Leishmania Parasites Activate Alternative Telomere Maintenance When Key Proteins Are Lost
Removing Ku80 or TERT from Leishmania mexicana triggers alternative telomere lengthening, revealing unique genome stability strategies in this neglected-disease pathogen.
Summary
Researchers used Oxford Nanopore long-read sequencing to study telomere biology in Leishmania mexicana at unprecedented resolution. They found that deleting the DNA-repair protein Ku80 causes chromosome-specific telomere elongation driven by sequence-dependent recombination rates. This elongation was accompanied by elevated C-circles, a hallmark biomarker of Alternative Lengthening of Telomeres (ALT), a telomerase-independent maintenance pathway. Deletion of the telomerase catalytic subunit TERT, or both Ku80 and TERT together, produced similar ALT signatures. The findings reveal that L. mexicana promastigotes have an inherent capacity to activate ALT, and that Ku80 and TERT normally work together to suppress recombination-based telomere maintenance. This mechanism differs fundamentally from the related parasite Trypanosoma brucei, suggesting evolutionary divergence in telomere regulation within Trypanosomatidae.
Detailed Summary
Telomeres, the protective caps at chromosome ends, must be maintained to ensure genome stability across cell divisions. Most eukaryotes rely on telomerase, but some organisms activate Alternative Lengthening of Telomeres (ALT), a recombination-based backup mechanism. Understanding which proteins govern telomere maintenance in Leishmania mexicana—a protozoan parasite causing cutaneous leishmaniasis in over a million people annually—has direct implications for identifying drug targets and understanding parasite genome plasticity.
This study employed third-generation Oxford Nanopore Technologies (ONT) long-read sequencing on five L. mexicana cell lines: wild-type (WT), Ku80-null (ΔKu80), Ku80 addback (rescue), TERT-null (ΔTERT), and double-null (ΔTERTΔKu80). CRISPR-Cas9 was used to generate the knockouts. Genomes were assembled de novo with Flye, polished with Pilon, and annotated via Companion. Telomere-containing reads were extracted, mapped to 43 chromosome ends using 10-kb flanking sequences as queries, and telomere lengths were measured precisely beyond the subtelomeric homology boundary. C-circle assays and RT-qPCR for Rad51 expression were used to assess ALT activity.
Deletion of Ku80 produced telomere elongation that varied substantially between individual chromosomes, rather than a uniform genome-wide effect. This heterogeneity correlated with the sequence content and structure of the TTAGGG tandem repeats at each chromosome end, suggesting that local recombination rates—driven by sequence composition—determine elongation magnitude. Critically, elevated levels of C-circles were detected in ΔKu80 cells, directly implicating ALT pathway activation. ΔTERT cells showed a distinct pattern: telomere shortening was expected but ALT signatures, including C-circles, were also elevated, indicating that even without telomerase, recombination-based mechanisms can be engaged. The double ΔTERTΔKu80 mutants exhibited further enhancement of ALT markers, suggesting that the two proteins act in partially independent but overlapping suppressive roles.
A key finding is that even wild-type L. mexicana promastigotes exhibit low-level ALT activity, implying this is not a purely compensatory emergency response but rather a latent, readily activatable feature of Leishmania telomere biology. This stands in stark contrast to the related kinetoplastid Trypanosoma brucei, where Ku loss leads to telomere shortening rather than elongation, highlighting meaningful evolutionary divergence within the Trypanosomatidae family.
These results suggest that Ku80 and TERT together form a regulatory axis that suppresses recombinational telomere maintenance in Leishmania, and their loss de-represses ALT in a chromosome-specific and sequence-dependent manner. While this work is conducted in the promastigote (insect-stage) form and may not fully represent the amastigote (mammalian-stage) biology, it opens new avenues for targeting telomere maintenance as an antiparasitic strategy.
Key Findings
- Ku80 deletion causes chromosome-specific, not uniform, telomere elongation in L. mexicana, linked to local sequence-dependent recombination rates.
- Elevated C-circles—a highly specific ALT biomarker—accompany Ku80 loss, confirming ALT pathway activation.
- TERT deletion also triggers ALT signatures; double Ku80/TERT knockouts amplify this effect, suggesting additive suppression roles.
- Wild-type L. mexicana promastigotes show baseline ALT activity, indicating ALT is an inherent latent feature of Leishmania biology.
- Leishmania telomere regulation diverges fundamentally from T. brucei, where Ku loss causes shortening rather than elongation.
Methodology
Five L. mexicana CRISPR-Cas9-generated cell lines (WT, ΔKu80, addback, ΔTERT, ΔTERTΔKu80) were sequenced with Oxford Nanopore long-read technology; genomes were de novo assembled and telomere lengths measured per chromosome end from 43 mapped chromosome termini. ALT activity was assessed via C-circle assays and RT-qPCR for Rad51 expression.
Study Limitations
Experiments were conducted exclusively in promastigote (sandfly-stage) cells and may not reflect telomere dynamics in the clinically relevant amastigote (mammalian-stage) form. The study used a single L. mexicana strain, limiting generalizability across Leishmania species. Long-read sequencing error rates in repetitive telomeric sequences, though mitigated by specialized basecalling models, may introduce some measurement imprecision.
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