An inner membrane complex protein IMC1g in Plasmodium berghei plays an essential role in asexual erythrocytic proliferation and transmission

The inner membrane complex (IMC), a double-membrane organelle underneath the plasma membrane in apicomplexan parasites, plays a significant role in motility and invasion and confers shape to the cell. We characterized the function of PbIMC1g, a component of the IMC1 family member in Plasmodium berghei. PbIMC1g is recruited to the IMC in late schizonts, activated gametocytes, and ookinetes. Pairwise yeast two-hybrid assays demonstrate that PbIMC1g interacts with IMC1c, a component of the PHIL1 complex, and the core sub-repeat motif 'EKI(V)V(I)EVP' in PbIMC1g is essential for this interaction. Localization of PbIMC1g to the IMC was dependent on its IMCp domain, while its C-terminus and palmitoylation sites were required for the full efficiency of proper IMC targeting. PbIMC1g is required for asexual stage development, and its conditional knockdown resulted in a defect in schizogony. Additionally, PbIMC1g was also important for male gametogenesis and ookinete development. As an IMC component that assists in anchoring the glideosome to the subpellicular network, PbIMC1g was also involved in ookinete motility and mosquito midgut invasion. IMC1g from the human parasite P. Plasmodium vivax could functionally replace PbIMC1g in P. berghei, confirming the evolutionary conservation of IMC1g proteins in Plasmodium spp. Together, this work reveals an essential role of IMC1g in the parasite life cycle and suggests that IMC1 family members likely contribute to parasite gliding and invasion. Overall design: About 108 purified activated gametocytes from PbIMC1gcKD [+]/[-] TMP parasites were lysed in 10 × volumes of TRIzol (Thermofisher) and used for total RNA extraction. The experiment was repeated at three-week intervals to obtain samples as biological duplicates. Sample preparations were performed as described]. Briefly, mRNA was enriched using poly-T oligo-attached magnetic beads. Reverse transcription was performed with SuperScript II (Invitrogen) using random primers. The reaction was then treated with RNaseH to remove the RNA template and cleaned using DNA Clean and Concentrator Kit (Zymo Research). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced using the Illumina platform. RNA-seq raw data were initially filtered by fastq to obtain high-quality, clean data. Adapter trimming was performed using cutadapt version 1.13 (http://cutadapt.readthedocs.io/en/stable/guide.html). Processed reads were aligned to the P. berghei ANA genome (assembly PlasmoDB-67) with Hisat2 v2.0.4 [108] and deduplicated according to UMI using UMI-tools (v1.0.0) [109]. Reads counts were obtained with HTSeq v0.9.1. Differential expression analysis of genes between the PbIMC1gcKD parasites cultured in both [+] and [-] TMP conditions were determined using the DESeq R package (1.18.0). The p-values were adjusted using the Benjamini-Hochberg procedure, which controls the false discovery rate (FDR). Genes with an adjusted P value <0.05 found by DESeq were assigned as differentially expressed. GO enrichment analysis was implemented using the GOseq R package.