Physiological febrile heat stress increases cytoadhesion through increased protein trafficking of Plasmodium falciparum surface proteins into the red blood cell
- Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, United Kingdom
- The Cell Biology of Host-Pathogen Interactions Lab, Gulbenkian Institute for Molecular Medicine, Portugal
Figures
Figure 1 with 3 supplements
A physiologically relevant, non-destructive febrile heat stress increases the percentage of P. falciparum infected red blood cells (iRBCs) 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., 2015 and Abdi et al., 2023) 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 iRBCs were exposed to heat stress for different 8 hr 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 Figure 1—figure supplement 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.
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Figure 1—source data 1
Total patient temperature data from Abdi et al. and Mok et al.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data1-v1.xlsx
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Figure 1—source data 2
Only feverish (>38 °C) patient temperature data from Abdi et al. and Mok et al.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data2-v1.xlsx
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Figure 1—source data 3
Patient data from Mok et al., correlating the temperature (°C) with the estimated parasite age (hpi).
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data3-v1.xlsx
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Figure 1—source data 4
Raw data file of next cycle parasitaemia fold change following different temperature conditions.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data4-v1.xlsx
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Figure 1—source data 5
Raw data file of infected RBCs positive for cell surface PfEMP1 over 40 hours post-invasion when cultured at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data5-v1.xlsx
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Figure 1—source data 6
Raw data file of the relative cytoadhesion of iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data6-v1.xlsx
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Figure 1—source data 7
Raw data file of the percentage of iRBC positive for cell surface PfEMP1 (VAR2CSA) following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-data7-v1.xlsx
Figure 1—figure supplement 1
Heat stress applied between 16 and 24 hours post-invasion (hpi) does not accelerate P. falciparum asexual development.
(A) Schematic overview of the experimental design. (B) Median fluorescent intensity (MFI) of Hoechst-stained infected red blood cells (iRBCs) at 24 hpi was not significantly altered by heat stress. (C) High DNA content iRBCs, identified as schizonts, showed no significant difference in proportion remaining at 44 hpi following heat stress. (D) Fold increase in parasitaemia in the subsequent asexual cycle was comparable between heat-stressed samples and the 37 °C control. Data represent N=3 biological replicates. Error bars indicate ± 1 SD. Statistical comparisons were made using unpaired t-tests with Welch’s correction. For panels (C) and (D), data were log-transformed prior to analysis.
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Figure 1—figure supplement 1—source data 1
Raw data file of infected RBCs Hoechst (DNA stain) MFI following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Raw data file of percentage of infected RBCs which had egressed at 40 hours post-invasion following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-figsupp1-data2-v1.xlsx
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Figure 1—figure supplement 1—source data 3
Raw data file of the fold change in parasitaemia in the next cycle (72 hpi) following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-figsupp1-data3-v1.xlsx
Figure 1—figure supplement 2
Flow cytometry gating strategy for detection of surface-trafficked VAR2CSA on iRBCs.
(A) Example flow cytometry gating strategy for NF54::DiCre parasites. Singlets were gated, followed by DNA staining to distinguish uninfected and infected red blood cells (iRBCs). iRBCs were further gated into young (lower DNA content) and late stage (higher DNA content) iRBCs. (B) iRBCs show increased signal following incubation with human anti-VAR2CSA antibody (PAM7.5) and phycoerythrin-conjugated secondary antibody. (C) A negative control, in the absence of primary antibody but with the secondary fluorophore, shows reduced signal on iRBCs. The iRBC gate, including all stages, was used to determine the percentage of cells positive for PfEMP1 surface labelling.
Figure 1—figure supplement 3
Temperature probe within cell culture flasks accurately measures heat stress conditions applied to cell cultures.
(A) Thermal profile of the in vitro heat stress protocol. A 10 mL water-filled flask was transferred from a 37 °C to a 39 °C incubator, and temperature was recorded every 30 s using a probe (±0.2 °C) placed in the liquid. The flask remained at 39 °C for 8 hr before being returned to 37 °C.
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Figure 1—figure supplement 3—source data 1
Raw data file of the temperature in a T25 tissue culture flask that has been heat stressed by transferring the flask from a 37 °C incubator to a 39 °C incubator.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig1-figsupp3-data1-v1.xlsx
Figure 2 with 2 supplements
Heat stress increases sorbitol sensitivity in RBCs infected with four P. falciparum strains from diverse geographic origins.
(A) At 24 hpi following an 8 hr 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 Preston et al., 2014). (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.
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Figure 2—source data 1
Raw data file of iRBCs (NF54 strain) sorbitol sensitivity following heat stress compared to when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-data1-v1.xlsx
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Figure 2—source data 2
Raw data file of iRBCs (HB3 strain) sorbitol sensitivity following heat stress compared to when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-data2-v1.xlsx
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Figure 2—source data 3
Raw data file of iRBCs (Cam3.II strain) sorbitol sensitivity following heat stress compared to when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-data3-v1.xlsx
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Figure 2—source data 4
Raw data file of iRBCs (HL2208 strain) sorbitol sensitivity following heat stress compared to when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
Sorbitol lysis of infected red blood cells (iRBCs) is reduced by the PSAC inhibitor furosemide regardless of temperature treatment.
