Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites

Abstract

During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum, and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group. In other organisms, farnesylation of Hsp40 orthologs controls their localization and function in resisting environmental stress. In this work, we find that plastidial isopentenyl pyrophosphate (IPP) synthesis and protein farnesylation are required for malaria parasite survival after cold and heat shock. Furthermore, loss of HSP40 farnesylation alters its membrane attachment and interaction with proteins in essential pathways in the parasite. Together, this work reveals that farnesylation is essential for parasite survival during temperature stress. Farnesylation of HSP40 may promote thermotolerance by guiding distinct chaperone-client protein interactions.

Keywords: Plasmodium falciparum; farnesylation; heat shock; isoprenoids; malaria; protein chaperone.

FIG 1

Growth under temperature stress requires IPP synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the nonmevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP), serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase inhibitors (FTI or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors (FTI [10 μM] and BMS [200 nM]), or geranylgeranyltransferase inhibitor GGTI (2 μM) for 24 h prior to a 6-h heat (40°C) or cold (25°C) shock. (C and D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C to E) n = 6; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C and D) 2-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. (E) Within each treatment group, the normalized control was compared to temperature shock sample by unpaired t test with Welch’s correction. Abbreviations: ctrl, control, hs, heat shock, cs, cold shock.

FIG 2

Supplementation with IPP and F-OL rescues growth in FSM-treated parasites after temperature stress. (A) Parasites were treated with FSM (5 μM) for 24 h prior to a 6-h heat (40°C) or cold (25°C) shock. Cultures were supplemented with isoprenoid products (IPP [250 μM], F-OL [10 μM], or GG-OL [10 μM]) for the entire length of experiment. (B) FSM-treated parasites are sensitive to heat shock. (C and D) Supplementation with IPP (C) or F-OL (D) rescues heat sensitivity. (E) GG-OL supplementation is unable to rescue growth after heat stress. (F to I) IPP or F-OL, but not GG-OL, supplementation is similarly able to rescue FSM-treated parasite growth after cold shock. (B to I) n = 3 to 6; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Two-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. Abbreviations: ctrl, control, hs, heat shock, cs, cold shock.

FIG 3

Localization of HSP40 in P. falciparum. (A) Immunofluorescence confocal microscopy of trophozoite, stained with anti-HSP40 (1:5,000) and Hoechst 33258 nuclear stain. HSP40 appears cytosolic. (B) Electron micrograph of immunolabeling: primary, rabbit anti-HSP40 (1:250), mouse anti-PDI (1:100); secondary, goat anti-rabbit IgG 18 nm colloidal gold, goat anti-mouse 12 nm. HSP40 (orange arrowheads) looks cytosolic in the parasites, with some apparent membrane association. A portion of HSP40 colocalizes with PDI, an established ER marker (white arrowheads). Scale, 500 nm.

FIG 4

Inhibition of either IPP synthesis or protein farnesylation results in reduced membrane association of HSP40. (A and B) Representative anti-HSP40 immunoblots of control- and FSM (20 μM)-treated (A) or FTI (10 μM)-treated (B) P. falciparum total lysate and membrane fractions. (C and D) Quantification of several immunoblots adjusted to loading control. HSP40 is significantly reduced in the membrane fraction after inhibition of IPP synthesis (C) and inhibition of farnesylation (D). Anti-HAD1 and anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. **, P ≤ 0.01; ***, P ≤ 0.001 unpaired t test with Welch’s correction. (E and F) HSP40 membrane association is reduced after FTI treatment. Apparent membrane-associated HSP40 (10 nm gold particles) is reduced after inhibition of farnesylation. The number of membrane-associated HSP40 per micrograph is quantified for control and treated parasites (F). A single control cohort was quantified. A decrease in the number of membrane-associated HSP40 particles is observed. *, P ≤ 0.05, unpaired t test with Welch’s correction. Scale, 500 nm.

FIG 5

Both farnesylation and palmitoylation contribute to HSP40 membrane association, but only farnesylation is required for thermotolerance. (A) Representative anti-HSP40 immunoblots of control, FTI (10 μM), 2BP (100 μM), and combination of FTI- and 2BP-treated P. falciparum total lysate and membrane fractions. (B) Quantification of several immunoblots adjusted with loading control. The membrane-associated proportion of HSP40 is significantly reduced upon inhibition of farnesylation (FTI) or palmitoylation (2BP). Inhibition of both farnesylation and palmitoylation (FTI + 2BP) reduces HSP40 membrane association further than does single inhibitor treatment. Anti-HAD1 and anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. n = 3; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 unpaired t test with Welch’s correction. (C to J) Parasites were treated with FTI (10 μM), 2BP (100 μM), or both prior to heat (40°C) or cold (25°C) shock. FTI-treated parasite growth is significantly reduced after heat (D) and cold shock (H). Growth in 2BP-treated parasites is unchanged after heat or cold shock (E and I). Parasites treated with both FTI and 2BP were sensitive to temperature stress (F and J). n = 3; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001, 2-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. Abbreviations: ctrl, control; hs, heat shock; cs, cold shock.

FIG 6

Both IPP synthesis and protein farnesylation influence HSP40 protein-protein interactions. Candidate protein interactors were determined by mass spectrometry after IP of parasite lysate with anti-HSP40. Results for FSM (5 μM)- and FTI (10 μM)-treated parasites are compared to untreated controls (n = 3). UniProt and GeneIDs are provided in Table S2. Heat map of normalized log₂-transformed data was generated using NG-CHM Heat Map Builder. Gene Ontology (GO) annotations are indicated by colored bars.

FIG 7

Localization of GAPDH, but not its glycolytic function, is IPP- or farnesylation-dependent. (A to C) Inhibition of IPP synthesis and farnesylation reduced membrane association of GAPDH. (A) Representative anti-GAPDH immunoblots of control-, FSM (20 μM)-, and FTI (10 μM)-treated P. falciparum. (B and C) Quantification of several immunoblots adjusted with loading control. Anti-Hsp70 (1:5,000) and anti-PM-V (1:500) were used as loading controls for total lysate and membrane fractions, respectively. n = 5 to 6; ****, P ≤ 0.0001, unpaired t test with Welch’s correction.

FIG 8

Glycolytic and pentose phosphate pathway metabolite levels remain constant under IPP- and farnesylation-deficient conditions. Levels of glycolytic and pentose phosphate pathway intermediates were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. No significant changes are observed after treatment with FSM (5 μM) or FTI (10 μM). n = 3, unpaired t test with Welch’s correction.

FIG 9

Isoprenoid biosynthesis and farnesylation affect membrane association and client protein assembly of HSP40.