Proteomic analysis of extracellular vesicles and extracellular vesicle-depleted excretory-secretory products of Toxocara canis and Toxocara cati larval cultures

Toxocara canis and Toxocara cati are parasitic nematodes in the order Ascaridida, which inhabit the small intestines of dogs and cats, respectively, as adults. Although often nonpathogenic as adults, nematodes within this genus are capable of causing widespread disease throughout the host while in a larval stage, during which time larvae migrate throughout the body in a process termed larva migrans. Larvae are also capable of surviving within host tissues in an encysted arrested stage, without immune clearance by the host. The ability of larvae to survive within host tissues during migration and encystment may be attributed to immunomodulatory molecules released by the excretory cells of larvae in excretory-secretory (ES) products. ES products of parasites contain a variety of molecules, including proteins, lipids, and extracellular vesicles (EVs). Toxocara excretory-secretory (TES) products have been studied to some degree, with proteomic analysis of TES proteins described previously; however, investigation of the EVs within TES is lacking, despite the suggested role for these molecules in host interaction and potential immunomodulation. To further characterize the protein cargo within EVs in TES, EVs were isolated from larval cultures of T. canis and T. cati via ultrafiltration, with concurrent collection of EVdepleted TES filtrate for additional study. Isolated EVs and EV-depleted TES from both T. canis and T. cati were submitted for proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteomic identification results revealed 140 proteins across all samples, with 16 shared by all samples, and 76 total proteins shared between T. canis and T. cati, present within EVs and EV-depleted TES. There were 17 proteins shared exclusively by EV samples, and 15 were shared exclusively between EV-depleted TES samples. Many shared proteins were associated with the host immune response. Several proteins were specific to either T. canis or T. cati, highlighting the potential use of these proteins as diagnostic tools in the differentiation of etiologic agents in cases of toxocariasis. The results of this study build upon previously reported proteomic evaluations of TES, contributing new information in regards to newly identified proteins, EV protein cargo within TES, and potential immunomodulatory functions of these proteins.