Untargeted Multimodal Metabolomics Investigation of the Haemonchus contortus Exsheathment Secretome

Abstract

In nematodes that invade the gastro-intestinal tract of the ruminant, the process of larval exsheathment marks the transition from the free-living to the parasitic stages of these parasites. To investigate the secretome associated with larval exsheathment, a closed in vitro system that effectively reproduces the two basic components of an anaerobic rumen environment (CO₂ and 39 °C) was developed to trigger exsheathment in one of the most pathogenic and model gastrointestinal parasitic nematodes, Haemonchus contortus (barber's pole worm). This study reports the use of multimodal untargeted metabolomics and lipidomics methodologies to identify the metabolic signatures and compounds secreted during in vitro larval exsheathment in the H. contortus infective third-stage larva (iL3). A combination of statistical and chemoinformatic analyses using three analytical platforms revealed a panel of metabolites detected post exsheathment and associated with amino acids, purines, as well as select organic compounds. The major lipid classes identified by the non-targeted lipidomics method applied were lysophosphatidylglycerols, diglycerides, fatty acyls, glycerophospholipids, and a triglyceride. The identified metabolites may serve as metabolic signatures to improve tractability of parasitic nematodes for characterizing small molecule host-parasite interactions related to pathogenesis, vaccine and drug design, as well as the discovery of metabolic biomarkers.

Keywords: Haemonchus contortus; exsheathment; helminth; lipidomics; metabolomics; parasite.

Figure 1

Developmental life cycle of the parasitic nematode Haemonchus contortus.

Figure 2

Overview of the experimental procedure and multimodal metabolomics workflow. Schematic diagram of the closed in vitro system that effectively reproduces the two basic components of an anaerobic rumen environment (CO₂ and 39 °C) was used to trigger exsheathment (xL3) in H. contortus third-stage infective larvae (iL3) in O₂-free CO₂ saturated saline solution (left). Multi-modal metabolomics workflow and statistical analyses used to process data integrated from multiple analytical approaches through to pathway mapping (right).

Figure 3

H. contortus infective larvae during in vitro exsheathment. Time series analysis of the exsheathment activity of H. contortus L3 using the anaerobic in vitro system. Mean of the total exsheathment percentage (±SEM) at each time point across replicates (n = 5). Significant (p < 0.001) exsheathment was obtained resulting in 75% and 100% of larvae exsheathing after 70 min and 5 h, respectively, post trigger exposure. No exsheathment activity was observed in the absence of anaerobic treatment conditions. Exsheathment up to 6 h post trigger application shown.

Figure 4

Scanning electron micrographs of the exsheathment process in H. contortus. SEMs are shown for seven time points post incubation (t = 0, 2, 4, 7, 9, 10 and 25 min). Each image depicts the earliest observations of key morphological changes that occur during the exsheathment process and is representative of the larval population for each biological sample. Scale bars differ and have been adjusted according to magnification of each image.