A physiologically relevant, non-destructive febrile heat stress increases the percentage of P. falciparum infected red blood cells expressing PfEMP1 on their surface and enhances cytoadhesion to the cognate receptor.

(A) Relative distribution of patient body temperature from two studies (Mok et al. 29 and Abdi et al. 30) that included accessible temperature data from P. falciparum infected patients (n = 1043 (blue) and n = 827 (black), respectively). (B) Average febrile temperature, defined as >38 °C, was 38.9 °C and 39.1 °C in Mok et al., and Abdi et al., respectively. (C) Estimated average age of circulating parasites from Mok et al. microarray data plotted against recorded patient temperature. (D) Synchronised infected red blood cells (iRBCs) were exposed to heat stress for different 8-hour windows during the first 48 hours post invasion (hpi). When not heat-stressed, parasites were cultured under normal conditions at 37°C. Parasitaemia was measured by flow cytometry in the following cycle at 72 hpi. N=3 (technical triplicates) for heat-stressed cultures; n=6 (biological replicates) for 37°C control cultures. Dashed lines represent the average next cycle parasitaemia fold change of control cultures. (E) Percentage of iRBC positive for PfEMP1 (VAR2CSA) surface expression from 12 to 40 hpi when cultured normally at 37 °C. Flow cytometry gating strategy is shown in Supplementary Figure 2. (F) Schematic representation of the heat stress application to synchronous NF54 DiCre parasites. (G) Following heat stress, more iRBCs bound to CSA under flow (1 dyne/cm2). (H) Significantly more iRBCs were positive for PfEMP1 (VAR2CSA) after heat stress. For G and H n=3 biological replicates. Error bars displayed are ± 1 SD. Statistical significance was determined using unpaired t-tests of log-transformed data with Welch’s correction. Flow cytometry and cytoadhesion assays were performed at room temperature.

Heat stress increases sorbitol sensitivity in red blood cells infected with four P. falciparum strains from diverse geographic origins.

(A) At 24 hpi following an eight-hour heat stress (39 °C), parasite cultures were treated with PBS and sorbitol, and the parasitaemia was determined and compared to that of a 37 °C control. Sorbitol sensitivity is conferred by a functional nutrient channel (PSAC) trafficked by the parasite onto the surface of iRBCs. (B) As sorbitol sensitive parasites rupture during treatment, the percentage of sorbitol sensitive parasites is calculated by comparing parasitaemia following sorbitol treatment to parasitaemia following PBS treatment. (C) Geographic origins of P. falciparum isolates (*NF54 is predicted to have an African origin but was isolated in the Netherlands 41). (D–G) After heat stress, NF54 (D), HB3 (E), Cam3.II (F), and HL2208 (G) strains exhibited significantly higher sensitivity to sorbitol treatment. Data represents three biological replicates (N=3). Statistical significance was assessed using unpaired t-tests on log-transformed data with Welch’s correction. All assays were performed at room temperature.

Febrile heat stress results in increased phosphorylation of host cell exported proteins.

(A) Overview of infected red blood cell lysate preparation for phosphoproteomics. (B) Histogram of 12654 unique P. falciparum phosphopeptides, showing enrichment of phosphopeptides from exported parasite proteins following heat stress. (C) Percentage of unique phosphopeptides that are from parasite exported proteins (red). Following heat stress the significantly more abundant phosphopeptides (>1 Log2FC) are enriched for exported proteins (71%) compared to the 12564 phosphopeptides not changing (7.5%). N=2 Biological Replicates. (D) Comparing the 102 exported phosphopeptides more abundant following heat stress against confident FIKK substrates identified FIKK10.2 and FIKK4.1 as likely major contributors to heat stress phosphorylation. The remaining phosphopeptides had not previously been linked to a specific kinase (No Identified Kinase) by Davies et al. 52. Neither FIKK4.1 nor FIKK10.2 are required for the heat stress-induced increase in PfEMP1 surface expression (E) or for the increased sorbitol sensitivity of iRBCs following heat stress (F), as shown using synchronised kinase conditional knockout (RAP-treated) parasite lines. N=3 biological replicates. Error bars displayed are ± 1 SD. Statistical significance was determined using the one-way ANOVA test with the Benjamini, Krieger and Yekutieli FDR correction. Antibody staining, flow cytometry and sorbitol treatment was performed at room temperature. For (E) and (F) parasites were heat stressed between 16-24 hpi at 39 °C, sorbitol treatment and antibody staining were performed at 24 hpi.

