Abstract
Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a ‘footprint’ of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.
Highlights
The neglected tropical disease human schistosomiasis caused by Schistosoma blood parasites is an enormous global public health concern [1]
Proteins for phosphoproteomic analysis were extracted from a mixed population of ~400 adult male and female S. mansoni, obtained from three separate batches of mouse infections each passaged though different batches of Biomphalaria glabrata snails
Western blotting was performed using a panel of three phospho-motif antibodies to confirm that each of the three separate batches of extracted protein were of suitable quality for phosphoproteomic profiling (S1 Fig)
Summary
The neglected tropical disease human schistosomiasis (bilharzia) caused by Schistosoma blood parasites is an enormous global public health concern [1]. This parasitic disease affects almost 240 million people across 78 countries, with ~0.8 billion at risk of infection [2,3]. The eggs are destined for expulsion from the host in excreta facilitating life cycle transmission via the snail intermediate host, many become trapped in host tissues eliciting immunopathological reactions [5] These immunological responses lead to liver fibrosis, hepatosplenic inflammation and intestinal disease in the case of S. mansoni/S. japonicum, or obstructive and inflammatory disease in the urinary system in the case of S. haematobium [7]. The exquisite biology of the schistosome underpins its fecundity and longevity—surviving on average for five to 10 years [16], but possibly up to 30 years [7]—in the human host
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