(A) Schematic overview of the experimental workflow. NF54 P. falciparum iRBCs were synchronised to a 2 hr window (>10% parasitaemia). At 20 hr post-infection, cultures were either maintained at 37 °C or exposed to heat stress at 39 °C for 8 hr. At 28 hr post-infection, all cells were washed with room-temperature phosphate-buffered saline, and a subset of samples was pre-incubated with 200 µM furosemide for 10 min at 37 °C. Samples were then resuspended in sorbitol or sorbitol plus furosemide and incubated for 10 min at 37 °C. Following incubation, cells were centrifuged, and supernatant absorbance at 415 nm was measured as an indicator of haemoglobin release and therefore cell lysis. Uninfected red blood cells (uRBCs) were processed in parallel. Sorbitol uptake is proposed to occur through the plasmodial surface anion channel (PSAC), which is inhibited by furosemide. (B) Supernatant absorbance at 415 nm of sorbitol-treated cultures following heat stress compared with the non-heat-stressed 37 °C control. Negative and positive supernatant controls were generated from iRBCs treated without sorbitol and from iRBCs lysed with saponin (supernatant diluted 1:5), respectively. Only iRBCs showed increased supernatant absorbance after sorbitol treatment, and this response was blocked by furosemide. uRBCs showed no increase in absorbance, indicating that sorbitol-mediated lysis requires parasite infection. Data represent at least two biological replicates. Error bars indicate ± 1 SD.
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Figure 2—figure supplement 1—source data 1
Raw data file of the cell supernatant absorbance (415 nm) following the different treatment of uninfected and infected RBCs.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
P. falciparum parasites are not more susceptible to heat stress with reduced nutrient availability.
(A) NF54 DiCre parasites were synchronised to a 2 hr invasion window and adjusted to 1% parasitaemia before culture with complete media (supplemented RPMI-1640), complete media containing 150 µM furosemide, or reduced nutrient media (composition detailed in Supplementary file 7). Between 16 and 24 hpi, iRBCs were heat stressed at 40 °C or maintained at 37 °C. In the following cycle (72 hpi), parasitaemia was determined. (B) Culturing with 150 µM furosemide, an inhibitor that blocks nutrient uptake, or with reduced nutrient media resulted in a significant reduction in replication compared to the complete media control. However, these reductions were not further decreased during heat stress. Data represent N=3 biological replicates. Error bars indicate ± 1 SD. Both the normal media control and the reduced nutrient media were treated with a DMSO vehicle control. Statistical significance was assessed using two-way ANOVA of log-transformed data, followed by FDR correction for multiple comparisons by Šidák’s correction.
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Figure 2—figure supplement 2—source data 1
Raw data showing the percentage of infected red blood cells under normal or reduced nutrient conditions with defined media composition or nutrient channel inhibitors, following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 12 supplements
Febrile heat stress results in increased phosphorylation of host cell exported proteins.
(A) Overview of infected RBC lysate preparation for phosphoproteomics. (B) Histogram of 12,654 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 12,564 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., 2020. Neither FIKK4.1 nor FIKK10.2 is 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 were performed at room temperature. For (E) and (F), parasites were heat stressed between 16 and 24 hpi at 39 °C, sorbitol treatment and antibody staining were performed at 24 hpi.
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Figure 3—source data 1
Raw data file of the distribution of P. falciparum phosphopeptide abundance Log2 fold change following heat stress compared to when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-data1-v1.xlsx
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Figure 3—source data 2
Raw data file of the four categories of previously identified FIKK substrates of the P. falciparum phosphopeptides more abundant following heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-data2-v1.xlsx
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Figure 3—source data 3
Raw data file of the percentage of FIKK4.1 and FIKK10.2 knockout iRBCs positive for cell surface PfEMP1 (VAR2CSA) following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-data3-v1.xlsx
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Figure 3—source data 4
Raw data file of the percentage of FIKK4.1 and FIKK10.2 knockout iRBCs which are sensitive to sorbitol following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-data4-v1.xlsx
Figure 3—figure supplement 1
The host and parasite proteomes are not substantially altered following febrile heat stress.
(A) Normalised protein abundance of human (n = 212) and Plasmodium falciparum (n = 624) proteins from heat-stressed and 37 °C control infected red blood cells (iRBCs), totalling 836 proteins. Pearson correlation analysis between conditions is shown (R² = 0.98). (B) Distribution of log₂ fold changes in protein abundance for human (n = 212) and P. falciparum (n = 624) proteins comparing heat-stressed versus 37 °C control iRBCs. Data are from two biological replicates. Violin plots display the median and interquartile range (25th and 75th percentiles, indicated by dotted lines).
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Figure 3—figure supplement 1—source data 1
Raw data file containing normalised protein abundance values of parasite proteins under control conditions and following febrile heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
Raw data file containing normalised protein abundance values for host and parasite proteins under control conditions and following febrile heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp1-data2-v1.xlsx
Figure 3—figure supplement 2
Repeated heat stress phosphoproteome with FIKK knockout (KO) strains identifies FIKK10.2 as the major contributor of heat-stress-dependent phosphorylation of host-cell exported parasite proteins.