FIKK10.2-TurboID reveals changes to the Maurer’s cleft protein environment during heat stress.

(A) In the presence of biotin, transgenic parasites expressing FIKK10.2, a Maurer’s cleft residing protein, fused to TurboID were used to biotinylate proximal proteins. (B) FIKK10.2-TurboID parasites were cultured under three different conditions: biotin was either present throughout the life cycle, or added as an eight-hour pulse (16–24 hpi) in the presence or absence of heat stress at 39 °C. At 40 hpi, late-stage iRBCs were enriched by Percoll density separation and lysed. Proteins were subsequently digested, and biotinylated peptides were enriched and identified. (C) Proportional Venn diagram of detected peptides across the three conditions. (D) Proportional Venn diagram of detected proteins in each condition. (E) Heat map of biotinylated peptide intensity across the three tested conditions. N = 3 biological replicates. For (C) and (D), valid peptides were defined as those detected in at least two replicates, and valid proteins were defined as those with at least two different valid peptides detected.

Febrile heat stress enhances trafficking of endogenously tagged PfEMP1 (VAR2CSA) to the RBC cytosol and surface.

(A) A transgenic P. falciparum strain expressing NanoLuc fused to the C-terminus of VAR2CSA was generated. (B) Following heat stress, a greater proportion of VAR2CSA-NanoLuc expressing parasites displayed PfEMP1 on the iRBC surface compared to parasites maintained at 37 °C. N = 3 biological replicates. Error bars represent ±1 SD. Statistical significance was determined using an unpaired t-test on log-transformed data with Welch’s correction. (C) At 24 hpi, following heat stress or continuous culture at 37 °C, cells were differentially permeabilised using three treatments. NanoLuc activity was measured in the supernatants. (D) Overview of the compartments released by each permeabilisation treatment: EqtII permeabilises the RBC membrane while preserving the PVM, SAP releases proteins from the RBC cytosol and PV lumen, and NP40 solubilises all non-nuclear proteins from the parasite, PV, and RBC cytosol. (E) Significantly higher NanoLuc activity was detected in the supernatant of heat-stressed parasites only following EqtII permeabilisation. Assays were performed at room temperature. Data represent the mean of three biological replicates, each averaged from three technical replicates. Statistical significance was determined using the one-way ANOVA test with the Benjamini, Krieger and Yekutieli FDR correction.

Constitutively expressed exported NanoLuc strains identify transmembrane domains as key determinants for increased export into the host cell during febrile temperatures.

(A) Tightly synchronised P. falciparum parasites constitutively expressing one of four NanoLuc reporter constructs were exposed to heat stress (39 °C) or maintained at 37 °C between 16–24 hpi. At 24 hpi, cells were subjected to three different differential permeabilisation treatments. NanoLuc activity in the resulting supernatants was measured. (B) Schematic representation of the cellular compartments released by each permeabilisation method. All assays were performed at room temperature. (C) The constitutively expressed PF3D7_0702500-NanoLuc reporter, but not D) the REX3-NanoLuc reporter, showed significantly increased NanoLuc activity in the EqtII-permeabilised supernatant following heat stress. REX3-NanoLuc contains the first 61 amino acids of REX3, which are sufficient to mediate export 59,60. (E) Fusion of the PF3D7_0702500 TMD to the REX3-NanoLuc reporter reduced luminescence in the EqtII-permeabilised supernatant under non-stress conditions. However, increased luminescence was observed following heat stress. (F) Reporters lacking an N-terminal fusion to an exported P. falciparum protein showed negligible luminescence in the supernatant following SAP or EqtII treatment. Across all four constructs, no significant differences in luminescence were observed between SAP and NP40-permeabilised samples when comparing conditions with and without heat stress. Data represent the mean of three biological replicates, each averaged from three technical replicates. Statistical significance was determined using the one-way ANOVA test with the Benjamini, Krieger and Yekutieli FDR correction.