An external sensing system in Plasmodium falciparum-infected erythrocytes
© Wu et al. 2016
Received: 4 December 2015
Accepted: 4 February 2016
Published: 19 February 2016
A number of experiments have previously indicated that Plasmodium falciparum-infected erythrocytes (pRBC) were able to sense host environment. The basis of this ability to detect external cues is not known but in screening signalling molecules from pRBC using commercial antibodies, a 34 kDa phosphorylated molecule that possesses such ability was identified.
The pRBC were exposed to different culture conditions and proteins were extracted for 1D or 2D gel electrophoresis followed by Western blot. The localization of 34 kDa protein was examined by biochemical fractionation followed by Western blot. High-resolution mass spectrometric analysis of immune precipitants was used to identify this protein and real-time quantitative reverse transcriptase polymerase chain reaction was used for detecting mRNA expression level.
The 34 kDa protein was called PfAB4 has immediate responses (dephosphorylation and rapid turnover) to host environmental stimuli such as serum depletion, osmolality change and cytokine addition. PfAB4 is expressed constitutively throughout the erythrocytic lifecycle with dominant expression in trophozoites 30 h post-infection. Tumour necrosis factor (TNF) treatment induced a transient detectable dephosphorylation of PfAB4 in the ItG strain (2 min after addition) and the level of expression and phosphorylation returned to normal within 1–2 h. PfAB4 localized dominantly in pRBC cytoplasm, with a transient shift to the nucleus under TNF stimulation as shown by biochemical fractionation. High-resolution mass spectrometric analysis of immune precipitants of AB4 antibodies revealed a 34 kDa PfAB4 component as a mixture of proliferating cellular nuclear antigen-1 (PCNA1) and exported protein-2 (EXP2), along with a small number of other inconsistently identified peptides. Different parasite strains have different PfAB4 expression levels, but no significant association between mRNA and PfAB4 levels was seen, indicating that the differences may be at the post-transcriptional, presumably phosphorylation, level. A triple serine phosphorylated PCNA1 peptide was identified from the PfAB4 high expression strain only, providing further evidence that the identity of PfAB4 is PCNA1 in P. falciparum.
A protein element in the human malaria parasite that responds to external cues, including the pro-inflammatory cytokine TNF have been discovered. Treatment results in a transient change in phosphorylation status of the response element, which also migrates from the parasite cytoplasm to the nucleus. The response element has been identified as PfPCNA1. This sensing response could be regulated by a parasite checkpoint system and be analogous to bacterial two-component signal transduction systems.
Protozoan parasites are capable of withstanding relatively large alterations in their external environments. Signalling is perhaps a primal requirement to respond to stimuli, the signal transduction systems being able to convert an extracellular stimulus into a chemical signal that the cell can sense/perceive and recognize, and can quickly give responses to the changing environment. The malaria parasite has invested in the machinery to transduce signals  and a number of experiments, such as those carried out by Cohen et al., have indicated that Plasmodium falciparum-infected erythrocytes (pRBC) may be able to sense host environment, where passive transfer of polyclonal sera or purified immune globulin from immune adults into P. falciparum-infected individuals resulted in a significant reduction in blood-stage parasitaemia and recovery from clinical symptoms, but also boosted gametocyte production . Plasmodium falciparum is considered a micro-aerophilic organism growing in an environment of limited oxygen content 0.5–5.0 %. The malaria parasite is under constant oxidative stress caused by exogenous reactive oxidant species and reactive nitrogen species (RNS) produced by the host immune system and generated by the parasite’s metabolism. Accordingly, parasites have already adapted to cope with the external oxidative stress by adapting to the host environment through superoxide dismutases and thioredoxin-dependent peroxidases, as part of an antioxidant defence system of the pRBC . Additionally, reversible phosphorylation has been shown to play an important role in parasite invasion  and intra-erythrocytic growth and development , through serine/threonine kinases [6–9].
pRBC are known to be able to activate several signalling pathways in the host: for example, MAPK and PI3 kinases/AKT pathways have been shown to be associated with malaria infection and pathogenesis via cytoadherence [10–12]. It was found that one of NF-kB family members REL-1A (P65), almost completely translocated into the nucleus within 10 min of pRBC-EC interaction, suggesting a direct role for parasite factors in NF-kB activation . Src-family signalling mediated ectophosphorylation of CD36 on endothelium has also been demonstrated, whereby treating HDMEC with a Src-family kinase-selective inhibitor PP1 resulted in a significant reduction of pRBC adhesion in a flow-chamber adhesion assay . However, all of these belong to the host responses to parasite infection, whereas there are only limited numbers of studies about signalling events in parasites themselves, despite the existence of an extensive kinase gene family. For example, signal transduction inside Plasmodium has been shown to be a major mechanism to control parasite development  with regulation in P. falciparum by calcium-dependent protein kinase 7 (PfCDPK7) being reported . PfCDPK1 has been identified as a Ca2+-dependent effector that plays a role in microneme secretion during erythrocyte invasion . Mutai and Waitumbi also suggested the existence of P. falciparum quorum sensing (the ability to detect conditions of overcrowding), which is more frequently seen in small-molecule signalling pathways in bacteria , to keep the parasite population under check . However, the signalling pathways controlling parasite growth and defence have not been well studied. Therefore, research on signalling molecules from the parasite were undertaken to understand their pathways and function in parasite growth, response and defence against the host immune system.
