Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector.

Nisha Philip,Andrew P Waters

doi:10.1016/j.chom.2015.05.018

Abstract

SummaryFunctional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle.

Highlights

While completing its complex life cycle, Plasmodium, the causative agent of malaria, experiences diverse host environments and undergoes remarkable variation in shape, size, and motility
We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin
Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions

Summary

Introduction

While completing its complex life cycle, Plasmodium, the causative agent of malaria, experiences diverse host environments and undergoes remarkable variation in shape, size, and motility. In contrast to six or seven CDPKs (depending on species), only two Plasmodium phosphatases, protein phosphatase 7 and calcineurin, have calcium-binding motifs, suggesting they modulate several calcium-dependent biological processes (Wilkes and Doerig, 2008). Calcineurin is a heterodimeric protein comprising of a catalytic (CnA) and calcium-binding regulatory (CnB) subunit (Rusnak and Mertz, 2000). Gene expression of both subunits is observed at specific life cycle points in both host and vector stages of the parasite, strongly suggesting a multistage functional profile for this phosphatase and a role in parasite transitions through the life cycle (Otto et al, 2014)

Results

Discussion

Conclusion