Research ArticleMalaria

Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria

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Science Translational Medicine  27 Jun 2018:
Vol. 10, Issue 447, eaar3619
DOI: 10.1126/scitranslmed.aar3619
  • Fig. 1 Whole-blood dual RNA sequencing and deconvolution.

    (A) Uniquely mapped reads from human (red) and P. falciparum (blue) from subjects with severe malaria (SM; n = 25) and uncomplicated malaria (UM; n = 21). (B and C) Heatmaps showing signature gene expression in counts per million (CPM) for different leukocyte (B) and parasite developmental stage (C) populations and their relative intensity in individual subjects with SM, including different SM phenotypes (CH, cerebral malaria plus hyperlactatemia; CM, cerebral malaria; HL, hyperlactatemia), and UM. (D) Surrogate proportion variables (SPVs) for parasite developmental stages compared between severe malaria and uncomplicated malaria using the Mann-Whitney test (bold line, box, and whiskers indicate median, IQR, and up to 1.5 times the IQR from the lower and upper ends of the box, respectively). (E and F) Principal component (PC) plots showing the effect of deconvolution on the segregation of subjects with UM and SM, adjusting human (E) and parasite (F) gene expression for differences in proportions of leukocytes or parasite developmental stages, respectively. Analyses of human gene expression (B and E): SM, n = 25; UM, n = 21. Analyses of parasite gene expression (C, D, and F): SM, n = 23; UM, n = 20.

  • Fig. 2 Association of gene expression with features of severe malaria and parasite load.

    (A) Volcano plot showing extent and significance of up- or down-regulation of human gene expression in severe malaria (SM) compared with uncomplicated malaria (UM) (red and blue, P < 0.05 after Benjamini-Hochberg adjustment for FDR; orange and blue, absolute log2 fold change (log2FC) in expression > 1; SM, n = 25; UM, n = 21). (B) Comparison of selected neutrophil-related gene expression multiplied by absolute neutrophil count in blood between SM (n = 21) and UM (n = 20) [bold line, box, and whiskers indicate median, IQR, and up to 1.5 times IQR from the lower and upper ends of the box, respectively; units are fragments per kilobase of transcript per million mapped reads/liter (FPKM/L); P for Mann-Whitney test]. (C) Heatmap comparing enrichment of GO terms for human genes significantly differentially expressed between severe malaria and uncomplicated malaria or significantly associated with blood lactate, platelet count, or BCS. TAP, transporter associated with antigen processing. (D) P. falciparum differential gene expression in severe malaria compared to uncomplicated malaria [color coding as in (A); SM, n = 23; UM, n = 20]. (E) Heatmap comparing enrichment of GO terms for parasite genes significantly differentially expressed between severe malaria and uncomplicated malaria and or significantly associated with blood lactate or BCS. (F) Heatmap comparing GO terms for human genes significantly associated with log parasite density and log PfHRP2.

  • Fig. 3 Interspecies gene expression modules and their associations with severity.

    Circos plot showing gene expression modules obtained from whole-genome correlation network analysis using expression of all human and parasite genes from each subject (severe malaria, n = 22; uncomplicated malaria, n = 19) as the input. From outside to inside: labels, hub gene, and most enriched GO term (with P value) for each module; track 1, module eigengene value for each subject; track 2, clinical phenotype (red, CH; orange, CM; green, HL; yellow, uncomplicated malaria); track 3, hub gene expression (log CPM) for each subject; track 4, heatmap for correlation with laboratory measurements (clockwise, blocks: log parasite density, log PfHRP2, lactate, platelets, hemoglobin, BCS; color intensity represents correlation coefficient as shown in color key); track 5, module size and composition (length proportional to number of genes in module; red, human genes; blue, parasite genes); polygons connect modules with significant (FDR P < 0.01) Pearson correlation between eigengene values (width proportional to −log10 FDR P value; red, positive correlation; blue, negative correlation). ER, endoplasmic reticulum.

  • Fig. 4 Severity-associated differential coexpression within the interspecies gene expression network.

