A heat-shock response regulated by the PfAP2-HS transcription factor protects human malaria parasites from febrile temperatures

Abstract

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates the transcription of hsp70-1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70-1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37 °C but not at 35 °C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to the maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.

Extended Data Fig. 1. Transcriptional analysis of heatshock-adapted and control lines.

a, Microarray-based transcriptomic comparison across the asexual blood cycle of 3D7-A cultures adapted to heat-shock after five cycles of selection with a 3 h heat-shock at 41.5°C at the trophozoite stage (3D7-A-HS r1 and r2) and control parasite lines maintained in parallel without heat-shock (3D7-A r1 and r2). Values are the log₂ of the maximum expression fold-change (from the average of all lines compared) across a time interval corresponding to half the length of the asexual cycle, calculated using the aMAFC score as previously described. Genes with a >1.5-foldchange in expression in two independent 3D7-A heat-shock-adapted lines (3D7-A-HS r1 and r2) relative to their respective controls (3D7-A r1 and r2) are shown. Data for parasite lines 10G (heat-shock-sensitive subclone), 1.2B (heat-shock-resistant subclone) and 3D7-A (right panel) is from RoviraGraells et al.. b, Time-course expression of genes in panel a that showed a concordant change in expression between heat-shock-adapted and control cultures, and between the heat-shock-resistant subclone 1.2B and the heat-shock-sensitive subclone 10G. Based on the predicted function of the three genes, clag2 was considered the most plausible candidate to play a role in heat-shock resistance. c, Expression of clag2 is neither necessary nor sufficient for heat-shock resistance. RT-qPCR analysis of clag2 transcript levels (normalized against rhoph2) in schizonts of heatshock sensitive (S) and heat-shock resistant (R) 3D7-A subclones (see Fig. 1f), and of the heatshock-adapted and control lines (n=1 biological replicates).

Extended Data Fig. 2. Generation and characterization of transgenic parasite lines edited at the pfap2-hs locus.

a, Schematic of the CRISPR/Cas9 strategy used to knockout pfap2-hs, using two guide RNAs. b-d, Tagging of endogenous PfAP2-HS using CRISPRCas9 technology. The tags used were a C-terminal 3xHA (b), a C-terminal eYFP (c) and an N-terminal eYFP (d). e, C-terminal tagging of endogenous PfAP2-HS by single homologous recombination with a tag consisting of a 2xHA epitope and an FKBP destabilization domain (DD domain). In all panels, the position of the primers used for analytical PCR (arrowheads), guide RNA and AP2 domains (blue vertical bars) is indicated. The electrophoresis images at the right are the analytical PCR validation of the genetic edition (single genomic DNA extraction and PCR analysis), showing correct edition and absence of wild-type locus in all cases except for the 8A subclone of 1.2B-ddFKBP (8A and 12E are subclones obtained after drug cycling). The bar charts at the right show the level of survival (mean of n=2 independent biological replicates) of sorbitol-synchronized cultures of the transgenic lines upon heat-shock (HS) exposure at the trophozoite stage, with heat-shock-resistant (10E) and heatshock-sensitive (10G, expressing PfAP2-HSΔD3) subclones as controls. Addition of a C-terminal eYFP or HA-FKBP tag did not affect growth at 37°C but resulted in high heat-shock sensitivity, similar to the 10G line. In contrast, C-terminal addition of the smaller 3xHA tag or addition of an N-terminal eYFP did not affect growth at 37°C or heat-shock sensitivity. In all cases, tagged PfAP2-HS was not detectable by immunofluorescence or Western blot analysis, probably as a consequence of the very low abundance of this transcription factor (see proteomic data in www.PlasmoDB.org).

Extended Data Fig. 3. Validation of the transcriptomic changes upon heat-shock and distribution of the tandem G-box motif.

a, RT-qPCR analysis of transcript levels (normalized against serine--tRNA ligase) of the genes selected for validation, using biological samples independent from the samples used for microarray analysis. Values are the average of triplicate reactions. The log₂ expression fold-change [heat-shock (HS) relative to control (35°C) conditions] for these genes in the microarray analysis (Fig. 2a) is shown in a heatmap to facilitate comparison. b, Genes in the P. falciparum genome containing tandem arrangements (maximum distance between the two: 9 nucleotides) of the Gbox [(A/G)NGGGG(C/A)] motif in their regulatory regions (defined as the 2 kb upstream of the start codon or until the neighbour gene, when it is closer). The sequence of the G-box in each gene is shown in blue, and the level of concordance with the consensus G-box motif is expressed as the P value of the match (determined using the FIMO v5.0.5 function in the MEME suite). Expression changes upon heat-shock for these genes are shown as in panel a.

