Abstract

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Highlights

  • Apicomplexa are a phylum of single-celled eukaryotes

  • Identification of candidate apicoplast proteins based on their gene expression profile To more fully understand the many functions of the apicoplast, we wished to establish a comprehensive catalog of organellar proteins

  • Similar time-ordered relationships leading to shared transcription patterns had previously been observed for organellar biogenesis during replication of Plasmodium merozoites in the red blood cell [34]

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Summary

Introduction

Apicomplexa are a phylum of single-celled eukaryotes. All members of the phylum are parasites and a number of species are responsible for important human (malaria and toxoplasmosis) and livestock (babesiosis, theileriosis and coccidiosis) diseases. Malaria is the most significant global health problem with a billion people at risk of infection and millions of cases annually. Treatment of malaria is constantly threatened by the ability of the parasite to rapidly develop drug resistance. Toxoplasma infections are even more common but do not usually result in overt disease. Severe toxoplasmosis occurs in immunocompromised individuals and during congenital infection. Toxoplasma has proven to be a reliable model for the study of those aspects of parasite biology that are shared among the members of the phylum

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