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. 2019 Oct 8;10(5):e01972-19.
doi: 10.1128/mBio.01972-19.

Protein Kinase A Is Essential for Invasion of Plasmodium Falciparum Into Human Erythrocytes

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Free PMC article

Protein Kinase A Is Essential for Invasion of Plasmodium Falciparum Into Human Erythrocytes

Mary-Louise Wilde et al. mBio. .
Free PMC article

Abstract

Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610.IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

Keywords: AMP-activated kinases; Plasmodium falciparum; host cell invasion; malaria.

Figures

FIG 1
FIG 1
PfPKAc is essential for parasite viability. (A) Schematic of PfPKAc gene disruption (ΔPfPKAc). (B) Schematic of conditional knockout line generation (PfPKAc:loxP). The same backbone was used for generation of both constructs used for transfection. (C) Following transfection, parasites were monitored and given viability scores each day. Tables summarize outcomes of each transfection. ×, no parasite survival; ✓, parasite survival; N.D., not done. In both NF54 and 3D7, gene disruption was unsuccessful as no parasites were observed after 30 days in culture (ΔPfPKAc, n = 3). PfPKAc:loxP transfection was successful in both 3D7 and NF54 and was not repeated.
FIG 2
FIG 2
Conditional regulation of PfPKAc. (A) Parental parasite lines expressed DiCre, which was inserted into the rh3 locus using Cas9-stimulated repair with pDiCre-CAM, a plasmid encoding the two halves of Cre recombinase as well as a blasticidin resistance cassette. Conditional knockout PfPKAc:loxP parasites were generated in the DiCre-expressing line. (B) Schematic of the dimerizable Cre recombinase (DiCre) system. The N-terminal and C-terminal fragments of Cre recombinase were conjugated to one of two rapamycin binding proteins, the FK506-binding protein (FKBP12) or the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin-associated protein (FRAP). Addition of rapamycin enables heterodimerization of the two inactive Cre components via interactions of rapamycin-binding proteins to restore Cre activity. Cre recombinase recognizes the two 34-bp loxP sites inserted into the pkac locus and excises the gene sequence between them, rendering pkac inactive. Restriction sites for Southern blotting (NcoI and AvaII) are shown as well as the resulting sizes of the expected RNA fragments expressed in kilobase pairs (kb). (C) Integration of DiCre and of both loxP sites was confirmed by PCR. A fragment (605 bp) at the 5′ end of the pka gene was amplified as a loading control. (D) Southern blot analysis of NcoI/AvaII digests confirmed excision of PfPKAc following the addition of rapamycin. NF54 PfPKAc:loxP parasites were cultured for 48 h in the presence of DMSO, GlcN, or rapamycin prior to genomic DNA harvest. (E) Western blot showing PfPKAc-HA expression levels following rapamycin treatment. NF54 DiCre or PfPKAc:loxP parasites were cultured for 72 h in the presence of DMSO or GlcN. Lysates were prepared from late-stage schizonts. Anti-HA was used to detect PfPKAc expression, and anti-PfHSP70 was used as a loading control. (F) Widefield imaging of NF54 PfPKAc:loxP schizonts labeled with anti-GAP45, anti-HA, and DAPI. PfPKAc-HA shows partial peripheral and cytoplasmic localization. Scale bars represent 2.5 μm.
FIG 3
FIG 3
PfPKAc is required for P. falciparum growth. (A) Immunoblot showing PfPKA-HA expression in NF54 PfPKAc:loxP rings (R), early trophozoites (ET), late trophozoites (LT), and schizonts (S). Anti-AMA1 was used as a late-stage-specific control, while anti-aldolase was used as a loading control. (B) Growth of parasites in the presence of rapamycin and GlcN. NF54 WT or PfPKAc:loxP parasites were cultured for 72 h under standard conditions or with the addition of DMSO, rapamycin, or GlcN or of both rapamycin and GlcN. Trophozoites were harvested and stained with ethidium bromide, and parasitemia was determined by FACS analysis. Growth is expressed relative to NF54 PfPKAc:loxP parasites grown under standard conditions. Data are expressed as means ± standard deviations (SD). ****, P < 0.0005 (Sidak’s multiple-comparison test). (C) (Panel i) Growth of NF54 PfPKAc:loxP parasites over 4 cycles compared to wild-type parasites in the presence of rapamycin. At the end of each intraerythrocytic growth cycle, late schizonts were harvested for FACS analysis to determine parasitemia levels. Growth is expressed relative to DMSO controls. (Panel ii) Parasites were added to fresh erythrocytes at 0.2% parasitemia, and growth was monitored over 4 cycles. The arrow indicates where the parasites were split under the DMSO treatment conditions. Data are presented as means ± SD (n = 3). (D) Light microscopy of Giemsa-stained NF54 PfPKAc:loxP parasites shows that rapamycin-treated parasites developed normally to schizonts within the first cycle but failed to progress into the next intraerythrocytic cycle. The parasites that remained after a second cycle of growth appeared to be dying or undergoing gametocytogenesis (black arrowhead).
FIG 4
FIG 4
PfPKAc is required for successful merozoite invasion. (A) NF54 PfPKAc:loxP parasites were treated with rapamycin for one growth cycle, and merozoites were purified and added to fresh red blood cells in an invasion assay. Data represent results determined for pooled technical replicates from 4 independent experiments. ****, P < 0.0005 (unpaired t test). (B) Parasites undergoing egress were imaged by live microscopy. “Normal egress” was defined as complete release of motile merozoites in less than 3 s, with no clumping of merozoites. (C) Live imaging revealed no defect in schizont development. Schizont morphology was determined by counting merozoites per schizont undergoing egress in live-imaging experiments. (D) (Panel i) Selected still images from Video S1 showing a PfPKAc:loxP DMSO-treated merozoite successfully deforming the erythrocyte membrane (black arrow), invading, and triggering echinocytosis (white arrowhead). (Panel ii) Stills from Video S2. (Panel iii) Stills from Video S3. Rapamycin-treated merozoites can still deform the erythrocyte membrane and stimulate echinocytosis (black arrow and white arrowhead, respectively); however, they remain attached for prolonged periods without invading (black arrowheads). (E) PfPKAc-deficient parasites attached to erythrocytes at a lower frequency than controls. Data are presented as means ± SEM, *=P < 0.05 (unpaired t test). (F) Outcomes were tracked for all merozoites that made initial attachment, showing prolonged attachment durations and reduced invasion frequencies by PfPKAc-deficient parasites. *, P < 0.05; ****, P < 0.00005 (chi-square test). Quantification of live-imaging experiments was performed on pooled data from 14 independent experiments for each set of conditions, collected over three different days.
FIG 5
FIG 5
PfPKAc is responsible for PfAMA1 Ser610 phosphorylation. (A) Western blot showing unchanged expression levels of AMA1 following PfPKAc knockdown in PfPKAc:loxP late-schizont-stage parasites. (B) Late-schizont-stage parasites were harvested and lysed following DMSO or rapamycin treatment and incubated with recombinant PfAMA1 cytoplasmic tails. Phosphorylation of Ser610 was detected in an ELISA format using anti-PfAMA1Ser610p antibody (23). Dose-response curves representing cAMP responses were generated, and data were normalized to DMSO-treated controls. Data are represented as average percentages of phosphorylation relative to DMSO-treated controls at 4 μM cAMP. Data are presented as means ± SD; ****, P < 0.00005 (unpaired t test).

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