A novel Plasmodium yoelii pseudokinase, PypPK1, is involved in erythrocyte invasion and exflagellation center formation

cAMP-dependent protein kinase regulates secretion of apical membrane antigen 1 (AMA1) in Plasmodium yoelii

“…The promoter to drive FRB-Cre60 and FKBP-Cre59 was replaced with the P. yoelii elongation factor 1 alpha (ef-1α) bi-directional promoter (pBS-DC-Pyef-1α). The DiCre expression cassette was PCRamplified from pBS-DC-Pyef-1α using oligonucleotide primers DiCre.F and DiCre.R, and ligated into the pDC2-cam-Cas9-PyU6-hDHFR plasmid using an In-Fusion HD cloning kit (Takara Bio Inc., Shiga, Japan), yielding pDC2-Cas9-DC-Pyef-1α [16]. A guide RNA (gRNA) sequence to introduce a double strand break in the p230p locus (PY17X_0306600) was designed using EupaGDT and ligated using T4 ligase (New England Biolabs, Ipswich, MA, USA).…”

“…Recodonization was done to avoid unwanted integration to this region in the genome by homologous recombination, instead of the 5'-HR. A DNA fragment containing hDHFR-yFCU open reading frame was PCR-amplified from pDC2-Cas9-PyU6-PypPK1-myc plasmid with primers PKAc-iKOhDHFR/ yFCU⋅F and PKAc-iKO-hDHFR/yFCU.R [16]. All PCR products were purified using a gel extraction kit and ligated into pDC2-Cas9-PyU6-hDHFR plasmid using an In-Fusion HD cloning kit, yielding pDC2-Cas9-PyU6-PKAc-iKO-hDHFR/yFCU.…”

“…Parasite-infected erythrocytes were collected at 24 and 48 h after RAP administration, treated with 0.1% saponin, and proteins were extracted with 1% Triton X-100 in PBS containing protease inhibitor cocktail (cOmplete™; Sigma-Aidrich). SDS-PAGE and Western blot were performed as described [16]. Membranes were probed for 1 h at room temperature (RT) with mouse anti-Cre monoclonal antibody (1:1000; 2D8; Sigma-Aldrich), rabbit anti-FKBP12 antibody (1:1000; ab2918; Abcam, Cambridge, UK), mouse anti-Myc monoclonal antibody (1:1000; 9B11; Cell Signaling Technology, Danvers, MA, USA), or rabbit anti-PyAMA1 polyclonal antibody (1:300) [20].…”

“…Secondary hybridization was with HRP-conjugated anti-mouse IgG or anti-rabbit IgG (1:12000; Promega) for 1 h at RT. Bands were detected as described [16]. The band intensities were quantified with ImageJ software [21].…”

“…To evaluate protein secretion from organelles to the merozoite surface, a secretion assay was performed basically as described [16]. In brief, merozoites were filtrated at 15 • C and incubated for 0 or 10 min at 37 • C, then were fixed with PFA-GTA for 15 min and transferred onto poly-L-lysine-coated cover slips.…”