Proteomic Investigation of <em>Falciparum</em> and <em>Vivax</em> Malaria for Identification of Surrogate Protein Markers

<div><p>This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites <em>Plasmodium falciparum</em> and <em>P. vivax</em> to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with <em>falciparum</em> malaria (FM) (n = 20), <em>vivax</em> malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (<em>p</em><0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates the potential of this analytical approach for the detection of malaria as well as other human diseases.</p> </div

Discrimination of <i>falciparum</i> and <i>vivax</i> malaria patients from healthy and febrile controls.

<p>(A) PLS-DA scores plot for FM (red spheres, n = 6), VM (blue spheres, n = 5) and FC (leptospirosis) (gray spheres, n = 6) samples based on 6 differentially expressed proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041751#pone.0041751.s017" target="_blank">Table S8</a>.3A) identified using 2D-DIGE. The axes of the plot indicate PLS-DA latent variables. (B–D) Receiver operating characteristic (ROC) curves depicting accuracy of 3 classifier proteins; apolipoprotein A-I (B), haptoglobin (C) and retinol-binding protein (D) for malaria prediction. The area under the ROC curve (AUC) signifies the accuracy of the classifier proteins for distinguishing FM, VM and leptospirosis from healthy controls. AUC value close to 1 indicates an excellent prediction of the disease. The reference line denotes an uninformative test, with an AUC of 0.50.</p

Differentially expressed serum proteins in <i>falciparum</i> malaria identified using 2DE and 2D-DIGE.

<p>(A) Representative 2D gels of serum from healthy controls and FM patients. 600 µg of total serum proteins were focused on linear pH 4–7 IPG strips (18 cm) and then separated on 12.5% polyacrylamide gels, which were stained with Gel Code Blue Stain. (B) The 3D images of some selected statistically significant (<i>p</i><0.05) differentially expressed proteins identified in 2DE. Data is represented as mean ± SE (where n = 20). (C) Representative 2D-DIGE image to compare serum proteome of HC and FM patients. FM and HC samples were labeled with Cy3 and Cy5 respectively, while the protein reference pool (internal standard) was labeled with Cy2. (D) Graphical and 3D fluorescence intensity representations of few selected statistically significant (<i>p</i><0.05) differentially expressed proteins in FM patients identified in biological variation analysis (BVA) using DeCyder 2D software. Graphs showing the decrease/increase in the standardized log abundance of spot intensity in FM compared to the HC cohort of the study (n = 8).</p

Modulation of essential physiological pathways in <i>falciparum</i> and <i>vivax</i> malaria.

<p>Members of multiple vital physiological processes including acute phase response signaling, chemokine and cytokine signaling, complement cascades, lipid transport and metabolism, and blood coagulation exhibited differential expression in response to <i>Pf</i> and <i>Pv</i> infection. Differential expression of serum proteins (up-regulated in red, down-regulated in green and no differences in expression level in yellow) are depicted in both plasmodial infections (FM and VM).</p

Demographics and clinical details of the malaria patients and febrile controls (Leptospirosis patients).

#<p>Data is represented as median (interquartile-range).</p>*<p>Not suffering from mix infections [multiple <i>Plasmodium</i> species] and not treated with any antimalarial drugs prior to the sample collection process.</p><p>•Not available.</p

Comparative analysis of <i>falciparum</i> and <i>vivax</i> malaria.

<p>(A) Venn diagram showing the number of proteins differentially expressed in FM and VM, among which 46.2% were found to be common. (B) PCA analysis shows the clustering of different spot maps (green-HC, red-FM, blue-VM group). The PC1 component separates the control group from the rest, and the PC2 clusters the diseased groups separately. Proteins which participated in PCA analysis were present in at least 80% of the spot maps and passed the filter of one-way ANOVA (<i>p</i><0.01) test. (C) The dendrogram showing the separation of different experimental groups after the hierarchical cluster analysis (red - up-regulated, green - down-regulated and black – no significant change in expression level). The spot maps are clustered together for each experimental group (HC, FM and VM). (D) Trend of differentially expressed proteins in malaria patients represented as standardized log abundance of spot intensity in FM, VM and HC cohort of the study. Compared to HC, all of the identified proteins (except serotransferrin and alpha-2-HS-glycoprotein) exhibited similar trend of differential expression in FM and VM; however, fold-change values are different.</p

Immunoassay-based validation of differentially expressed proteins.

<p>Immunoturbidimetric measurement of serum haptoglobin (A) and Apo A−1 (B) levels in healthy subjects (n = 20), <i>falciparum</i> malaria (n = 20), <i>vivax</i> malaria (n = 17) and febrile controls (n = 6). Histograms depicting the haptoglobin and Apo A−1 concentrations determined by immunoturbidimetry. Patients with malaria infection found to have lower serum level of haptoglobin and Apo A−1 compared to the HC and FC (<i>p</i><0.0001; Mann-Whitney test). (C) Retinol-binding protein 4 (RBP4) level was measured in sera derived from malaria patients (n = 24), healthy subjects (n = 20) and febrile controls (n = 6) by ELISA and results are shown as bar-diagrams (mean ± SE), which indicates down-regulation of RBP4 in FM and VM patients (<i>p</i><0.01) compared to HC, while serum level of RBP4 found to be unaltered in FC (leptospirosis patients) (D &E) Western blot analysis of haptoglobin (HP) serum amyloid A (SAA), clusterin (CLU) and retinol-binding protein (RBP) from serum samples of HC (n = 12), FM (n = 12),VM (n = 12) and leptospirosis patients (n = 6). Representative blots of the target proteins (D) are depicted along with their respective relative intensities (X 10⁴) (E). Western blot analysis revealed up-regulation of serum amyloid A and down-regulation of haptoglobin, clusterin and retinol-binding protein in FM and VM patients (<i>p</i><0.01) compared to the controls (HC and FC). All data are represented as mean ± SE.</p