Plasmodium vivax inhibits erythroid cell growth through altered phosphorylation of the cytoskeletal protein ezrin
Malaria Journal volume 14, Article number: 138 (2015)
The underlying causes of severe malarial anaemia are multifactorial. In previously reports, Plasmodium vivax was found to be able to directly inhibited erythroid cell proliferation and differentiation. The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and remain unclear. In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development.
This study describes the first comparative phosphoproteome analysis of growing erythroid cells (gECs), derived from human haematopoietic stem cells, exposed to lysates of infected erythrocytes (IE)/uninfected erythrocytes (UE) for 24, 48 and 72 h. This study utilized IMAC phosphoprotein isolation directly coupled with LC MS/MS analysis.
Lysed IE significantly inhibited gEC growth at 48 and 72 h and cell division resulting in the accumulation of cells in G0 phase. The relative levels of forty four phosphoproteins were determined from gECs exposed to IE/UE for 24-72 h and compared with the media control using the label-free quantitation technique. Interestingly, the levels of three phosphoproteins: ezrin, alpha actinin-1, and Rho kinase were significantly (p < 0.05) altered. These proteins display interactions and are involved in the regulation of the cellular cytoskeleton. Particularly affected was ezrin (phosphorylated at Thr567), which is normally localized to gEC cell extension peripheral processes. Following exposure to IE, for 48-72 h, the ezrin signal intensity was weak or absent. This result suggests that phospho-ezrin is important for actin cytoskeleton regulation during erythroid cell growth and division.
These findings suggest that parasite proteins are able to inhibit erythroid cell growth by down-regulation of ezrin phosphorylation, leading to ineffective erythropoiesis ultimately resulting in severe malarial anaemia. A better understanding of the mechanisms of ineffective erythropoiesis may be beneficial in the development of therapeutic strategies to prevent severe malarial anaemia.
Plasmodium vivax is a risk factor for severe anaemia, among patients in vivax-endemic areas [1-7]. Increasing evidence has established an association between vivax malaria, severe anemia, and death [8-16]. The pathogenesis of severe anaemia in vivax-malaria remains unclear and is likely caused by multiple underlying factors. These include the destruction of parasitized erythrocytes, ineffective erythropoiesis or dyserythropoiesis, and immunity associated with disease. Evidence for dyserythropoiesis, pancytopenia and degradation of erythroblasts was found in bone marrow from patients infected with P. vivax parasites [17-21]. Moreover, in vitro cultures of erythroid cells derived from haematopoietic stem cells has demonstrated that P. vivax is able to directly inhibit erythroid cell proliferation and differentiation . The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and poorly understood.
The phosphoproteome strategy is alternative proteomic method that allows investigation into the molecular mechanisms of signal transduction pathways . The separation and enrichment of phosphoproteins utilizes metal ion or TiO2 embedded columns prior to the identification and determination of phosphoproteins under liquid chromatography–mass spectrometry (LC-MS) based techniques [24,25]. Many molecular pathways in eukaryotic cells are modulated by specific signaling proteins that are controlled, by phosphorylation and dephosphorylation, through the activity of kinase and phosphatase enzymes. This post-translational control of eukaryotic cellular machinery is a hallmark of pathways that respond to different stimuli. The level of protein phosphorylation at specific sites varies from less than 1% to greater than 90%, depending on conditions . The regulation of complex and dynamic signal transduction proteins contributes to the destination of targeting proteins and the signal transduction of cell growth, and exposure to parasites can also influence signaling pathways. This occurs through specific modulation of regulatory proteins during the host-pathogen interaction, especially proteins with roles in pathogenesis . The specific mechanism involved in the suppression of erythroid development by P. vivax has not been elucidated. However, it is known that during parasite exposure, suppressed erythroid development is a key aspect in the pathophysiology of anaemia. Here, this study describes the first comparative phosphoproteome of erythroid cells, derived from human haematopoietic stem cells, exposed to proteins of P. vivax. This analysis utilized IMAC phosphoprotein isolation, directly coupled with LC MS/MS analysis. Interestingly, the phosphoprotein ezrin, involved in the regulation of cytoskeleton during cell development, was identified using this procedure. Ezrin, a member of the ezrin, radixin, and moesin (ERM) subfamily of cytoskeletal proteins and is conserved both functionally and structurally in mammalian cells . Through the regulation of the cytoskeleton ezrin has a role in several cellular processes, such as maintenance of survival, cytokinesis, adhesion, membrane dynamics, motility, and integration of membrane transport with signaling pathways . Usually, ezrin is present in an inactive conformation and functions as a crosslinker between the plasma membrane and cytoskeleton . Two regions of ezrin, the N-terminal and C-terminal domains are involved in the active conformation. The N-terminal domain binds to phosphatidylinositol 4,5-biphosphate, which facilitates phosphorylations of threonine 567 (Thr567) in the C-terminal domain . Rho kinase and protein kinase C (PKC) are capable of phosphorylating the C-terminal threonine in ezrin, promoting the formation of the F-actin binding site in active conformation [31-35]. This study has identified the involvement of the cytoskeleton protein ezrin in the inhibitory effect of P. vivax on erythroid cell growth, leading to ineffective erythropoiesis. The molecular mechanism characterized in this study, relevant to ineffective erythropoiesis, should have utility in the development of therapeutic strategies for severe malarial anaemia.
Plasmodium vivax parasites from patient blood with 0.05-0.2% parasitaemia, as determined by examining thick and thin blood smears, were collected. The ethical and methodological aspects of this study for parasite collection from patients attending the malaria clinic in Tha Sae, Chumpon Province, Thailand (MU-IRB 2012/170.2511) have been approved by the Mahidol University Institutional Review Board, Mahidol University, Bangkok, Thailand. Infected erythrocytes (IE) were separated from patient blood using a 60% Percoll solution as previously described . Briefly, 20 ml of whole blood from patients were collected and passed through a Plasmodipur filter (Euro-Diagnostic B.V., Netherlands) to remove white blood cells. To separate IE, patient blood, after filtration, was diluted 1:20 with RPMI1640 (Invitrogen®, CA, USA), layered on 60% Percoll, and centrifuged at 1,200 g for 20 min at 20°C. The purity of IE after isolation was 95%, containing 80% schizonts and 20% other stages. The isolated IEs were used as lysed cells prepared by freezing and thawing as previously described .