(A) Heat map of 64 exported phosphopeptides that were significantly more abundant following heat stress in wild-type–like NF54 DiCre parasites (log₂ fold change > 1; heat stress vs 37 °C control). Fold changes for these phosphopeptides are shown across several exported FIKK kinase conditional knockouts (KOs). The FIKK9s strain represents the conditional deletion of seven FIKK kinases (FIKK9.1 to FIKK9.7). In the absence of FIKK10.2, 52 of the 64 phosphopeptides that were significantly more phosphorylated under heat stress in the wild-type strain no longer show increased phosphorylation. N = 1 for each condition (temperature and strain).
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Figure 3—figure supplement 2—source data 1
Raw data file containing abundance values for exported phosphopeptides that were significantly more abundant following heat stress in wild-type NF54 DiCre parasites, with corresponding values for the FIKK kinase knockout strains under control and heat stress conditions.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp2-data1-v1.xlsx
Figure 3—figure supplement 3
Generation of HSP70x-3xHA (PF3D7_0831700) conditional knockout P. falciparum strain.
(A) Generation of a conditional knockout strain of HA-tagged HSP70x using CRISPR/Cas9-mediated excision and homologous repair. Upon rapamycin (+RAP) treatment, Cre recombinase excises DNA flanked by loxP sites to remove HSP70x. Primer binding sites indicated by the red and blue arrows are outside the 5’ and 3’ repair template homology regions. (B) PCR confirmed 5′ and 3′ integration, reduction in gene-specific amplification following deletion, and absence of the original wild-type (WT) allele. Predicted amplicon sizes are indicated in white. DNA size marker values (kbp) are indicated to the left of the gel. (C) Western blot using anti-HA antibody of Percoll-enriched HSP70x-3xHA schizonts previously treated with DMSO (−RAP) or rapamycin (+RAP), compared to WT control (NF54). Total protein staining was used as a loading control. The predicted size of HSP70x-3xHA is 80.4 kDa.
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Figure 3—figure supplement 3—source data 1
Raw images of the DNA gel assessing the correct integration of the HSP70x-3xHA transgenic parasite strain, the efficiency of the DiCre-mediated conditional knockout and the absence of the parental strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
Raw images of the α-HA immunoblot of HSP70x-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data2-v1.zip
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Figure 3—figure supplement 3—source data 3
Raw images of the total protein content control of the α-HA immunoblot of HSP70x-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data3-v1.zip
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Figure 3—figure supplement 3—source data 4
PDF containing the raw images of the DNA gel assessing the correct integration of the HSP70x-3xHA transgenic parasite strain, the efficiency of the DiCre-mediated conditional knockout and the absence of the parental strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data4-v1.pdf
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Figure 3—figure supplement 3—source data 5
PDF containing the raw images of the α-HA immunoblot of HSP70x-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data5-v1.pdf
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Figure 3—figure supplement 3—source data 6
PDF containing the raw images of the total protein content control of the α-HA immunoblot of HSP70x-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp3-data6-v1.pdf
Figure 3—figure supplement 4
HSP70x-3xHA (PF3D7_0831700) does not strongly co-localise with KAHRP or SBP1.
Representative immunofluorescence microscopy images showing HSP70x-3xHA (anti-HA), parasite DNA (DAPI) merged with differential interference contrast (DIC), and either KAHRP (A) or SBP1 (B). Neither KAHRP (a RBC cytoskeleton-associated protein) nor SBP1 (a Maurer’s cleft-resident protein) co-localised with HA-tagged HSP70x. Scale bar = 5 μm.
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Figure 3—figure supplement 4—source data 1
Raw images.
Raw microscopy images of transgenic HSP70x-3xHA parasites probed with α-HA and α-SBP1.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
Raw images.
Raw microscopy images of transgenic HSP70x-3xHA parasites probed with α-HA and α-KAHRP.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp4-data2-v1.zip
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Figure 3—figure supplement 4—source data 3
PDF containing the raw microscopy images of transgenic HSP70x-3xHA parasites probed with α-HA and α-SBP1.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp4-data3-v1.pdf
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Figure 3—figure supplement 4—source data 4
PDF containing the raw microscopy images of transgenic HSP70x-3xHA parasites probed with α-HA and α-KAHRP.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp4-data4-v1.pdf
Figure 3—figure supplement 5
Generation and validation of PF3D7_0702500-3xHA conditional knockout P. falciparum strain.
(A) Generation of a conditional knockout strain of HA-tagged PF3D7_0702500 using CRISPR/Cas9-mediated excision and homologous repair. Upon rapamycin (+RAP) treatment, Cre recombinase excises DNA flanked by loxP sites to remove PF3D7_0702500. Primer binding sites indicated by the red and blue arrows are outside the 5’ and 3’ repair template homology regions. (B) PCR confirmed 5′ and 3′ integration, reduction in gene-specific amplification following deletion, and absence of the original wild-type (WT) allele. Predicted amplicon sizes are indicated in white. DNA size marker values (kbp) are indicated to the left of the gel. (C) Western blot using anti-HA antibody of Percoll-enriched PF3D7_0702500-3xHA schizonts previously treated with DMSO (−RAP) or rapamycin (+RAP), compared to WT control (NF54). Total protein staining was used as a loading control. The predicted size of PF3D7_0702500-3xHA is 33.0 kDa. The observed discrepancies in apparent molecular weight may result from extensive post-translational modifications and are consistent with findings previously reported by Heiber et al., 2013. Additionally, the faint larger band likely represents a population in which the T2A skip peptide failed to cleave efficiently, resulting in a fusion with the neomycin resistance cassette. This fusion protein is predicted to be 62.1 kDa (an additional 29.1 kDa).