Extended Data Fig. 4. Changes in hsp70–1, hsp90 and PF3D7_1421800 transcript levels in parasites lacking the entire PfAP2-HS or D3.

Fold-increase in transcript levels (determined by RT-qPCR, normalized against serine--tRNA ligase) during and after heat-shock (HS) starting at 33–35 (a) or 30–35 (b) hpi, relative to cultures maintained in parallel without heat-shock (37 or 35°C). In panel a, values for three individual 3D7-A subclones carrying or not the Q3417X mutation are shown as dotted lines, whereas the average of the three subclones is shown as a continuous line. In panel b, the mean of n=2 independent biological replicates is shown.

Extended Data Fig. 5. Transcript level changes upon heat-shock in chaperone-encoding genes.

Log₂ expression foldchange [heat-shock (HS) relative to control (35°C) conditions, as in Fig. 2a] for all chaperone-encoding genes described by Pavithra and colleagues. Columns at the left indicate presence of the G-box or tandem G-box (TdGbox) in the upstream region, and log₂ fold-change during heat-shock in a previous study (Oakley).

Extended Data Fig. 6. Transcriptomic characterization of the heatshock response in parasites expressing complete PfAP2-HS (10E line).

Log₂ expression fold-change [heat-shock (HS) relative to control (35°C) conditions] in the wild-type 10E line determined by microarray analysis. Genes with a fold-change ≥2 at any of the time points analysed are shown. The mean log₂ expression fold-change (with 95% confidence interval) and representative enriched GO terms are shown for each cluster. Columns at the left indicate fold-change during heatshock in a previous study (Oakley), and annotation as chaperone. Ten genes had values out of the range displayed (actual range: −3.89 to +4.03).

Extended Data Fig. 7. ChIP analysis of the chromosomal distribution of PfAP2-HS.

a, ChIP-seq analysis of HA-tagged PfAP2-HS. Number of reads of ChIP (IP) and input (in) tracks, and log₂-transformed ChIP/input ratio tracks (IP/in) for five independent biological replicates [three including heat-shock (HS) and 37°C conditions, two including only the 37°C condition]. Snapshots are shown for the three genes in cluster I (Fig. 2a) and snoR04. Binding at the hsp70–1 and hsp90 promoters coincides with the position of a tandem G-box motif, whereas PF3D7_1421800 and snoR04 lack a G-box. b, Peaks present in ≥3 out of 5 replicate ChIP-seq experiments (37°C) or ≥2 out of 3 replicate experiments (heat-shock) and with a MACS score >100 in each positive replicate. c, ChIP-qPCR analysis of HA-tagged PfAP2-HS binding at selected loci, in cultures exposed to heat-shock (HS) or control (37°C) conditions (mean and s.e.m. of % input in n=3 independent biological replicates). No significant difference (P<0.05) was observed between 37°C and heat-shock using a two-sided unpaired t-test.

Extended Data Fig. 8. Transcriptional changes associated with PfAP2HS deletion under basal (no heat-shock) conditions.

a, Changes in transcript levels in the absence of heat-shock for genes with an average expression fold-change >2 between 10E_Δpfap2-hs and 10E. Values are the log₂ of the average expression fold-change relative to 10E across the time period compared (~27–30.5 hpi). Genes artificially modified or introduced in the knockout line, which serve as controls, are shown at the bottom (their values are out of the range displayed). The column at the left indicates the presence of the G-box. b, Expression plots for selected genes under basal conditions. Expression values are plotted against statistically-estimated parasite age, expressed in h post-invasion (hpi). Grey shading marks the interval used to calculate the average expression fold-change. c, RT-qPCR analysis of hsp70–1 and hsp90 transcript levels in pfap2-hs knockout (KO) lines compared to their wild type (WT) controls (the parental line for each knockout line) under basal conditions. Expression values are normalized against serine--tRNA ligase, and expressed as the fold-change (FC) in the knockout versus control lines. The mean of n=3 (10E, 30–35 hpi) or n=2 (others) independent biological replicates is shown.