Isolation of cord blood CD 34+ cells and culture conditions
Haematopoietic stem cells (HSCs)/CD 34+ cells were isolated from normal human umbilical cord blood as previously described . Cord blood collected from normal full-term deliveries at Ramathibodi Hospital (ID 04-52-39) was approved by the Ethical Committee of Research on Human Beings of the Ramathibodi Hospital, Faculty of Medicine, Mahidol University. Briefly, HSCs/CD34+ cells from cord blood were isolated using a CD 34 isolation kit with magnetic microbead Mini-MACS columns (Miltenyi Biotech, Geramany). The purity of CD34+ cells after isolation was 97% as determined by flow cytometry analysis.
Growing erythroid cells (gECs), derived from HSCs/CD34+ cells, at a density of 5 x 105 cell/well in 24-well tissue culture plates (Corning Incorporated Costar®, NY, USA) were cultured in 1 ml of complete medium containing Stemline II medium (Sigma-Aldrich Corporation, Missouri, USA) supplemented with cytokines as previously described . Lysates of IE or uninfected erythrocytes (UE) from normal donor blood were added to gEC cultures, at a ratio of 1:10 (gEC:IE/UE), on day 5 and cultured at 37°C in 5% CO2 for 24, 48, 72 h. Viable cells were determined by trypan blue dye exclusion and dead cells were stained with 2 μg/ml propidium iodide (eBioscience) and analysed by flow cytometry.
Cell cycle analysis
gECs exposed to IE/UE for 48 and 72 h were washed with PBS and fixed with 70% cold ethanol overnight at -20°C. After fixed cells were washed with PBS, cells were added with 5 μg/ml RNase A (Geneaid Biotech Ltd., Taiwan), stained with 20 μg/ml propidium iodide (eBioscience) and incubated for 30 min at 37°C in dark. DNA analysis was performed on a FACS Canto™ flow cytometer (Becton Dickinson). Cell cycle analysed by BD FACSDiva software version 4.1 (Becton Dickinson) was used to determine the percentages of cells in the different cell cycle phases. Experiments were performed five independent times and values are shown as means ± S.D.
Preparation of phosphoprotiens
Phosphoproteins were prepared from gECs, after exposure to IE/UE lysates, using Pierce Phosphoprotein enrichment kit (Thermo Scientific, Pierce Biotechnology, IL, USA) followed the manufacturer’s instructions. Briefly, 5 x 106 cells were added to 200 μl of lysis/binding/wash buffer with CHAPS and 1X phosphatase inhibitors (phosphatase inhibitor cocktail 100X, Roche, UK) and lysed on ice by sonication (Sonics Vibra cell,sonics & materials INC., USA) with amplitude 70% utilizing 15 cycles of 2 sec disruption followed by 2 sec on ice. Following centrifugation (Tomy MX-305 high speed refrigerated microcentrifuge, CA, USA), 10,000 g at 4°C for 10 min, supernatants from cell lysates were collected and proteins concentrations were determined by Lowry method .
For phosphoprotein enrichment, cell lysates were adjusted to a concentration to 0.5 mg/ml in lysis/binding/wash buffer without CHAPS and applied to the column. Columns were inverted to mix for 30 min at 4°C and then washed three times with lysis/binding/wash buffer with CHAPS using centrifugation (Allegra X-22R centrifuge, Beckman Coulter, Inc, USA) at 1,000 g for 1 min at 4°C. One millimetre of elution buffer, consisting of 75 mM sodium phosphate, 500 mM sodium chloride, pH 7.5, was added to each column and then incubated at room temperature, with agitation for 3 min. The eluted proteins were collected by centrifugation at 1,000 g for 1 min at 4°C, and this step was repeated four times. The pool of eluted proteins was concentrated using a Pierce concentrator (Thermo Scientific). Briefly, 4 ml of each sample of eluted proteins was placed into the upper chamber of the concentrator followed by centrifugation at 7,000 g for 30 min at 4°C. The concentrated proteins, approximately 100-200 μl, were collected from the upper chamber and stored at -80°C. In the final step, salts and other molecules (<1,000 Da) were removed using Zeba™ spin desalting columns (Thermo Scientific). One hundred microlitres of each sample was applied to columns and then centrifuged at 700 g for 30 sec. The desalted samples were collected and the proteins content of each sample was determined by the Lowry method and used for phosphoprotein analysis.
After desalting, 4 μg of phosphoproteins in 10 mM ammonium bicarbonate were reduced with 10 mM DTT (Dithiotheitol) for 30 min at 60°C, alkylated with 15 mM IAA (iodoacetamide) at room temperature for 30 min, and digested with sequencing grade trypsin (Promega, Geramany) for 16 h at 37°C. Tryptic phosphopeptides were diluted with 0.1% formic acid to final concentration of 0.25 μg/μl and centrifuged 10,000 rpm at room temperature for 10 min. Phosphopeptide samples were injected into a NanoAcquity system (Waters Corp., Milford, MA) equipped with a Symmetry C18 5 μm, 180-μm x 20-mm Trap column and a BEH130 C18 1.7 μm, 100-μm x 100-mm analytical reversed phase column (Waters Corp., Milford, MA). The samples were initially transferred with an aqueous 0.1% formic acid solution (mobile phase A) to the trap column with a flow rate of 15 μl/min for 1 min. The peptides were separated with a gradient of 15-50% mobile phase B in acetonitrile with 0.1% formic acid over 15 min at a flow rate of 600 nl/min followed by a 3 min rinse with 80% of mobile phase B. The column temperature was maintained at 35°C. The lock mass was delivered from the auxiliary pump of the NanoAcquity system with a constant flow rate of 500 nl/min with 200 fmol/μl of [Glu1] fibrinopeptide B delivered to the reference sprayer of the NanoLockSpray source of the mass spectrometer. All samples of tryptic peptides were analysed by using a SYNAPT™ HDMS mass spectrometer (Waters Corp., Manchester, UK), which was operated in the V-mode of analysis with a resolution of at least 10,000 full-width half-maximum, using positive nanoelectrospray ion mode. The time-of-flight analyzer of the mass spectrometer was externally calibrated with [Glu1] fibrinopeptide B from m/z 50 to 1600 with acquisition lock mass corrected using the monoisotopic mass of the doubly charged precursor of [Glu1] fibrinopeptide B. The reference sprayer was sampled with every 20 sec. Accurate mass LC-MS data were acquired with data direct acquisition mode. The energy trap was set at the collision energy of 6 V. In the transfer collision energy control, low energy was set at 4 V. The quadrupole mass analyzer was adjusted such that ions from m/z 300 to 1800 were efficiently transmitted. The MS\MS survey was over range from 50 to 1990 Da and scan time was 0.5 sec.
