To Each His Own: Fever Induces Different Suicide Mechanisms in Early and Late Stage Plasmodium falciparum Malaria Parasites in vitro
Authors: Coetzer Theresa L. , Engelbrecht Dewaldt 
Published: October 2019

Abstract
Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41°C, last for 2-6 hours and recur every 48 hours, in accordance with the parasite life cycle. Febrile episodes typically have a deleterious effect on parasites, and probably benefit the host by aiding parasite clearance; however, a reduction in parasitemia may also be an advantage for the parasite by limiting the burden of infection on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Although programmed cell death (PCD) has been identified in the mosquito stages of the parasite, data regarding the erythrocytic stages are limited and often conflicting and thus the occurrence of PCD and the phenotype remain controversial. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress similar to fever periods experienced during malaria paroxysms.
Synchronised early and late stage P. falciparum 3D7 cultures were exposed to 41°C for 2 hours or maintained as controls at 37°C. Parasitemia was monitored with thiazole orange flow cytometry. Numerous biochemical markers of PCD were assessed by flow cytometric assays immediately after heat stress and 24 hours later. DNA fragmentation was measured with the Terminal deoxynucleotidyl transferase-mediated Nick End Labelling (TUNEL) assay; mitochondrial membrane polarisation was measured with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]; and phosphatidylserine (PS) externalisation of infected erythrocytes was quantified by Annexin V binding. Morphological studies of Giemsa-stained thin smears and real-time microscopy were also utilised to characterise the parasite response to heat stress.
Exposure to 41°C decreased the parasitemia of both early and late stage P. falciparum in vitro. Ring stage parasites were more affected than expected, although some ring stage parasites developed into trophozoites similar to control parasites maintained at 37°C. Biochemical markers showed evidence of DNA fragmentation and mitochondrial depolarisation suggesting that ring stage parasites exhibited an apoptosis-like phenotype of PCD, although infected erythrocytes showed no significant increase in PS externalisation.
Late stage control parasites maintained at 37°C replicated within 24 hours to form newly-invaded rings with increased parasitemia. In contrast, heat stress caused a reduction in the number of late stage parasites and the remaining parasites failed to invade new erythrocytes. Although infected erythrocytes exhibited increased PS externalisation, the parasites exhibited very little DNA fragmentation and no mitochondrial dysregulation. Giemsa-stained thin smears showed some late stage parasites with cytoplasmic vacuolisation, which was suggestive of an autophagy-like form of PCD. Real-time microscopy revealed the movement of many late stage parasites inside host erythrocytes after heat stress, indicating that these parasites were alive, consistent with a recovery in parasitemia after two cycles of continued culture at 37°C.
Data regarding the possible induction of PCD by heat stress in P. falciparum are scarce and conflicting. Our results showed biochemical and morphological markers of PCD that varied with intra-erythrocytic parasite development and suggested that P. falciparum exhibited both apoptosis- and autophagy-like phenotypes of PCD after exposure to febrile temperatures. However, these cell death markers may also represent different facets of a PCD pathway in P. falciparum that is distinct from that in metazoans. The identification and exploitation of an intrinsic cell death pathway unique to P. falciparum may provide novel targets for eliminating the parasite in malaria patients.

Disclosures:
No relevant conflicts of interest to declare.