Using a panel of commercial antibodies to several signalling transduction pathways of different species, only one molecule from P. falciparum parasites with the appropriate molecular weight was identified. The antibody was against human phospho-I-kappaB-α (IκB), an important component in the NF kappa B (NFκB) pathway of mammals. The NFκB pathway is found in almost all animal cell types, although not in P. falciparum, and is involved in cellular responses to stimuli such as cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. It mediates stress and innate immunity and plays a key role in regulating the immune response to infection [20, 21].
Activation of NFκB is initiated by the signal-induced degradation of IκBα proteins (a negative feedback regulator). IĸBα functions rapidly and is primarily involved in determining the temporal profiles of NFκB signalling in response to cytokines that serve intercellular communication. Rapid IкBα turnover has been implicated in the high basal NFκB activity in WEHI 231 B immature IgM+ B cells , primarily via activation of the IκB kinase.
PfAB4 molecule identified in this investigation has several interesting features which could resemble the responses of IĸBα/NFκB in mammalian species. However, there are no NFκB and IκB homologues per se in P. falciparum, therefore, the identity of the PfAB4 antibody (phosphorylated IκBα antibody)-reacting epitope (called PfAB4 in this study) was one of the first questions. In this paper, the discovery of a sensing system in P. falciparum and an investigation into the identity of PfAB4 are reported.
Plasmodium falciparum isolates used in this study were mainly 3D7 , ItG  and Dd2 , as well as a number of patient isolates PO69, PCM-7, BC12, BC31, and GL-6 recently characterized in our laboratory . Parasites were cultured in vitro in group O+ human erythrocytes using previously described conditions [27, 28]. To minimize the effect of antigenic switching in culture, a batch of stabilates was prepared from a post-selection culture and used for no more than three weeks. Mycoplasma contamination of the parasite culture was checked (Universal mycoplasma detecting kit, ATCC, UK). pRBC were regularly synchronized by 5 % sorbitol treatment or Plasmion-gel flotation.
Sample preparation from infected erythrocytes and immunoblotting
To study the PfAB4 expression profile in the parasites, saponin was added to the parasite culture to a final concentration of 0.05 % and kept on ice for 8 min to lyse the erythrocytes. Following centrifugation at 5000×g at 4 °C for 10 min, erythrocyte ghosts were removed and the free parasite pellets were washed twice using RPMI 1640 without serum. The pellet was dissolved in SDS sample buffer (final: 3 % [w/v] SDS, 62 mM Tris–HCl pH 6.8, 15 % [v/v] glycerol) containing 5 % ß-mercaptoethanol), vortexed and concentrated for 5 min at 13,000 RPM to remove any insoluble material, and this was subjected to gel electrophoresis (used as total lysate). This part of pRBC was also further extracted by the fractionation method described by Voss et al.  with modifications. Briefly, free parasite pellets were disrupted with ice-cold lysis buffer (20 mM Hepes, pH 7.8, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.65 % Nonidet P-40) and incubated for 5 min on ice. Nuclei were pelleted at 2500×g for 5 min, the supernatants were used as parasite cytosol. The nuclear pellet was washed twice in lysis buffer, then re-suspended in 2× pellet volume of nuclear extraction buffer (20 mM Hepes, pH 7.8, 800 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) and put on ice for 30 min with vigorous shaking every 5 min. The extract was cleared by centrifugation at 13,000×g for 30 min. The supernatant was used as the parasite nuclear fraction. The insoluble pellet was further washed twice with nuclear extraction buffer, pelleted and used as parasite insoluble fraction. The protein quantity was controlled by using starting material with equal amounts of culture containing the same parasitaemia and haematocrit. Cross-contamination of the nuclear fraction with other fractions was investigated using a number of markers, for example, histone-nuclear localization and localization of other molecules, e.g., HSP90 and PGK should not be in nuclear fraction. All of these fractions were diluted with 2× SDS gel sample buffer. After boiling for 5 min, insoluble material was removed by centrifugation for 5 min at 13,000×g and the supernatants were run on Thermo Scientific Precise™ precast polyacrylamide gels for protein electrophoresis using Tris-HEPES running buffer. These fractions were also precipitated by acetone-methanol method  and were subjected to two-dimensional (2D) electrophoresis performed as described previously . Briefly, the lysate was solubilized in 2-DE rehydration buffer [8 M urea, 2 M thiourea, 2 % CHAPS, 65 mM dithiothreitol (DTT), and 0.5 % ampholyte pH 4–7 or 3–10]. The sample was vortexed and sonicated on ice ten times for 5 s followed by centrifugation at 15,000×g for 10 min. The supernatant was subjected to 2-DE and the isoelectric focusing (IEF) by running on precast Amersham 11 cm pH 3–10 immobiline Drystrip gels using IPG phor IEF Unit (Amersham). The narrow pH precast IPG-strips of pH 3.9–4.9 were from GENETIX. The running programme consists of 10 h for 30 V, 40 min for 200 V, 1 h for 500 V, 4 h for 2000 V and finally 8 h for 8000 V. The voltage was increased gradually until a total of 80,000 vh was reached. The focused strips were equilibrated in 10 ml equilibration solution (50 mM Tris–HCl, pH 6.8, 6 M urea, 30 % glycerol, 2 % SDS) with reducing agent of 1 % DTT for 10 min, and 10 ml equilibration solution with 4.5 % iodoacetamide for another 10 min. The strips were then briefly washed twice with 1× SDS gel running buffer and loaded on 10 or 12.5 % SDS-PAGE gels for second dimension separation. The gels were run at constant current 40 mA in a Laemmli’s buffer system  until the dye front reached the bottom of the gel. For Western blot analysis, the gel-separated proteins were transferred electrophoretically to nitrocellulose using glycine-tris-methanol buffer. The nitrocellulose membranes were blocked by 1-hour incubation in 5 % skim milk in TST buffer (0.01 M Tris pH 8.5/0.15 M sodium chloride/0.1 % Tween 20) and washed with TST buffer briefly, then probed with different primary antibodies: anti-phospho-IκB-α (Cell signalling); anti-EXP2 mAb 7.7 (kindly provided by Brendan S Crabb); Anti-HSP90α and HSP90β (Cell signalling); Anti-GAPDH (Enzo); Anti-calcineurin (Abgent); Anti-PGK1/2 (Santa Cruz). All primary antibodies were diluted in Calbiochem Signal Boost immunoreaction enhancer solution 1. Goat anti-rabbit or anti-mouse (as appropriate) IgG (H + L) horseradish peroxidase conjugate (Nordic, 1:2000) was used in enhancer solution 2 to localize antibody-antigen complexes and visualized with Pierce chemiluminescent (ECL2) systems (GE Healthcare).