    (A to C) Cytoscape visualization of merged coexpression networks derived separately from severe malaria (n = 22) and uncomplicated malaria (n = 19). Networks were merged such that genes found in both subnetworks (represented as arrow-shaped, larger-sized nodes) are connected to genes found in only one subnetwork (represented as circular-shaped and smaller-sized nodes). (A) Genes and gene clusters are colored and annotated by module, species, most enriched GO terms, and conservation between subnetworks. Preserved, module pairs from severe malaria and uncomplicated malaria subnetworks overlap with each other but not with other modules; partially preserved, module clusters in one subnetwork overlap with two or more modules in the other subnetwork; unique, gene clustering only found in one subnetwork. Genes in black do not belong to any characterized module. TCA, tricarboxylic acid. (B) Identical network layout with genes colored by species (red, human; blue, P. falciparum). (C) Identical network layout with genes colored by whether they are significantly differentially expressed in severe malaria versus uncomplicated malaria (red, human; blue, P. falciparum; black, not differentially expressed).

  • Table 1 Characteristics of study subjects (n = 46).

    CM, cerebral malaria; CH, cerebral malaria plus hyperlactatemia; HL, hyperlactatemia (CM, CH, and HL are all subgroups of severe malaria); UM, uncomplicated malaria; PfHRP2, P. falciparum histidine-rich protein 2; Hb, hemoglobin concentration; WBC, white blood cell count. Data are median [interquartile range (IQR)], and superscripts indicate the number of subjects with data for each variable if less than the total. P for Kruskal-Wallis test comparing all groups (df = 3) except for sex where P is for Fisher’s exact test. Leukocyte population numbers and proportions were measured by a clinical hematology analyzer.

    CM (n = 5)CH (n = 12)HL (n = 8)UM (n = 21)P
    Age (years)4.3 (4.2–4.8)4.9 (3.6–5.7)5.0 (3.8–8.3)6.0 (4.0–9.0)0.51
    Male (%)3 (60)5 (42)7 (88)13 (62)0.24
    Parasitemia (%)8.3 (5.3–9.0)412.6 (9.4–19.0)9.6 (1.8–12.2)5.1 (3.8–7.0)0.008
    Parasites (×105/μl)2.3 (1.7–3.1)33.5 (2.7–8.4)112.8 (0.7–5.0)2.3 (1.6–3.2)0.062
    Clones2 (1.5–2.5)42 (1–2)91 (1–2)52 (1–2)150.67
    PfHRP2 (ng/ml)202 (93–528)4763 (374–1750)470 (164–2214)163 (128–227)0.004
    Duration of illness (days)2.0 (1.7–3.0)2.0 (2.0–2.5)2.0 (2.0–3.5)2.7 (2.0–3.0)0.22
    Hb (g/dl)9.7 (7.4–10.4)9.3 (7.8–11.5)119.1 (7.4–11.0)10.8 (9.9–12.1)0.12
    WBC (×109/liter)9.8 (8.2–12.9)48.8 (6.4–9.4)1115.3 (7.9–16.8)79.5 (7.7–11.8)0.41
    Platelets (×109/liter)41 (40–82)436 (23–65)1159 (33–132)122 (96–132)0.013
    Lymphocytes (×109/liter)2.7 (2.1–3.6)42.9 (2.4–3.6)113.1 (1.8–5.2)72.4 (1.4–3.1)200.57
    Lymphocyte (%)29.8 (20.6–37.3)437.8 (29.9–49.9)1122.3 (14.7–37.3)723.9 (16.0–33.5)200.087
    Neutrophils (×109/liter)6.4 (4.0–8.7)44.0 (2.9–4.3)106.5 (5.8–10.4)77.0 (5.3–7.7)200.045
    Neutrophil (%)55.1 (49.0–69.6)448.3 (39.6–56.2)1061.5 (55.6–74.9)768.0 (59.9–79.6)200.016
    Monocytes (×109/liter)0.6 (0.6–0.7)40.6 (0.5–0.9)100.8 (0.6–1.3)70.7 (0.4–0.9)200.58
    Monocyte (%)7.1 (6.0–7.7)47.8 (6.8–8.6)106.6 (5.1–7.8)76.7 (4.8–7.3)200.12
  • Table 2 Numbers of differentially expressed genes before and after adjustment for parasite load.