The differential quantitation of proteins and peptides based on the MS signal intensities of individual LC-MS samples was analysed using DeCyder MS differential analysis software (DeCyderMS, GE Healthcare) [38,39]. Acquired LC-MS raw data were converted and the PepDetect module was used for automated peptide detection, charge state assignments, and quantitation based on the peptide ions signal intensities in MS mode. The analysed MS/MS data from DeCyderMS were submitted for database search using the Mascot software (Matrix Science, London, UK) . For protein identification, data was compared against the NCBI Homo sapiens database with the following parameters: enzyme (trypsin); variable modifications (carbamidomethyl, oxidation of methionine residues); mass values (monoisotopic); protein mass (unrestricted); peptide mass tolerance (1.2 Da); fragment mass tolerance (±0.6 Da), peptide charge state (1+, 2+ and 3+) and max missed cleavages.
All MS/MS data were compared against the human protein set using Swissprot protein database. The heatmap visualization was constructed using the web-based analysis MEV program  and the protein cluster was analysed by distance metric selection with Pearson correlation parameters calculate K-Means. Different levels of phosphoproteins were analysed using the significant t-test at P-value ≤ 0.05. The gene ontology analysis was performed with UniprotKB (protein knowledgebase)  and PANTHER (classification system)  databases for biological processes, molecular function and classification. The mapping of protein-protein interactions was conducted using data visualization with statistical analysis at a low confidence score in the STRING database (known and predicted protein-protein interactions) . Gene biological categorization was performed and selected at P-value ≤ 0.01. Analysis of the protein-protein interaction network was performed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAY database [45,46].
The distribution of threonine-phosphorylated ezrin and ezrin in gECs after exposure to IE/UE and in control media was verified by immunofluorescence. Cells were fixed in 4% formaldehyde in PBS (phosphate buffer saline) at 4°C for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked in 2% human serum in PBS for 20 min at room temperature. Mouse anti-ezrin (Abcam®, Cambridge, UK) and rabbit anti-phospho-ezrin (Thr567)/radixin (Thr564)/mosin (Thr41A3) ERM (Cell Signaling Technology, Danvers, MA, USA) antibodies, diluted in 1% human serum/PBS at a ratio 1:100, were added and incubated at room temperature for 1.5 h. Cells were incubated with goat anti-rabbit and anti-mouse antibodies, conjugated with Alexa Fluor green 488 and Alexa Fluor Red 594 (Molecular probes), respectively, diluted in 2% human serum/PBS were for 2 h. After washing with PBS, stained cells were mounted with anti-fade medium containing DAPI (Molecular probes). Cells were examined using a laser scanning confocal microscope (LSM 510 Meta, Zeiss, Jena, Germany) with a 63x objective at zoom 2. Immunofluoresence intensity of 1,000 cells from each culture condition was determined using ImageJ 1.48v/Java software . The mean of intensity from 1,000 cells were presented as intensity ratios, calculated from the intensity of IE/UE-exposed gECs divided by the intensity of gECs in control cultures.
Data for cell growth was analysed using the SPSS program (version 17). The unpaired Mann-Whitney-Wilcoxon test was used to compare means between independent groups for significantly statistic evaluation of cell growth, P-value < 0.01.
Inhibition of erythroid cell growth in in vitro cultures by P. vivax
It was previously reported that P. vivax parasites inhibited erythroid cell growth and perturbed erythroid cell division in three-day in vitro cultures . However, the molecular mechanisms underlying the inhibition of erythroid development by P. vivax were unclear. In this study, the underlining mechanism of the inhibitory effect on gEC growth by P.vivax was examined using growing erythroid cells (gECs), derived from human cord blood HSCs/CD34+, and infected erythrocytes (IE), isolated from patient blood. gECs from 5-day old cultures were exposed for 24, 48 and 72 h to lysed IE or UE, at a ratio of IE/UE: gEC 10:1. Lysed IE significantly inhibited gEC growth at 48 and 72 h (P-value < 0.01), compared with lysed UE and media controls (Figure 1a). No difference in cell death at 24, 48 and 72 h was observed in cultures with and without IE/UE, as shown in Figure 1b. Interestingly, lysed IE dramatically increased the number of cells in the G0 phase and decreased the number of cells in the mitotic fraction (G2/M and > 4n) at 48 and 72 h (Figure 1c). This indicates that parasite proteins were able to inhibit erythroid cell growth and division resulting in the accumulation of cells in a resting phase but did not induce cell death.
Characterization of phosphoproteins from erythroid cells after exposure to P. vivax
To analyse the mechanism of P. vivax inhibition on gEC growth, following exposure to IE/UE for 24, 48 and 72 h, phosphoproteins were enriched and analysed by LC-MS/MS. The DecyderMS program module was used for quantitation of the MS/MS intensity in each culture condition and results were compared to the human protein database. Phosphoproteins were classified based on biological function, molecular function, and protein class. Forty four phosphoproteins were identified from gECs exposed to IE/UE and media control at 24, 48 and 72 h (Figure 2). The molecular functions of the 44 phosphoproteins were classified according to the gene ontology analysis (Figure 2a) and include those involved in catalysis (40%), binding (28.6%), structural molecules (17%), enzyme regulators (5.7%), translation regulators (2.9%), transcription regulators (2.9%) and transporters (2.9%). The biological processes of these phosphoproteins (Figure 2b) were found to be predominantly in metabolic processes (23.3%), cellular processes (15.6%), transport (8.9%), and cell cycle (4.4%). In addition, protein class categories were analysed for these phosphoproteins indicating functions in cytoskeleton (10.6%), proteases (4.3%), kinases (4.3%) and structural proteins (2.1%) (Figure 2c).