RNA extraction and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Primer sequences for qRT-PCR analysis
Forward: 5′ AGTAGGTGATGCTGAAGTAGC 3′
Reverse: 5′ CCTAAGACAACGACATCTGCT 3′
Forward: 5′ AGGTGTGATAAAAATTGCGTTT 3′
Reverse: 5′ TCGTCAGCACTTGATTGTTC 3′
Forward: 5′ AGATGGTCACGTATGTGGTG 3′
Reverse: 5′TGGTTTGGATTCTACGGCAT 3′
Forward: 5′ ACCAAGAAGAGGAGAAGTTGG 3′
Reverse: 5′ GCTCTTTGTTCTTCTGCACT 3′
Forward: 5′ ACAGACCTTAGAAAATATCCCATCA 3′
Reverse: 5′ TGGTTTCCACCTATTTCAAGGG 3′
Immunoprecipitation (IP) and co-IP using AB4 antibody with Dynabeads®
This is a powerful technique to specifically pull down molecules reacting directly with antibodies (IP) and to indirectly capture proteins that are bound to a specific target protein (co-IP). 3D7-infected and uninfected erythrocytes (used as control) were disrupted by saponin lysis at 4 °C and washed with RPMI medium without serum. The parasite pellet was then disrupted with IP lysis buffer (50 mM Tris–HCl pH 7.4/150 mM NaCl/0.5 % NP-40/1.5 mM MgCl2) by vortexing and centrifuged at 13,000×g for 20 min at 4 °C. Supernatants were cleaned using pre-immune rabbit sera to remove any non-specific reaction and then incubated with primary antibody against PfAB4 diluted 1:200 for overnight under gentle rotation (15 rpm) at 4 °C. Dynabeads® protein A or G (Invitrogen) washed three times in cell lysis NP-40 buffer were added into the lysate-antibody mixture and incubated at room temperature for 2 h with gentle rotation. Beads containing antibody-antigen complexes were washed three times in NP-40 buffer using magnetic isolation and reacted proteins were eluted from beads by adding 2D gel-electrophoresis buffer, vortexed, centrifuged and subjected to 2D gel electrophoresis.
Protein in-gel digestion, phosphopeptide enrichment, nanoflow LC/MS/MS analysis and database searching
Spot picking of interesting proteins in 2D gels was guided by immunoblot images on duplicate gels. Immunoprecipitated samples were stained with Coomassie blue and the most abundant spots were picked. In-gel digestion was performed as described below: the excised protein spot was put into an Eppendorf Ultra-Pure 1.5 ml centrifuge tube. The band was then cut into 1-cubic mm cubes and rinsed twice in 200 µl MilliQ water for 15 min. The gel slices were dehydrated by the addition of 100 µl of 50 % (v/v) acetonitrile/water and incubated at room temperature for 10 min. One-hundred µl of ammonium bicarbonate (50 mM) was then added to each sample and incubated again at room temperature for 10 min. These last two steps were repeated. After removal of the ammonium bicarbonate, 10 µl of sequence grade trypsin (Promega Southampton, UK) (10 µg ml−1 in 50 mM ammonium bicarbonate) was then added to the gel fragments and incubated at 37 °C for 15 h (overnight), after which the supernatant was removed and kept. Twenty µl of 70 % acetonitrile (v/v in water) was added to the gel and incubated for 10 min at room temperature. The supernatant was then removed and pooled with the previous supernatant. The combined supernatant was dried in a speed-vac, re-suspended in 12 µl of 0.1 % formic acid.
