Authors

H Northcote²; P Wititkornkul¹; PM Brophy¹; RE Wonfor¹; RM Morphew¹;  ¹ Aberystwyth University, UK;  ² Holly Northcote, UK

Discussion

Gastrointestinal (GI) helminths have been observed to create significant changes to the GI microbiome they share their environment with. Helminths, largely nematodes, have also been shown to create a plethora of antimicrobial peptides (AMPs) secreted through secretory products (ESP, which contain extracellular vesicles (EVs) which has been hypothesised as a mechanism for host-microbiome changes. Anoplocephala perfoliata (Aper), an equine tapeworm, has often been neglected in molecular research. However, the recent generation of a transcriptome and secretory (ESP and EV) proteomes provides a foundation for a greater understanding of its host-parasite relationships. Utilising a comparative bioinformatic investigation the current work has identify AMPs within A. perfoliata andtwo additional flatworms and localised them in EVs. AperEV-free ESP (AperESP) and the cytosolic proteome (AperCY) were also further investigated for AMPs.Furthermore, Aper peptidomes (Somatic, ESP, EVs) were extracted, using an acidic methanol treatment, and identified via MSMS. Finally, AperEVs and AperESP antimicrobial activity was assessed following incubation with Escherichia coli and Bacillus megaterium and the optical density (OD) monitored over 24 hours. In total, 34 unique IDs were identified in A. perfoliata as AMPs in contrast to 130, and 13 from a rumen fluke and a liver fluke respectively. Notably, 6, 8, and 4 of these retrieved IDs were localised in the parasite's EV proteomes. Within A. perfoliata, a further four and 25 potential AMP IDs were resolved in the AperCY and AperESP, respectively. Preliminary peptidome analysis of the same fractions within A. perfoliatademonstrates the potential to identify a plethora of novel peptides and possible AMP agents and for the first time the identification of peptides within the EVs. Despite promise of AMP activity through bioinformatic, proteomic and peptidomic analysis, initial optical density assays of A. perfoliata EVs demonstrated no antimicrobial activity. The current data suggests that A. perfoliata can produce a range of different potential AMP proteins and/or peptides and these can be localised within their EVs. Whilst initial assays have not demonstrated antimicrobial activity, bioinformatic data suggests A. perfoliata may have the ability to affect/alter the microbial environment around them through AMPs. Alternatively, proteins similar to potential AMPs may not possess antimicrobial activity similar to helminth defence proteins. previous studies. Thus, further microbial investigation is needed.