Extended Data Fig. 9. Analysis of proteome stress and unfolded protein response (UPR) markers in pfap2-hs mutants.

a-b, Western blot analysis (representative of n=4) of polyubiquitinated proteins (Ub) (a) or phosphorylated eIF2α (eIF2α-P) (b) immediately after a 3 h heat-shock (3 h HS) and 2 h later (2 h post HS). Histone H3 is a loading control. DHA was used as a positive control, as it is a known inducer of the UPR^,. The Log₂ of histone H3-normalized signal in heat-shock or DHA-treated cultures versus control cultures is shown at the bottom (mean and s.e.m. of n=4, except for the DHA control mean of n=2 independent biological replicates). P values were calculated using a two-sided unpaired t-test. Only significant P values (P<0.05) are shown. The position of molecular weight markers is shown (in kDa).

Extended Data Fig. 10. Sequence alignment of the three AP2 domains (D1-D3) present in AP2-HS orthologs in Plasmodium spp.

Dots indicate identity with the amino acid in the first sequence.

Fig 1.. Mutations in PfAP2-HS and sensitivity to heat-shock.

a, Schematic of the parasite lines used in this study. Colours indicate wild type PfAP2-HS or truncated forms lacking AP2 domain 3 (ΔD3), the three AP2 domains (ΔD1–3), or virtually the full protein (KO). Parasite lines shown with a colour gradient consist of a mixture of individual parasites expressing different versions of the protein. An asterisk indicates a heat-shock (HS) sensitive phenotype, and r1 and r2 are independent replicates of the selection of 3D7-A with periodic heat-shock (3D7-A-HS r1 and r2 are the selected lines, whereas 3D7-A r1 and r2 are controls maintained in parallel at 37°C). b, Proportion of Illumina reads with (Alt) or without (Ref) a nonsense mutation in pfap2-hs in two independently selected heat-shock-adapted cultures (3D7-A-HS r1 and r2) and their controls (3D7-A r1 and r2). c, Sanger sequencing confirmation of the mutation (in the r1 replicate, representative of r1 and r2). d, Schematic of wild-type PfAP2-HS, PfAP2-HS_ΔD3 and ΔPfAP2-HS. The position of the AP2 domains is indicated (D1–3). e, Survival of tightly synchronized cultures exposed to heat-shock at different ages (in h post-invasion, hpi) for two heat-shock-sensitive (3D7-A r2 and 10G) and two heat-shock-resistant (3D7-A-HS r2 and 1.2B) lines (mean of n=2 independent biological replicates). f, Heat-shock survival at the trophozoite stage of 3D7-A subclones carrying or not the Q3417X mutation (mean of n=2 independent biological replicates). g, Heat-shock survival of tightly synchronized cultures of parasite lines expressing wild-type or mutated PfAP2-HS. Values are the mean and s.e.m. of n=5 (lines of 3D7 origin) or n=3 (HB3 and D10 lines) independent biological replicates. P values were calculated using a two-sided unpaired t-test.

Fig 2.. Global transcriptional alterations in parasites exposed to heat-shock.

a, Hierarchical clustering of genes with altered transcript levels (≥4 fold-change at any of the time points analysed) during (1.5 and 3 h) or 2 h after finishing (2 h post) heat-shock (HS). Values are the log₂ of the expression fold-change in heat-shock versus control cultures. 13 genes had values out of the range displayed (actual range: −4.78 to +4.93). For each cluster, mean values (with 95% confidence interval) for the genes in the cluster and representative enriched GO terms are shown. Columns at the left indicate annotation as chaperone, presence of the G-box or tandem G-box (TdGbox) in the upstream region, and log₂ fold-change after heat-shock in a previous study (Oakley). b, Venn diagrams of the genes altered upon heat-shock in the three parasite lines. c, Pearson correlation of the genome-wide transcript levels of each culture versus the most similar time point of a high-density time-course reference transcriptome. d, Age progression during the assay, statistically estimated from the transcriptomic data. e, ChIP-seq analysis of HA-tagged PfAP2-HS, representative of n=5 and n=3 independent biological replicates for 35°C and heat-shock, respectively. The log₂-transformed ChIP/input ratio at the hsp70–1 and hsp90 loci is shown. The position of the tandem G-box is indicated.

