A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes

Abstract

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Figure 1. PfCDPK5 is an essential calcium-dependent protein kinase

A) Schematic of PfCDPK5. Scale bar, 100 amino acids. B) Recombinant GST-PfCDPK5–6His (94 kDa) was incubated with or without substrate (myelin basic protein, 18 kDa), with 1.1 mM Ca²⁺ or 1 mM EGTA, and ³²P-g-ATP. In the presence of Ca²⁺, PfCDPK5 phosphorylates itself (double arrowhead) and substrate (arrowhead). C) PfCDPK5 fused to DD_TM is targeted for degradation, but is stabilized by Shld1. D) Protein lysates from D10-PfCDPK5-HA-DD_TM ring (0–20h), trophozoite (20–36h), and schizont-stage (36–48h) parasites cultured with Shld1 and probed with anti-HA (PfCDPK5) or anti-Histone H3 (loading control). E) Na₂CO₃-extracted schizonts were probed with anti-PfLDH (cytoplasmic fraction), anti-PfAMA1 (membrane fraction), or anti-HA. F) D10- PfCDPK5-HA-DD_TM parasites were grown [+] or [−] Shld1 until 44 h, incubated with E- 64 for 10 h, harvested, fractionated with 0.6% NP-40, and probed with anti-HA and anti- Histone H3. G) Representative replication curves. D10-PfCDPK5-DD_TM and D10- PfCDPK4-DD_TM parasites were cultured [+] and [−] Shld1.

Figure 2. PfCDPK5-deficient parasite arrest prior to egress

A) Light microscopy of synchronized D10-PfCDPK5-DD_TM parasites grown [+] or [−] Shld1 at indicated hours post invasion (h.p.i.). Pie charts show relative proportions of rings, trophozoites, and schizonts (N=100). B) D10-PfCDPK5-DD_TM parasites were grown [−]Shld1 until 56 h.p.i. and visualized by electron microscopy. The erythrocyte PM (black triangle), PVMe), and the parasite PM (grey triangle) were intact. C) Ring-stage D10-PfCDPK5-DD_TM parasites were grown [+] or [−] Shld1 until 48 h.p.i.. In parallel [-]Shld1cultures, Shld1 was added back at the indicated times (49, 52, 56 h.p.i). Rings and schizonts were counted 4 h later (mean +/− S.D., N=4). D) RNA expression patterns from 3D7-PfCDPK5-DD_TM parasites at 48 h.p.i. [+] and [−] Shld1 and 54 h.p.i. [−]Shld1. Log₂- transformed expression pattern shown for 15 late schizont-stage and 15 early ring-stage genes.

Figure 3. PfCDPK5-deficient parasites are fully mature and invasion-competent

A) D10-PfCDPK5-HA-DD_TM parasites were grown [+] and [−] Shld1, harvested by Percoll-purification at 48 h.p.i., and analyzed by western blot with anti-PfSUB1, anti-PfSERA5, and anti-PfMSP1₄₂. B) D10-PfCDPK5-DD_TM parasites were grown until 48 h.p.i. ([+]/[−]0.5 μM Shld1, [+]/[−] 4 μM compound 1). Subsets of parasites were sheared through a 28.5G needle. Ring parasitemia was determined 4 h later. (mean, +/− S.D. [or data range], N=2–7, *p < 0.05, unpaired t-test.)