Mutant/mutation
The mutant lacks expression of both Mei2 and LINUP

Protein (function)
Mei2 is a member of the largest family of RNA binding proteins (RBPs) – those that contain a RNA recognition motif (RRM), a stretch of 70-90 amino acids that contain two consensus RNA-interacting motifs, RNP1 and RNP2. RRM-containing proteins are subdivided into ten separate families (RRM_1 thru RRM_10) based on shared amino acid identities between members of each family and Mei2 contains a C-terminal RRM_2, thought to be unique to fungi and plants. Plasmodium contains a single Mei2-like gene.

LINUP is a single exon gene encoding a 746 amino acid protein and is conserved among Plasmodium species. The overall amino acid identity between the P. yoelii, P. falciparum and P. vivax syntenic orthologs was 40%, whilst amino acid similarity was 60%. Identity in a 122 amino acid stretch near to the N-terminus (amino acids 44-161) was 89%. In addition, comprehensive protein BLAST searches revealed that the gene has no orthologs in other Apicomplexa or any other eukaryote and is thus unique to Plasmodium. LINUP contains a conserved N-terminal 24 amino acid nuclear localization sequence (NLS) in Py, Pf and Pv that was nearly identical in amino acid sequence among the three species.

Phenotype
Py mei2–/linup– (PyLARC2) shows complete growth arrest (attenuation) of liver stage development.
To determine whether PyLARC2 undergoes complete late LS arrest, PyLARC2 and wildtype (PyWT) salivary gland sporozoites 30  BALB/cByJ mice were intravenously (i.v.) challenged with a high dose of 250,000 PyLARC2 and 3 mice with PyWT sporozoites, None of the 30 mice challenged with PyLARC2 sporozoites developed blood stage infections while blood-stage parasites were detected in the mice injected with PyWT sporozoites 3 days after infection. These results indicate that PyLARC2 parasites suffer a complete defect during pre-erythrocytic infection and cannot initiate blood stage infection.

Additional information
To further investigate PyLARC2 liver infection with focus on late liver stage (LS) development at the cellular level, BALB/cByJ mice were intravenously infected with 250,000 sporozoites of either PyLARC2 or PyWT and livers were analyzed at 36 and 48h post sporozoite infection (hpi). Measurement of LS size revealed no difference in size between PyWT and PyLARC2 LS schizonts at 36 hpi. However, at 48 hpi, PyLARC2 late LS schizonts were significantly smaller compared to PyWT.
To visualize organelle segregation, PyWT and PyLARC2 LS at 48 hpi were labeled with anti-ACP antibodies, which marks the parasite apicoplast. DNA segregation was visualized by DAPI staining. The late LS plasma membrane and LS merozoite plasma membrane was delineated by staining for Py merozoite surface protein 1 (MSP1). PyWT late LS showed MSP1 localization to the cytomeres, which had partitioned the LS cytoplasm. The LS apicoplast network displayed well differentiated branches that were in the final stages of segregation. In addition, multiple punctate DNA centers, indicative of complete DNA segregation, were partitioned along defined cytomere boundaries. In contrast, PyLARC2 late LS schizonts displayed severe abnormalities in differentiation. They lacked PyMSP-1 expression, indicating a block of LS maturation and LS merozoite formation. The apicoplast remained branched, tubular, and lacked organized distribution. Furthermore, PyLARC2 LS showed fewer DNA centers that appeared aggregated, indicating that both DNA replication and segregation were affected. The inner membrane complex protein myosin A tail domain-interacting protein (mTIP) is an additional differentiation marker for LS merozoites. MTIP localized to the inner membrane complex of nascent LS merozoites of PyWT LS at 48 hpi. In contrast, no mTIP expression was observed in  PyLARC2 LS at 48 hpi, further substantiating that PyLARC2 does not form LS merozoites.

From the Abstract: 'Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2–/linup– or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice'.

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