AP2XII-1 and AP2XI-2 Suppress Schizogony Gene Expression in Toxoplasma gondii

Toxoplasma gondii is a medically and veterinary important intracellular parasite that undergoes distinct developmental transitions in its intermediate and definitive hosts. The switch between stages of T. gondii is meticulously regulated by a variety of factors. Previous studies have explored the role of the microrchidia (MORC) protein complex as a transcriptional suppressor of sexual commitment. By utilizing immunoprecipitation and mass spectrometry, constituents of this protein complex have been identified, including MORC, Histone Deacetylase 3 (HDAC3), and several ApiApiAP2 transcription factors. Conditional knockout of MORC or inhibition of HDAC3 results in upregulation of a set of genes associated with schizogony and sexual stages in T. gondii tachyzoites. Here, our focus extends to three primary ApiAP2s: ApiAP2XII-1, ApiAP2XI-2, and ApiAP2V-2, demonstrating their significant impact on the fitness of asexual tachyzoites and their target genes. Notably, the targeted disruption of ApiAP2XII-1 and ApiAP2XI-2 resulted in a more profound alteration in merozoite-specific genes targeted by the MORC/HDAC3 complex compared to ApiAP2V-2. Additionally, considerable overlap was observed in downstream gene profiles between ApiAP2XII-1 and ApiAP2XI-2, with ApiAP2XII-1 specifically binding to a subset of ApiAP2 transcription factors, including ApiAP2XI-2. These findings reveal an intricate cascade of ApiAP2 regulatory networks involved in T. gondii schizogony development, orchestrated by ApiAP2XII-1 and ApiAP2XI-2. This study provides valuable insights into the transcriptional regulation of T. gondii growth and development, shedding light on the intricate life cycle of this parasitic pathogen. Transgenic parasite strains cultured in Vero cells were treated with either 500 μM IAA or vehicle for 12 h. Total RNAs from T. gondii tachyzoites were then extracted using the M5 Total RNA Extraction Reagent (Mei5 Biotechnology Co., Ltd, Beijing) according to the manufacturer’s protocol. Each treatment consisted of three biological replicates. The purity, concentration and integrity of RNAs were tested using the NanoPhotometer® (IMPLEN, CA, USA), the Qubit® RNA Assay Kit in the Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. Only qualified samples were used for library preparation. Illumina sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations. Sequencing was performed using the Illumina Novaseq 6000 platform from Shanghai Personal Biotechnology Co., Ltd. to generate 150 bp paired-end reads. The original sequencing data can be found in the Sequence Read Archive database under the accession number PRJNA1009254. RNA-seq reads were uploaded to the BMKCloud (www.biocloud.net) platform for analysis based on the reference genome of Toxoplasma Type II ME49 strain (ToxoDB-57). Briefly, Paired-end clean reads were aligned to the reference genome using Hisat242. Read counts for each gene were calculated using the sorted bam files using StringTie43. Differentially expressed genes (DEGs) between treated and untreated parasites were calculated by edgeR44. Gene ontology enrichment analysis was performed in ToxoDB. Gene expression with a fold change >2 or < -2 and FDR < 0.05 was defined as significantly differentially expressed. TPM (Transcripts per kilobase million) was calculated for each gene and used for clustered heatmap drawing. The transgenic strains were inoculated in 25 cm2 cell culture flasks and washed after 4 h to remove non-invasive parasites. Transgenic strains (1 x 105) were harvested after 12 h of incubation with or without IAA (500 μM). Library construction was performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, Nanjing, China) according to the manufacturer's instructions. Briefly, fresh tachyzoites were bound to activated concanavalin A beads (10 ml/sample) and incubated for 10 min at room temperature. The mixture was resuspended and incubated with primary antibody (1:50, mice anti-HA) at 4°C overnight. After several washes, the parasites were incubated with secondary antibody (1:100, goat anti-mouse IgG) for 1 h at room temperature. The parasites were then resuspended with 300 ml of pA-Tn5 buffer (0.04 mM) and incubated for 1 h at room temperature on a rotator. Tagmentation was stopped by proteinase K treatment and DNA extraction was performed using DNA extract beads (Vazyme, Jiangsu, China)45. Illumina sequencing libraries were generated by PCR amplification with specific adaptors according to the manufacturer’s recommendations (TruePrep Index Kit V4 for Illumina, Vazyme, Nanjing, China). Cut-Tag libraries were sequenced using the Illumina Novaseq 6000 platform (Shanghai Personal Biotechnology Co., Ltd). The paired-end reads were filtered and then aligned to the T. gondii reference genome using Bowtie246 (v.2.1.0). The resulting sam files were transformed into bam files. PCR duplicates were removed from the sorted bam files using Sambamba47. The filtered reads were then employed to identify Cut-Tag peaks using MACS248. The overlapped peaks in two biological replicates were identified by the Irreproducibility Discovery Rate (IDR)49. The final peaks were annotated against the latest T. gondii data in ToxoDB. The sorted and filtered bam files of Cut-Tag peaks and RNA-seq reads were normalized to RPKM with a resolution of 10 bp (bin size) and transformed into bigwig files for direct visualization in IGV (Integrative Genomics Viewer)50. All raw and processed files can be accessed on the Gene Expression Omnibus (GEO).