SRA

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 (?pbgpr180) 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 ?pbgpr180 gametocytes had assigned functions in cyclic nucleotide signaling transduction. In the ?pbgpr180 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. Overall design: Total RNA was extracted from the 48% Nycodenz purified activated gametocytes (incubation at 25? for 15 min ) of the WT and ?pbgpr180 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.