The trypsin-digested peptide sample or phosphopeptides, enriched by Magnetic Titanium Dioxide Phosphopeptide Enrichment kit (Pierce) according to manufacturer’s instructions, was centrifuged at 13,000×g for 15 min to remove any insoluble material. The supernatant was placed in a new tube and diluted using 0.1 % (v/v) formic acid according to loading requirements. For LC-MSMS analysis: peptides were initially separated by reverse-phase chromatography using a DIONEX UltiMate™ 3000LC chromatography system. For MS proteomics, 10 µl of peptides were injected onto a C18 reverse-phase column [2 µm particle size (100), 75 µm diameter × 150 mm long] at nanoflow rate (0.3 µl min−1) and separated over linear chromatographic gradients. The gradients employed for chromatographic separation were composed of buffer A (2.5 % acetonitrile: 0.1 % formic acid) and buffer B (90 % acetonitrile: 0.1 % formic acid). For in-gel proteolysis we employed a 60-min linear chromatographic gradient. Following chromatographic separation, MS analysis was performed on an LTQ Orbitrap Velos mass spectrometer using Xcalibur (version 2.1) software (Thermo Scientific, UK). Ions were scanned between 350–2000 m/z in positive polarity mode. The ion-trap operated with CID MS/MS (with wide band activation) on the 20 most intense ions. Dynamic exclusion was enabled to avoid repeatedly selecting intense ions for fragmentation and this was set at 500 with an exclusion duration of 20.0 s. Charge states of 1 were rejected. The minimum MS signal threshold was set at 500 counts and the MS/MS default charge state was 2 with a 1.2 m/z isolation width, normalized CID at 35 V and an activation time of 10 min. The resulting MS/MS spectra were submitted to Proteome Discoverer (Thermo Scientific, UK) version 1.2. Searching was against NCBI P. falciparum and human databases separately and was performed using fixed carbamidomethyl and variable phosphorylation modifications. Peptide tolerance was set at 0.5 Da, MS/MS tolerance was set at 0.1 Da. Phosphorylated protein identities were considered significant if the protein score was over the 95 % confidence limit and at least one phosphorylated site was unambiguously identified when a phosphorylated residue existed (matched) y- or b-ions in the peak lists of the fragment ions [providing evidence of observed neutral loss of H3PO4 from the precursor or identified intact phosphorylated residues of serine (pS), threonine (pT) and tyrosine (pY)]. If the protein score reached a significant level but the ion score of phosphorylated peptide was under the 95 % confidence limit, these were referred to as potential-phosphorylated proteins.
Using ten commercial antibodies to five different signalling transduction pathways, only one positive molecule from P. falciparum parasites was identified with an anti-mammalian phospho-IĸBα antibody, with a molecular weight (34 kDa) (Additional file 1). This molecule was named PfAB4 and further characterization of PfAB4 in several P. falciparum laboratory parasite lines was undertaken.
Mass spectrometry identifications of AB4 immno-precipitated material
The low molecular weight 34kDa PF-proteins from IP with AB4 antibody
Exported protein 2
Proliferating cell nuclear antigen 1
DNA/RNA-binding protein α
Proliferating cell nuclear antigen 2
The high molecular weight 90kDa PF-proteins from IP with AB4 antibody
Heat shock protein 70
Myo-inositol 1-phosphate synthase, putative
Heat shock protein 86
Heat shock 70 kDa protein homologue
Kelch protein, putative
Vacuolar ATP synthase, catalytic subunit a
Erythrocyte membrane protein, putative
Mass spectrometry identifications from phospho-peptide enrichment
Dd2 before enrichment
Dd2 after enrichment
ItG before enrichment
ItG after enrichment
34 kDa/no treatment
Heat shock 70 kDa 187
78 kDa glucose-regulated protein
Elongation factor 1-alpha merozoite surface protein P12
Putative E3 ubiquitin-protein ligase protein
Heat shock 70 kDa protein
Proliferating cell nuclear antigen
Aspartic acid-rich prot
Elongation factor 1-alpha
Merozoite surface protein P12
Dynein heavy chain-like protein
Acidic leucine-rich nuclear phosphoprotein 32-related 40S ribosomal protein S3a
Proliferating cell nuclear antigen
Aspartic acid-rich protein
Aspartic acid-rich protein
Knob-associated histidine-rich protein
Reticulocyte-binding protein 3
Dynein heavy chain-like protein
Reticulocyte-binding protein 2
MATH and LRR domain-containing protein
Putative E3 ubiquitin-protein ligase protein
Knob-associated histidine-rich protein
101 kDa malaria antigen
Probable cathepsin C
Heat shock 70 kDa protein
78 kDa glucose-regulated protein homologue
Knob-associated histidine-rich protein
The intention of this work was to look for parasite signalling molecules and understand how malaria parasites might sense the host environment. To exist in a wide range of environmental niches, organisms must sense and respond to a variety of external signals.
A primary means by which external sensing occurs in bacteria is through two-component signal transduction pathways, typically composed of a sensor histidine kinase that receives the input stimuli and then phosphorylates a response regulator that effects an appropriate change in cellular physiology. The sensing system described in this paper could be part of a two-component signal transduction pathway in P. falciparum, which would be the first time such a system has been recognized and reported. The results indicate that PCNA1 is most likely a component of this sensing system. PCNA1 is an auxiliary protein of DNA polymerase-delta, forms a trimer ring around a DNA double-helix and is a member of the DNA sliding clamp family . It is absent or present in very low amounts in normal non-dividing cells and tissues, but synthesizes in variable amounts by proliferating cells [38, 39].
