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Regulation of stage conversion in the malaria parasite: molecular insights for novel vaccine strategies

Final Report Summary - STACOMAL (Regulation of stage conversion in the malaria parasite: molecular insights for novel vaccine strategies)

Malaria, a mosquito-borne disease caused by Plasmodium parasites, remains a major health problem in many tropical countries. Parasite multiplication inside red blood cells is responsible for the clinical manifestations of malaria, and is preceded by an obligate and clinically silent phase of parasite replication in the liver. Malaria liver stages are considered ideal targets for anti-malarial prophylactic approaches. However, the development of an efficacious vaccine has proven extremely difficult, notably due to the challenge of identifying target antigens among more than 5,000 different parasite-encoded proteins. The project STACOMAL ‘Regulation of stage conversion in the malaria parasite: molecular insights for novel vaccine strategies’ employs genetic approaches to investigate the role of candidate liver stage factors and elucidate how the parasite remodels its antigenic repertoire during multiplication in the liver. The project’s goal is to provide novel insights into the molecular mechanisms driving parasite stage conversion and to identify potential new malaria therapeutic or vaccine targets.

Malaria liver stages remain the least characterized stages of the Plasmodium life cycle due to their limited accessibility. We previously identified a master regulator of Plasmodium liver stage development, called SLARP (sporozoite and liver stage asparagine-rich protein). SLARP encodes a large protein of unknown function and is conserved among Plasmodium species, including the human malaria parasites P. falciparum and P. vivax. In the rodent malaria parasites P. berghei and P. yoelii, SLARP-knockout parasites show a complete developmental arrest inside infected hepatocytes, associated with the down-regulation of a subset of genes in sporozoites and liver stages. Interestingly, SLARP-knockout parasites, although perfectly attenuated, confer only limited protection in immunized animals as compared to other attenuated parasites, suggesting that some of the genes under SLARP control may contribute to protective immunity, either directly as target antigens or indirectly through their role during parasite development in the liver.

During transition from sporozoites to liver stages there is a profound remodelling of the parasite antigenic repertoire, a process that is regulated in part by the parasite protein SLARP and may have a profound impact on the host immune responses. The project STACOMAL investigated the mechanisms of regulation of parasite antigen expression during liver stage development and its impact on protective immunity against liver stages. Specifically, our project aimed at answering the following questions:
1. What is the functional importance of SLARP-regulated genes during LS development?
Depletion of SLARP causes an early arrest during LS development, a phenotype that is associated with the down-regulation of a specific subset of genes. We propose to investigate the functional importance of SLARP-regulated genes, using a reverse genetics approach. For this purpose, we have developed a novel ‘Gene Out Marker Out’ (GOMO) strategy, combining positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites, for the rapid generation of drug-selectable marker-free mutant malaria parasites expressing a GFP or a GFP-luciferase cassette. This strategy should greatly facilitate the identification of new genes with vital functions during Plasmodium liver stage development, and thus potential targets for novel anti-malarial drugs.

2. How does SLARP control expression of target genes?
The absence of nucleic acid binding domains in SLARP protein sequence suggests an indirect role of SLARP in target gene regulation. To investigate the mechanisms of gene control by SLARP, we used a combination of genetic tools (including promoter exchange, epitope tagging and a novel plasmodial artificial chromosome system for reporter assays in sporozoites) to characterize the cis-acting factors involved in SLARP regulation. In particular, we discovered that expression of a SLARP-dependent gene is controlled by translational repression in sporozoites, where mRNAs accumulate but are not translated until the parasite infects the liver. Through an in-depth characterization of the mechanism of action of SLARP, we hope to improve our understanding of gene regulation during LS development. The data may also help understanding hypnozoite formation and reactivation in P. vivax, where SLARP is also present.
One major roadblock for the development of pre-erythrocytic immune intervention strategies is the remodelling of the parasite antigenic make-up during stage conversion upon host switch. Generation of parasite lines with defined defects in stage conversion, ranging from early transformation to onset of DNA replication and parasite growth, will aid in prioritizing candidate protective antigens, which together can elicit lasting protection against reinfection. Characterization of gene regulation in early liver stages is particularly relevant to understand the phenomenon of parasite dormancy in the liver, a hallmark of P. vivax human malaria. A better understanding at the molecular level of the process of parasite differentiation in the liver is a pre-requisite for the rational development of novel therapeutic approaches targeting malaria pre-erythrocytic stages.