Malaria: Understanding Infection and Pathogenicity
Summary: Alan Cowman is investigating how the malaria parasite invades human erythrocytes and develops within this protected environment so that it can grow and hide from the host's protective responses.
In my laboratory, the ultimate goal is to understand how the malaria parasite infects humans and causes disease and to use these insights to develop new therapies. Malaria caused by Plasmodium falciparum is one of the most serious and widespread parasitic diseases of humans. Each year, several hundred million people become infected, and up to 1 million die. An essential step in survival of the malaria parasite is the requirement of the blood-stage merozoite form to invade erythrocytes. This is a complex multistep process involving sequential receptor-ligand interactions and signal transduction events. Merozoite invasion is a point in the parasite lifecycle that is vulnerable to host responses and drug intervention. Once the malaria parasite is inside the host red blood cell, it begins a remarkable process of remodeling that turns a terminally differentiated cell lacking most functions of normal mammalian cells, such as protein-trafficking machinery, into one in which the parasite can grow and hide from host responses. Central to its survival is the ability of the infected red blood cells to sequester in and obstruct the microvasculature of organs. Sequestration helps avoid destruction of parasitized red blood cells by the reticuloendothelial system and allows the microaerophilic parasite to mature in a relatively hypoxic environment.
Invasion of Host Erythrocytes
Invasion of the erythrocyte by merozoites can be separated into an ~11-second preinvasion stage, which is characterized by long-distance interactions and deformation of the host cell, and an ~17-second invasion step, in which the parasite forms a moving tight junction. The preinvasion stage is poorly understood, regarding both receptor-ligand interactions and the communication events that link the subsequent stages of invasion. It appears likely that during invasion, signaling cascades are activated to initiate subsequent invasion steps, such as apical organelle secretion and the function of the actomyosin motor. Understanding merozoite invasion of erythrocytes is a core component of our research.
To unravel key events in this rapid and complex process, we are overlaying molecular details onto the kinetic and morphological framework of invasion. We have identified proteins on the merozoite surface important for interaction with the host cell, and some of these are related to the erythrocyte-binding-like (EBL) protein family, whose members function after initial binding of the invasive parasite form. We have made important inroads into understanding the function of key invasion proteins, especially the EBL and reticulocyte-binding-like homologue (PfRh) families, including PfRh4, and we have identified other important proteins, such as PfRh5 and PfRipr. We are analyzing the function of these key proteins by identifying partners to which they bind and by conducting structural studies to determine their three-dimensional shape. In addition, current evidence suggests that the EBL and PfRh proteins are involved in signaling events during erythrocyte invasion. The large extracellular domain of these proteins bind erythrocytes, and the cytoplasmic regions may be involved in events such as rhoptry release and activation of the parasite actomyosin motor by initiating a complex cascade of events within the parasite for successful entry of malaria parasites. Our aim is to understand the role of these proteins in binding to the host erythrocyte and signaling subsequent events in the invasion process.
In preclinical studies, we are focusing on our lead candidates: the EBL and PfRh proteins. We are collaborating with the PATH/Malaria Vaccine Initiative, USAID, and Gennova Biopharmaceuticals to determine the best proteins for a combination vaccine. This work involves studies on immunization of small animals, human immune responses, and identification of functional domains. Our preclinical studies will guide further development and testing of these antigens as well as provide crucial knowledge that more broadly informs the development of other candidate antigens.
Another important direction in my laboratory is to understand the extensive remodeling of the erythrocyte that occurs during the blood stage, as this is central to the intracellular survival of P. falciparum and pathogenesis of malaria. Remodeling occurs in the absence of a host secretory network and enables nutrient uptake and surface exposure of the major adhesin, PfEMP1 (P. falciparum erythrocyte membrane protein 1), which mediates cytoadherence to microvascular endothelia and placental trophoblasts and facilitates immune evasion. P. falciparum remodels the infected erythrocyte by exporting effector proteins, initially via the endoplasmic reticulum (ER) and the parasite membrane and across the parasitophorous vacuole (PV) and vacuole membrane (PVM) into the host cell. The virulence protein PfEMP1 is trafficked to the PVM and packaged into Maurer’s clefts, structures that bud off and transport protein cargo across the erythrocyte, followed by insertion into the erythrocyte membrane at the electron-dense knobs.
A conserved export motif, termed PEXEL (Plasmodium export element), or vacuolar transfer signal, is required for export of many proteins beyond the PVM to the erythrocyte. The PEXEL consists of a pentameric sequence, RxLxE/Q/D, and is present in more than 200 P. falciparum proteins. The PEXEL is a bifunctional export motif containing a protease recognition sequence cleaved in the ER, and the remaining component of the PEXEL residue directs the mature protein to the host cell. In addition to identifying this motif in exported proteins, we, in collaboration with Brendan Crabb's laboratory, have identified a complex that appears to be responsible for export of PEXEL motif proteins across the PVM. This complex (PTEX) is ATP powered and comprises HSP101, which is a ClpA/B-like AAA+ ATPase, a protein termed PTEX150, and EXP2, the putative channel, as well as two other proteins, PTEX88 and a thioredoxin known as TRX2. We are determining the steps in export and identifying the processes involved. In addition, we are analyzing the function of exported proteins, as they are important for virulence and pathogenesis of P. falciparum in the host.
Plasmepsin V cleaves PEXEL and reveals xE/Q/D, and this conserved fifth amino acid is required for export but not cleavage. In addition, cleavage by Plasmepsin V is required for export, since constructs that lack a PEXEL motif that are cleaved by the canonical signal peptidase are not exported despite cleavage revealing an xQ. Recently, it has been suggested that phosphatidylinositol-3-phosphate (PI(3)P) is involved in export of proteins to the parasite-infected erythrocyte. Currently, we have little understanding of the molecular events after plasmepsin V or PI(3)P recognition of proteins to be exported. We believe that protein cargo selected for export is packaged in vesicles for trafficking via a specialized pathway to domains in the PV where they can be recognized by the putative translocon machine (Figure).
We are tracking PEXEL-exported proteins as well as non-PEXEL-containing proteins, including PfEMP1, to identify events between ER exit and recognition by the translocon machinery.
These projects are funded in part by grants from the National Health and Medical Research Council of Australia.
As of September 26, 2012