Phosphorylation/dephosphorylation, ubiquitination, sumoylation, and acetylation have all been described for nuclear PCNA and offer a range of options to modulate PCNA activities . Post-translational modification of cytosolic PCNA has been assumed as a key factor in neutrophil survival. Neutrophils express high levels of PCNA localized exclusively in cytosol, which is ubiquitinated and degraded via the proteasome during apoptosis. Furthermore, PCNA nuclear to cytoplasmic delocalization at the end of granulocyte differentiation has also been demonstrated [41, 42]. These post-translational modifications of PCNA may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis in order to maintain genomic stability. A recent study has shown that both Pf-PCNA1 and Pf-PCNA2 participate in an active DNA-damage-response pathway with significant accumulation in the parasite upon DNA damage induction, but Pf-PCNA-mediated regulation was not at the level of transcription, but presumably at the protein stability level . It has been suggested by this study that one feature of regulation is via post-translational modification, and that phosphorylation/dephosphorylation controls some functions of Pf-PCNA1.
Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that plays a critical role in diverse cellular events, including cell proliferation, differentiation, and apoptosis. Following stimuli by pro-inflammatory cytokines such as TNF and interleukin-1, IkBs are phosphorylated by IkB kinase (IKK) in mammalian species resulting in their rapid, proteasome-dependent degradation [44, 45]. IkBs are regulatory proteins that inhibit NFkB by complexing with and trapping it in the cytoplasm. They are involved in events including cell adhesion, immune and pro-inflammatory responses, apoptosis, differentiation, and growth. Although this machinery is not thought to exist in Plasmodium parasites, an analogous system may have evolved to detect this important host protein. Signal transduction regulation inside Plasmodium has been shown as a major mechanism to control parasite development . In the mammalian host, studies have shown that TNF induces extensive alterations in host microvascular endothelium, including morphological re-organization , release of membrane microparticles , production of other pro-inflammatory cytokines, up-regulation of receptors and apoptosis [48, 49]. Interestingly, inhibitors of TNF production reduced infected erythrocyte cytoadherence [50, 51]. However, TNF signalling in the Plasmodium parasite itself has not been previously reported, therefore, understanding the mechanisms of sensing TNF stimulation seen in this study could improve the molecular description of how Plasmodium adapts to its host environment.
In a parallel study looking for TNF effects on parasite calcium-dependent signalling, the work showed the ability of the parasite to increase intracellular calcium concentration after exposure to TNF, thus corroborating the hypothesis of an external sensing system allowing the parasite to sense the environment (Cruz et al., unpublished data). This behaviour is also linked to the parasite synchronicity to host circadian rhythms  being previously shown that the parasite cell cycle can be modulated by external signals such as melatonin and products of tryptophan catabolism, leading to intracellular calcium increase thought a PLC-IP3 mechanism [53–55]. Plasmodium is also able to sense external ATP through rise in cytosolic calcium [15, 56]. Moreover, evidence has also been provided for K−/Ca2+ signalling mechanisms in P. falciparum .
The pRBC ghosts contain haemoglobin-depleted erythrocyte material that has lost most of its internal proteins, therefore, this fraction represents an enriched erythrocyte membrane compartment containing molecules attached/associated with the membrane. Parasite-encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurer’s clefts), play a key role in the asexual lifecycle. How might all these membrane proteins fit together in the parasite’s ‘sensing apparatus’? Many studies have reported that heat shock proteins HSP70 and HSP90 regulate a number of signalling cascades to maintain cellular homeostasis several species [58, 59]. Recent studies have revealed that HSP70 and HSP90 proteins regulate the function of the IKK complex, which is the major activator of the NF-κB complex . It has been shown that the histidine kinase auto-phosphorylates in the presence of an intra-or extra-cellular environment stimulus or stress, which could be one of the triggers. HSP90 plays an important role in TNF-mediated NF-kB activation by modulating the stability and solubility of receptor interacting protein (RIP) . In this study, antibody AB4 recognized a 90 kDa protein consistent with being HSP90, which was supported by Western blot with an anti-HSP90 antibody (Additional file 4). This was also confirmed by co-immunoprecipitation followed by mass spectrum. HSP90 inhibitor Gambogic acid not only inhibited the expression of this 90 kDa protein, but also abrogated the effects of TNF on PfAB4. This suggests that PfPCNA1 may be one of the HSP90 client proteins, which may explain its identification by AB4 immuno-precipitation and co-migration in the gel electrophoresis. Such an association has been reported previously in several cancer cell lines . The proteins in the insoluble fraction are probably cell membrane components or have high affinity binding to the cytoskeleton, and these potentially could be the ‘perception proteins’ of pRBC. Although data are incomplete they suggest, for the first time, a link between PCNA1 and HSP90 in a parasite sensing system.
An attempt was made to investigate the outcome of the TNF regulatory mechanism of P. falciparum parasite strains by analysing transcriptomes of the 3D7 and Dd2 strains (representing high and low PfAB4 phenotypes) with and without TNF treatment by RNAseq analysis. A remarkable conservation of the normal transcriptional programme between these strains has previously been observed . The results showed no transcriptional differences associated with TNF treatment. This suggests that post-transcriptional mechanisms may drive the signalling processes and changes to the global transcription pattern in the parasite may only occur in subsequent cycles, which was not investigated. Indeed it is tempting to speculate that changes in var gene expression might be linked to the TNF status in the host to align with altered host adhesion profiles, and might not be detected until one or more cycles after exposure of pRBC to this cytokine.
