The impact of knocking out the Leishmania major telomerase RNA (LeishTER): from altered cell proliferation to decreased parasite infectivity

The telomerase RNA, TER, is an intrinsic component of the telomerase ribonucleoprotein complex. It contains the telomere template sequence copied by the enzyme during telomere elongation. This unique molecule shows divergent nucleotide sequences but a more conserved secondary structure containing domains involved with telomerase assembly and biogenesis. The present work aims to characterize the biological roles played by the Leishmania TER component (LeishTER) in parasite homeostasis. We generated double knockout (LmTER-/-) parasites, which showed a distinct growth pattern at early passages, characterized by lower density and an extended stationary phase compared to the control. Although this pattern normalized after multiple in vitro passages, ablation of LeishTER affected cell division and proliferation, with cells arrested at the G0/G1 phase. Progressive telomere shortening was also observed during continuous passages, along with a reduction in the expression of TERRA29. Complementation with the episomal expression of LeishTER did not restore telomere length to the control levels, corroborating preliminary results showing that the overexpression of TER has a dominant negative effect on parasite lifespan. LmTER-/- also presented a higher percentage of gamma-H2A phosphorylation, likely due to stalled replication forks since no DNA damage was observed. Also, no plasma membrane modifications were detected, but pro-survival autophagic signals were present. Intriguingly, LmTER-/- retained the ability to transform into metacyclic forms, although its in vitro infectivity and growth inside the host cell were compromised. Together, these results highlight the importance of TER in parasite lifespan and open a discussion about its potential as a drug target against Leishmania.