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 C₂H₂ 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 < 0.0001) than mice infected with wild-type (WT) parasites. In mosquito transmission experiments, the infectivity of KO and Trunc parasites was severely compromised (>95% 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 C₂H₂ 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 ZnF-containing 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.

Keywords: DNA damage; epigenetic modification; malaria transmission; zinc finger proteins.

FIG 1

Schematic of PbZfp and gene-targeting plasmids. PbZfp was identified by PSI-BLAST sequence profiling for C2H2 ZnF and BLAST analysis of PlasmoDB database entries using the protein sequence of mouse PRDM9 as a query. (A) Schematic of PbZfp protein (1,248 amino acids) and relative positions of 11 zinc fingers (boundaries are indicated in the text box), identified by PSI-BLAST analysis (panel I). A schematic of truncated PbZfp protein lacking Zfd expression is shown in panel II. (B) Schematic of targeting plasmids and parasite genomic locus, before and after transfection. Details of sequential steps used to construct plasmids are given in Materials and Methods. Key features of the plasmids used to generate PbZfp knockout and truncated parasites are shown in panels I and II, respectively. In the truncation plasmid shown in panel II, a stop codon (*) was inserted near the 5′ end of the sequence coding for the Zfd. Various restriction sites are also identified. The wild-type Pbzfp genomic locus is represented in panel III. Panel IV shows the genomic locus in Pbzfp knockout (KO) parasites after homologous recombination events at the 5′utr and 3′utr sequences present in the targeting plasmid (panel I) and the wild-type genomic locus (panel III). Panel V shows the expected genomic locus after homologous recombination events at the PbZfp coding sequence and 3′utr sequences present in the plasmid (panel II) and wild-type genomic locus (panel III). Various oligonucleotide primers used for PCR amplification and characterization are shown by numbers (panels I and III). Forward primers are indicated in black numbers and reverse primers in red.

FIG 2

RT-PCR demonstration of pbzfp expression. Total RNA from WT, KO (A), and truncated (B and C) parasites was reverse transcribed (+RT lanes), and cDNAs were used as the templates for PCR amplification. RNAs without reverse transcription (−RT lanes) were used as negative controls. Primer pairs used to confirm PbZfp expression by RT-PCR were 811/812 (A), 807/808 (B), and 807/810 (C). Primers 610/857 were used for PbRad51expression in the WT, KO, and Trunc parasites (panels A, B, and C). Sequences of various primers used are described in Table S1. All the samples were also tested using primers for an unrelated gene (pbRad51) to establish the specificity and quality of RNA samples.

FIG 3

Comparison of in vivo asexual growth kinetics (A) and survival (B) of mice infected with WT, KO, and truncated P. berghei. Data shown are pooled from 12 mice per infection group (4 independent experiments, 3 mice per group for each parasite type). Blood smears prepared daily, beginning day 3 postinfection (10⁵ infected red blood cells injected i.v.), were subjected to Giemsa staining for microscopy. The mice started to die beginning day 8 or 9, and the cages were observed twice daily to record death. A minimum of 500 to 1,000 red blood cells were counted to determine percent parasitemia [(infected RBC/total RBC) × 100]. (A) Mean percentages of parasitemia + standard errors of the means (SEM) for all surviving mice on any given day. (B) Kaplan-Meier survival plots generated using GraphPad Prism software.

FIG 4

Comparison of transmission competence of WT, KO, and truncated parasites. Mice (n = 3 per group) were infected with WT, KO, and Trunc parasites and on day 4 postinfection were used to feed A. stephensi mosquitoes as described in Materials and Methods. Blood-fed mosquitoes were dissected (day 9 to day 11 post-blood feed), and midguts were examined microscopically (total magnification, ×100) to determine the oocyst number in each midgut. Each column shows the oocyst distribution in the mosquitoes fed on an individual mouse. (A) Results from 3 mice per group infected with WT and one of the two clones of KO and Trunc parasites. (B) Repeat of analysis of transmission data using mice infected with WT and a second independent clonal line of KO and Trunc parasites. (C) Results obtained with WT and transfection control (Tc) parasites (parasites that possessed the WT locus but went through all the experimental manipulations used to obtain two different clonal KO and Trunc parasites). Blood smears on the day of mosquito infection were examined to determine gametocyte sex (female/male) ratios. Immediately after a mosquito feed, mice were bled to compare the characteristics of morphological development of ookinetes in vitro. (D) Results of analyses of female/male (F/M) gametocyte ratios and ookinete development (Ook), each averaged for observations pooled for 6 mice + SEM. (E) Representative Giemsa-stained ookinetes of WT, KO, and Trunc parasites.

FIG 5

Histone methylation and acetylation patterns in WT, KO, and Trunc parasites. Blood from mice infected with WT (lanes W), KO (lanes K), and truncated (lanes T) parasites was passed through a cellulose column to remove leucocytes prior to saponin lysis and Western blot analysis. Samples (10⁶ parasite equivalents per lane) were fractionated by 15% SDS-PAGE followed by transfer to a nitrocellulose membrane and probing with specific antibodies. (A and B) Reactivity patterns were determined using lysates of infected red blood cells with the indicated histone trimethylation (A)- and acetylation (B)-specific antibodies. We further purified histones from leucocyte depleted normal mouse red blood cells (N), WT (W), KO (K), and Trunc (T) parasites using an acid extraction procedure. (C) SDS-PAGE results obtained using a Coomassie blue-stained gel of each purified preparation. (D and E) Reactivity patterns determined using purified histones with the indicated histone trimethylation (D)- and acetylation (E)-specific antibodies.

FIG 6

Susceptibility of WT, KO, and truncated P. berghei parasites in MMS-induced DNA damage response assay. Blood from infected mice was processed as described in Materials and Methods and tested in a MMS-induced DMA damage response assay previously optimized for similar studies in P. falciparum (36). Parasites in duplicate wells were treated with the indicated concentrations of MMS for 6 h followed by Comet assay analysis. The concentrations of MMS used were 0 (bars 1), 0.05% (bars 2), 0.005% (bars 3), and 0.0005% (bars 4). (Top panel) Olive tail moment (OTM) values were calculated as previously described (35) and averaged for each sample. (Bottom panel) Comets from each sample were further analyzed visually to estimate the proportion of damaged nuclei compared to the total number. Data were analyzed using GraphPad Prism software. Statistically significant differences (P < 0.05) between WT and KO parasites are indicated by an asterisk.

FIG 7

Transcriptional differences in WT parasites (left panel), KO parasites (middle panel), and truncated parasites (right panel) after exposure to MMS. An aliquot of parasite samples analyzed in Comet assays (Fig. 6) was used for transcriptional analysis by real-time RT-PCR. RNA was purified from parasites that were left untreated or were exposed to the indicated concentrations of MMS in duplicate wells, and purified RNA (1.0 μg) was reverse transcribed as described in Materials and Methods. Three different amounts of cDNA (undiluted, diluted 1:10, and diluted 1:100) from each were analyzed by PCR using specific primers for PbZfp, PbSpo11, PbRad51, and PbDmc1 (Table S1) and a BioRad IQ5 real-time PCR detection system. Pb18rRNA gene served as an internal control. Relative C_T values from real-time PCR in the control and treated parasites were calculated by the Pfaff method, and fold change between untreated and treated samples was determined as previously described (35).