Forty four phosphoproteins, identified from gECs exposed to IE/UE for 24 – 72 h, are presented using a heatmap visualization (Figure 3). The relative levels of these phosphoproteins was evaluated as high and low compared with level of phosphoprotein present in gECs from media control. The patterns of phosphoproteins from gECs exposed to IE/UE or in media for 24, 48 and 72 h were analysed in five clusters and categorized using the K-mean clustering method (Figure 3). Interestingly, the relative level pattern of phosphoproteins in cluster 1 was low from gECs exposed to IE for 24 – 72 h. To identify potential phosphoproteins from five clusters involved in inhibition of gEC growth and division by IE, the relative levels of phosphoproteins in each condition and time point were determined using statistical analysis with paired t-test at P-value ≤ 0.05. The lists of selected phosphoproteins with significant differences in relative level (P-value ≤ 0.05) were evaluated for biological processes and molecular functions using the KEGG PATHWAY database, as shown in Table 1. Interestingly, three of the phosphoproteins identified, ezrin, alpha actinin-1 and Rho kinase were reported to function in the regulation of the cellular cytoskeleton (Table 1). Ezrin, alpha actinin-1 and Rho kinase from gECs were observed to be all included in cluster 1 and the relative levels of these three phosphoproteins were significantly low from gECs exposed to IE for 72 h, compared with gECs in media only. These findings suggests that ezrin, alpha actinin-1 and rho kinase in the phosphoproteome of gECs may have roles relevant to the cellular pathology caused by P. vivax during erythroid cell growth.
Exposure to a pathogen, like parasites, has the ability to disturb the cellular processes in particular cell types. The alteration of protein expression and function inside the cells are the result of host-pathogen interactions. The exploring protein-protein interactions of gECs exposed to IE lysate were analysed using web based free database, STRING and three significant relative levels of phosphoproteins including ezrin, alpha actinin-1and Rho kinase were specifically found in the networks (Figure 4a). Moreover, the finding of phosphatidylinositol-4-phosphate 5-kinase type II beta in cluster 5 (Figure 3) also links the association of three proteins (Figure 4a). Interestingly, the protein-protein interaction of ezrin, alpha actinin-1, Rho kinase and phosphatidylinositol-4-phosphate 5-kinase type II beta was found in a specific relevant interaction pathway of IE-exposed gEC. Likewise, the alteration of defined phosphoprotein levels in gECs during exposure to IE for 24 to 72 h was performed as the dynamic relative level of phosphoproteins as shown in Figure 4b. Levels of the phosphoproteins ezrin, alpha actinin-1 and rho kinase are reduced following 48 to 72 h of IE exposure to gECs, compared with media controls. In contrast, abundance of phosphatidylinositol-4-phosphate 5-kinase type II beta was elevated under the same conditions. This showed that the altered relative level of these 4 phosphoproteins was a result of the interactions between IE and gEC proteins. To investigate whether reduced abundance of ezrin is in-volved in the mechanism of ineffective erythropoiesis in malaria, the ezrin protein-protein interaction network was analysed using the KEGG PATHWAY database. The ezrin interactome and its involvement in various cellular functions is shown in Figure 4c. This analysis revealed that the ezrin protein has important roles in various pathways though its function in the regulation of actin cytoskeleton may be the most relevant to malaria. While ezrin is associated with several pathways, only regulation of the actin cytoskeleton is shared among alpha actinin-1, Rho kinase and phosphatidylinositol-4-phosphate 5-kinase type II beta (Figure 4c).
Inhibitory effect on erythroid cell growth by P. vivax is regulated by ezrin phosphorylation
Protein phosphorylation is involved in several regulatory functions in living cells. Using phosphoproteomic and bioinformatics analysis, ezrin was identified and determined to have to function in regulation of actin cytoskeleton. One ezrin phosphopeptide from gECs contained the amino acid sequences DKYKTLRQIR, corresponding to residues 563-572. The phosphorylation site of this peptide analysed from MS/MS data was predicted to be tyrosine 565 (Y565). Ezrin, and phospho-ezrin were evaluated by immunofluorescence using specific antibodies against ezrin and phospho-ezrin Thr567 , as shown in Figure 5. Unfortunately, the antibody against phospho-ezrin Y565 is not available and data for phosphorylation at this ezrin residue was not reported. Results of immunofluorescence showed that ezrin and phospho-ezrin Thr567 localized to cell extensions peripheral processes of gECs. gECs in culture with UE or in media displayed strong signals for phospho-ezrin Thr567. In contrast, the signal strength for phospho-ezrin Thr567 was markedly reduced following exposure to IE for 48 and 72 h (Figure 5a and b). Quantitation of signals using ImageJ software for ezrin and phospho-ezrin Thr567 from 1,000 gECs from each culture, confirm the decreased level of ezrin proteins in cells exposed to IE (Additional file 1). The intensity ratios of phospho-ezrin Thr567 in IE-exposed gECs compared to media controls were less than 1 (0.17 and 0.26, at exposure times 48 and 72 h, respectively). In contrast, the same analysis using UE-exposed gECs gave intensity ratios for phospho-ezrin Thr567 of approximately 1 (0.99 and 0.93, at exposure time 48 and 72 h, respectively). Ezrin signals in IE/UE-exposed gECs compared to gECs in media at 48 and 72 h were nearly 1 (0.86 and 0.95 for IE-exposed gECs, and 0.9 and 0.98 for UE-exposed gECs, respectively), as shown in Additional file 1. These results indicate that ezrin phosphorylation at the carboxy-terminal threonine residue 567 was decreased in gECs exposed to IE. This suggests that parasite proteins were able to inhibit erythroid cell growth by preventing the phosphorylation of the C-terminal region of ezrin.
