Date Published: January 8, 2015
Publisher: Public Library of Science
Author(s): Douglas P. Jasmer, Bruce A. Rosa, Makedonka Mitreva, John Pius Dalton. http://doi.org/10.1371/journal.pntd.0003375
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.
Partial Text: Parasitic nematodes cause major diseases of humans, directly affecting more than two billion people on a global scale . Diseases they cause in food animal species also pose significant constraints to agricultural production and thus, indirectly impact human health in regions of the world where nutritional resources are limited. Acquisition of resistance to contemporary anthelmintics by many species of parasitic nematodes is on the rise , . Hence, the need for research to identify new targets for therapies to treat and control infections by these pathogens has never been greater. The nematode intestine is one tissue of importance in this context.
Additional details are provided in Supporting Information (S1 Text).
Parasitic nematodes present numerous challenges for conducting research on individual worm tissues, and even more so on individual cells. Such is the case for biological questions pertaining to the intestine and intestinal cells of these pathogens. Here we describe progress towards comprehensive identification of proteins that function in the IL and/or on the AIM of adult female A. suum, which is one of a relatively few parasitic nematodes with sufficient size to support the progress reported by using relatively straightforward perfusion methods. Knowledge of H. contortus AIM peptidases gained from past research guided efforts to identify A. suum homologs (likely orthologs or paralogs) that were detected in ConA binding fractions and intestinal perfusates. Collective observations reported here indicated that most of the predicted aspartic, metallo and serine carboxy peptidases identified in Table 1, are indeed A. suum glycoproteins that function in the IL and/or on the AIM. Moreover, testing of this hypothesis led to identification of a greatly expanded set of apparent peptidases that function in these compartments, while also identifying many proteins with other functions that are likely sited in these compartments.