The cell cycle checkpoints are signal transduction pathways that respond to damaged DNA by inhibiting cell cycle progression . The post-translational modifications of PCNA are considered to be the most relevant during the S phase cell cycle checkpoint (surveillance system) . PCNA interacts with several eukaryotic cell cycle proteins, binds to cyclin-CDK complexes  and CDK inhibitor p21 [67, 68]. PCNA forms complexes with critical checkpoint proteins, transducing both positive and negative signals . It is possible that cell cycle checkpoints may serve as switch points for choosing between cell proliferation and apoptosis, or even have a broader role in parasite replication. The ability to sense the external environment and, for example, move a proportion of the population into a quiescent phase could be beneficial to the parasite in avoiding killing by drugs and a protein such as PfPCNA1 could play a significant role in this.
This study reported a novel sensing system and some potential regulation mechanisms by which parasites respond to external stimuli including inflammatory cytokines, such as TNF. The sensing and regulating system/rapid responding system in P. falciparum described here might be an equivalent of the two-component regulatory system of bacterial signal transduction. HSP90 could be a component of a membrane-bound histidine kinase that receives the input stimuli from as yet uncharacterized external receptors, and reversible phosphorylated PfPCNA1 could be a response regulator that effects an appropriate change in cellular physiology from this event. This sensing system might also be shared with cell cycle checkpoints, the stimuli which triggered PCNA1 dephosphorylation/degradation may also be implicated in DNA replication stress and damage. Further work is needed to identify the sensor proteins necessary for interacting with specific external signals and the outcomes for the parasite from this system.
YW, CRSG and AGC designed the study. YW, LNC and TS conducted parasite culture, experimental and laboratory work. YW, GL and GRM conducted mass spectrometric analysis and Bioinformatics. YW, LNC, CRSG, and AGC analysed the data and wrote the manuscript. All authors contributed to the manuscript. All authors read and approved the final manuscript.
This work was supported by The Wellcome Trust (grant reference 095507) and by grants and funding from Fundação de Amparo à Pesquisa (FAPESP process number 2011/51295-5). LNC received a FAPESP Fellowship.
The authors declared that they have no competing interests.
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- Doerig C, Rayner JC, Scherf A, Tobin AB. Post-translational protein modifications in malaria parasites. Nat Rev Microbiol. 2015;13:160–72.View ArticlePubMedGoogle Scholar
- Cohen S, McGregor IA, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961;192:733–7.View ArticlePubMedGoogle Scholar
- Becker K, Tilley L, Vennerstrom JL, Roberts D, Rogerson S, Ginsburg H. Oxidative stress in malaria parasite-infected erythrocytes: host-parasite interactions. Int J Parasitol. 2004;34:163–89.View ArticlePubMedGoogle Scholar
- Rangachari K, Dluzewski A, Wilson RJ, Gratzer WB. Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton. Nature. 1986;324:364–5.View ArticlePubMedGoogle Scholar
- Yuthavong Y, Limpaiboon T. The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium berghei-infected mouse erythrocytes. Biochim Biophys Acta. 1987;929:278–87.View ArticlePubMedGoogle Scholar
- Doerig C, Horrocks P, Coyle J, Carlton J, Sultan A, Arnot D, et al. Pfcrk-1, a developmentally regulated cdc2-related protein kinase of Plasmodium falciparum. Mol Biochem Parasitol. 1995;70:167–74.View ArticlePubMedGoogle Scholar
- Bracchi V, Langsley G, Thelu J, Eling W, Ambroise-Thomas P. PfKIN, an SNF1 type protein kinase of Plasmodium falciparum predominantly expressed in gametocytes. Mol Biochem Parasitol. 1996;76:299–303.View ArticlePubMedGoogle Scholar
- Li JL, Robson KJ, Chen JL, Targett GA, Baker DA. Pfmrk, a MO15-related protein kinase from Plasmodium falciparum. Gene cloning, sequence, stage-specific expression and chromosome localization. Eur J Biochem. 1996;241:805–13.View ArticlePubMedGoogle Scholar
- Lin DT, Goldman ND, Syin C. Stage-specific expression of a Plasmodium falciparum protein related to the eukaryotic mitogen-activated protein kinases. Mol Biochem Parasitol. 1996;78:67–77.View ArticlePubMedGoogle Scholar
- Wu Y, Szestak T, Stins M, Craig AG. Amplification of P. falciparum cytoadherence through induction of a pro-adhesive state in host endothelium. PLoS ONE. 2011;6:e24784.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu Y, Nelson MM, Quaile A, Xia D, Wastling JM, Craig A. Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum. Malar J. 2009;8:105.PubMed CentralView ArticlePubMedGoogle Scholar
- Jenkins N, Wu Y, Chakravorty S, Kai O, Marsh K, Craig A. Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells. J Infect Dis. 2007;196:321–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Tripathi AK, Sha W, Shulaev V, Stins MF, Sullivan DJ Jr. Plasmodium falciparum-infected erythrocytes induce NF-kappaB regulated inflammatory pathways in human cerebral endothelium. Blood. 2009;114:4243–52.PubMed CentralView ArticlePubMedGoogle Scholar
