Modulation of Rumen Microbes Through Extracellular Vesicle Released by the Rumen Fluke Calicophoron daubneyi

Abstract

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.

Keywords: Calicophoron daubneyi; extracellular vesicle; mass spectrometry; proteomics; rumen microbiome.

Figure 1

Representative developed TEM micrographs identifying extracellular vesicles secreted by C. daubneyi in vitro through DC and SEC isolation. (A) DC purified samples with visible aggregation of vesicles (circled) (B) SEC purified samples demonstrating a reduction in EV aggregation.

Figure 2

(A) EV proteome arrays of lysed C. daubneyi EVs (n=3). EVs released in vitro were lysed and subjected to 12.5% 1D polyacrylamide gel analysis and colloidal Coomassie blue stained. All biological replicates produced a highly reproducible profile. (B) Categorization of all sequences returned from the C. daubneyi EV proteome. Proteins consistent across three replicates were submitted to Interpro and GeneOntology searches and assigned to 9 functional categories as defined by Cwiklinski et al. (2015). Proteins that did not fit any of the nine categories were placed into a final category classified as ‘other’. Cytoskeleton associated proteins accounted for 13% of the sequences resolved, Proteases 14%, Enzymes 8%, Chaperones 7%, Transporters 4%, Exosome biogenesis 3%, Metabolism 5%, Carriers 1% with Others filling the remaining 36%.

Figure 3

Bacterial qPCR analysis following in vitro culture of rumen fluid incubated with C. daubneyi SEC purified EVs (p) and without EVs (n). Data shown for (A) Total bacteria, (B) Fibrobacter succinogenes, (C) Ruminococcus albus and (D) Prevotella spp. *indicates a significant difference between treatment means.