A dominance of Mu class glutathione transferases within the equine tapeworm Anoplocephala perfoliata.

The most common equine tapeworm, Anoplocephala perfoliata, has often been neglected amongst molecular investigations and has been faced with limited treatment options. However, the recent release of a transcriptome dataset has now provided opportunities for in-depth analysis of A. perfoliata protein expression. Here, global, and sub-proteomic approaches were utilized to provide a comprehensive characterization of the A. perfoliata soluble glutathione transferases (GST) (ApGST). Utilizing both bioinformatics and gel-based proteomics, GeLC and 2D-SDS PAGE, the A. perfoliata 'GST-ome' was observed to be dominated with Mu class GST representatives. In addition, both Sigma and Omega class GSTs were identified, albeit to a lesser extent and absent from affinity chromatography approaches. Moreover, 51 ApGSTs were localized across somatic (47 GSTs), extracellular vesicles (EVs) (Whole: 1 GST, Surface: 2 GSTs) and EV depleted excretory secretory product (ESP) (9 GSTs) proteomes. In related helminths, GSTs have shown promise as novel anthelmintic or vaccine targets for improved helminth control. Thus, provides potential targets for understanding A. perfoliata novel infection mechanisms, host­parasite relationships and anthelmintic treatments.

Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata.

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host-parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host-parasite interaction within the horse host.

Interaction of preimplantation factor with the global bovine endometrial transcriptome.

Preimplantation factor (PIF) is an embryo derived peptide which exerts an immune modulatory effect on human endometrium, promoting immune tolerance to the embryo whilst maintaining the immune response to invading pathogens. While bovine embryos secrete PIF, the effect on the bovine endometrium is unknown. Maternal recognition of pregnancy is driven by an embryo-maternal cross talk, however the process differs between humans and cattle. As many embryos are lost during the early part of pregnancy in cattle, a greater knowledge of factors affecting the embryo-maternal crosstalk, such as PIF, is needed to improve fertility. Therefore, for the first time, we demonstrate the effect of synthetic PIF (sPIF) on the bovine transcriptome in an ex vivo bovine endometrial tissue culture model. Explants were cultured for 30h with sPIF (100nM) or in control media. Total RNA was analysed via RNA-sequencing. As a result of sPIF treatment, 102 genes were differentially expressed compared to the control (Padj<0.1), although none by more than 2-fold. The majority of genes (78) were downregulated. Pathway analysis revealed targeting of several immune based pathways. Genes for the TNF, NF-κB, IL-17, MAPK and TLR signalling pathways were down-regulated by sPIF. However, some immune genes were demonstrated to be upregulated following sPIF treatment, including C3. Steroid biosynthesis was the only over-represented pathway with all genes upregulated. We demonstrate that sPIF can modulate the bovine endometrial transcriptome in an immune modulatory manner, like that in the human endometrium, however, the regulation of genes was much weaker than in previous human work.

Effects of preimplantation factor on interleukin-6 and prostaglandin F2α and E2 in the bovine endometrium.

Preimplantation factor (PIF) is a pregnancy specific peptide with immune modulatory properties exerted on the human endometrium. Viable bovine embryos secrete PIF, but its effect on the bovine endometrial immune response is unknown, both in native and inflammatory stimulated endometrial tissue. An ex vivo bovine endometrial tissue culture model was used with lipopolysaccharide (LPS) as an inflammatory stimulant. The effect of synthetic PIF (sPIF) was assessed, in three separate experiments, on the secretion or mRNA expression of essential prostaglandins and cytokines. Radioimmunoassays were used to assess prostaglandin secretion and ELISA for IL-6 secretion from endometrial explants. mRNA expression of IL6 and IL8 was analysed from endometrial explants with real-time PCR. Synthetic PIF reduced native IL-6 secretion from explants when pre-treated for 24 h. There was no effect of sPIF on IL-6 secretion from LPS challenged explants; however, sPIF increased IL6 mRNA expression when challenged with 500 ng/mL LPS. There was no effect of sPIF on prostaglandin secretion or mRNA expression of IL8. Therefore, sPIF is able to modulate the native IL-6 production pathway in the bovine endometrium, yet demonstrates no effect on prostaglandin secretion or IL8 expression. Unlike in human studies, effects of sPIF were minimal, thus sPIF is not an effective modulator of the immune targets investigated in the bovine endometrium.