Identification of Cdc45-interacting partners uncovers two Leishmania donovani proteins involved in DNA replication

Among fatalities due to parasitic diseases, deaths due to Leishmaniases rank second only to those caused by malaria. Endemic to 90 countries, more than a billion people are at risk of Leishmania infection. While there are no vaccines against Leishmaniases, treatments are available. However, the complexities of the treatment regimens, their high costs and toxic side effects, and most importantly the emerging problem of drug resistance, have continued to thrust forward research into this parasite's cellular processes. The parasite is digenetic, reproducing by binary fission in both hosts, with DNA replication being central to the process. The absence of many conserved DNA replication proteins in Leishmania suggests the possibility of parasite-specific proteins being involved in the process. This investigation aimed to identify such proteins.

Using Leishmania donovani Cdc45, a component of the eukaryotic replicative helicase, as bait in immunoprecipitation reactions coupled to mass spectrometry, we looked for proteins that are a part of the Leishmania replisome. One of the promising candidates, annotated "hypothetical protein" in the Leishmania genome database, shared partial structural homology with Timeless of human, C. elegans and Drosophila, and hence we named it "LdTIM-like". A second candidate, annotated "PIF1-like" in the genome database, was named "LdPIF6" based on sequence homology with T.brucei PIF6. Their possible involvement in DNA replication was examined by creating genomic knockouts and analyzing resultant phenotypes.

LdTIM-like and LdPIF6 were constitutively nuclear, in keeping with a possible role in DNA replication. We found tim-like and pif6 were essential for parasite survival. Single allele knockouts lowered tim-like/pif6 expression significantly, and resulted in slower parasite growth. The mutant parasites took considerably longer to navigate S phase, and pulse-labelling with EdU revealed that the time taken by the mutant cell populations to complete DNA replication was significantly extended. The mutant parasites survived poorly in host macrophages as well.

Considering that these two proteins: LdTIM-like and LdPIF6, are essential to the parasite, coupled to their limited homology with human proteins, they could be investigated as potential sites for therapeutic intervention in future in-depth studies.