A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle

Abstract

SummaryA family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle.

Systematic CRISPR-Cas9-Mediated Modifications of Plasmodium yoelii ApiAP2 Genes Reveal Functional Insights into Parasite Development

“…The ortholog of this gene is resistant to genetic disruption in P. berghei (9), and its role in parasite development has not been investigated previously, although its putative function in gametocytogenesis was implicated in a previous study of P. falciparum parasites (24,25). Here, we successfully disrupted the Pyap2-g3 gene and showed that disruption of this gene significantly reduced the numbers of male and female gametocytes (Fig.…”

“…We were able to knock out 12 of the 24 genes (see Fig. S1A to Q and Table S1 in the supplemental material), including three PyApiAP2 genes (PY17X_1317000, PY17X_ 1417400, and PY17X_0523100) whose orthologs in P. berghei were either resistant to disruption or not attempted (9). We carefully evaluated the morphologies of asexual/ sexual stages in ICR mice and sexual stages in Anopheles stephensi mosquitoes for the 12 gene KO mutants.…”

“…There were 12 PyApiAP2 genes (PY17X_0104500, PY17X_1231600, PY17X_1209100, PY17X_0941600, PY17X_0911000, PY17X_1361700, PY17X_1456200, PY17X_0111100, PY17X_0113700, PY17X_0838600, PY17X_0934300, and PY17X_ 1405400) that could not be disrupted even after 4 to 12 independent transfections and selections (Table S1). The orthologs of these 12 genes in P. berghei also resisted disruption attempts (9) and are likely to be essential for parasite viability or affect the growth of these rodent parasites in the mouse. Further investigations are necessary to dissect the functional roles of these genes, including tagging the genes for protein expression analysis.…”

“…Two ApiAP2 genes (Pfap2-g/Pbap2-g and Pbap2-g2) have been shown to play a critical role in gametocytogenesis in P. falciparum and/or P. berghei (9,(12)(13)(14). To investigate whether the genes are also critical to P. yoelii gametocytogenesis, we used the CRISPR-Cas9 method to disrupt the Pyap2-g and Pyap2-g2 genes.…”

“…In this study, we attempted to disrupt 24 of the 26 ApiAP2 genes in the mouse malaria parasite P. yoelii using the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9)-based methods we described previously (22), and we were able to knock out 12 of these 24 genes, including 3 genes homologous to genes that were not disrupted in P. berghei (9). We infected mice and mosquitoes with the mutant parasites to characterize phenotypic changes in parasite development, leading to functional characterization of two new P. yoelii ApiAP2 (PyApiAP2) genes that play important roles in gametocytogenesis and ookinete development, respectively.…”