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.

Excretory-secretory products from the brown stomach worm, Teladorsagia circumcincta, exert antimicrobial activity in in vitro growth assays

“…Helminth EVs are defined as lipid membrane-enclosed packages, rich in nucleic acids, peptide and protein cargo [ 19 ], with several functions in host-parasite interactions, including helminth migration through host tissues, nutrition (via the activity of protein-degrading enzymes) and modulation of host immune responses [ 19 , 20 ]. Crucially, in a recent study, exposure of rumen fluids to EVs derived from the rumen fluke, Calicophoron daubneyi , resulted in increased bacterial diversity, which led the authors to speculate that C. daubneyi EVs might exert bactericidal and/or bacteriostatic activities that be (at least partially) responsible for the changes in gut microbial profiles observed over the course of parasite infection and establishment [ 21 ].…”

“…In spite of emerging evidence of the role(s) of helminth ESPs and EVs in parasite-microbiota crosstalk, little is known of the putative antimicrobial properties of molecules contained in ESPs of livestock helminths, including those enclosed in vesicles [ 21 – 23 ]. Whilst previous investigations have comprehensively elucidated the proteomic profiles of ESPs derived from third- and fourth-stage larvae of T. circumcincta (L3s and L4s, respectively) [ 24 ], only a single study has undertaken a preliminary proteomic analysis of EV-enriched ESPs (obtained by ultracentrifugation) from L4s of this parasite [ 25 ].…”

Parasitic helminths and the host microbiome – a missing ‘extracellular vesicle-sized’ link?

“…However, it is important to note that, while such increase in total bacterial DNA was interpreted as a proxy for increased bacterial species diversity, no statistically significant differences in the relative abundances of Ruminococcus albus, Fibrobacter succinogenes, and Prevotella spp. were detected over time between colonies incubated with C. daubneyi EVs and PBS negative controls [55]. Nevertheless, in a follow-up study, classical colony counting of P. aeruginosa and Bacillus subtilis following incubation with C. daubneyi EVs resulted in 12.4% and 12.1% colony-forming-unit (CFU) reductions, respectively, compared to control colonies (Nathan Allen, PhD Thesis, Aberystwyth University, 2019, https://pure.aber.ac.uk/portal/files/49906429/Allen_Nathan.pdf), which suggests that the observed alterations might be linked, at least in part, to the antimicrobial activity of rumen fluke vesicles.…”

“…Figure 1). A preliminary study conducted by Allen et al [55], in which exposure of rumen fluid to EVs isolated from the rumen fluke Calicophoron daubneyi led to a significant overall increase in total bacterial DNA yields, provides support to this hypothesis [55]. However, it is important to note that, while such increase in total bacterial DNA was interpreted as a proxy for increased bacterial species diversity, no statistically significant differences in the relative abundances of Ruminococcus albus, Fibrobacter succinogenes, and Prevotella spp.…”

“…Antibodies can then be obtained against surface proteins of interest and utilised in further antibody neutralisation experiments to assess mechanisms of EV-bacteria interactions (Figure I). collected to date (e.g., [55]) serve as proof-of-concept of the direct and/or immune-mediated involvement of helminth EVs in host-parasite-microbiota interactions; mechanistic studies investigating such crosstalk are urgently needed in order to shed light on the relative contribution(s) of worm EVs in host-pathogen relationships and dynamics of helminth infection and establishment in the vertebrate hosts. Inevitably, such studies rely on the availability of standardised techniques to isolate, purify, and characterise populations of EVs often from minute amounts of parasite material (Box 1).…”