Targeting topoisomerases for antileishmanial chemotherapeutics and deciphering the role of Mre11 in topoisomerase induced DNA damage repair in Leishmania donovani

Visceral Leishmaniasis or Kala‐azar, caused by Leishmania donovani , is the second largest cause of human death tally due to parasitic protozoan infection. Researchers across the globe are engaged in identifying and validating novel drug targets specific for Leishmania. Contextually, DNA topoisomerase IB of L. donovani (LdTopIB) has emerged as a promising candidate. Its unique bisubunit structure, completely different from single subunit structure of human topoisomerase I (HTopI), offers an interesting attribute to be exploited to develop novel therapeutics. In a programme to search for novel antileishmanials, our group has been probing into large array of compounds, both from natural and synthetic origin, to identify potential lead molecules. However, when subjected to topoisomerase stressors, the response machineries in trypanosomatid parasites including Leishmania remain largely unknown till date. Parasite death caused by LdTopIB poisons is fundamentally due to the generation of stabilized DNA‐enzyme covalent complex (Top1cc). When replication or transcription machineries collide with this Top1cc, it leads to DNA strand breaks. This molecular threat is detrimental for the parasite. In order to survive, the parasites need to repair this Top1cc and thereby rescue topoisomerase. We found that, among many factors, the Meiotic Recombination Protein 11 (Mre11) got over‐expressed in the parasite when it was subjected to camptothecin treatment. However, reports sugesting direct evidence of Mre11 to resolve Top1cc in vivo remain scarce till date. In the present investigation, we have identified, cloned and sequenced Mre11 gene (LdMre11) from L. donovani for the first time. The LdMre11 protein was expressed and the activity was assayed. We found that the recombinant protein could successfully cleave a phosphotyrosyl substrate in a magnesium dependent way due to its phosphodiesterase activity. The protein also possessed an exonuclease activity. This report is first of its kind to elucidate the possible phosphodiesterase activity of Mre11 protein from a member of trypanosomatid parasite. In order to study the biological significance of Mre11, Mre11 knockout parasites (LdMre11 −/− ) were next generated by targeted gene deletion method. Mre11 null parasites became hypersensitive to camptothecin. Ectopic expression of wildtype Mre11 in a null background reverted the mutant phenotype. Mre11 null parasites showed perturbed cell cycle progression with a clear arrest in G0/G1 phase in contrast to control cells. Results from immunoband depletion experiments using an antibody raised against the small subunit of LdTopIB clearly indicated that Mre11 null parasites accumulated larger amount of in vivo Top1cc in response to topoisomerase poisoning which was rescued in ectopically overexpressed Mre11 mutants. In addition, we have also found that Mre11 is overexpressed in drug resistant field isolates of L. donovani isolated from endemic zones of India. Interestingly, flow cytometry based experiments showed that Mre11 null parasites were hypersensitive to classical antileishmanial drugs viz. sodium antimony gluconate and miltefosine. Probably this is one of the probable mechanisms in Leishmania to achieve drug resistance. Cumulatively, the study provides the first mechanistic insight into the functioning of Mre11 in Leishmania donovani in response to topoisomerase IB poisoning and probably Mre11 also has a role in drug resistance. Support or Funding Information Department of Science and Technology(DST, Govt. of India) joint Indo‐Brazil project (DST/INT/Brazil/RPO‐01/2009/2) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .