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PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection.

ABSTRACT: Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

SUBMITTER: Bansal A

PROVIDER: S-EPMC5789930 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection.

Bansal Abhisheka A Molina-Cruz Alvaro A Brzostowski Joseph J Liu Poching P Luo Yan Y Gunalan Karthigayan K Li Yuesheng Y Ribeiro José M C JMC Miller Louis H LH

Proceedings of the National Academy of Sciences of the United States of America 20180108 4

Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent ...[more]

PMID: 29311293

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Project description:Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines. We identified a conserved protein (PBANKA_0305900) in Plasmodium berghei, which encodes a protein of 22 kDa (thus named Pb22) and is expressed in both asexual stages and gametocytes. Its homologues are present in all Plasmodium species and its closely related, Hepatocystis, but not in other apicomplexans. Pb22 protein was localised in the cytosols of schizonts, as well as male and female gametocytes. During gamete-to-ookinete development, Pb22 became localised on the plasma membranes of gametes and ookinetes. Compared to the wild-type (WT) parasites, P. berghei with pb22 knockout (KO) showed a significant reduction in exflagellation (~89%) of male gametocytes and ookinete number (~97%) during in vitro ookinete culture. Mosquito feeding assays showed that ookinete and oocyst formation of the pb22-KO line in mosquito midguts was almost completely abolished. These defects were rescued in parasites where pb22 was restored. Cross-fertilisation experiments with parasite lines defective in either male or female gametes confirmed that the defects in the pb22-KO line were restricted to the male gametes, whereas female gametes in the pb22-KO line were fertile at the WT level. Detailed analysis of male gametogenesis showed that 30% of the male gametocytes in the pb22-KO line failed to assemble the axonemes, whereas ~48.9% of the male gametocytes formed flagella but failed to egress from the host erythrocyte. To explore its transmission-blocking potential, recombinant Pb22 (rPb22) was expressed and used to immunise mice. in vitro assays showed that the rPb22-antisera significantly inhibited exflagellation by ~64.8% and ookinete formation by ~93.4%. Mosquitoes after feeding on rPb22-immunised mice also showed significant decreases in infection prevalence (83.3-93.3%) and oocyst density (93.5-99.6%). Further studies of the Pb22 orthologues in human malaria parasites are warranted.

Project description:UnlabelledTransmission of malaria occurs during Anopheles mosquito vector blood meals, when Plasmodium sporozoites that have invaded the mosquito salivary glands are delivered to the mammalian host. Sporozoites display a unique form of motility that is essential for their movement across cellular host barriers and invasion of hepatocytes. While the molecular machinery powering motility and invasion is increasingly well defined, the signaling events that control these essential parasite activities have not been clearly delineated. Here, we identify a phosphodiesterase (PDEγ) in Plasmodium, a regulator of signaling through cyclic nucleotide second messengers. Reverse transcriptase PCR (RT-PCR) analysis and epitope tagging of endogenous PDEγ detected its expression in blood stages and sporozoites of Plasmodium yoelii. Deletion of PDEγ (pdeγ(-)) rendered sporozoites nonmotile, and they failed to invade the mosquito salivary glands. Consequently, PDEγ deletion completely blocked parasite transmission by mosquito bite. Strikingly, pdeγ(-) sporozoites showed dramatically elevated levels of cyclic GMP (cGMP), indicating that a perturbation in cyclic nucleotide balance is involved in the observed phenotypic defects. Transcriptome sequencing (RNA-Seq) analysis of pdeγ(-) sporozoites revealed reduced transcript abundance of genes that encode key components of the motility and invasion apparatus. Our data reveal a crucial role for PDEγ in maintaining the cyclic nucleotide balance in the malaria parasite sporozoite stage, which in turn is essential for parasite transmission from mosquito to mammal.ImportanceMalaria is a formidable threat to human health worldwide, and there is an urgent need to identify novel drug targets for this parasitic disease. The parasite is transmitted by mosquito bite, inoculating the host with infectious sporozoite stages. We show that cellular signaling by cyclic nucleotides is critical for transmission of the parasite from the mosquito vector to the mammalian host. Parasite phosphodiesterase γ is essential for maintaining cyclic nucleotide balance, and its deletion blocks transmission of sporozoites. A deeper understanding of the signaling mechanisms involved in transmission might inform the discovery of novel drugs that interrupt this essential step in the parasite life cycle.

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PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection.

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<i>Pf</i>CDPK1 is critical for malaria parasite gametogenesis and mosquito infection.

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