Malaria Parasite Morphology And Ph Modulation At The Time Of Egress From Host-Red Blood Cells

To egress from its host erythrocyte, the malaria parasite Plasmodium falciparum breaches two enveloping membranes, the vacuolar (PVM) and erythrocyte membrane, in an orchestrated multi-step sequence. Following the natural parasite egress steps under a fluorescence microscope using endogenously mNeonGreen-tagged vacuolar membrane resident protein EXP2, we made two remarkable observations: (1) signal from the EXP2 was found intruding far into the segmented parasite, and (2) the fluorescent signal of EXP2 decreases stepwise at the moment of PVM rupture. We hypothesize that both observations may be related. The intrusion may act as connection of the acidic compartments inside the parasite, such as digestive vacuole (DV), to the vacuolar lumen and RBC cytoplasm. A subsequent decrease of pH in the PV and the red cell could be a part of or a trigger for the egress mechanism. Using focused ion beam scanning electron miscopy (FIBSEM) and conventional thin-section EM we found that indeed tubular extensions of the PVM filled with hemoglobin protrude deep into the segmented parasite. The extensions terminate in a cystotome-like structure frequently contacting the digestive vacuole. Next, to interrogate the requirement of acidification for parasite egress, segmented parasites were treated with weak bases and an ionophore to increase or dissipate the pH in acidic organelles. We found that 30 min acute treatment of schizonts with ammonium chloride, chloroquine and monensin inhibit parasite egress in a dose-dependent manner. We conclude that pH may play a role in parasite egress mechanism and that PVM tubules offer a putative delivery pathway to acidify the extra-parasitic compartment of the infected RBC.