Phosphatidic acid homeostasis regulated by a type-2 phosphatidic acid phosphatase represents a novel druggable target in malaria intervention

a Domain architecture of PfPAP2. PfPAP2 harbors all the canonical domains of PAP2 superfamily. It contains 5 trans-membrane domains marked in black (T1-T5) and an acid phosphatase domain (APD) highlighted in light green towards end of C-terminal. Acid phosphatase domain possesses the conserved signature acid phosphatase motif. b Homology model of PfPAP2 three-dimensional structure. Predicted 3D structure of PfPAP2 generated by Phyre2 server. The model was further processed for energy minimization and to reduce steric hindrances. APD lies between T2-T5, which are clustered together. c Characteristic signature motif of acid phosphatase domain (APD) of PAP2 superfamily is conserved across different species of Plasmodium. KXXXXXXRP-PSGH-SRXXXXXHXXXD is the characteristic signature motif of APD. Multiple sequence alignment of APD of PfPAP2 with APDs of other Plasmodium species shows that the consensus sequence is highly conserved across different Plasmodium species. d PfPAP2 transcript is present in the blood stage. An amplicon of 460？bp amplified using primer probes specific to PfPAP2 was detected in the cDNA isolated from blood stage parasite. Intron specific primers were used in RT-PCR analysis with cDNA as a negative control for genomic DNA contamination RNA samples isolated from unsynchronized blood stage parasites were reverse-transcribed (+RT) and used for detection of transcripts for PfPAP2, EBA175 and 18S rRNA by PCR using specific primers. These primers were also used for PCR with RNA without reverse transcription (？RT) to control for genomic DNA contamination and with P. falciparum genomic DNA (G) as a positive control. e Expression of PfPAP2 was also detected at protein level in P. falciparum merozoites by Western blotting. Mouse serum raised against rAPD detects a protein of ~36？kDa in merozoite lysates by Western blotting, corresponding to native PfPAP2. Pre-immune sera (PIS) did not recognize any proteins in the merozoite lysate. f Detection of PfPAP2 by IFA. i PfPAP2 is expressed throughout the erythrocytic schizogony. The localization of PfPAP2 was investigated in blood stages of P. falciparum by IFA using confocal microscope, Nikon A1. In the ring stage, PfPAP2 (green) was found around the nucleus stained with DAPI (blue). In trophozoites and late schizont stage, PfPAP2 (green) was found to give a honeycomb-staining pattern. g In the schizont and merozoite stage, colocalization of PfPAP2 (green) with PfMSP1 (red), a merozoite surface marker protein, revealed localization of PfPAP2 below cell membrane towards parasite cytoso