Anaemia has been frequently observed to be a consequence of vivax infection [4,7,10,48-58]. Recently, transmission electron microscope analysis of two vivax malarial cases with symptom of anaemia demonstrated the presence of P. vivax-infected erythroblasts and subsequent degradation of erythroblasts. Interestingly, P. vivax parasites were found in bone marrow, but could not be detected in peripheral blood . Consistent with this observation, an in vitro study has revealed that P. vivax can directly inhibit erythroid cell proliferation and differentiation through altered division of erythroid cells . However, the mechanisms underlying the suppression of erythroid development by P. vivax appear to be complex and poorly characterized. In this study, phosphoprotein analysis was used to investigate the underlying the suppression of erythroid development by P. vivax. Using LC-MS/MS analysis in combination with gene ontology information from the human protein database, 44 phosphoproteins from gECs exposed and non-exposed to IE/UE were identified and categorized according to molecular function, biological process and protein class. Interestingly, the relative level of phosphoproteins was significantly lower following exposure of gECs to parasite proteins, compared to non-exposed cells. Phosphoproteins that displayed significant decreases include ezrin, alpha actinin-1 and Rho kinase. These phosphoproteins were determined to be in the same cluster 1 with significantly low level pattern of phosphoproteins and exhibited a similar pattern of abundance in response to IE exposure. These results suggest that the phosphoproteins ezrin, alpha actinin-1 and Rho kinase may have roles in the inhibitory effect on erythroid cell growth and division following P. vivax exposure. The protein-protein interaction networks determined using the KEGG human protein database revealed that ezrin functions in several cellular pathways, including regulation of actin cytoskeleton.
Ezrin, a member of ERM (ezrin, radixin and moesin) proteins, is localized to cell extension peripheral processes and able to interact with transmembrane proteins and the cytoskeleton. Ezrin functions to regulate cytoskeletal assembly, membrane-protein function and membrane transport [29,59,60]. Recent studies, have implicated ezrin as a signal transducer involved in a wide variety of cellular functions, including cell survival, adhesion, morphogenesis, motility, cytokinesis and cellular proliferation [61,62]. Increasing evidence indicates a direct relationship between cell proliferation and the level of ezrin expression in cells [63-65]. Microinjection of anti-ezrin antibodies into the cytoplasm blocked the entry of mouse fibroblasts into S phase, confirming the function of ezrin in proliferation . In response to TNF, ezrin inhibits endothelial cell proliferation through transcriptional repression of cyclin A, a cell cycle regulatory protein . This study also found that ezrin in erythroid cells appears to function in the regulation of cell growth and division. Erythroid cells with growth inhibited by P. vivax display low levels of ezrin phosphoprotein.
The phosphorylation of a threonine at residue 567 (T567) is necessary for the active conformation of ezrin and enhances binding with F-actin . The results of this study also find that phosphorylation of ezrin, possibly at Thr567, is involved in the regulation of erythroid cell growth. Although our analysis identified the position of the phosphorylation site in an ezrin-phosphopeptide as tyrosine 565, the phosphorylation of ezrin threonine 567 was also detected using a specific antibody. A source of error in phosphorylation site localization on peptides is the presence and intensity of ions for these species in the MS/MS spectra . However, both residues 565 and 567 are contained in the phosphopeptide 563-572 from ezrin, which was detected in this study. It is also possible that the anti-ezrin Thr567 monoclonal antibody may recognize the phosphorylation of Tyr565 as well as Thr567. This antibody was produced using phosphopeptide of human ezrin containing residues 564-568 (KYKTL)  and contains both Tyr565 and Thr567. Previous reports have demonstrated the specificity of this antibody for phospho-ezrin Thr567 [69-71] but it is not known if it can recognize phosphorylation at other sites.
Three phosphoproteins in this study, ezrin, Rho kinase and alpha actinin-1 are present at significantly low levels in parasite-exposed cells. Investigation of the KEGG PATHWAY database demonstrates that ezrin has important roles in various pathways, particularly, regulation of actin cytoskeleton, which has implications to malaria, as shown in Figure 4c. Ezrin exerts its biological functions through protein-protein interactions and its active form is regulated by Rho kinase/ROCK2, which directly interacts with membrane proteins [31,72]. Ezrin binds to adhesion-related proteins with single transmembrane domains such as ICAM-1, CD44 and CD43 through their cytoplasmic tails to modulate cell morphology [61,73]. Cytoadhesion molecules such as ICAM1 contribute to cytoadhesive phenotype/rosetting and high intensity of rosetting is found in anaemia cases with P. vivax infection . Both ICAM-1 and VCAM-1, soluble adhesion molecules, are detected at high levels in serum from falciparum patients with severe malaria . This study also found abnormal erythroid cell aggregation in culture of gECs exposed to IE lysates (Additional file 2). Taken together, this suggests that during interaction of P. vivax proteins with adhesion molecules on erythroid cells, ezrin regulates cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. This erythroid cell adhesion may contribute to down regulation of erythroid cell production leading to the development of anaemia. For other adhesion molecules, integrin alpha L chain (ITGAL) and integrin α2β1 has been found a higher probability to involve in severe thrombocytopenia in vivax malaria . The expression of molecules sharing an epitope with human ITGB2/LFA-1 integrin, or CD18 leukocyte integrin on the Plasmodium falciparum-parasitized erythrocyte surface could be involved in the pathogenesis of severe disease . However, further studies are need to explore the association of these intercellular adhesion molecules with ezrin in vivax malaria.
In addition, ERM proteins are members of the band 4.1 superfamily, FERM (four-point one, ezrin, redixin, moesin) [78-80]. Phosphorylation of erythrocyte protein 4.1 is involved with the modification of the host erythrocyte membrane by P. falciparum . Moreover, tyrosine phosphorylation of band 3, band 4.2, catalase and actin in P. falciparum-infected erythrocytes are predicted to be part of the regulatory mechanism to modify the erythrocyte membrane . This study found low levels of alpha actinin-1 in gECs in the presence of IE. The subsequent reassembly of actin structure in response to parasite suppression is currently being investigated. This poorly understood aspect of ezrin function suggests that parasites inhibit the ezrin protein allowing the assembly of complex cellular structures in erythroid cells leading to dyserythropoiesis, inhibition of erythroid cell growth and division resulting in the accumulation of cells in a resting G0 phase. This study demonstrates that vivax parasites suppress development of erythroid progenitor cells through a mechanism that includes decreased ezrin phosphorylation. Plasmodium vivax is able to enter bone marrow, as previously reported [17,20,57,83], and parasites or its products bind to erythroid progenitor cells, resulting in decreased ezrin phosphorylation, leading to suppression of erythroid development, and ultimately anaemia. This is the first analysis suggesting that ezrin contributes to the suppression of erythroid cell growth by P. vivax. Further investigation of this mechanism should help to better understand pathogenesis of anaemia in acute or chronic P. vivax infection.
