Date Published: January 6, 2016
Publisher: Public Library of Science
Author(s): Min Zhang, Satish Mishra, Ramanavelan Sakthivel, Beatriz M. A. Fontoura, Victor Nussenzweig, Christian Doerig.
Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.
Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasitic protozoans of the genus Plasmodium. In 2013, there were 198 million cases of malaria and 584,000 fatalities (WHO world malaria report 2014), underscoring its role as a major pathogen. Sporozoites are the infectious and quiescent forms of the malaria parasite residing in the salivary glands of Anopheles mosquitoes. Malaria transmission begins with the injection of salivary sporozoites (Ssp) into the skin of a vertebrate host by infected mosquitoes. The parasites enter the blood circulation and rapidly invade hepatocytes where the crescent-shaped sporozoite progressively transforms into a spherical liver stage (or exo-erythrocytic stage, EEF). Many genes required for the Ssp transformation into liver stages are transcribed in the Ssp [1–3]. However, translation is repressed by phosphorylation of eIF2α by eIK2 kinase, also named UIS1 (UIS, Upregulated in Infective Sporozoites) [4, 5]. If the eIK2 kinase uis1 is knocked out, the Ssp transcripts are translated prematurely while sporozoites are still inside the salivary glands of mosquitoes . During the normal parasite cycle, liver-stage transcripts are only translated when Ssp enter hepatocytes and eIF2α-P is de-phosphorylated . Thus, Ssp quiescence is regulated by phosphorylation and de-phosphorylation of eIF2α.
We show here that UIS2 is the phosphatase that controls the development of the dormant Ssp into the liver stages. Its activity is enhanced by Mn2+, inhibited by Cd2+, but is not affected by okadaic acid, a powerful inhibitor of PP1 and PP2A phosphatases. Thus, UIS2 belongs to the PP2C/PPM family of phosphatases. The human genome encodes ~ 500 protein kinases, ~2/3 of which are serine/threonine kinases, and approximately 40 serine/threonine phosphatases [30, 31]. In Plasmodium, there are ~ 80 protein kinases and ~30 protein phosphatases [13, 32, 33]. These disparate numbers raise the question of how few phosphatases recognize specifically the very large number of phosphorylated proteins. In the case of the PP1 mammalian phosphatase, enzyme specificity and localization are regulated by a large number of multiple co-factors. However, this is not the case for Plasmodium UIS2 that encompasses catalytic and regulatory domains within the same polypeptide chain . The N-terminal domain of UIS2 interacts stably with the eIF2α-P substrate placing it in close proximity to the catalytic site. We showed that eIF2α-P interacted with endogenous UIS2 from lysates of blood stage parasites. We did not use lysates from sporozoites because of the repression of uis2 expression by Puf2. The interaction of UIS2/ eIF2α-P is in sharp contrast to other phosphatases whose interaction with the substrate is unstable and is enhanced by co-factors. In the blood stage, the mRNA level of uis2 is very low but UIS2 is translated and is essential for the parasite’s blood stage development. However, the function of uis2 in the blood stage is unknown.