Discovery of Schistosoma mekongi circulating proteins and antigens in infected mouse sera

Abstract

Schistosomiasis is a neglected tropical disease caused by an infection of the parasitic flatworms schistosomes. Schistosoma mekongi is a restricted Schistosoma species found near the Mekong River, mainly in southern Laos and northern Cambodia. Because there is no vaccine or effective early diagnosis available for S. mekongi, additional biomarkers are required. In this study, serum biomarkers associated with S. mekongi-infected mice were identified at 14-, 28-, 42-, and 56-days post-infection. Circulating proteins and antigens of S. mekongi in mouse sera were analyzed using mass spectrometry-based proteomics. Serine protease inhibitors and macrophage erythroblast attacher were down-regulated in mouse sera at all infection timepoints. In addition, 54 circulating proteins and 55 antigens of S. mekongi were identified. Notable circulating proteins included kyphoscoliosis peptidase and putative tuberin, and antigens were detected at all four infection timepoints, particularly in the early stages (12 days). The putative tuberin sequence of S. mekongi was highly similar to homologs found in other members of the genus Schistosoma and less similar to human and murine sequences. Our study provided the identity of promising diagnostic biomarkers that could be applicable in early schistosomiasis diagnosis and vaccine development.

Fig 1. SDS-PAGE analysis of S. mekongi-infected mouse serum at 0- (pre-infection), 14-, 28-, 42-, and 56-days post-infection.

M: marker; No. 1–3: mice number 1–3. The 12 horizontal sections represent excised protein bands used in mass spectrometric analysis.

Fig 2. Comparison of protein expression in uninfected and infected mouse sera at 14- (D14), 28- (D28), 42- (D42), and 56- (D56) days post-infection with S. mekongi using volcano plots.

Two vertical red lines represent differences as a minimum 2-fold changes relative to pre-infection conditions. The horizontal red line represents statistically significant at p-value <0.05. Dots above the horizontal red line with the difference more than 1 and less than -1 indicate up-regulated and down-regulated mouse serum proteins, respectively. Arrows indicate differential proteins found in all four post-infection time points.

Fig 3

Up-regulated (A) and down-regulated (B) mouse serum proteins 14-, 28-, 42-, and 56-days post-infection with S. mekongi.

Fig 4. Protein-protein interactions of up-regulated and down-regulated S. mekongi-infected mouse serum proteins at 14-, 28-, 42-, and 56-days post-infection.

The protein-protein interaction network was created using String database. Red and blue nodes indicate proteins in each pathway predicted to be altered with S. mekongi infection. Color nodes (red and blue) represent query proteins with first shell of interactors. White nodes represent proteins with second shell of interactors. Empty nodes indicate unknown 3D structural protein. Filled nodes indicate proteins with known or predicted 3D structure. Edges represent protein-protein associations with different types; red line: presence of fusion evidence; green line: neighborhood evidence; blue line: cooccurrence evidence; purple line: experimental evidence; yellow line: textmining evidence; light blue line: database evidence; black line: co-expression evidence. Acta1: actin alpha 1 skeletal muscle; Gsn: gelsolin; Cfl1: cofilin 1; Vasp: vasodilator stimulated phosphoprotein; Cyp27b1: cytochrome P450 family 27 subfamily B member 1; Cyp2r1: cytochrome P450 family 2 subfamily R member 1; Vdr: vitamin D receptor; C3: complement C3; Cfp: complement factor properdin; Cfh: complement factor H; Cfb: complement factor B; Cfd: complement factor D; Ufd1: ubiquitin recognition factor in ER associated degradation 1; Vcp: valosin containing protein; Nploc4: NPL4 homolog, ubiquitin recognition factor; Serpina3f: serpin clade A member 3F; Serpina3n: serpin clade A member 3N; Serpina3g: serpin clade A member 3G; Ithi3: inter-alpha-trypsin inhibitor heavy chain 3; Krt: keratin.

Fig 5. Circulating proteins of S. mekongi identified in infected mouse sera at 14-, 28-, 42-, and 56-days post-infection using mass-spectrometric analysis.

Numbers represent the number of S. mekongi circulating proteins identified by mass spectrometry.

Fig 6. SDS-PAGE analysis of circulating antigens from immune complexes in sera from mice with and without S. mekongi infection.

Immune complexes were enriched using protein A/G magnetic beads and separated with 12% SDS-PAGE. M: Marker; NI: Uninfected; 14-, 28-, 42-, and 56-days post-infection. The 10 horizontal sections represent regions excised for mass spectrometric analysis.

Fig 7. Circulating antigens of S. mekongi identified in infected mouse sera at 14-, 28-, 42-, and 56-days post-infection using mass spectrometric analysis.

Numbers represent number of S. mekongi circulating antigens identified by mass spectrometry.

Fig 8. Percentage similarity in protein sequence alignment of putative tuberin among Schistosoma spp., Mus musculus, and Homo sapiens.

All protein sequences were obtained from the non-redundant protein sequence databases of NCBI. Sequence alignment and identity calculations were performed using Clustal Omega software. Regions of highest similarity (green), high similarity (yellow), and lower similarity (red) are indicated.