A G-protein-coupled receptor modulates gametogenesis via PKG-mediated signaling cascade in Plasmodium berghei

Gametogenesis is essential for malaria parasite transmission, but the molecular mechanism of this process remains to be refined. Here, we identified a G-protein-coupled receptor 180 (GPR180) that plays a critical role in signal transduction during gametogenesis in Plasmodium. P. berghei GPR180 was predominantly expressed in gametocytes and ookinetes and associated with the plasma membrane in female gametes and ookinetes. Knockout of pbgpr180 (Δpbgpr180) had no noticeable effect on blood-stage development but impaired gamete formation and reduced transmission of the parasites to mosquitoes. Transcriptome analysis revealed that a large proportion of the dysregulated genes in the Δpbgpr180 gametocytes had assigned functions in cyclic nucleotide signaling transduction. In the Δpbgpr180 gametocytes, the intracellular cGMP level was significantly reduced, and the cytosolic Ca2+ mobilization showed a delay and a reduction in the magnitude during gametocyte activation. These results suggest that PbGPR180 functions upstream of the cGMP-protein kinase G-Ca2+ signaling pathway. In line with this functional prediction, the PbGPR180 protein was found to interact with several transmembrane transporter proteins and the small GTPase Rab6 in activated gametocytes. Allele replacement of pbgpr180 with the P. vivax ortholog pvgpr180 showed equal competence of the transgenic parasite in sexual development, suggesting functional conservation of this gene in Plasmodium spp. Furthermore, an anti-PbGPR180 monoclonal antibody and the anti-PvGPR180 serum possessed robust transmission-blocking activities. These results indicate that GPR180 is involved in signal transduction during gametogenesis in malaria parasites and is a promising target for blocking parasite transmission. Total RNA was extracted from the 48% Nycodenz purified activated gametocytes (incubation at 25℃ for 15 min ) of the WT and Δpbgpr180 K1 parasites using the Qiagen RNeasy kit (Qiagen, Dusseldorf, Germany). The purity of RNA was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 1 µg total RNA was used to purify the mRNAs using poly-T oligo-attached magnetic beads, followed by fragmentation using divalent cations under elevated temperature in the first-strand synthesis reaction buffer. First-strand cDNA was synthesized using random hexamer primer and RNase H. To select cDNA fragments of 100~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Adapters were ligated at 25°C for 10 min before PCR. PCR was performed with the Phusion High-Fidelity DNA polymerase, universal PCR primers, and the index (X) primer. PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, USA). After cluster generation, the libraries were sequenced on an Illumina platform, and 150 bp paired-end reads were generated. Raw reads in the fastq format were firstly processed through in-house Perl scripts to remove low-quality reads, reads containing adapter, and ploy-N. Meanwhile, the Q20, Q30 and GC content of the clean data were calculated. The UMI (Unique Molecular Identifiers) was extracted by the UMI-tools (v2.0.4). Only clean UMI reads were kept for further analysis. RNA-seq reads from each sample were mapped to the P. berghei ANKA genome obtained from the NCBI reference genome (PbANKA01) using the Hisat2 (v2.0.4) (68). The UMI-tools (v1.0.0) were used to deduplicate reads based on the mapping coordinates and the UMI attached to the read. Cuffdiff v2.1 was used as the default method for normalization (69), while differential expression analysis was conducted using DESeq package (1.18.0) in R (70). The resulting P-values were adjusted using Benjamini and Hochberg’s approach for controlling the FDR. Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.

配子发生（Gametogenesis）对于疟原虫的传播至关重要，但该过程的分子机制仍有待进一步阐明。本研究鉴定出一种G蛋白偶联受体180（G-protein-coupled receptor 180, GPR180），其在疟原虫配子发生过程中的信号转导中发挥关键作用。伯氏疟原虫（Plasmodium berghei, P. berghei）GPR180主要在配子体（gametocyte）和动合子（ookinete）中表达，并定位于雌性配子及动合子的质膜。敲除pbgpr180（Δpbgpr180）对疟原虫的红内期发育无显著影响，但会损害配子形成能力，并降低其向蚊子的传播效率。转录组分析显示，Δpbgpr180配子体中大量表达失调的基因，其功能均与环核苷酸信号转导相关。在Δpbgpr180配子体中，细胞内环磷酸鸟苷（cGMP）水平显著降低，且配子体激活过程中胞质钙离子（Ca²+）动员出现延迟，峰值幅度也有所下降。上述结果表明，PbGPR180作用于cGMP-蛋白激酶G-Ca²+信号通路的上游。与该功能预测相符，活化的配子体中PbGPR180蛋白可与多种跨膜转运蛋白以及小GTP酶Rab6发生相互作用。用间日疟原虫（Plasmodium vivax, P. vivax）的同源基因pvgpr180替换pbgpr180后，转基因疟原虫的有性发育能力未发生显著变化，提示该基因在疟原虫属（Plasmodium spp.）中功能保守。此外，抗PbGPR180单克隆抗体以及抗PvGPR180血清均表现出较强的传播阻断活性。上述结果表明，GPR180参与疟原虫配子发生过程中的信号转导，是阻断疟原虫传播的潜在靶点。本研究使用Qiagen RNeasy试剂盒（Qiagen，德国杜塞尔多夫），从野生型（WT）和Δpbgpr180 K1株疟原虫经48%尼科登（Nycodenz）密度梯度离心纯化的活化配子体（25℃孵育15分钟）中提取总RNA。使用NanoPhotometer®分光光度计（IMPLEN，美国加利福尼亚州）检测RNA纯度；使用安捷伦2100生物分析仪（Agilent Technologies，美国加利福尼亚州）的RNA Nano 6000检测试剂盒评估RNA完整性。取1 µg总RNA，使用结合有poly-T寡核苷酸的磁珠纯化mRNA，随后在高温下以二价阳离子在第一链合成反应缓冲液中进行片段化处理。以随机六聚体引物和RNase H合成第一链cDNA。为筛选长度为100~200 bp的cDNA片段，使用AMPure XP磁珠纯化系统（Beckman Coulter，美国贝弗利）对文库片段进行纯化。PCR前于25℃连接接头10分钟；使用Phusion高保真DNA聚合酶、通用PCR引物以及索引（X）引物进行PCR扩增。纯化PCR产物（AMPure XP系统），并通过安捷伦2100生物分析仪评估文库质量。使用TruSeq PE Cluster Kit v3-cBot-HS试剂盒（Illumina，美国圣迭戈），在cBot簇生成系统上对带有索引的样本进行簇扩增。簇扩增完成后，在Illumina平台上对文库进行测序，生成150 bp双端读段。首先使用自研Perl脚本处理fastq格式的原始读段，去除低质量读段、含接头的读段以及包含poly-N的读段；同时计算clean数据的Q20、Q30值以及GC含量。使用UMI-tools（v2.0.4）提取唯一分子标识符（Unique Molecular Identifiers, UMI），仅保留带有有效UMI的clean读段用于后续分析。使用Hisat2（v2.0.4）（68）将每个样本的RNA-seq读段比对到从NCBI参考基因组（PbANKA01）获取的伯氏疟原虫ANKA株基因组；使用UMI-tools（v1.0.0）根据比对坐标以及读段附带的UMI信息对读段进行去重。使用Cuffdiff v2.1作为默认的标准化方法（69），同时使用R语言中的DESeq软件包（1.18.0）进行差异表达分析（70）；采用Benjamini和Hochberg方法对得到的P值进行校正，以控制错误发现率（FDR）。经DESeq分析得到的校正后P值<0.05的基因被定义为差异表达基因。