting with the TERT TRB domain and a C/D box snoRNA domain found in other TER. LeishTER is expressed at similar levels in procyclic and metacyclic promastigote forms. The mature molecule coimmunoprecipitates and colocalizes with the TERT component in a cell cycle-dependent manner. Its secondary structure prediction shows the template sequence (5’->3’) in a single-stranded form localized near the 5’ end of the RNA molecule. Its double knockout (KO) led to partial cell cycle arrest and increased apoptosis in procyclic promastigotes. TER KO also triggers a progressive telomere shortening during continuous parasite passages (unpublished data). A similar effect was observed in T. brucei TER knockouts.