Abstract
Protein phosphorylation is the most important post-translational event in the regulation of various essential signaling pathways in a cell. Here, we show the functional characterization of a FIKK family protein kinase of the rodent malaria parasite (PbMLFK), which is expressed only in mosquito and liver stages and contains two functional C-terminal PEXEL motifs. We demonstrate that this protein plays a role in mosquito and liver stages of parasite growth. The oocysts of PbMLFK-deficient parasites produced 4-fold fewer sporozoites. In the liver of infected mice, PbMLFK-deficient parasites grew 100-fold less than did wild type parasites. We also show that the C-terminal domain of this protein has a functional serine-threonine kinase and that its activity was inhibited by a known PKA inhibitor. Transcriptome analysis of infected host cells suggests that in absence of this protein expression of the 288 host mRNAs are perturbed which are primarily associated with the immune system, cell cycle and metabolism.
Highlights
(d) Anti-PbMLFK peptide mouse polyclonal antibody detects PbMLFK in wild type (WT) sporozoite lysate but not in PbMLFK-KO sporozoite lysates or PbA blood stage lysate
Because PbMLFK protein does not express in blood stage parasites [ transcript was detected in blood stage schizonts24], one is able to delete this gene in the blood stage without affecting parasite growth/development
To find out protein expression when analyzing its expression in oocysts via immunofluorescence assay (IFA), we found that PbMLFK expressed in the oocyst stage (Fig. 2b)
Summary
(d) Anti-PbMLFK peptide mouse polyclonal antibody detects PbMLFK in WT sporozoite lysate (arrow marks) but not in PbMLFK-KO sporozoite lysates or PbA blood stage lysate. Lane[1], PbA sporozoite (5 × 10e6) lysate; Lane 2, PbMLFK-KO (described later, refer to Fig. 4) sporozoite (5 × 10e6) lysate; Lane 3, PbA blood stage (5 × 10e6) lysate; Lane 4, positive control (E. coli recombinant and partially purified C-terminal PbMLFK), Right side to lane 4-protein size markers. (e) Western blot analysis of full-length PbMLFK: IP lysate from 150 infected mosquito midguts (left side lane, oocyst) and 150 uninfected mosquito midgut IP lysate (right side lane, control). We named it PbMLFK (P. berghei Mosquito and Liver stage specific FIKK Kinase). We showed that PbMLFK has a serine-threonine kinase activity and that it is expressed from the early oocyst stage (day 4) in mosquitos to the late liver stage in mammalian hosts
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