Fig 3.. Phenotypes of parasite lines lacking PfAP2-HS.

a, Growth rate of Δpfap2-hs and parental lines of 3D7 genetic background at different temperatures (mean and s.e.m. of n=4 independent biological replicates). P values were calculated using a two-sided unpaired t-test (10E_Δpfap2-hs: 37 vs. 35°C, P=2.3 ×10⁻³; 37.5 vs. 35°C, P=1.7 ×10^-4. 10G_Δpfap2-hs: 37 vs. 35°C, P=0.011; 37.5 vs. 35°C, P=0.001). Only significant P values (P<0.05) are shown. b, Same as in panel a for parasite lines of HB3 and D10 genetic background (mean and s.e.m. of n=4 independent biological replicates). c, Number of merozoites per schizont (median and quartiles box with 10–90 percentile whiskers). Values were obtained from 100 schizonts for each parasite line and condition. d, Duration of the asexual blood cycle. The cumulative percent of new rings formed at each time point is shown (mean of n=2 independent biological replicates).

Fig 4.. Characterisation of a cultured-adapted field isolate with mutations in pfap2-hs.

a, Schematic of wild-type PfAP2-HS and PfAP2-HS_ΔD1–3 occurring in Line 1 from The Gambia after culture adaptation (C to G mutation at codon 931, S931X). The position of the AP2 domains is indicated (D1–3). b, Frequency of the mutation (as determined by Sanger sequencing) in culture-adapted Line 1 before (Pre) and after (Post) performing a heat-shock (HS) at the trophozoite stage and culturing for an additional cycle (mean of n=2 independent biological replicates). c, Frequency of the mutation during culture at different temperatures. Day 0 is when the frozen stock from The Gambia (culture-adapted for 91 days) was placed back in culture. d, Sanger sequencing determination of the presence or absence of the mutation at codon 931 in Line 1 subclones 4E and 1H. e, Heat-shock survival of tightly synchronised 4E and 1H cultures (mean and s.e.m. of n=4 independent biological replicates). The P value was calculated using a two-sided unpaired t-test. f, Growth rate of 4E and 1H at different temperatures (mean and s.e.m. of n=5 independent biological replicates). No significant difference (P<0.05) was observed between growth at 35°C and 37°C using a two-sided unpaired t-test.

Fig 5.. Sensitivity of parasites lacking PfAP2-HS to proteotoxic conditions.

a, Survival (%) after a 3 h dihydroartemisinin (DHA) pulse at the ring or trophozoite (troph.) stage. Values are the mean and s.e.m. of n=3 (3D7-A and Line 1 genetic backgrounds) or mean of n=2 (HB3 and D10 genetic backgrounds) independent biological replicates. Mean IC₅₀ for each line is shown (same colour code as the plots). b, Survival (%) after a 3 h epoxomicin pulse at the trophozoite stage. Values are the mean and s.e.m. of n=4 (100 nM) or n=3 (150 nM) independent biological replicates. In all panels, P values were calculated using a two-sided unpaired t-test (only for experiments with n≥3). Only significant P values (P<0.05) are shown.

Fig 6.. Model of the P. falciparum heat-shock response and phylogenetic analysis of AP2-HS.

a, The P. falciparum heat-shock response involves rapid upregulation of the expression of a very restricted set of chaperones by PfAP2-HS. The PF3D7_1421800 gene (in brackets) shows PfAP2-HS-dependent increased transcript levels upon heat-shock, but PfAP2-HS binding was not detected in its promoter, and it lacks a G-box. The main defects associated with PfAP2-HS deletion or truncation, under heat-shock or basal conditions, are listed. b, Phylogenetic analysis of the protein sequence of AP2-HS orthologs in Plasmodium spp. c, Schematic of the domain structure of AP2-HS orthologs in Plasmodium spp. The position of the AP2 domains (D1–3) is based on domains identified in PlasmoDB release 50, except for those marked with an asterisk that were annotated manually based on sequence alignments.