A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Abstract

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.

Introduction

Intracellular pathogens have evolved to reside and proliferate within a multitude of host-cell types. Pore-forming proteins (PFPs) are emerging as a major part of the tool set used by a variety of prokaryotic and eukaryotic pathogens. PFPs play important roles in host-cell entry, nutrient acquisition, escape from the phagolysosomal system and final egress from the host cell [1]. Pathogenic bacteria employ PFPs to deliver activators of actin-mediated uptake mechanisms into the host cell leading to engulfment and internalization of the pathogen [2]. Protozoan pathogens, however, do not employ this strategy of induced passive uptake by non-phagocytic cells. While the protozoan parasite Leishmania is taken up passively by its phagocytic host cells [3] others like most apicomplexan parasites use a unique acto/myosin motor to actively enter their non-phagocytic host cells [4] and PFP involvement in this mode of entry has not been described. After host-cell entry the pathogen may grow and replicate in a vacuolar compartment [5], [6]. Life in a vacuole poses new challenges and PFPs may be used for the efficient uptake of nutrients across the vacuolar membrane [7] in order to sustain growth and replication or to avoid destruction by the host-cell phagolysosomal system [8]. Finally, after completion of the intracellular phase the pathogen must exit its host cell (egress) [9], [10] and PFPs are employed by some pathogens for egress [10].

The malaria parasite Plasmodium is a single-cell obligate intracellular parasite that cycles between a mosquito vector and a vertebrate host. Distinct stages of the parasite actively invade, replicate and egress from a number of different host cells. Considering their multiple interactions with target cells Plasmodia should be considered as prime suspect PFP producers. The merozoite stage invades red blood cells, replicates inside a vacuolar compartment, the parasitophorous vacuole (PV) and egresses to rapidly invade new red blood cells [11]. The motile ookinete stage penetrates the mosquito midgut epithelium and lyses the epithelial cell barrier to reach the basal lamina where it transforms into an oocyst [12]. Sporozoite stages that are released from a mature oocyst invade salivary gland cells, transmigrate through the cytoplasm and exit into the secretory cavity. When an infected mosquito bites, motile salivary gland sporozoite stages are then deposited into the skin of the host were they must traverse cellular barriers before entering the bloodstream that allows their transport to the liver. Here, they exit from the blood vessel, invade hepatocytes and transform into hepatic stages that reside in a PV where they grow and replicate, producing thousands of first generation merozoites [13]. Upon maturity merozoites must egress from the hepatocyte to invade red blood cells. It is conceivable that each parasite stage may utilize PFPs for its distinct interactions with host cells. Pores that may serve nutrient acquisition have been described in the PV of blood stages [14] yet the constituents of this pore remain to be characterized. Thus, we speculated that the Plasmodium genome [15], [16] should encode proteins with predicted pore-forming activity and the identification of such PFPs is an important step towards understanding the parasites interactions with target membranes.

Here, we describe five related genes, highly conserved among Plasmodium species, each encoding a putative secreted protein bearing a membrane-attack complex/perforin (MACPF)-like domain. This domain is well characterized in pore-forming proteins such as components of the mammalian complement system [17] and perforin [18]. We show that one Plasmodium Perforin-Like Protein (PPLP) is expressed in the secretory micronemes of the sporozoite stage and may thus function in its interaction with the mosquito and/or during the initial infection of the mammalian host.