Novel functions of rhomboid proteases during development of the rodent malaria parasite

Abstract

Plasmodium, a protozoan parasite in the phylum Apicomplexa is the etiological agent of malaria, an infectious disease that causes major morbidity and mortality. The Plasmodium life cycle is split between two hosts: the sexual cycle within an Anopheles mosquito and the asexual cycle within a vertebrate host. During transfer from vertebrate host to the mosquito, parasites replicate in the mosquito midgut within an encapsulated oocyst to produce thousands of sporozoites. The sporozoite is the infective form of the parasite that is transmitted from the mosquito to the vertebrate host. The sporozoites invade hepatocytes and replicate into thousands of merozoites within a non-fusigenic membrane-bound compartment called the parasitophorous vacuole (PV). Rhomboid proteins are intramembrane serine proteases that cleave substrate transmembrane domains within the lipid bilayer. The Plasmodium genome encodes eight rhomboid proteases. Three of these, ROM1, ROM3, and ROM4, are conserved in the Apicomplexa. We used the rodent malaria model, Plasmodium yoelii, to dissect function of ROM1 and ROM3 throughout the entire life cycle using a reverse genetics approach. ROM1 localizes to secretory organelles involved during invasion called the micronemes and and transcripts are expressed in all stages of the life cycle with a 20-fold upregulation in the salivary gland sporozoites. Pyrom1(-) parasites are attenuated in erythrocytic and hepatic stages. Intracellular development of pyrom1(-) sporozoites within hepatocytes decreases gradually in the first 24 hours. The decrease in pyrom1(-) parasite fitness is due to the improper establishment and modification of the PV. A fraction of the developing pyroml (-) parasites exhibit morphological differences at the ultrastructural level and display decreased targeting of a PV protein, UIS4. ROM3 is sex-stage specific and pyrom3(-) parasites cannot transmit infection to the mammalian host. Mutant oocysts are unable to undergo sporulation and appear empty and degenerate. Ultrastructural analysis reveals that pyrom3(-) oocysts are defective in sporoblast formation and the cytoplasm displays an abundance of acidocalcisomes, membranous whorls, and enlarged nuclei. The pyrom3(-) phenotype is likely a defect in the oocyst secretory pathway. Thus, we shed new light on novel functions of two Rhomboid proteases, ROM1 and ROM3, during development of the Plasmodium parasite replicative stages.