Poster Presentation First Malaria World Congress 2018

Plasmodium falciparum conditional knockout parasites expressing exported NanoLuc luciferase allow investigation of events that precede merozoite egress (#411)

Hamille R. Melo 1 , Emily T. Faria 1 , Caroline L. Santos 1 , Gerhard Wunderlich 2 , Marcos L. Gazarini 1 , Brendan S. Crabb 3 4 5 , Paul R. Gilson 3 5 , Mauro F. Azevedo 1
  1. Federal University of Sao Paulo, Santos, SP, Brazil
  2. University of Sao Paulo, Sao Paulo, SP, Brazil
  3. Burnet Institute, Melbourne, Victoria, Australia
  4. University of Melbourne, Melbourne, Victoria, Australia
  5. Monash Unversity, Melbourne, Victoria, Australia

After growing and multiplying inside human erythrocytes, Plasmodium falciparum merozoites must exit the host cell through a tightly regulated process called egress. Parasite enzymes are known to act in concert dismantling parasitophorous vacuole (PV) and red blood cell (RBC) membranes, but the hierarchy of their action is not completely understood. The aim of this study was to establish a method that can be applied to identify compounds that affect egress and to investigate the sequence of the signalling pathways involved. In order to synchronize parasites in two key moments that precede egress, P. falciparum 3D7 parasites were engineered to express endogenous calcium dependent protein kinase 5 (CDPK5) or protein kinase G (PKG) in fusion with a destabilization domain (DD). Expression of each DD tagged kinase is dependent on the ligand Shld-1 and its removal reversibly blocks egress. Expression of the sensitive reporter nanoluc (NLuc) exported to the RBC was applied to quantify egress. As expected, removal of Shld-1 nearly completely blocked egress in both transgenic lines and when Shld-1 was reintroduced to cultures synchronized at 45-48 hours post invasion, Nluc activity in the supernatant increased in CDPK5-DD and PKG-DD cultures after 1 and 2 hours, respectively. Microscopic analysis confirmed the increase in reporter activity correlated to egress. Then, compounds known to inhibit egress were evaluated. The PLC inhibitor U73122, the calcium chelator BAPTA-AM and the promiscuous kinase inhibitor staurosporine, among others, completely blocked egress and reduced supernatant reporter activity in both lines to levels similar to parasites cultured without Shld-1. In contrast, other compounds such as the PkA inhibitor H-89 decreased reporter activity in PKG-DD but not in CDPK5-DD parasites. Application of this protocol might allow the identification of compounds that affect egress and elucidating when their targets are required respectively to PKG and CDPK5 activation.