Stress-responsive Entamoeba topoisomerase II: a potential anti-amoebic target

Abstract

18Topoisomerases are a group of ubiquitous enzymes, involved in all key DNA processes and are 19 often stress responsive across the biological world. As a result, these enzymes are targets for 20 various anticancer and antibacterial agents. The causative organism of amoebiasis, E. histolytica 21 (Eh), has seven unexplored genes annotated as putative topoisomerases. One of the seven 22 topoisomerases in this parasite was found to be highly up-regulated during heat shock and 23 oxidative stress. The bioinformatic analysis shows that it is a eukaryotic type IIA topoisomerase. 24 Its ortholog in E. invadens (Ei), the encystation model of E. histolytica was also highly up-25 regulated during the late hours of encystation. Immunoprecipitated endogenous EhTopo II 26 showed topoisomerase II activity in vitro. Immunolocalization studies show that this enzyme 27 colocalized with newly forming nuclei during encystation, which is one of the significant events 28 of mature cysts. Double-stranded RNA mediated downregulation of the Topo II both in Eh and 29 Ei reduced the viability of actively growing trophozoites and also reduced the encystation 30 efficiency in Ei. Drugs, targeting eukaryotic topoisomerase II e.g., etoposide, ICRF193, and 31 amsacrine, show 3-5 times higher EC 50 in Eh than that of mammalian cells, although EhTopo II 32 is eukaryotic. Sequence comparison with human TopoIIα showed that key amino acid residues 33 involved in the interactions with ICRF193 and etoposide are naturally substituted in EhTopo II. 34 Interestingly, ciprofloxacin an inhibitor of prokaryotic DNA gyrase showed about six times less 35 EC 50 value in case of Eh in comparison to human cells. The parasite's notable susceptibility to 36 prokaryotic topoisomerase drugs in comparison to human cells opens up future scope to study 37 this invaluable enzyme in the light of an antiamoebic target. 38 39 40 2 Author Summary 41Amoebiasis, a globally prevalent parasitic infection, claims about 100,000 lives annually. The 42 disease is caused by the protozoan parasite E. histolytica, which can alternate between a 43 pathogenic trophozoite and a dormant cyst stage, within and outside the human host respectively. 44 Stage conversion to the resistant cyst i.e., encystation is the primary reason for the disease 45 transmission. There are no vaccines available to date, and broad spectrum nitroimidazole 46 derivatives are still the primary mode of treatment for this epidemic. However, the toxicity and 47 side effects of these drugs as well as increasing drug resistance in the parasites emphasize the 48 urgency for better treatment methods. Topoisomerases are vital enzymes required for all DNA 49 process and are extensively studied as targets for antibacterial and anticancer drugs. E. 50 histolytica has seven putative topoisomerases. Here, we identified that topoisomerase II of 51 Entamoeba is crucial not only for the growth of the trophozoite form but also for the mature cyst 52 formation. Additionally, we found that this enzyme is hig...