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Figure 3—figure supplement 5—source data 1
Raw images of the DNA gel assessing the correct integration of the PF3D7_0702500-3xHA transgenic parasite strain, the efficiency of the DiCre-mediated conditional knockout and the absence of the parental strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data1-v1.zip
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Figure 3—figure supplement 5—source data 2
Raw images of the α-HA immunoblot of PF3D7_0702500-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data2-v1.zip
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Figure 3—figure supplement 5—source data 3
Raw images of the total protein content control of the α-HA immunoblot of PF3D7_0702500-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data3-v1.zip
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Figure 3—figure supplement 5—source data 4
PDF containing the raw images of the DNA gel assessing the correct integration of the PF3D7_0702500-3xHA transgenic parasite strain, the efficiency of the DiCre-mediated conditional knockout and the absence of the parental strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data4-v1.pdf
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Figure 3—figure supplement 5—source data 5
PDF containing the raw images of the α-HA immunoblot of PF3D7_0702500-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data5-v1.pdf
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Figure 3—figure supplement 5—source data 6
PDF containing the raw images of the total protein content control of the α-HA immunoblot of PF3D7_0702500-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp5-data6-v1.pdf
Figure 3—figure supplement 6
PF3D7_0702500-3xHA co-localises with SBP1.
Representative immunofluorescence microscopy images showing PF3D7_0702500-3xHA (anti-HA), parasite DNA (DAPI) merged with differential interference contrast (DIC), and either KAHRP (A) or SBP1 (B). PF3D7_0702500-3xHA does not co-localise with KAHRP (a RBC cytoskeleton-associated protein) but does co-localise with SBP1 (a Maurer’s cleft-resident protein). Scale bar = 5 μm.
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Figure 3—figure supplement 6—source data 1
Raw images.
Raw microscopy images of transgenic PF3D7_0702500-3xHA parasites probed with α-HA and α-SBP1.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp6-data1-v1.zip
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Figure 3—figure supplement 6—source data 2
Raw images.
Raw microscopy images of transgenic PF3D7_0702500-3xHA parasites probed with α-HA and α-KAHRP.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp6-data2-v1.zip
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Figure 3—figure supplement 6—source data 3
PDF containing the raw microscopy images of transgenic PF3D7_0702500-3xHA parasites probed with α-HA and α-SBP1.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp6-data3-v1.pdf
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Figure 3—figure supplement 6—source data 4
PDF containing the raw microscopy images of transgenic PF3D7_0702500-3xHA parasites probed with α-HA and α-KAHRP.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp6-data4-v1.pdf
Figure 3—figure supplement 7
HSP70x conditional knockout parasites have normal growth at 37 °C and elevated temperatures.
Parasites were either DMSO or rapamycin (RAP) treated (four hours) to conditionally knockout HSP70x (PF3D7_0831700), and parasites were later synchronised to a 2 hr invasion window. Parasites were heat stressed at 39 °C or 40 °C between 16 and 24, 24 and 32 hpi, or kept at 37 °C for the full duration. 40 °C heat stress between 24 and 32 hpi significantly reduced growth as shown in the fold change in parasitaemia in the first (A) and second cycle (B) following heat stress. However, the absence of HSP70x (RAP-treated) did not have a significant impact on the growth of the parasites following any of the heat stress conditions tested compared to the wild-type resembling (DMSO-treated) strain. Data represent N=3 biological replicates, with error bars showing ± 1 SD. Statistical significance was assessed using two-way ANOVA of log-transformed data, followed by FDR correction for multiple comparisons.
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Figure 3—figure supplement 7—source data 1
Raw data file of the parasitaemia fold change of HSP70x knockout infected RBCs at 72 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp7-data1-v1.xlsx
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Figure 3—figure supplement 7—source data 2
Raw data file of the parasitaemia fold change of HSP70x knockout infected RBCs at 120 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp7-data2-v1.xlsx
Figure 3—figure supplement 8
The exported protein HSP70x is not required for the trafficking of PfEMP1 onto the infected cell’s surface under normal or febrile temperatures.
(A) Parasites were either DMSO or rapamycin (RAP) treated (4 hr) to conditionally knockout HSP70x (PF3D7_0831700), parasites were later synchronised to a 2 hr invasion window. Parasites were heat stressed at 39 °C between 16 and 24 hpi or kept at 37 °C for the full duration. At 24 and 40 hpi, the presence of VAR2CSA was measured on the infected RBCs (iRBCs) surface by flow cytometry. (B) At 24 hpi, there was no change in the percentage of PfEMP1-positive iRBCs in the RAP compared to DMSO. There was a significant increase in the number of iRBCs positive for VAR2CSA following heat stress regardless of DMSO or RAP treatment. (D) At 40 hpi, there was no difference in percentage VAR2CSA-positive iRBCs regardless of RAP or heat treatment. The α-VAR2CSA median fluorescent intensity (MFI) was not significantly different with heat or rapamycin treatment but did increase 10-fold from 24 hpi (C) to 40 hpi (E). N=3 biological replicates. Error bars displayed are ±1 SD. Statistical significance was determined using two-way ANOVA on log-transformed data with FDR correction.
