Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus.

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DDI1 proteins are conserved in eukaryotes and involved in a variety of cellular processes, including proteasomal degradation of specific proteins and DNA-protein crosslink repair. All DDI1 proteins contain ubiquitin-like (UBL) and retroviral aspartyl protease (RVP) domains, and some also contain ubiquitin-associated (UBA) domain, which mediate distinct activities of these proteins. We investigated the Plasmodium DDI1 to identify its roles during parasite development and potential as a therapeutic target. The DDI1 proteins of Plasmodium and other Apicomplexan parasites vary in domain architecture, share UBL and RVP domains, and the majority of proteins contain the UBA domain. Plasmodium DDI1 is expressed across all major life stages and is essential, as conditional depletion of DDI1 protein in P. berghei and P. falciparum drastically reduced the asexual stage parasite development. Infection of mice with DDI1 knock-down P. berghei parasites was self-limiting and protected from the subsequent infection with both homologous and heterologous parasites, indicating potential of DDI1 knock-down parasites as a whole organism vaccine. P. falciparum DDI1 (PfDDI1) is associated with chromatin and DNA- protein crosslinks, and PfDDI1 knock-down parasites showed increased DNA-protein crosslinks and susceptibility to DNA damaging chemicals, indicating an important role for DDI1 in repair of DNA-protein crosslinks. The knock-down of PfDDI1 increased susceptibility to retroviral protease inhibitors, epoxomicin and artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these inhibitors or drugs. Hence, the essentiality, ability of DDI1 knock-down parasites to confer protective immunity and increased susceptibility to inhibitors support Plasmodium DDI1 as a dual-target therapeutic candidate.