Peptidases Compartmentalized to the Ascaris suum Intestinal Lumen and Apical Intestinal Membrane

Abstract

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.

Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

“…A tuberculin syringe was attached to the needle via the syringe hub to deliver experimental treatments. In contrast to previous perfusion experiments (Jasmer et al, 2014), the posterior end of worms remained intact to conduct dsRNA experiments. Cannulas were slid into a slot cut into a plastic microscope slide, allowing suspension and culture of worms (three for each treatment) in a 250 ml oak ridge tube filled with PBS (37°C) for the duration of the experiment (Fig.…”

“…Adult female A. suum were collected from pigs after 60–65 days oral infection with infective eggs (Rosa et al, 2014), decapitated just below the esophagus and fitted with a blunt-end 25 gauge needle inserted into the intestinal lumen, which was superglued in place (Jasmer et al, 2014). A tuberculin syringe was attached to the needle via the syringe hub to deliver experimental treatments.…”

“…The small size, biological complexity, and experimentally intractable nature of most parasitic nematodes hinder research aimed at elucidating essential functions in these pathogens. However, recently developed ‘omics’ resources (Jex et al, 2011; Jasmer et al, 2014; Rosa et al, 2014, 2015; Wang et al, 2015; Gao et al, 2016) and experimental capabilities (Chen et al, 2011; Jasmer et al, 2014; McCoy et al, 2015) established for the large round worm of swine and humans, Ascaris suum , continue to build on this global pathogen’s importance as a facile model system to conduct research on essential cellular functions in parasitic nematodes.…”

“…Nevertheless, limitations to knowledge and experimental methods have impeded research on this and other tissues of parasitic nematodes. Multiomics databases have been established for the intestine and other tissues of A. suum (Jasmer et al, 2014; Rosa et al, 2014; Gao et al, 2016), including proteomics distinguishing multiple intestinal cell compartments (Rosa et al, 2015) and pan-Nematoda intestinal transcriptome comparisons (Wang et al, 2015). This knowledge base helps to guide experimental design and interpret results from studies using the model organism A. suum .…”

“…Here we advance the A. suum experimental model by extending our intestinal cannulation-perfusion method (used in previous proteomic research (Jasmer et al, 2014; Rosa et al, 2015)) to directly manipulate intestinal cells in situ by using controlled delivery of experimental treatments. No comparable methods are known to exist for controlled delivery of this kind to nematode intestinal cells.…”