Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response

Abstract

In sexually reproducing organisms, meiosis is an essential step responsible for generation of haploid gametes from diploid somatic cells. The quest for understanding regulatory mechanisms of meiotic recombination in Plasmodium led to identification of a gene encoding a protein that contains 11 copies of C 2 H 2 zinc fingers (ZnF). Reverse genetic approaches were used to create Plasmodium berghei parasites either lacking expression of full-length Plasmodium berghei zinc finger protein (PbZfp) (knockout [KO]) or expressing PbZfp lacking C-terminal zinc finger region (truncated [Trunc]). Mice infected with KO parasites survived two times longer (P Ͻ 0.0001) than mice infected with wild-type (WT) parasites. In mosquito transmission experiments, the infectivity of KO and Trunc parasites was severely compromised (Ͼ95% oocyst reduction). KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C 2 H 2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination.IMPORTANCE Malaria parasites are haploid throughout their life cycle except for a brief time period when zygotes are produced as a result of fertilization between male and female gametes during transmission through the mosquito vector. The reciprocal recombination events that follow zygote formation ensure orderly segregation of homologous chromosomes during meiosis, creating genetic diversity among offspring. Studies presented in the current manuscript identify a novel C2H2 ZnFcontaining protein exhibiting multifunctional roles in parasite virulence, mosquito transmission, and homologous recombination during meiosis. Understanding the transmission biology of malaria will result in the identification of novel targets for transmission-blocking intervention approaches.

PRDM9 and Its Role in Genetic Recombination

“…In what appears to be a remarkable case of horizontal gene transfer, the malarial parasite Plasmodium berghei , possesses a single zinc finger protein (PbZfp) that lacks a SET domain, but is nevertheless required for methylation of histone H3 at K4, K27 and K36, presumably by complexing with another histone methyltransferase [71]. Mice infected with parasites carrying a knockout of PbZfp survive longer than controls and show severely reduced mosquito infectivity.…”

“…Mice infected with parasites carrying a knockout of PbZfp survive longer than controls and show severely reduced mosquito infectivity. PbZfp-deficient parasites also have a markedly reduced ability to produce plasmodial oocysts, implicating a role in genetic recombination and the possibility that mammalian PRDM9 may have served as the original source of this gene [71].…”

Checks and balances? DNA replication and the cell cycle in Plasmodium

“…Accordingly, HR is the prime source of DSB repair in the parasite [ 79 – 81 ] (by contrast, T. gondii possesses a functional NHEJ pathway, indicating loss of this pathway in Plasmodium [ 82 , 83 ]). HR, indeed, appears to be essential for the completion of the parasite life-cycle because the knockout of a zinc finger protein, Pb Zfp, in P. berghei leads to a loss of transmission competence in mosquitoes due to a failure to recruit the topisomerase-like enzyme Spo11 to recombination hotspots [ 80 ]. During all haploid growth phases the parasite must therefore rely upon alternative end joining pathways such as microhomology-mediated end joining (MMEJ) to repair DSBs within the core genome, because no repair template exists to allow HR [ 84 ].…”

Zinc finger proteins of Plasmodium falciparum

“…PfTRZ further binds to 5S rDNA genes to regulate 5S rRNA synthesis. A similar loss-of-function analysis of the P. berghei homologue PbZfp did not reveal any defect during intraerythrocytic development, while the transmission to the mosquito was reduced (Gopalakrishnan, Aly, Aravind, & Kumar, 2017).…”