Falciparum Heat Shock Protein Research Articles

PFB0595w is a Plasmodium falciparum J protein that co-localizes with PfHsp70-1 and can stimulate its in vitro ATP hydrolysis activity

Heat shock proteins, many of which function as molecular chaperones, play important roles in the lifecycle and pathogenesis of the malaria parasite, Plasmodium falciparum. The P. falciparum heat shock protein 70 (PfHsp70) family of chaperones is potentially regulated by a large complement of J proteins that localize to various intracellular compartments including the infected erythrocyte cytosol. While PfHsp70-1 has been shown to be an abundant cytosolic chaperone, its regulation by J proteins is poorly understood. In this study, we characterized the J protein PFB0595w, a homologue of the well-studied yeast cytosolic J protein, Sis1. PFB0595w, similarly to PfHsp70-1, was localized to the parasite cytosol and its expression was upregulated by heat shock. Additionally, recombinant PFB0595w was shown to be dimeric and to stimulate the in vitro ATPase activity of PfHsp70-1. Overall, the expression, localization and biochemical data for PFB0595w suggest that it may function as a cochaperone of PfHsp70-1, and advances current knowledge on the chaperone machinery of the parasite.

Plasmodial Hsp40 and Hsp70 chaperones: current and future perspectives

Plasmodium falciparum displays a large and remarkable variety of heat shock protein 40 family members (PfHsp40s). The majority of the PfHsp40s are poorly characterized, and although the functions of some of them have been suggested, their exact mechanism of action is still elusive and their interacting partners and client proteins are unknown. The P. falciparum heat shock protein 70 family members (PfHsp70s) have been more extensively characterized than the PfHsp40s, with certain members shown to function as molecular chaperones. However, little is known about the PfHsp70-PfHsp40 chaperone partnerships. There is mounting evidence that these chaperones are important not only in protein homoeostasis and cytoprotection, but also in protein trafficking across the parasitophorous vacuole (PV) and into the infected erythrocyte. We propose that certain members of these chaperone families work together to maintain exported proteins in an unfolded state until they reach their final destination. In this review, we critically evaluate what is known and not known about PfHsp40s and PfHsp70s.

A secretory multifunctional serine protease, DegP of Plasmodium falciparum, plays an important role in thermo‐oxidative stress, parasite growth and development

Plasmodium falciparum heat shock proteins and proteases are known for their indispensable roles in parasite virulence and survival in the host cell. They neutralize various host-derived stress responses that are deleterious for parasite growth and invasion. We report identification and functional characterization of the first DegP from an apicomplexan (P. falciparum). To determine the molecular identity and functions of the parasite-encoded DegP, we complemented the Escherichia coli degP null mutant with a putative PfdegP gene, and the results showed that PfDegP complements the growth defect of the temperature sensitive DegP-deficient mutant and imparts resistance to non-permissive temperatures and oxidative stress. Molecular interaction studies showed that PfDegP exists as a complex with parasite-encoded heat shock protein 70, iron superoxide dismutase and enolase. DegP expression is significantly induced in parasite culture upon heat shock/oxidative stress. Our data suggest that the PfDegP protein may play a role in the growth and development of P. falciparum through its ability to confer protection against thermal/oxidative stress. Antibody against DegP showed anti-plasmodial activity against blood-stage parasites in vitro, suggesting that PfDegP and its associated complex may be a potential focus for new anti-malarial therapies. ●PfDegP physically interacts with PfHsp70 and PfEno by anti-bait co-immunoprecipitation (View interaction) ●PfDegP physically interacts with PfEno, PfSod, PfOat, PfHsp70, PfLDH and PfGpi by anti-bait co-immunoprecipitation (View interaction) ●PfHsp-70 and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP physically interacts with PfOat, PfHsp70, PfEno, PfSod, PfGpi and PfLDH by surface plasmon resonance (View interaction) ●PfEno and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP and PfHsp70 co-localize by co-sedimentation through density gradient (View interaction).

Harmine Is a Potent Antimalarial Targeting Hsp90 and Synergizes with Chloroquine and Artemisinin

Previous studies have shown an antimalarial effect of total alkaloids extracted from leaves of Guiera senegalensis from Mali in West Africa. We independently observed that the beta-carboline alkaloid harmine obtained from a natural product library screen inhibited Plasmodium falciparum heat shock protein 90 (PfHsp90) ATP-binding domain. In this study, we confirmed harmine-PfHsp90-specific affinity using surface plasmon resonance analysis (dissociation constant [K(d)] of 40 μM). In contrast, the related compound harmalol bound human Hsp90 (HsHsp90) (K(d) of 224 μM) more tightly than PfHsp90 (K(d) of 7,010 μM). Site-directed mutagenesis revealed that Arg98 in PfHsp90 is essential for harmine selectivity. In keeping with our model indicating that Hsp90 inhibition affords synergistic combinations with existing antimalarials, we demonstrated that harmine potentiates the effect of chloroquine and artemisinin in vitro and in the Plasmodium berghei mouse model. These findings have implications for the development of novel therapeutic combinations that are synergistic with existing antimalarials.

Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers.

One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1

Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

BackgroundPlasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.MethodsStage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.ResultsIn parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.ConclusionResults indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns

Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.

