Hirst, Natasha (2019) 'Schistosoma mansoni' : integration of phosphoproteomics, kinomics and CaMKII. (PhD thesis), Kingston University, .
Abstract
'Schistosoma mansoni' is a parasitic trematode and the causative agent of the debilitating disease human schistosomiasis. This study focused on global signalling processes which occur in the parasite and on Ca2+/Calmodulin Kinase II (CaMKII) signalling. Phosphoproteomics of a mixed population of adult worms was performed, identifying a total of 15,844 phosphopeptides, from 3,176 proteins with 12,936 unique phosphorylation sites. Analysis of the phosphorylated sites/proteins provided valuable insights into the complexities of signalling in this parasite, including insights into overrepresented and novel phosphorylation site motifs, putative upstream protein kinases, molecular function, and interactions between phosphoproteins (1,586 nodes and 12,733 edges). Highly connected interaction clusters highlighted the pivotal role of phosphorylated ribosomal and ribosomal biogenesis proteins, proteasome, and spliceosome proteins within S. mansoni. Furthermore, analysis of the protein kinases revealed 808 phosphorylation sites amongst 136 protein kinases representing 54% of the S. mansoni kinome. This novel, deep, phosphoproteomic dataset supported the development of the first parasite-specific kinomic array comprising 96 peptides with many upstream kinases predicted. The array was deployed to identify eight peptides (proteins) which were significantly differentially phosphorylated by homogenates from male and female adult worms: the serine/threonine protein kinases CDK/PITSLRE, AMPK, AKT, SmTK4, insulin receptor, protein phosphatase 2c gamma, Rho2 GTPase, and vacuolar protein sorting 26. CaMKII was identified as a protein of interest in the phosphoproteomic analysis, further investigations were carried out. Using anti-phospho-CaMKII antibodies, CaMKII was detected at approximately 57 kDa in cercariae, somules, and adult worms. Activated CaMKII was located visually in the nervous system, excretory system, sensory structures, and the tegument as well as in the female vitellaria. Inhibition assays determined that CaMKII phosphorylation (activation) in somules could be suppressed by KN93, and further assays found that the anti-schistosomal drug praziquantel had a significant effect on CaMKII activation in S. mansoni. Finally, suppression of CaMKII expression by small interfering RNA (siRNA) resulted in a 66% and 52% reduction in males and females respectively. Collectively these studies show that phosphorylation plays a wide reaching and crucial role in the complex signalling pathways that ultimately control S. mansoni from highlighting their involvement in reproduction and proliferation, host location and movement to commanding cell death, the elucidation of these phosphorylating pathways are vital to understanding this parasites biology.
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