- Yipp BG, Robbins SM, Resek ME, Baruch DI, Looareesuwan S, Ho M. Src-family kinase signaling modulates the adhesion of Plasmodium falciparum on human microvascular endothelium under flow. Blood. 2003;101:2850–7.View ArticlePubMedGoogle Scholar
- Cruz LN, Wu Y, Craig AG, Garcia CR. Signal transduction in Plasmodium-red blood cells interactions and in cytoadherence. An Acad Bras Cienc. 2012;84:555–72.View ArticlePubMedGoogle Scholar
- Kumar P, Tripathi A, Ranjan R, Halbert J, Gilberger T, Doerig C, et al. Regulation of Plasmodium falciparum development by calcium-dependent protein kinase 7 (PfCDPK7). J Biol Chem. 2014;289:20386–95.PubMed CentralView ArticlePubMedGoogle Scholar
- Bansal A, Singh S, More KR, Hans D, Nangalia K, Yogavel M, et al. Characterization of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) and its role in microneme secretion during erythrocyte invasion. J Biol Chem. 2013;288:1590–602.PubMed CentralView ArticlePubMedGoogle Scholar
- Camilli A, Bassler BL. Bacterial small-molecule signaling pathways. Science. 2006;311:1113–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Mutai BK, Waitumbi JN. Apoptosis stalks Plasmodium falciparum maintained in continuous culture condition. Malar J. 2010;9(Suppl 3):S6.PubMed CentralView ArticlePubMedGoogle Scholar
- Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 1999;18:6853–66.View ArticlePubMedGoogle Scholar
- Tergaonkar V. NFkappaB pathway: a good signaling paradigm and therapeutic target. Int J Biochem Cell Biol. 2006;38:1647–53.View ArticlePubMedGoogle Scholar
- Shen J, Channavajhala P, Seldin DC, Sonenshein GE. Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha. J Immunol. 2001;167:4919–25.View ArticlePubMedGoogle Scholar
- Walliker D, Quakyi IA, Wellems TE, McCutchan TF, Szarfman A, London WT, et al. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science. 1987;236:1661–6.View ArticlePubMedGoogle Scholar
- Ockenhouse CF, Betageri R, Springer TA, Staunton DE. Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus. Cell. 1992;68:63–9.View ArticlePubMedGoogle Scholar
- Wellems TE, Panton LJ, Gluzman IY, do Rosario VE, Gwadz RW, Walker-Jonah A, et al. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature. 1990;345:253–5.View ArticlePubMedGoogle Scholar
- Madkhali AM, Alkurbi MO, Szestak T, Bengtsson A, Patil PR, Wu Y, et al. An analysis of the binding characteristics of a panel of recently selected ICAM-1 binding Plasmodium falciparum patient isolates. PLoS One. 2014;9:e111518.PubMed CentralView ArticlePubMedGoogle Scholar
- Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193:673–5.View ArticlePubMedGoogle Scholar
- Dolan SA, Miller LH, Wellems TE. Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum. J Clin Invest. 1990;86:618–24.PubMed CentralView ArticlePubMedGoogle Scholar
- Voss TS, Mini T, Jenoe P, Beck HP. Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript. J Biol Chem. 2002;277:17493–501.View ArticlePubMedGoogle Scholar
- Yuan X, Russell T, Wood G, Desiderio DM. Analysis of the human lumbar cerebrospinal fluid proteome. Electrophoresis. 2002;23:1185–96.View ArticlePubMedGoogle Scholar
- Nirmalan N, Sims PF, Hyde JE. Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition. Mol Microbiol. 2004;52:1187–99.View ArticlePubMedGoogle Scholar
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.View ArticlePubMedGoogle Scholar
- Viebig NK, Levin E, Dechavanne S, Rogerson SJ, Gysin J, Smith JD, et al. Disruption of var2csa gene impairs placental malaria associated adhesion phenotype. PLoS One. 2007;2:e910.PubMed CentralView ArticlePubMedGoogle Scholar
- Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science. 2002;298:1241–5.View ArticlePubMedGoogle Scholar
- Maniatis T. A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev. 1999;13:505–10.View ArticlePubMedGoogle Scholar
- Zhang L, Yi Y, Chen J, Sun Y, Guo Q, Zheng Z, Song S. Gambogic acid inhibits Hsp90 and deregulates TNF-alpha/NF-kappaB in HeLa cells. Biochem Biophys Res Commun. 2010;403:282–7.View ArticlePubMedGoogle Scholar
- Bravo R, Frank R, Blundell PA, Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987;326:515–7.View ArticlePubMedGoogle Scholar
- Celis JE, Madsen P, Celis A, Nielsen HV, Gesser B. Cyclin (PCNA, auxiliary protein of DNA polymerase delta) is a central component of the pathway(s) leading to DNA replication and cell division. FEBS Lett. 1987;220:1–7.View ArticlePubMedGoogle Scholar
- Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell. 2007;129:665–79.View ArticlePubMedGoogle Scholar
- Naryzhny SN, Lee H. The post-translational modifications of proliferating cell nuclear antigen: acetylation, not phosphorylation, plays an important role in the regulation of its function. J Biol Chem. 2004;279:20194–9.View ArticlePubMedGoogle Scholar
- Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA, Candalh C, et al. Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival. J Exp Med. 2010;207:2631–45.PubMed CentralView ArticlePubMedGoogle Scholar