This analysis demonstrates that the pathogenesis of anaemia in vivax malaria is mediated by parasite suppression of human erythroid cell growth and division. Inactivation of the ezrin protein, leading to ineffective erythropoiesis and dyserythropoiesis, appears to be a key event resulting in the development of severe anaemia. The understanding of the pathogenesis of anaemia in vivax malaria should help in the development of therapeutic strategies to treat severe anaemia malaria.
Growing erythroid cells
Ezrin ezrin radixin and moesin
Haematopoietic stem cells
Liquid chromatography mass spectrometry
Rodríguez-Morales AJ, Sánchez E, Vargas M, Piccolo C, Colina R, Arria M, et al. Is anemia in Plasmodium vivax malaria more frequent and severe than in Plasmodium falciparum? Am J Med. 2006;119:9–10.
Genton B, D’Acremont V, Rare L, Baea K, Reeder JC, Alpers MP, et al. Plasmodium vivax and mixed infections are associated with severe malaria in children: a prospective cohort study from Papua New Guinea. PLoS Med. 2008;5:e127.
Kochar DK, Tanwar GS, Khatri PC, Kochar SK, Sengar GS, Gupta A, et al. Clinical features of children hospitalized with malaria–a study from Bikaner, northwest India. Am J Trop Med Hyg. 2010;83:981–9.
Ketema T, Bacha K. Plasmodium vivax associated severe malaria complications among children in some malaria endemic areas of Ethiopia. BMC Public Health. 2013;13:637.
Dhingra N, Jha P, Sharma VP, Cohen AA, Jotkar RM, Rodriguez PS, et al. Adult and child malaria mortality in India: a nationally representative mortality survey. Lancet. 2010;376:1768–74.
Sharma S, Aggarwal KC, Deswal S, Raut D, Roy N, Kapoor R. The unusual presentation of a usual organism - the changing spectrum of the clinical manifestations of Plasmodium vivax malaria in children: a retrospective study. J Clin Diagn Res. 2013;7:1964–7.
Bhattacharjee P, Dubey S, Gupta VK, Agarwal P, Mahato MP. The clinicopathologic manifestations of Plasmodium vivax malaria in children: a growing menace. J Clin Diagn Res. 2013;7:861–7.
Barcus MJ, Basri H, Picarima H, Manyakori C, Sekartuti, Elyazar I, et al. Demographic risk factors for severe and fatal vivax and falciparum malaria among hospital admissions in northeastern Indonesian Papua. Am J Trop Med Hyg. 2007;77:984–91.
Tjitra E, Anstey NM, Sugiarto P, Warikar N, Kenangalem E, Karyana M, et al. Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a prospective study in Papua. Indonesia PLoS Med. 2008;5:e128.
Kochar DK, Das A, Kochar SK, Saxena V, Sirohi P, Garg S, et al. Severe Plasmodium vivax malaria: a report on serial cases from Bikaner in northwestern India. Am J Trop Med Hyg. 2009;80:194–8.
Alexandre MA, Ferreira CO, Siqueira AM, Magalhães BL, Mourão MP, Lacerda MV, et al. Severe Plasmodium vivax malaria, Brazilian Amazon. Emerg Infect Dis. 2010;16:1611–4.
Raposo CC, Santos JB, Santos GM, Gonçalves Eda G, Silva AR. Plasmodium vivax malaria: related factors to severity in the State of Maranhão, Brazil. Rev Soc Bras Med Trop. 2013;46:67–72.
Quispe AM, Pozo E, Guerrero E, Durand S, Baldeviano GC, Edgel KA, et al. Plasmodium vivax hospitalizations in a monoendemic malaria region: severe vivax malaria? Am J Trop Med Hyg. 2014;91:11–7.
Douglas NM, Lampah DA, Kenangalem E, Simpson JA, Poespoprodjo JR, Sugiarto P, et al. Major burden of severe anemia from non-falciparum malaria species in Southern Papua: a hospital-based surveillance study. PLoS Med. 2013;10:e1001575.
Baird JK. Evidence and implication of mortality with acute Plasmodium vivax malaria. Clin Microbiol Rev. 2013;26:36–57.
McGready R, Wongsaen K, Chu CS, Tun NW, Chotivanich K, White NJ, et al. Uncomplicated Plasmodium vivax malaria in pregnancy associated with mortality from acute respiratory distress syndrome. Malar J. 2014;13:191.
Ru YX, Mao BY, Zhang FK, Pang TX, Zhao SX, Liu JH, et al. Invasion of erythroblasts by Plasmodium vivax: a new mechanism contributing to malarial anemia. Ultrastruct Pathol. 2009;33:236–42.
Raina V, Sharma A, Gujral S, Kumar R. Plasmodium vivax causing pancytopenia after allogeneic blood stem cell transplantation in CML. Bone Marrow Transplant. 1998;22:205–6.
Yamakawa H, Kiyotaki M, Hattori Y, Obana M, Matsuoka Y, Irimajiri S. A case of Plasmodium vivax malaria complicated with pancytopenia due to hypoplasia of the bone marrow. Kansenshogaku Zasshi. 1989;63:1043–6.
Wickramasinghe SN, Abdalla SH. Blood and bone marrow changes in malaria. Baillieres Best Pract Res Clin Haematol. 2000;13:277–99.
Wickramasinghe SN, Looareesuwan S, Nagachinta B, White NJ. Dyserythropoiesis and ineffective erythropoiesis in Plasmodium vivax malaria. Br J Haematol. 1989;72:91–9.
Panichakul T, Payuhakrit W, Panburana P, Wongborisuth C, Hongeng S, Udomsangpetch R. Suppression of erythroid cell development in vitro by Plasmodium vivax. Malar J. 2012;11:173.
Choudhary C, Mann M. Decoding signaling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol. 2010;11:427–39.
