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

Abstract

Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate hosts, and different developmental stages express unique sets of genes. Unexpectedly, many transcription factors (TFs) commonly found in eukaryotic organisms are absent in malaria parasites; instead, a family of genes encoding proteins similar to the plant Apetala2 (ApiAP2) transcription factors is expanded in the parasites. Several malaria ApiAP2 genes have been shown to play a critical role in parasite development; however, the functions of the majority of the ApiAP2 genes remain to be elucidated. In particular, no study on the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family has been reported so far. This study systematically investigated the functional roles of PyApiAP2 genes in parasite development. Twenty-four of the 26 PyApiAP2 genes were selected for disruption, and 12 were successfully knocked out using the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) method. The effects of gene knockout (KO) on parasite development in mouse and mosquito stages were evaluated. Ten of 12 successfully disrupted genes, including two genes that have not been functionally characterized in any Plasmodium species previously, were shown to be critical for P. yoelii development of sexual and mosquito stages. Additionally, seven of the genes were labeled for protein expression analysis, revealing important information supporting their functions. This study represents the first systematic functional characterization of the P. yoelii ApiAP2 gene family and discovers important insights on the roles of the ApiAP2 genes in parasite development.IMPORTANCE Malaria is a parasitic disease that infects hundreds of millions of people, leading to an estimated 0.35 million deaths in 2015. A better understanding of the mechanism of gene expression regulation during parasite development may provide important clues for disease control and prevention. In this study, systematic gene disruption experiments were performed to study the functional roles of members of the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family in parasite development. Genes that are critical for the development of male and female gametocytes, oocysts, and sporozoites were characterized. The protein expression profiles for seven of the PyApiAP2 gene products were also analyzed, revealing important information on their functions. This study provides expression and functional information for many PyApiAP2 genes, which can be explored for disease management.

Extensive Transcriptional and Translational Regulation Occur During the Maturation of Malaria Parasite Sporozoites

“…For instance, in both P. yoelii and P. falciparum , CelTOS, GEST and SPELD were not detected or were only weakly expressed in oocyst sporozoites, but were among the most abundant proteins in salivary gland sporozoites (Table 2, Table S1). Moreover, the presence/absence of cellular regulators such as specific ApiAP2s, histone modifiers, RNA-binding proteins and other proteins (Table S1) agree with previous reports describing how these types of regulation may be used by the sporozoite 32, 40, 48 .…”

“…Beyond the previously defined UIS mRNAs, several other transcripts are notable as their protein products are or may be important to the function of salivary gland sporozoites: FAS-II pathway proteins, fatty acid modifiers, plasma membrane transporters, adhesins/surface proteins ( p113 , speld , tlp ), traversal-related proteins ( celtos ), heat shock proteins, and ApiAP2 specific transcription factors ( e.g. py17x_0523100, pf3d7_0420300 ) 40–47 . Together, these transcripts encode for proteins that encompass many essential attributes necessary for sporozoite development, transmission, and infectivity.…”

Regulation of sexual differentiation is linked to invasion in malaria parasites

“…One critical regulator of sexual commitment is AP2-G, which is a member of the ApiAP2 family of DNA-binding proteins 8,9 . Forward and reverse genetics in P. falciparum, P. berghei, and P. yoelii has demonstrated that AP2-G is a master regulator of gametocytogenesis [8][9][10][11] . Cells that do not express ap2-g will continue asexual development, whereas those that do are able to commit and enter gametocytogenesis.…”

Female‐specific gene regulation in malaria parasites by an AP2‐family transcription factor

“…More recently, its ortholog in P . yoelii (PY17X_1417400), named AP2‐G3, was reported to be expressed in both sexual and asexual stages and to play a critical role in gametocytogenesis (Zhang et al , ). Here, we report that this transcription factor is female‐specific and essential for the development (thus designated as AP2‐FG).…”