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Figure 3—figure supplement 8—source data 1
Raw data file of the percentage of HSP70x knockout iRBCs positive for PfEMP1 at 24 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp8-data1-v1.xlsx
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Figure 3—figure supplement 8—source data 2
Raw data file of the MFI corresponding to surface PfEMP1 levels of HSP70x knockout iRBCs positive for PfEMP1 at 24 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp8-data2-v1.xlsx
-
Figure 3—figure supplement 8—source data 3
Raw data file of the percentage of HSP70x knockout iRBCs positive for PfEMP1 at 40 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp8-data3-v1.xlsx
-
Figure 3—figure supplement 8—source data 4
Raw data file of the MFI corresponding to surface PfEMP1 levels of HSP70x knockout iRBCs positive for PfEMP1 at 40 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp8-data4-v1.xlsx
Figure 3—figure supplement 9
HSP70x is not required for functional PSAC activity at 24 hpi under normal or elevated temperatures.
(A) Parasites were either DMSO or rapamycin (RAP) treated (4 hr) to conditionally knockout HSP70x (PF3D7_0831700), parasites were later synchronised to a 2 hr invasion window. Parasites were heat stressed at 39 °C between 16 and 24 hpi or kept at 37 °C for the full duration. At 24 hpi, aliquots of cells were treated with PBS or sorbitol before parasitaemia was measured by flow cytometry. (B) The reduction in parasitaemia with sorbitol treatment (Sorbitol/PBS) was measured and showed that in the absence of HSP70x (RAP), there was no change in percentage of sorbitol-sensitive cells at 37 °C. Furthermore, there was an increase in the percentage of sorbitol-sensitive following heat stress. Data represent n = 3 biological replicates. Error bars indicate ± 1 SD. Statistical significance was determined using two-way ANOVA on log-transformed data with FDR correction.
-
Figure 3—figure supplement 9—source data 1
Raw data file of the percentage of HSP70x knockout iRBCs which are sorbitol sensitive following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp9-data1-v1.xlsx
Figure 3—figure supplement 10
PF3D7_0702500 conditional knockout parasites have normal growth at 37 °C and elevated temperatures.
Parasites were either DMSO or rapamycin (RAP) treated (4 hr) to conditionally knockout PF3D7_0702500, and parasites were later synchronised to a 2 hr invasion window. Parasites were heat stressed at 39 °C or 40 °C between 16 and 24, 24 and 32 hpi, or kept at 37 °C for the full duration. 40 °C heat stress between 24 and 32 hpi significantly reduced growth as shown in the fold change in parasitaemia in the first (A) and second cycle (B) following heat stress. However, the absence of PF3D7_0702500 (RAP-treated) did not have a significant impact on the growth of the parasites following any of the heat stress conditions tested compared to the wild-type resembling (DMSO-treated) strain. Data represent N=3 biological replicates, with error bars showing ± 1 SD. Statistical significance was assessed using two-way ANOVA of log-transformed data, followed by FDR correction for multiple comparisons.
-
Figure 3—figure supplement 10—source data 1
Raw data file of the parasitaemia fold change of PF3D7_0702500 knockout infected RBCs at 72 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp10-data1-v1.xlsx
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Figure 3—figure supplement 10—source data 2
Raw data file of the parasitaemia fold change of PF3D7_0702500 knockout infected RBCs at 120 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp10-data2-v1.xlsx
Figure 3—figure supplement 11
The exported protein PF3D7_0702500 is not required for the trafficking of PfEMP1 onto the infected cell’s surface under normal or febrile temperatures.
(A) Parasites were treated with either DMSO or rapamycin (RAP) for 4 hr to conditionally knock out PF3D7_0702500. Parasites were subsequently synchronised to a 2 hr invasion window. Heat stress was applied at 39 °C from 16 to 24 hours post-invasion (hpi), while control parasites were maintained at 37 °C. The presence of VAR2CSA on the iRBC surface was assessed by flow cytometry at 24 and 40 hpi. (B) At 24 hpi, there was no change in the percentage of PfEMP1-positive iRBCs in RAP-treated parasites compared to DMSO. Heat stress significantly increased the proportion of VAR2CSA-positive iRBCs, irrespective of treatment. (D) At 40 hpi, there was no difference in the percentage of VAR2CSA-positive iRBCs with either RAP or heat treatment. (C, E) Median fluorescent intensity (MFI) of anti-VAR2CSA staining was not significantly affected by heat or RAP treatment. Data represent n = 3 biological replicates. Error bars indicate ± 1 SD. Statistical analysis was performed using two-way ANOVA with FDR correction. Data were log-transformed prior to analysis.
-
Figure 3—figure supplement 11—source data 1
Raw data file of the percentage of PF3D7_0702500 knockout iRBCs positive for PfEMP1 at 24 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp11-data1-v1.xlsx
-
Figure 3—figure supplement 11—source data 2
Raw data file of the MFI corresponding to surface PfEMP1 levels of PF3D7_0702500 knockout iRBCs positive for PfEMP1 at 24 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp11-data2-v1.xlsx
-
Figure 3—figure supplement 11—source data 3
Raw data file of the percentage of PF3D7_0702500 knockout iRBCs positive for PfEMP1 at 40 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp11-data3-v1.xlsx
-
Figure 3—figure supplement 11—source data 4
Raw data file of the MFI corresponding to surface PfEMP1 levels of PF3D7_0702500 knockout iRBCs positive for PfEMP1 at 40 hpi following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp11-data4-v1.xlsx
Figure 3—figure supplement 12
PF3D7_0702500 is not required for functional PSAC activity at 24 hours post-invasion (hpi) under normal or elevated temperatures.