    Number of genes associated with severity category or laboratory marker of severity before and after adjustment for parasite load (% of number in unadjusted analysis where applicable). SM, severe malaria; UM, uncomplicated malaria.

    Human genesParasite genes
    n*UnadjustedLog parasite densityLog PfHRP2n*UnadjustedLog parasite densityLog PfHRP2
    SM versus UM43907914 (101%)13 (1.4%)41516562 (109%)329 (64%)
    BCS43738491 (67%)12 (1.6%)41445340 (76%)148 (33%)
    Lactate401012526 (52%)51 (5.0%)38100109 (109%)47 (47%)
    Platelets4317866 (37%)46 (25%)4111 (100%)1 (100%)
    Hemoglobin430004165 (83%)4 (67%)

    *Only subjects with complete data for every parameter are included.

    • Table 3 Parasite genes correlated with human gene coexpression modules after adjustment for parasite load.

      “+/−” indicates the number of parasite genes positively/negatively correlated with each human module eigengene value. All “top genes” in the table are positively correlated with the module eigengene value. RTP4, receptor transporter protein 4; TNRC6B, trinucleotide repeat containing 6B; ATPase, adenosine triphosphatase; PPIB, peptidylprolyl isomerase B.

      Human moduleCorrelated parasite genes after adjustment for parasite load
      Hub geneTop GO termn (+/−)Top genesFDR PTop GO termsP
      RTP4GO:0034340 response to type 1 IFN2 (2/0)PF3D7_0827500 (apicoplast ribosomal protein L21 precursor)0.014GO:0006412 translation0.018
      PF3D7_0111800 (eukaryotic translation initiation factor 4E)0.035
      TNRC6BGO:0016569 chromatin modification97 (29/68)PF3D7_1119200 (unknown function)0.00012GO:0008380 RNA splicing4.15 × 10−6
      PF3D7_1309100 (60S ribosomal protein L24)0.00027GO:0006396 RNA processing3.88 × 10−5
      PF3D7_0825500 (protein KRI1)0.00036
      HSPH1GO:0006457 protein folding21 (17/4)PF3D7_0933100 (unknown function)0.0071GO:0000338 protein deneddylation0.0038
      PF3D7_1118400 (haloacid dehalogenase-like hydrolase)0.0079
      PF3D7_0521800 (ATPase family gene 1-like ATPase)0.01
      MMP8GO:0009617 response to bacterium18 (16/2)PF3D7_1356200 (mitochondrial import inner membrane translocase subunit TIM23)0.0045GO:0019219 regulation of nucleobase-containing compound metabolic process0.0065
      PF3D7_1119100 (transfer RNA m(1)G methyltransferase)0.0071
      PF3D7_0823100 (RWD domain–containing protein)0.0073
      PPIBGO:0034976 response to endoplasmic reticulum stress24 (24/0)PF3D7_1420300 (DNL-type zinc finger protein)0.0022GO:0006364 rRNA processing0.0058
      PF3D7_0821200 (unknown function)0.0057
      PF3D7_1119200 (unknown function)0.0057
      RPL24GO:0006614 signal recognition particle-dependent cotranslational protein targeting to membrane93 (78/15)PF3D7_0530600 (XAP-5 DNA binding protein)5.3 × 10−6GO:0006396 RNA processing6.92 × 10−4
      PF3D7_1309100 (60S ribosomal protein L24)5.3 × 10−6GO:0008380 RNA splicing1.10 × 10−3
      PF3D7_0821200 (unknown function)3.3 × 10−5

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/447/eaar3619/DC1

      Materials and Methods

      Fig. S1. Estimates of the relative proportions of leukocyte subpopulations in subjects with severe and uncomplicated malaria.

      Fig. S2. Validation of the gene-signature approach to estimate parasite developmental stage proportions.

