Distinct Roles of Plasmodium Rhomboid 1 in Parasite Development and Malaria Pathogenesis

Jacobs-Lorena M (2009) Distinct Roles of Plasmodium Rhomboid 1 in Parasite Development and Malaria Pathogenesis. PLoS Pathog 5(1): e1000262. doi:10.1371/journal.ppat.1000262 Distinct Roles of Plasmodium Rhomboid 1 in Parasite Development and Malaria Pathogenesis Prakash Srinivasan 0 Isabelle Coppens 0 Marcelo Jacobs-Lorena 0 Kirk Deitsch, Weill Medical College of Cornell University, United States of America 0 Malaria Research Institute and Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health , Baltimore, Maryland , United States of America Invasion of host cells by the malaria parasite involves recognition and interaction with cell-surface receptors. A wide variety of parasite surface proteins participate in this process, most of which are specific to the parasite's particular invasive form. Upon entry, the parasite has to dissociate itself from the host-cell receptors. One mechanism by which it does so is by shedding its surface ligands using specific enzymes. Rhomboid belongs to a family of serine proteases that cleave cellsurface proteins within their transmembrane domains. Here we identify and partially characterize a Plasmodium berghei rhomboid protease (PbROM1) that plays distinct roles during parasite development. PbROM1 localizes to the surface of sporozoites after salivary gland invasion. In blood stage merozoites, PbROM1 localizes to the apical end where proteins involved in invasion are also present. Our genetic analysis suggests that PbROM1 functions in the invasive stages of parasite development. Whereas wild-type P. berghei is lethal to mice, animals infected with PbROM1 null mutants clear the parasites efficiently and develop long-lasting protective immunity. The results indicate that P. berghei Rhomboid 1 plays a nonessential but important role during parasite development and identify rhomboid proteases as potential targets for disease control. - Funding: This work was supported by Johns Hopkins Malaria Research Institute, The Bloomberg Family Foundation, and grant R01 AI 1031478 from the National Institute of Allergy and Infectious Diseases. Competing Interests: The authors have declared that no competing interests exist. For successful development and transmission, Plasmodium has to invade multiple cell types both in the mammalian host and in the mosquito vector. Much of our knowledge about the molecular mechanisms of invasion comes from the study of P. falciparum merozoite invasion of red blood cells (RBCs). RBC invasion involves an initial attachment followed by re-orientation and entry of the parasite into the host cell [1]. There are two main classes of parasite surface molecules, the GPI-anchored proteins such as the merozoite surface protein family (MSP) [2] and transmembrane domain-containing proteins such as AMA1 [3,4], erythrocyte binding-like family (EBL) [5,6] and reticulocyte binding-like family proteins (RBL) [7,8]. A few host-cell receptors to which these ligands bind have been identified [912]. In the mosquito, motility plays an important role in ookinete and sporozoite invasion. Motile ookinetes form within the mosquito blood meal and invade the midgut epithelium. After exiting on the basal side facing the hemocoel they differentiate into sessile oocysts [13]. Subsequently, sporozoites released from mature oocysts invade the salivary glands from where they are delivered to the vertebrate host by a mosquito bite. These sporozoites travel through the blood stream until they reach the liver, where they invade and infect hepatocytes. All three invasive forms (ookinetes, sporozoites in the mosquito and sporozoites in the mammalian host) utilize the same actin-based motor for entry into the host cell. Thrombospondin-related anonymous protein (TRAP) family homologues constitute one class of protein required for motility and host cell invasion [1416]. The extracellular domains of TRAP interact with host-cell receptors, while the cytoplasmic tail links to the actin-myosin cytoskeleton [17]. As the parasite glides, the parasite surface ligand-receptor complexes translocate towards the posterior end. Dissociation of these interactions by proteolytic processing is thought to be important, as this enables the parasite to move forward [1820]. In another Apicomplexan parasite-Toxoplasma-the TRAP homologue MIC2 is cleaved within its transmembrane domain releasing the receptorbinding domain from the parasite surface [18] and Plasmodium merozoite TRAP (MTRP) also appears to be cleaved in a similar manner [16]. Rhomboid-family (ROM) proteins are serine proteases that cleave their substrates within their membrane domain [21,22]. Multiple rhomboid-family proteins have been identified in the genomes of Plasmodium and Toxoplasma [23]. Cleavage requires the presence of helix-destabilizing residues within the membrane domain of substrates [24]. Indeed, Apicomplexan surface proteins such as EBL and RBL proteins, AMA1, TRAP and their homologues contain such helix-destabilizing residues [23]. Assays in cultured mammalian cells identified possible substrates for both Toxoplasma and Plasmodium falciparum rhomboid proteins [25,26]. Toxoplasma ROM5 localizes to the posterior end of the parasite and can cleave MIC2 within its transmembrane domain [25,27]. Malaria is one of the major infectious diseases and is responsible for the death of more than a million people, mostly children under the age of five. Plasmodium, the causative agent of malaria, is transmitted by female Anopheles mosquitoes. Successful development of the parasite requires efficient recognition, attachment, and invasion of host cells. Several parasite cell-surface molecules have been implicated in these processes and may require proteolytic processing in order for the parasite to complete invasion. Rhomboid family proteins are serine proteases that cleave within the transmembrane region of their substrates. Here, we use a genetic approach to study the function of Plasmodium berghei rhomboid 1 (PbROM1). PbROM1 is expressed in both vertebrate and mosquito stages of parasite development, and the protein is present in secretory organelles that contain other parasite molecules required for invasion. We find that PbROM1 is required for efficient infection of both the mosquito and the vertebrate host. Interestingly, we also find that mice infected with ROM1(2) parasites clear the infection efficiently and are protected upon subsequent wild-type parasite challenge. Our study suggests a role for PbROM1 throughout parasite development and identifies ROM1 as a target for disease intervention. Plasmodium does not have a ROM5 homologue but ROM4 is able to cleave EBA175 [28], an EBL family protein involved in binding to erythrocytes [10]. Processing of EBA175 within its membrane domain appears to be essential for parasite invasion [28]. Here we report on experiments investigating the role of Plasmodium berghei rhomboid 1 (PbROM1) during parasite development in the (...truncated)