Detection of Malaria Infection via Latex Agglutination Assay

A rapid test for malaria diagnosis based on specific agglutination of sensitive polystyrene particles containing carboxylic acid with antigen or antibody molecules in the presence of their corresponding antibody or antigen in human plasma has been achieved. The particle-malaria antigen conjugate (PAgC), particle-monoclonal IgG antibody to Plasmodium falciparum heat shock protein 70 conjugate (PmAbC), and particle-polyclonal IgG antibody to P. falciparum malaria conjugate (PpAbC) were prepared via adsorption process. The higher affinity of the malaria antigen adsorption onto particles was observed compared to that of the antibodies. Immunoagglutination of sensitive latex particles was monitored by measuring the change in turbidity, and the aggregate's formation was clearly observed under optical microscope. The efficacy in malaria diagnosis of the conjugated particles evaluated at an outpatient malaria clinic (Mae Sod, Thailand) indicated a success detection of antibody or antigen. Sensitivity of PAgC, PmAbC, and PpAbC for P. falciparum was 84%, 90%, and 90%, respectively, while specificity for malaria disease was 70% for PAgC and 80% for PmAbC and PpAbC. The rapid agglutination-based latex particles assay developed herein showed a great potential for diagnosis of malaria infection.

Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain

Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.

Identification of regulatory elements in the Plasmodium falciparum genome

There is little information regarding regulatory sequences in the newly sequenced genome of the malaria parasite, Plasmodium falciparum. Thus, for the first time, a bioinformatic strategy was utilized to identify regulatory elements in this genome using the P. falciparum heat shock protein (hsp) gene family as a model system. Our analysis indicates that the P. falciparum hsp genes do not contain standard eukaryotic regulatory elements. However, a novel G-rich regulatory element named the G-box was identified upstream of several P. falciparum hsp genes and the P. yoelii yoelii, P. berghei, and P. vivax hsp86 genes. Remarkably, the Plasmodium sp. G-boxes were required for maximal reporter gene expression in transient transfection assays. The G-box is not homologous to known eukaryotic elements, and is the best-defined functional element elucidated from Plasmodium sp. Our analysis also revealed several other elements necessary for reporter gene expression including an upstream sequence element, the region surrounding the transcription start site, and the 5' and 3' untranslated regions. These data demonstrate that unique regulatory elements are conserved in the genomes of Plasmodium sp., and demonstrate the feasibility of bioinformatic approaches for their identification.

Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His) 6-PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His) 6-PfHsp70 derivatives and improved yields of the full-length protein. (His) 6-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The V max, K m, and k cat for the basal ATPase activity of (His) 6-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 μM, and 1.03 min −1, respectively. Gel filtration studies indicated that (His) 6-PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.

Cellular and antibody responses to the Plasmodium falciparum heat shock protein Pf72/HSP70 during and after acute malaria in individuals from an endemic area of Brazil

Proliferative and antibody responses to three synthetic peptides corresponding to Pf72/HSP70 were followed-up in acute malaria patients from an endemic area of Brazil. In vitro lymphocyte responsiveness to all peptides was relatively low and short-lived and there was a considerable variation in the frequency and magnitude of the individual lymphoproliferative response to the peptides at different periods after the onset of infection. Although 96% of the patients had IgG antibodies to crude Plasmodium falciparum asexual blood stage antigens, specific IgG antibody responses to the peptides varied from 12.5 to 40% according to the tested peptides. No significant difference was observed in the proliferative or antibody responses to the peptides between individuals that remained parasitemic after treatment and those that recovered from malaria infection. The different frequencies of proliferative responses in peripheral blood T cells on different occasions after the onset of their infection show that, in order to be informative, evaluation of the in vitro cellular immune response to peptides requires longitudinal studies in which each individual is tested repeatedly.

Enhanced expression of Plasmodium falciparum heat shock protein PFHSP70-I at higher temperatures and parasite survival

The effect of various body temperatures, encountered during malaria fever, on the synthesis of Plasmodium falciparum heat-shock protein called PFHSP70-I and parasite growth rates among five different isolates are described. The results show that after the exposure of parasites at 39°C for 30 min the amount of PFHSP70-I in all five isolates increased markedly and significantly, whereas parasite growth rates and the amount of total blood stage antigens remained almost unaffected. This indicates that the PFHSP70-I gene responds to heat-shock by producing higher amounts of PFHSP70-I protein, presumably to protect the parasite from being killed during malaria fever.

Enhanced expression of Plasmodium falciparum heat shock protein PFHSP70-I at higher temperatures and parasite survival.

The effect of various body temperatures, encountered during malaria fever, on the synthesis of Plasmodium falciparum heat-shock protein called PFHSP70-I and parasite growth rates among five different isolates are described. The results show that after the exposure of parasites at 39 degrees C for 30 min the amount of PFHSP70-I in all five isolates increased markedly and significantly, whereas parasite growth rates and the amount of total blood stage antigens remained almost unaffected. This indicates that the PFHSP70-I gene responds to heat-shock by producing higher amounts of PFHSP70-I protein, presumably to protect the parasite from being killed during malaria fever.

Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.

Human response to a malaria vaccine candidate antigen

The Plasmodium falciparum heat-shock protein 70 (PFHSP70) has been proposed as a potential candidate antigen for a malaria vaccine. In this study the human immune response to this antigen has been evaluated. Enzyme-linked immunosorbent assays (ELISA) were performed on serum samples collected from individuals during and after malarial infection. Only 70% of falciparum malaria patients responded to this antigen during the natural course of infection and 54% of the recovered cases who had no parasitaemia at the time of blood collection lacked detectable antibody. The non-responders to PFHSP70 exhibited an antibody response to total schizont antigens. There was no correlation between the response against total schizont antigens and PFHSP70 antigen. The non-responsiveness against PFHSP70 was not related to age, sex or regional variations. These qualities are undesirable for a vaccine candidate antigen.