(A) Parasites were treated with either DMSO or rapamycin (RAP) for 4 hr to conditionally knock out PF3D7_0702500. Parasites were subsequently synchronised to a 2 hr invasion window. Heat stress was applied at 39 °C between 16 and 24 hpi, or parasites were maintained at 37 °C. At 24 hpi, aliquots of cells were treated with either PBS or sorbitol, and parasitaemia was assessed by flow cytometry. (B) The reduction in parasitaemia following sorbitol treatment (sorbitol/PBS) was a surrogate for functional PSAC activity. At 37 °C, deletion of PF3D7_0702500 (RAP treatment) had no effect on the percentage of sorbitol-sensitive cells. Heat stress led to an increased proportion of sorbitol-sensitive infected RBCs in both the absence and presence of PF3D7_0702500. Data represent n = 3 biological replicates. Error bars indicate ± 1 SD. Statistical significance was determined using two-way ANOVA on log-transformed data with FDR correction.
-
Figure 3—figure supplement 12—source data 1
Raw data file of the percentage of PF3D7_0702500 knockout iRBCs which are sorbitol sensitive following heat stress or when maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig3-figsupp12-data1-v1.xlsx
Figure 4 with 2 supplements
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 8 hr pulse (16–24 hours post-invasion [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.
-
Figure 4—source data 1
Raw data file of peptides detected following biotin enrichment of FIKK10.2-TurboID lysates after culturing of the FIKK10.2-TurboID strain in three different conditions.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Generation and validation of FIKK10.2-TurboID P. falciparum transgenic strain.
(A) An FIKK10.2-TurboID endogenous protein fusion was generated by CRISPR/Cas9 excision and homologous repair. Primer binding sites indicated by the red and blue arrows are outside the 5’ and 3’ repair template homology regions. (B) 5’ integration, 3’ integration and the absence of the original (WT) parental strain was confirmed by PCR. Predicted amplicon sizes are indicated in white. DNA size marker values (kbp) are indicated to the left of the gel. (C) Western blotting (α-Biotin) of percoll-enriched mature stages transgenic FIKK10.2-TurboID parasites and a WT control (parental strain) grown in the presence of biotin shows promiscuous biotinylation only in the FIKK10.2-TurboID strain. A total protein dye was included as a comparative loading control.
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Figure 4—figure supplement 1—source data 1
Raw images of the DNA gel assessing the correct 5’ integration of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Raw images of the DNA gel assessing the correct 3’ integration of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Raw images of the DNA gel assessing the absence of the parental strain in the culture of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data3-v1.zip
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Figure 4—figure supplement 1—source data 4
Raw images of the α-biotin immunoblot of FIKK10.2-TurboID lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data4-v1.zip
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Figure 4—figure supplement 1—source data 5
Raw images of the total protein content control of the α-biotin immunoblot of FIKK10.2-TurboID lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data5-v1.zip
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Figure 4—figure supplement 1—source data 6
PDF containing the raw images of the DNA gel assessing the correct 5’ integration of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data6-v1.pdf
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Figure 4—figure supplement 1—source data 7
PDF containing the raw images of the DNA gel assessing the correct 3’ integration of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data7-v1.pdf
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Figure 4—figure supplement 1—source data 8
PDF containing the raw images of the DNA gel assessing the absence of the parental strain in the culture of the FIKK10.2-TurboID transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data8-v1.pdf
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Figure 4—figure supplement 1—source data 9
PDF containing the raw images of the α-biotin immunoblot of FIKK10.2-TurboID lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data9-v1.pdf
-
Figure 4—figure supplement 1—source data 10
PDF containing the raw images of the total protein content control of the α-biotin immunoblot of FIKK10.2-TurboID lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig4-figsupp1-data10-v1.pdf
Figure 4—figure supplement 2
Predicted Maurer’s cleft protein topology from enriched phosphopeptides and biotinylated peptides.
Single letter amino acid layouts of the Maurer’s cleft residing proteins (A) PF3D7_0702400 (SEMP1), (B) PF3D7_1370300 (MAHRP1), and (C) PF3D7_0702500. Predicted transmembrane domains (TMDs) are shown within the Maurer’s cleft plasma membrane. N and C-termini are shown as H2N and COOH, respectively. Biotinylated lysines detected within all three replicates of the FIKK10.2-TurboID (full asexual cycle condition) are highlighted in red. Phosphorylated amino acids from phosphopeptides that are more abundant following heat stress are shown in orange. All three proteins are exported into the host cell without a PEXEL motif (PNEPs) and are predicted to contain only one TMD; however, only PF3D7_0702500 is predicted to have the N-terminus within the Maurer’s Cleft lumen. A single amino acid schematic was generated with the Protter Tool (Love et al., 2014).
Figure 5 with 1 supplement
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.