- De Chiara A, Pederzoli-Ribeil M, Mocek J, Candalh C, Mayeux P, Millet A, et al. Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer. J Leukoc Biol. 2013;94:723–31.View ArticlePubMedGoogle Scholar
- Mitra P, Banu K, Deshmukh AS, Subbarao N, Dhar SK. Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response. Biochem J. 2015;470:115–29.View ArticlePubMedGoogle Scholar
- Traenckner EB, Pahl HL, Henkel T, Schmidt KN, Wilk S, Baeuerle PA. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J. 1995;14:2876–83.PubMed CentralPubMedGoogle Scholar
- Webster MK, Goya L, Firestone GL. Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase. J Biol Chem. 1993;268:11482–5.PubMedGoogle Scholar
- Pober JS, Cotran RS. Cytokines and endothelial cell biology. Physiol Rev. 1990;70:427–51.PubMedGoogle Scholar
- Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood. 1997;89:1121–32.PubMedGoogle Scholar
- Gnant MF, Turner EM, Alexander HR Jr. Effects of hyperthermia and tumour necrosis factor on inflammatory cytokine secretion and procoagulant activity in endothelial cells. Cytokine. 2000;12:339–47.View ArticlePubMedGoogle Scholar
- Kimura H, Gules I, Meguro T, Zhang JH. Cytotoxicity of cytokines in cerebral microvascular endothelial cell. Brain Res. 2003;990:148–56.View ArticlePubMedGoogle Scholar
- Wassmer SC, Cianciolo GJ, Combes V, Grau GE. LMP-420, a new therapeutic approach for cerebral malaria? Med Sci (Paris). 2006;22:343–5 (in French).View ArticleGoogle Scholar
- Chakravorty SJ, Hughes KR, Craig AG. Host response to cytoadherence in Plasmodium falciparum. Biochem Soc Trans. 2008;36:221–8.View ArticlePubMedGoogle Scholar
- Hotta CT, Gazarini ML, Beraldo FH, Varotti FP, Lopes C, Markus RP, et al. Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites. Nat Cell Biol. 2000;2:466–8.View ArticlePubMedGoogle Scholar
- Budu A, Peres R, Bueno VB, Catalani LH, Garcia CR. N1-acetyl-N2-formyl-5-methoxykynuramine modulates the cell cycle of malaria parasites. J Pineal Res. 2007;42:261–6.View ArticlePubMedGoogle Scholar
- Alves E, Bartlett PJ, Garcia CR, Thomas AP. Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells. J Biol Chem. 2011;286:5905–12.PubMed CentralView ArticlePubMedGoogle Scholar
- Beraldo FH, Mikoshiba K, Garcia CR. Human malarial parasite, Plasmodium falciparum, displays capacitative calcium entry: 2-aminoethyl diphenylborinate blocks the signal transduction pathway of melatonin action on the P. falciparum cell cycle. J Pineal Res. 2007;43:360–4.View ArticlePubMedGoogle Scholar
- Levano-Garcia J, Dluzewski AR, Markus RP, Garcia CR. Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells. Purinergic Signal. 2010;6:365–72.PubMed CentralView ArticlePubMedGoogle Scholar
- Singh S, More KR, Chitnis CE. Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion. Cell Microbiol. 2014;16:50–63.View ArticlePubMedGoogle Scholar
- Citri A, Harari D, Shohat G, Ramakrishnan P, Gan J, Lavi S, et al. Hsp90 recognizes a common surface on client kinases. J Biol Chem. 2006;281:14361–9.View ArticlePubMedGoogle Scholar
- Chen Y, Voegeli TS, Liu PP, Noble EG, Currie RW. Heat shock paradox and a new role of heat shock proteins and their receptors as anti-inflammation targets. Inflamm Allergy Drug Targets. 2007;6:91–100.View ArticlePubMedGoogle Scholar
- Salminen A, Paimela T, Suuronen T, Kaarniranta K. Innate immunity meets with cellular stress at the IKK complex: regulation of the IKK complex by HSP70 and HSP90. Immunol Lett. 2008;117:9–15.View ArticlePubMedGoogle Scholar
- Lewis J, Devin A, Miller A, Lin Y, Rodriguez Y, Neckers L, et al. Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation. J Biol Chem. 2000;275:10519–26.View ArticlePubMedGoogle Scholar
- Wang X, Heuvelman DM, Carroll JA, Dufield DR, Masferrer JL. Geldanamycin-induced PCNA degradation in isolated Hsp90 complex from cancer cells. Cancer Invest. 2010;28:635–41.View ArticlePubMedGoogle Scholar
- Llinas M, Bozdech Z, Wong ED, Adai AT, DeRisi JL. Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains. Nucleic Acids Res. 2006;34:1166–73.PubMed CentralView ArticlePubMedGoogle Scholar
- Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature. 2004;432:316–23.View ArticlePubMedGoogle Scholar
- Zhu Q, Chang Y, Yang J, Wei Q. Post-translational modifications of proliferating cell nuclear antigen: a key signal integrator for DNA damage response (Review). Oncol Lett. 2014;7:1363–9.PubMed CentralPubMedGoogle Scholar
- Xiong Y, Zhang H, Beach D. D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell. 1992;71:505–14.View ArticlePubMedGoogle Scholar
- Gulbis JM, Kelman Z, Hurwitz J, O’Donnell M, Kuriyan J. Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell. 1996;87:297–306.View ArticlePubMedGoogle Scholar
- Knibiehler M, Goubin F, Escalas N, Jonsson ZO, Mazarguil H, Hubscher U, et al. Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance. FEBS Lett. 1996;391:66–70.View ArticlePubMedGoogle Scholar
- Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci. 2003;116:3051–60.View ArticlePubMedGoogle Scholar