Association of younger age (<10 years old) with reinfection with schistosomes.

<p>Presented here is the meta-analysis forest plot showing the pooled odds ratio and the corresponding 95% CI, subgroup analysis by species, and assessment of heterogeneity among studies. There was a strong statistically significant positive association between younger age (<10 years old) and reinfection with schistosomes.</p

Host determinants of reinfection with schistosomes identified in this study.

<p>NB: No = Number of included studies; OR = odds ratio; SMD = standardized mean difference; <i>p</i>-val. = <i>p</i>-value; PTI = Pre-treatment intensity of infection; Exposure = Exposure rate; HTA = High transmission area; SWA = Schistosoma adult worm antigen; SEA = Schistosoma egg antigen.</p><p>Host determinants of reinfection with schistosomes identified in this study.</p

Flow diagram for the search and systematic review process.

<p>Flow diagram for the search and systematic review process.</p

Association of gender (male) with reinfection with schistosomes.

<p>Presented here is the meta-analysis forest plot showing the pooled odds ratio and the corresponding 95% CI, subgroup analysis by species, and assessment of heterogeneity among studies. The observed positive association between reinfection and gender was only slightly significant.</p

Association of IgE levels with reinfection with schistosomes.

<p>(<b>A</b>) Meta-analysis forest plot for the association of reinfection with anti-SWA IgE levels showing the pooled standardized mean difference and the corresponding 95% CI and assessment of heterogeneity among studies. The observed negative overall effect was not statistically significant. (<b>B</b>) Meta-analysis forest plot for the association of reinfection with anti-SEA IgE levels showing the pooled standardized mean difference and the corresponding 95% CI and assessment of heterogeneity among studies. The observed negative overall effect was not statistically significant.</p

Association of IgG4 levels with reinfection with schistosomes.

<p>(<b>A</b>) Meta-analysis forest plot for the association of reinfection with anti-SWA IgG4 levels showing the pooled standardized mean difference and the corresponding 95% CI and assessment of heterogeneity among studies. IgG4 level was highly significantly associated with reinfection with schistosomes. (<b>B</b>) Meta-analysis forest plot for the association of reinfection with anti-SEA IgG4 levels showing the pooled standardized mean difference and the corresponding 95% CI and assessment of heterogeneity among studies. The observed positive overall effect was not statistically significant.</p

Protein expression and antigenicity of the candidate proteins.

<p>(<b>A</b>) SDS-PAGE of recombinant <i>E. coli</i> lysates (lane L) and purified protein without inclusion bodies (lane P). Arrows indicate expected molecular weights of oligomers. Other bands are expected SEA-domain cleavage products. (<b>B</b>) Western blots of recombinant protein expression as in (B), probed with anti-HisG antibody. (<b>C</b>) Anti-HisG probed western blots showing oligomerization of proteins with multiple bands of additive ∼30 kDa subunits, and tetramer as the most stable state. (<b>D</b>) Size exclusion gel filtration chromatography of SjCP3842 showed multiple elution peaks, another evidence of oligomerization. (<b>E</b>) Immunoblots showing reactivity of parasite crude antigen preparations (SEA and SWA) with immune sera. (<b>F</b>) The candidate proteins specifically reacted with infected miniature pig sera in IgG ELISA, indicating potential antigenicity during schistosomiasis. Bars represent standard deviation (SD). * = p<0.05, ** = p<0.01. n = 4 for each group.</p

Extracellular loop of the candidate proteins contain SEA-domains.

<p>(<b>A</b>) Modeled molecular structures of the extracellular domains with striking similarity with SEA-domain. Also shown for comparison is the SEA-domain of mouse TMPRSS2. Typical of SEA-domains, the secondary structure components showed an antiparallel arrangement of β-sheets. A summary of structural models of the entire transcripts in this gene family is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002644#pntd.0002644.s008" target="_blank">Table S1</a>. (<b>B</b>) Rigid body superposition of SjP3842 (blue) over the highest scoring template, PDB: 2e7v (olive). The graph is the Ramachandran plot (φ/ψ) showing conformational angles distribution of the residues. Over 98% of residues were in the favored regions while less than 2% were in the outlier region. (<b>C</b>) Alignments of SjCP3842 with two well defined SEA-domains (human MUC1 and mouse TMPRSS2). Putative SEA-domain consensus cleavage site (red arrow) was identified between β2 and β3.</p

<i>S. japonicum</i> SEA-domain proteins are heme-binding proteins.

<p>(<b>A</b>) Hemin-agarose binding assay showing potential of SjCP3842 to bind heme on hemin-agarose beads. (<b>B</b>) Hemin-agarose binding assay confirmed by immunoblotting using three candidates. ‘U’: unbound, ‘W’: last wash, ‘E’: eluates. (<b>C</b>) Identification of SjCP3842 in heme-binding protein fractions from parasite crude extracts (SWA). (<b>D</b>) Estimation of the amount of heme bound using peroxidase activity of bound heme. Standard curve (linear graph) of peroxidase activity of known concentrations of hemin was used to estimate the amount of bound heme. (<b>E</b>) Differential spectral titration of protein-heme interaction using 10 µM of heme and increasing concentrations of the protein (0 to 28 µM). Soret peak was red shifted from 388 nm to 412 nm, and absorption maximum increased with increasing accumulation of protein-heme complex. The inset is the heme-binding curve constructed by plotting ΔA₄₁₂ versus protein concentration, showing 1∶1 stoichiometry.</p

Heme-binding pocket of SjCP3842.

<p>(<b>A</b>) Heme-binding mode of SjCP3842 showing the hydrophobic vinyl end of the protoporphyrin heme inserted into a hydrophobic cavity, while hydrophilic propionate end of points away from the pocket. Heme is represented using spheres model colored by atoms (C: green, N: blue, O: red, Fe: brown). The protein is shown using cartoon model. (<b>B</b>) Heme-binding site showing the Connolly surface of the binding pocket (dots). (<b>C</b>) Heme iron (brown sphere) hexa-coordinated with His-149 and His-147 as axial ligands.</p