Carrascal M, Ovelleiro D, Casas V, Gay M, Abian J. Phosphorylation analysis of primary human T lymphocytes using sequential IMAC and titanium oxide enrichment. J Proteome Res. 2008;7:5167–76.
Ge F, Xiao CL, Yin XF, Lu C, Zeng H, He QY. Phosphoproteomic analysis of primary human multiple myeloma cells. J Proteomics. 2010;73:1381–90.
Macek B, Mann M, Olsen J. Global and site-specific quantitative phosphoproteomics: Principles and applications. Annu Rev Phormacol Toxicol. 2009;49:199–21.
Salinas JL, Kissinger JC, Jones DP, Galinski MR. Metabolomics in the fight against malaria. Mem Inst Oswaldo Cruz. 2014;109:589–97.
Fievet B, Louvard D, Arpin M. ERM proteins in epithelial cell organization and functions. Biochim Biophys Acta. 2007;1773:653–60.
Bretscher A, Edwards K, Fehon RG. ERM proteins and merin: integrators at the cell cortex. Nat Rev Mol Cell Biol. 2002;3:586–99.
Gary R, Bretscher A. Ezrin self-association involves binding of an N-terminal domain to a normal masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell. 1995;6:1061–75.
Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol. 1998;140:647–57.
Tran Quang C, Gautreau A, Arpin M, Treisman R. Ezrin function is required for ROCK-mediated fibroblast transformation by the net and dbl oncogenes. EMBO J. 2000;19:4565–76.
Ng T, Parsons M, Hughes WE, Monypenny J, Zicha D, Gautreau A, et al. Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J. 2001;20:2723–41.
Pietromoneco SF, Simons PC, Altman A, Elias L. Protein kinase C-zeta-phosphorylation of moesin in the actin-binding sequence. J Biol Chem. 1998;273:7594–603.
Simons PC, Pietromonaco SF, Reczek D, Bretscher A, Elias L. C-teminal threonine phosphorylation actives ERM proteins to link the cells cortical lipid bilayer to the cytoskeleton. Biochem Biophys Res Commun. 1998;253:561–6.
Panichakul T, Sattabongkot J, Chotivanich K, Sirichaisinthop J, Cui L, Udomsangpetch R. Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax. Int J Parasitol. 2007;37:1551–7.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75.
Johansson C, Samskog J, Sundstrom L, Wadensten H, Bjorkesten L, Flensburg J. Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data. Proteomics. 2006;6:4475–85.
Thorsell A, Portelius E, Blennow K, Westman-Brinkmalm A. Evaluation of sample fractionation using micro-scale liquid-phase isoelectric focusing on mass spectrometric identification and quantitation of proteins in a SILAC experiment. Rapid Commun Mass. 2007;21:771–8.
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999;20:3551–67.
Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003;34:374–8.
Magrane M, the UniProt consortium: UniProt knowledgebase: a hub of integrated protein data. Database 2011; bar009, doi:10.1093/database/bar009.
Mi H, Muruganujan A, Thomas PD. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 2013;41:D377–86.
Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013;41:D808–15.
Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014;42:D199–205.
Kanehisa M, Goto S, KEGG. Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.
Rasband WS: ImageJ US National Institutes of Health, Bethesda, Maryland, USA, 1997-2014. http://imagej.nih.gov/ij/. Accessed 1 Aug 2014.
Selvam R, Baskaran G. Hematological impairments in recurrent Plasmodium vivax infected patients. Jpn J Med Sci Biol. 1996;49:151–65.
Luxemburger C, Ricci F, Raimond D, Bather S, White NJ. The epidemiology of severe malaria in an area of low transmission in Thailand. Trans R Soc Med Hyg. 1997;91:256–62.
Mohapatra MK, Padhiary KN, Mishra DP, Sethy G. Atypical manifestations of Plasmodium vivax malaria. Indian J Malariol. 2002;39:18–25.
Song HH OSO, Kim SH, Moon SH, Kim JB, Yoon JW, et al. Clinical features of Plasmodium vivax malaria. Korean J Intern Med. 2003;18:220–4.
Collins WE, Jeffery GM, Roberts JM. A retrospective examination of anemia during infection of humans with Plasmodium vivax. Am J Trop Med Hyg. 2003;68:410–2.
Echeverri M, Tobón A, Alvarez G, Carmona J, Blair S. Clinical and Laboratory findings of Plasmodium vivax malaria in Colombia, 2001. Rev Inst Med Trop Sao Paulo. 2003;45:29–34.
Kochar DK, Saxena V, Singh N, Kochar SK, Kumar SV, Das A. Plasmodium vivax malaria. Emerg Infect Dis. 2005;11:132–4.
Rizvi I, Tripathi DK, Chughtai AM, Beg M, Zaman S, Zaidi N. Complications associated with Plasmodium vivax malaria: a retrospective study from a tertiary care hospital based in Western Uttar Pradesh, India. Ann Afr Med. 2013;12:155–9.
Gehlawat VK, Arya V, Kaushik JS, Gathwala G. Clinical spectrum and treatment outcome of severe malaria caused by Plasmodium vivax in 18 children from northern India. Pathog Glob Health. 2013;107:210–4.
Sharma P, Varma N. Pancytopenia following vivax malaria in a CLL patient. Blood. 2013;122:3098.
Abdallah TM, Abdeen MT, Ahmed IS, Hamdan HZ, Magzoub M, Adam I. Severe Plasmodium falciparum and Plasmodium vivax malaria among adults at Kassala Hospital, eastern Sudan. Malar J. 2013;12:148.
Vaheri A, Carpén O, Heiska L, Helander TS, Jääskeläinen J, Majander-Nordenswan P, et al. The ezrin protein family: membrane-cytoskeleton interactions and disease associations. Curr Opin Cell Biol. 1997;9:659–66.
Bretscher A, Reczek D, Berryman M. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J Cell Sci. 1997;110:3011–8.
Louvet-Vallee S. ERM proteins: from cellular architecture to cell signaling. Biol Cell. 2000;92:305–16.
Gautreau A, Louvard D, Arpin M. ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling. Curr Opin Cell Biol. 2002;2002(14):104–9.
