Our laboratory focuses on the pre-erythrocytic phase of malaria, which is defined by events that occur between the time the mosquito injects the parasite into the host and when the parasite infects the red blood cells. This phase of malaria is the most elusive, in part because of the small number of parasites injected during the mosquito bite and the speed with which the motile parasites (called sporozoites at this stage of their life cycle) reach the liver and transform into the red blood cell–infecting form. Nevertheless, the pre-erythrocytic phase of the malaria parasite has long been an attractive target for malaria vaccine development. When sporozoites do not complete their development in the liver and thus do not transform into the red blood cell–infecting form, after irradiation or genetic modification, the attenuated parasites can protect their host. Such vaccination has achieved protection rates that have not yet been matched by subunit vaccines.
Our major goal is to understand the interactions between parasite and host that occur during the parasite's pre-erythrocytic stage. To this end, we use a rodent malaria model and follow three main approaches: first, describe, using real-time imaging, the natural history of the pre-erythrocytic phase and its important host-parasite interactions; second, characterize at the molecular level how the parasite invades host cells inside a vacuole and to identify the parasite and host-cell molecular players involved in the process; and third, identify genes that are essential to the pre-erythrocytic stages of the parasite, with the dual goal of uncovering new function and creating new parasite mutants that can be used as probes of the host immune response.
Pre-Erythrocytic Phase of the Sporozoite
The journey of the sporozoite in a mammalian host is the least understood part of the parasite life cycle. Sporozoites are known to be injected into the skin in small numbers—usually fewer than 50—and to develop inside hepatocytes, but the intermediary steps have remained obscure. In vivo imaging has shown that the pre-erythrocytic phase is complex and has revealed many unsuspected host-parasite interactions. First, the parasites that the mosquito deposits in the dermis have several fates in a mammalian host. As expected, they invade blood capillaries to reach the bloodstream and ultimately the liver, but only about a third of the parasites follow this path. Two other fates are possible: about half the parasites remain in the dermis after loosing their motile capacity, and the remaining 15 percent invade lymphatic vessels. The latter parasites are arrested in the proximal draining lymph node, where most are degraded inside dendritic cells. However, a few of these lymph node parasites can partially develop into exo-erythrocytic forms, although they do not infect red blood cells. Lastly, during the pre-erythrocytic phase, the parasite uses a number of remarkable tricks to escape immune surveillance. For example, sporozoites traverse (migrate through) host phagocytes in the dermis, and probably also in the liver sinusoids, to resist phagocytosis during their journey to hepatocytes. Even more strikingly, thousands of newly formed red blood cell–infecting parasites manipulate the host hepatocyte so as to generate large, parasite-filled host cell buds called merosomes. These merosomes, which eventually detach from the mother host cell, transport the parasites directly into the bloodstream while protecting them from clearance by the numerous phagocytic cells present in the liver sinusoids.
Many questions regarding the pre-erythrocytic infection process remain unanswered. Our goal is to determine how sporozoites cross endothelial barriers, both in the dermis and in the liver, via a para-cellular or a trans-cellular route. We also plan to document the complete set of interactions between the parasite and the immune cells of the host in the dermis, lymph node, and liver. It will be important to test whether the conclusions we draw from the use of a rodent model are valid for the human parasite. This problem might be tackled by using humanized mice, including mice bearing a human immune system, which might also allow for dissecting and visualizing the response of human immune cells to the human malaria parasite.
Mechanisms of Sporozoite Invasion
Malaria sporozoites can invade host cells in two distinct ways. Like other invasive stages of Apicomplexa protozoa, they can do so inside a so-called parasitophorous vacuole formed by invagination of the host cell plasma membrane. This process, termed cell infection, is a prerequisite for complete parasite differentiation. It follows the formation of a tight junction between the parasite and the cell surface, on which the parasite exerts force to pull itself inside the cell using its own, submembranous motor. Sporozoites can also disrupt host membranes and migrate through and out of the cell.
So far, studies on cell infection and junction formation by the malaria sporozoite have been complicated by the speed of the process (which takes only a few seconds) and the lack of synchronization of these events, because of the dominant cell traversal activity of the parasite that retards cell infection. The lack of synchronization of entry events has been a major problem for understanding the cellular and molecular basis of the process. Sporozoites do not synchronously invade cells because they have an alternative way of interacting with a host cell, which is to traverse it. In other words, wild-type sporozoites infect cells progressively; they start by traversing cells and switch to the infection mode (invasion inside a vacuole) at different times during the one-hour infection periods (what triggers the switch remains unknown). Cell traversal–deficient mutants, in contrast, immediately invade cells inside a vacuole. Therefore, the lack of cell traversal synchronizes infection. We have recently shown that cell traversal–deficient sporozoites invade host cells rapidly and synchronously. Therefore, using such sporozoite mutants, it is now possible to study the structural and molecular bases of cell infection and junction formation. We are analyzing the contribution of several sporozoite surface proteins in host cell infection. We rely on a double approach of protein tagging and molecular imaging—to follow the protein dynamics during parasite entry—and conditional mutagenesis using a Flp/FRT-based technique recently devised in our laboratory—to assess the actual protein contribution to the process.
Identification of Malarial Genes Essential for the Sporozoite Phase
In recent years, numerous techniques have been used to analyze the transcription of parasite genes at different stages of the parasite life cycle. For large-scale sequencing of sporozoite cDNA, subtractive suppressive hybridizations as well as proteomic and microarray studies have been performed. We have undertaken serial analysis of gene expression of sporozoites and an in silico negative screen of the gene expression database of human and rodent parasites to identify genes that are specifically or preferentially activated in pre-erythrocytic stages. These techniques have revealed many genes of potential interest. We are now performing systematic mutagenesis of such genes further selected on the basis of gene expression levels and sequence annotation—for example, genes whose sequence suggests secretion of the gene product or the presence of cell-adhesive domains. Our goal is to create novel deficient or attenuated parasite mutants blocked at new steps of the infection process in the mammalian host. Characterization of relevant mutants, in addition to identifying new function, might also teach us about the host-parasite interactions and host-tissue infections that play a role in triggering an effective protective response against the pre-erythrocytic phase of malaria.
Last updated September 2008