-
Figure 5—source data 1
Raw data file of the percentage of VAR2CSA-NanoLuc expressing iRBCs which are positive for cell surface PfEMP1 (VAR2CSA) following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-data1-v1.xlsx
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Figure 5—source data 2
Raw data file of the NanoLuc luminescence detected in different lysis buffer treatments of VAR2CSA-NanoLuc expressing iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Generation and validation of VAR2CSA-NanoLuc-3xHA P. falciparum transgenic strain.
(A) VAR2CSA-NanoLuc-3xHA protein fusion was generated by CRISPR/Cas9 excision and homologous repair. Primer binding sites indicated by the red and blue arrows are outside the 5’ and 3’ repair template homology regions. Predicted amplicon sizes are indicated in white. (B) 5’ integration, 3’ integration and the absence of the original (WT) parental strain was confirmed by PCR. (C) Western blotting (α-HA Tag) of percoll-enriched mature stages transgenic VAR2CSA-NanoLuc-3xHA parasites and a WT control (parental strain). A total protein dye was included as a comparative loading control. VAR2CSA-NanoLuc-3xHA has a predicted molecular weight of 379.8 kDa.
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Figure 5—figure supplement 1—source data 1
Raw images of the DNA gel assessing the correct 5’ integration of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Raw images of the DNA gel assessing the correct 3’ integration of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Raw images of the DNA gel assessing the absence of the parental strain in the culture of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data3-v1.zip
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Figure 5—figure supplement 1—source data 4
Raw images of the α-HA immunoblot of VAR2CSA-NanoLuc-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data4-v1.zip
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Figure 5—figure supplement 1—source data 5
Raw images of the total protein content control of the α-HA immunoblot of VAR2CSA-NanoLuc-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data5-v1.zip
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Figure 5—figure supplement 1—source data 6
PDF containing the raw images of the DNA gel assessing the correct 5’ integration of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data6-v1.pdf
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Figure 5—figure supplement 1—source data 7
PDF containing the raw images of the DNA gel assessing the correct 3’ integration of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data7-v1.pdf
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Figure 5—figure supplement 1—source data 8
PDF containing the raw images of the DNA gel assessing the absence of the parental strain in the culture of the VAR2CSA-NanoLuc-3xHA transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data8-v1.pdf
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Figure 5—figure supplement 1—source data 9
PDF containing the raw images of the α-HA immunoblot of VAR2CSA-NanoLuc-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data9-v1.pdf
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Figure 5—figure supplement 1—source data 10
PDF containing the raw images of the total protein content control of the α-HA immunoblot of VAR2CSA-NanoLuc-3xHA lysates.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig5-figsupp1-data10-v1.pdf
Figure 6 with 1 supplement
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 and 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 (Sargeant et al., 2006; Tarr et al., 2014). (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.
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Figure 6—source data 1
Raw data file of the NanoLuc luminescence detected in different lysis buffer treatments of PF3D7_0702500-NanoLuc expressing iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-data1-v1.xlsx
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Figure 6—source data 2
Raw data file of the NanoLuc luminescence detected in different lysis buffer treatments of REX3-NanoLuc expressing iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-data2-v1.xlsx
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Figure 6—source data 3
Raw data file of the NanoLuc luminescence detected in different lysis buffer treatments of REX3-(TMD)-NanoLuc expressing iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-data3-v1.xlsx
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Figure 6—source data 4
Raw data file of the NanoLuc luminescence detected in different lysis buffer treatments of the NanoLuc only control expressing iRBCs following heat stress compared to iRBCs maintained at 37 °C.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-data4-v1.xlsx
Figure 6—figure supplement 1
Generation and validation of constitutively expressed NanoLuc reporter strains.
(A) Following a CRISPR/Cas9-directed double-stranded DNA break, reporter constructs were inserted into the EBA165 Pseudogene Locus (PF3D7_0424300). Reporter constructs consisted of a constitutive promoter (HSP90, PF3D7_0708400), a variable N-terminal domain, NanoLuc and a T2A skip peptide followed by a neomycin resistance cassette. Primer binding sites indicated by the red and blue arrows are outside the 5’ and 3’ repair template homology regions. (B) Four distinct N-terminal variants were generated. REX3-NanoLuc (NL) comprises the first 61 amino acids of REX3, which are sufficient to mediate export (Sargeant et al., 2006; Tarr et al., 2014). PF3D7_0702500 NL represents a fusion of the PF3D7_0702500 N-terminus with NL. REX3(TMD)-NL refers to REX3-NL incorporating the transmembrane domain (TMD) of PF3D7_0702500. Finally, NL alone, lacking any N-terminal domain, was included. (C) Successful 5′ and 3′ integration of all four constructs into the EBA165 locus was confirmed by PCR. Predicted amplicon sizes are indicated in white.
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Figure 6—figure supplement 1—source data 1
Raw images of the DNA gel assessing the correct 5’ integration of the four NanoLuc reporter transgenic parasite strains.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Raw images of the DNA gel assessing the correct 3’ integration of the four NanoLuc reporter transgenic parasite strains.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
PDF containing the raw images of the DNA gel assessing the correct 5’ integration of the four NanoLuc reporter transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-figsupp1-data3-v1.pdf
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Figure 6—figure supplement 1—source data 4
PDF containing the raw images of the DNA gel assessing the correct 3’ integration of the four NanoLuc reporter transgenic parasite strain.