      Fig. S3. Differential gene expression between severe malaria and uncomplicated malaria phenotypes.

      Fig. S4. Top functional network for the small LYSMD3 module.

      Fig. S5. Association between gene expression and plasma protein concentrations.

      Fig. S6. Host-pathogen interactions in severe malaria revealed through dual RNA sequencing.

      Table S1. Human genes differentially expressed between malaria disease phenotypes in unadjusted and parasite load–adjusted analyses.

      Table S2. GO terms associated with human differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.

      Table S3. Predicted upstream regulators associated with human differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.

      Table S4. Human genes significantly correlated with parasite load and pathophysiological variables in unadjusted and parasite load–adjusted analyses.

      Table S5. P. falciparum genes differentially expressed in unadjusted and parasite load–adjusted analyses.

      Table S6. GO terms associated with parasite differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.

      Table S7. P. falciparum genes significantly correlated with parasite load and pathophysiological variables in unadjusted and parasite load–adjusted analyses.

      Table S8. Summary of modules obtained from combined whole-genome correlation network.

      Table S9. Univariate and multivariate associations of module eigengene values and parasite load with severity.

      Table S10. Summary and overlap of whole-genome correlation subnetworks for severe and uncomplicated malaria.

      Table S11. Summary of modules obtained from human-only whole-genome correlation network.

      Data set S1. Subject-level clinical and laboratory data.

      References (8593)

    • Supplementary Material for:

      Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria

      Hyun Jae Lee, Athina Georgiadou, Michael Walther, Davis Nwakanma, Lindsay B. Stewart, Michael Levin, Thomas D. Otto, David J. Conway, Lachlan J. Coin, Aubrey J. Cunnington*

      *Corresponding author. Email: a.cunnington{at}imperial.ac.uk

      Published 27 June 2018, Sci. Transl. Med. 10, eaar3619 (2018)
      DOI: 10.1126/scitranslmed.aar3619

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Estimates of the relative proportions of leukocyte subpopulations in subjects with severe and uncomplicated malaria.
      • Fig. S2. Validation of the gene-signature approach to estimate parasite developmental stage proportions.
      • Fig. S3. Differential gene expression between severe malaria and uncomplicated malaria phenotypes.
      • Fig. S4. Top functional network for the small LYSMD3 module.
      • Fig. S5. Association between gene expression and plasma protein concentrations.
      • Fig. S6. Host-pathogen interactions in severe malaria revealed through dual RNA sequencing.
      • Table S9. Univariate and multivariate associations of module eigengene values and parasite load with severity.
      • Legends for tables S1 to S8, S10, and S11
      • Legends for data set S1
      • References (8593)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S1 (Microsoft Excel format). Human genes differentially expressed between malaria disease phenotypes in unadjusted and parasite load–adjusted analyses.
      • Table S2 (Microsoft Excel format). GO terms associated with human differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.
      • Table S3 (Microsoft Excel format). Predicted upstream regulators associated with human differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.
      • Table S4 (Microsoft Excel format). Human genes significantly correlated with parasite load and pathophysiological variables in unadjusted and parasite load– adjusted analyses.
      • Table S5 (Microsoft Excel format). P. falciparum genes differentially expressed in unadjusted and parasite load–adjusted analyses.
      • Table S6 (Microsoft Excel format). GO terms associated with parasite differentially expressed or significantly correlated genes in unadjusted and parasite load–adjusted analyses.
      • Table S7 (Microsoft Excel format). P. falciparum genes significantly correlated with parasite load and pathophysiological variables in unadjusted and parasite load–adjusted analyses.
      • Table S8 (Microsoft Excel format). Summary of modules obtained from combined whole-genome correlation network.
      • Table S10 (Microsoft Excel format). Summary and overlap of whole-genome correlation subnetworks for severe and uncomplicated malaria.
      • Table S11 (Microsoft Excel format). Summary of modules obtained from humanonly whole-genome correlation network.
      • Data set S1 (Microsoft Excel format). Subject-level clinical and laboratory data.

      [Download Supplementary Tables]

      [Download Dataset S1]

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