Chen Z, Fadiel A, Feng Y, Ohtani K, Rutherford T, Naftolin F. Ovarian epithelial carcinoma tyrosine phosphorylation, cell proliferation, and ezrin translocation are stimulated by interleukin 1 alpha and epidermal growth factor. Cancer. 2001;92:3068–75.
Ohtani K. Ezrin, a membrane-cytoskeletal linking protein, is highly expressed in atypical endometrial hyperplasia and uterine endometrioid adenocarcinoma. Cancer Lett. 2002;179:79–86.
Crepaldi T, Gautreau A, Comoglio PM, Louvard D, Arpin M. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol. 1997;138:423–34.
Kaul SC, Mitsui Y, Komatsu Y, Reddel RR, Wadhwa R. A highly expressed 81 kDa protein in immortalized mouse fibroblast: its proliferative function and identity with ezrin. Oncogene. 1996;13:1231–7.
Kishore R, Qin G, Luedemann C, Bord E, Hanley A, Silver M, et al. The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-a- induced transcriptional repression of cyclin A. J Clin Inves. 2005;115:1785–95.
Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol. 2006;24:1285–92.
Zheng S, Huang J, Zhou K, Zhang C, Xiang Q, Tan Z, et al. 17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin. PLoS One. 2011;6:e22439.
Zhu L, Zhou R, Mettler S, Wu T, Abbas A, Delaney J, et al. High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function. Am J Physiol Cell Physiol. 2007;293:874–84.
Chuan YC, Pang ST, Cedazo-Minguez A, Norstedt G, Pousette A, Flores-Morales A. Androgen induction of prostate cancer cell invasion is mediated by ezrin. J Biol Chem. 2006;281:29938–48.
Ren L, Hong SH, Cassavaugh J, Osborne T, Chou AJ, Kim SY, et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene. 2009;28:792–802.
Legg JW, Isacke CM. Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44. Curr Biol. 1998;8:705–8.
Marín-Menéndez A, Bardají A, Martínez-Espinosa FE, Bôtto-Menezes C, Lacerda MV, Ortiz J, et al. Rosetting in Plasmodium vivax: a cytoadhesion phenotype associated with anaemia. PLoS Negl Trop Dis. 2013;7:e2155.
Jakobsen PH, Morris-Jones S, Rønn A, Hviid L, Theander TG, Elhassan IM, et al. Increased plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 in patients with Plasmodium falciparum or P. vivax malaria and association with disease severity. Immunology. 1994;83:665–9.
Campos FM, Santos ML, Kano FS, Fontes CJ, Lacerda MV, Brito CF, et al. Genetic variability in platelet integrin α2β1 density: possible contributor to Plasmodium vivax-induced severe thrombocytopenia. Am J Trop Med Hyg. 2013;88:325–8.
Tacchini-Cottier F, Lou JN, Roberts DJ, Garcia AM, Grau GE. Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum. Immunology. 1995;2:205–13.
Gould KL, Bretscher A, Esch FS, Hunter T. cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO J. 1989;8:4133–42.
Lankes WT, Furthmayr H. Moesin: a member of the protein 4.1-talin-ezrin family of proteins. Proc Natl Acad Sci U S A. 1991;88:8297–301.
Funayama N, Nagafuchi A, Sato N, Tsukita S, Tsukita S. Radixin is a novel member of the band 4.1 family. J Cell Biol. 1991;115:1039–48.
Chishti AH, Maalouf GJ, Marfatia S, Palek J, Wang W, Fisher D, et al. Phosphorylation of protein 4.1 in Plasmodium falciparum-infected human red blood cells. Blood. 1994;83:3339–45.
Pantaleo A, Ferru E, Carta F, Mannu F, Giribaldi G, Vono R, et al. Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth. Proteomics. 2010;10:3469–79.
Imirzalioglu C, Soydan N, Schaller M, Bretzel RG, Chakraborty T, Domann E. Diagnosis of mixed Plasmodium malariae and P. vivax infection in a development aid volunteer by examination of bone-marrow specimens by real-time PCR. J Clin Microbiol. 2006;44:2307–10.
This study was supported by Thailand Research Fund, Commission on Higher Education, Ministry of Education and Suan Dusit Rajabhat University, (MRG5680064). I would like to thank the Department of Pathobiology, Faculty of Science for excellent technical assistance, Ramathibodi Hospital, Faculty of Medicine for collecting cord blood, the Division of Medicine Siriraj Hospital, Mahidol University for use of the laser scanning confocal microscope, Malaria Clinic in Tha Sae, Chumpon Province for parasite collection, Prof. Sumalee Tungpradubkul for scientific comments and suggestions and Dr. Laran Jensen for critical reading of this manuscript, Department of Biochemistry, Faculty of Science, Mahidol University.
The authors declare that they have no competing interests.
TP designed the study, collected parasites from patient blood, performed cell culture and other experiments, performed statistical analysis, and wrote the manuscript. SP performed bioinformatic analysis. SR supported laboratory facilities for proteomic analysis. AP and SK performed LC-MS/MS. SH and RU contributed substantially to design of experiments and gave advice for writing of the manuscript. All authors read and approved the final manuscript.
Intensity ratios of ezrin and phospho-ezrin Thr567 from gECs exposed to IE/UE relative to signals from gECs in medium control. The intensity of ezrin and phospho-ezrin Thr567, from 1,000 gECs cells in each condition, exposed to IE/UE or in media were determined using ImageJ software (http://imagej.nih.gov). Intensity ratios were calculated using the mean intensity of IE/UE-exposed gECs normalized to the mean intensity from gECs in media.
Erythroid cell aggregation in culture of gECs exposed to P. vivax . Erythroid cells, 5-day old, were cultured with IE/UE lysates at a ratio of 1:10 (gEC:IE/UE) for 24, 48 and 72 h. Cell aggregation was observed under inverted microscope with 200x magnification.
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Panichakul, T., Ponnikorn, S., Roytrakul, S. et al. Plasmodium vivax inhibits erythroid cell growth through altered phosphorylation of the cytoskeletal protein ezrin. Malar J 14, 138 (2015). https://doi.org/10.1186/s12936-015-0648-9
- Plasmodium vivax
- Ineffective erythropoiesis
- Haematopoiesis stem cells
- Erythroid cells