- https://cdn.elifesciences.org/articles/107860/elife-107860-fig6-figsupp1-data4-v1.pdf
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Plasmodium falciparum) | NF54::DiCre | Tibúrcio et al., PMID:31530668 | Parental transgenic line used throughout this manuscript. | |
| Strain, strain background (P. falciparum) | NF54 | Tibúrcio et al., PMID:31530668 | Isolate predicted to originate from West Africa. | |
| Strain, strain background (P. falciparum) | HB3 | Gift (Maria Bernabeu) | Honduras isolate | |
| Strain, strain background (P. falciparum) | Cam3.II | Gift (Colin Sutherland) | Cambodia isolate | |
| Strain, strain background (P. falciparum) | HL2208 | Gift (Colin Sutherland) PMID:38019958 | Uganda isolate | |
| Cell line (Homo sapiens) | Human erythrocytes | UK, NHS Blood and Transplant (NHSBT) | RBCs used for P. falciparum parasite culture | |
| Genetic reagent (P. falciparum) | FIKK1 conditional knockout strain | Davies et al., PMID:32284562 | HA-tagged DiCre excisable line | |
| Genetic reagent (P. falciparum) | FIKK4.1 conditional knockout strain | Davies et al., PMID:32284562 | HA-tagged DiCre excisable line | |
| Genetic reagent (P. falciparum) | FIKK4.2 conditional knockout strain | Davies et al., PMID:32284562 | HA-tagged DiCre excisable line | |
| Genetic reagent (P. falciparum) | FIKK9 family conditional knockout strain | Davies et al., PMID:32284562 | DiCre excisable line to knockout all FIKK9 genes | |
| Genetic reagent (P. falciparum) | FIKK10.1 conditional knockout strain | Davies et al., PMID:32284562 | HA-tagged DiCre excisable line | |
| Genetic reagent (P. falciparum) | FIKK10.2 conditional knockout strain | Davies et al., PMID:32284562 | HA-tagged DiCre excisable line | |
| Genetic reagent (P. falciparum) | FIKK10.2-TurboID-V5 strain | This paper | Endogenous FIKK10.2 C-terminally fused to TurboID-V5 (NF54::DiCre is the parental strain) | |
| Genetic reagent (P. falciparum) | HSP70x conditional knockout strain | This paper | HA-tagged DiCre excisable line (NF54::DiCre is the parental strain) | |
| Genetic reagent (P. falciparum) | PF3D7_0702500 conditional knockout strain | This paper | HA-tagged DiCre excisable line (NF54::DiCre is the parental strain) | |
| Antibody | Anti-VAR2CSA (human monoclonal, PAM7.5) | Gift (Lars Hviid) | 1:200 (flow cytometry) | |
| Antibody | Goat anti-human PE-conjugated secondary | Thermo Fisher Scientific | Cat#: 12-4998-82, RRID:AB_465926 | 1:200 (flow cytometry) |
| Antibody | Anti-HA (rat monoclonal, clone 3F10) | Merck | Cat#: 11867423001, RRID:AB_390918 | 1:3000 (western blot) |
| Antibody | Streptavidin-HRP | Pierce | Cat#: 21130 | 1:5000 (western blot) |
| Antibody | Anti-SBP1 | Gift (Tobias Spielmann) | Parasite protein detection, 1:5000 (Immunofluorescence Microscopy) | |
| Antibody | Anti-KAHRP | European Malaria Reagent Repository | Parasite protein detection, 1:2000 (Immunofluorescence Microscopy) | |
| Chemical compound, drug | WR99210 | Jacobus Pharmaceuticals | Parasite drug selection | |
| Chemical compound, drug | G418 | Gibco | Cat#: 10131027 | Parasite drug selection |
| Chemical compound, drug | Rapamycin (RAP) | Sigma | Cat#: 553210–5 MG | 100 nM (DiCre induction to induce conditional knockout) |
| Chemical compound, drug | Furosemide | Sigma | Cat#: F4381-10G | PSAC inhibitor |
Additional files
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Supplementary file 1
Heat stress phosphoproteome and proteome data from the 10-plex TMT from NF54::DiCre (wild type).
Includes P. falciparum and H. sapiens peptides and proteins.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp1-v1.xlsx
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Supplementary file 2
P. falciparum phosphosites that are more phosphorylated following heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp2-v1.xlsx
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Supplementary file 3
Heat stress phosphoproteome and proteome data from the 16-plex TMT with FIKK conditional knockouts.
Includes P. falciparum and H. sapiens peptides and proteins.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp3-v1.xlsx
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Supplementary file 4
FIKK10.2-TurboID proximal biotinylated proteins identified under various conditions, including the presence or absence of biotin and heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp4-v1.xlsx
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Supplementary file 5
FIKK10.2-TurboID proximal biotinylated proteins identified in the presence of biotin and in the absence of heat stress.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp5-v1.xlsx
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Supplementary file 6
Primer sequences and binding sites used for validation of transgenic P. falciparum strains.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp6-v1.xlsx
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Supplementary file 7
Composition of reduced nutrient media.
- https://cdn.elifesciences.org/articles/107860/elife-107860-supp7-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107860/elife-107860-mdarchecklist1-v1.docx
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Physiological febrile heat stress increases cytoadhesion through increased protein trafficking of Plasmodium falciparum surface proteins into the red blood cell
eLife 14:RP107860.
https://doi.org/10.7554/eLife.107860.3
