A ubiquitous Plasmodium protein displays a unique surface labeling pattern in sporozoites

Abstract

The Plasmodium sporozoite is infective for mosquito salivary glands and vertebrate host tissues. Although it is a key developmental stage of the malaria parasite, relatively few sporozoite surface or secreted proteins have been identified and characterized. Herein, we describe the molecular and cellular characterization of a novel surface molecule that is preferentially-expressed in salivary gland sporozoites as compared to oocyst and hemolymph sporozoites. This molecule, designated the sporozoite and erythrocytic stages (SES) protein (formerly known as Pg4), exhibits a spiral surface labeling pattern that spans over a known sporozoite surface antigen, the circumsporozoite protein, with only minor co-localization. SES consists of 551 amino acids encoding a putative 63.2 kDa protein that has been shown to be expressed not only on particular sporozoite stages, but also during the asexual and gametocyte stages. This novel protein also has three domains of unknown function that are conserved in at least eight Plasmodium spp. that represent human, avian, non-human primate, and rodent malarias.

Introduction

Malaria, which is exhibiting a resurgence, is a devastating disease caused by Plasmodium spp. that results in significant morbidity and mortality for millions of people throughout the world [1]. During the life cycle of the malaria parasite, the sporozoite is a key developmental stage that is infective for both the mosquito salivary glands and vertebrate host tissues. Although host tissue invasion is likely mediated by molecules expressed on the surface or secreted, there are few well-characterized sporozoite molecules of this type. The circumsporozoite (CS) protein and thrombospondin-related adhesive protein (TRAP)/sporozoite surface protein (SSP) are surface molecules that have been well-characterized and shown to play vital roles in sporozoite biology [2], [3], [4]. The CS protein has been demonstrated to not only be critical for sporozoite budding during oocyst differentiation, but it also has been implicated in the invasion of mosquito salivary glands and vertebrate liver tissue [5], [6], [7], [8]. Similarly, mutant sporozoites lacking the TRAP/SSP gene product have shown impaired gliding motility and poorly invaded host tissue [9]. In addition, MAEBL, an erythrocyte binding protein that originally was shown to be involved in merozoite invasion of erythrocytes, has recently been demonstrated to be abundantly expressed in the sporozoite stages and implicated in the invasion of mosquito salivary glands as well as vertebrate host tissue [10], [11], [12], [13]. Other sporozoite surface molecules, such as the Plasmodium falciparum sporozoite antigen Pfs16 [14], sporozoite and liver stage antigen (SALSA) [15], and the sporozoite threonine- and asparagine-rich protein (STARP) [16] also have been identified and characterized to varying degrees.

The MB2 protein is a P. falciparum sporozoite surface molecule that was isolated from a sporozoite expression library following screening with immune serum from a protected human volunteer [17]. The PfMB2 protein is differentially-expressed with a distribution on the surface of the sporozoite, in the cytoplasm and parasitophorous vacuole (PV) of the liver stages, and in the cytoplasm, nucleus, and PV of the asexual blood stages. On the sporozoites, the PfMB2 gene encodes a 120 kDa putative surface antigen with a transmembrane domain at the amino terminus and six copies of a nine-amino acid tandem repeat at the carboxyl terminus [17]. Although other Plasmodium spp. homologs recently have been identified [18], initial BLAST analyses of PfMB2 indicated a significant lack of homology with identified genes in the databases. Therefore, efforts were made to isolate its Plasmodium gallinaceum homolog and test its ability for mediating invasion of mosquito or vertebrate tissues in the accessible P. gallinaceum–chicken–Aedes aegypti model system. During this screening, another novel sporozoite gene, designated SES for sporozoite and erythrocytic stages gene, was identified and it was determined to have no sequence identity with the MB2 gene [17], [18].

Herein, we describe the molecular and cellular characterization of PgSES that has protein homologs, containing three conserved regions of identity/similarity with unknown function, in at least eight Plasmodium species including the human species, P. falciparum and P. vivax. The PgSES protein is expressed in the erythrocytic stages and is preferentially-expressed on the surface of salivary gland sporozoites as compared with other sporozoite populations. A particularly interesting feature of this protein is that in the salivary gland sporozoites it exhibits a unique surface labeling pattern that spirals around the sporozoite.