Changes in surface glycosylation and glycocalyx shedding in Trichobilharzia regenti (Schistosomatidae) during the transformation of cercaria to schistosomulum

RESEARCH ARTICLE Changes in surface glycosylation and glycocalyx shedding in Trichobilharzia regenti (Schistosomatidae) during the transformation of cercaria to schistosomulum Jana Řimnáčová, Libor Mikeš*, Libuše Turjanicová, Jana Bulantová, Petr Horák Department of Parasitology, Faculty of Science, Charles University, Viničná 7, Prague 2, Czech Republic a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Řimnáčová J, Mikeš L, Turjanicová L, Bulantová J, Horák P (2017) Changes in surface glycosylation and glycocalyx shedding in Trichobilharzia regenti (Schistosomatidae) during the transformation of cercaria to schistosomulum. PLoS ONE 12(3): e0173217. https://doi.org/ 10.1371/journal.pone.0173217 Editor: Josué de Moraes, Universidade Guarulhos, BRAZIL Received: January 8, 2016 Accepted: February 17, 2017 Published: March 15, 2017 Copyright: © 2017 Řimnáčová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funded by Czech Science Foundation (Grant No. 13-29577S), www.gacr.cz – (PH, LM, JŘ, JB, LT), Charles University grant support, www.cuni.cz/UKEN-65.html – GAUK No. 220214/ 2014 (LT, LM), UNCE No. 204017 (JB), PRVOUK No. P41 (PH, LM, JB, LT), SVV No. 260202/2015 (PH, LM, JB, LT). The funders had no role in study * Abstract The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host’s immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding. PLOS ONE | https://doi.org/10.1371/journal.pone.0173217 March 15, 2017 1 / 20 Glycocalyx shedding in Trichobilharzia regenti cercariae design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Trichobilharzia regenti (Trematoda, Schistosomatidae) is a neuropathogenic avian schistosome that migrates through the CNS to nasal areas of anatid birds. Its infective larvae, aquatic freeswimming cercariae, are well known as the causative agent of cercarial dermatitis in humans [1], a condition regarded as an emerging disease that currently requires attention in many countries all over the world [2,3]. Cercariae emerging from the snail intermediate host actively penetrate the skin of their definitive bird hosts or accidental (dead-end) mammalian hosts, including humans, and transform to schistosomula [4]. This process is accompanied by a detachment of the cercarial tail and emptying of the penetration glands. In schistosomes, cercarial bodies generally undergo extensive ultrastructural rebuilding that is accompanied by changes in biochemical processes and molecular (antigenic) composition of the tegumental glycocalyx. Transforming larvae form a double outer tegumental membrane with protective function, and shed the highly immunogenic glycocalyx which had protected them against the aquatic environment [5,6]. Much information about the structure of glycocalyx is available from human schistosomes, especially Schistosoma mansoni. The entire surface of the cercarial stage is covered by a 1–2 μm thick glycocalyx unusually rich in fucose residues [7,8]. Saccharide molecules represented by a heterogenous population of highly fucosylated glycans are bound to lipids and proteins on the membrane of the cercarial tegument by O-glycosidic bonds via sphingosine and serine or threonine, respectively [9–12]. Further detailed structural studies performed with cercariae and schistosomula have led to additional identification of a range of glycans, including smaller O-glycans with unusual core structures and xylosylated N-glycans, which both carry large amounts of Lewis-X antigen (LeX, CD15, 3-fucosyl-N-acetyl-lactosamine) also bound in glycolipids. The transformation of cercaria to schistosomulum is accompanied by extensive changes, characterized by a loss of multifucosylated GalNAcβ1-4GlcNAc (LDN) O-glycoproteins, which are replaced by LDN-rich glycosphingolipids [13–16]. Body surface glycosylation of schistosomes has been extensively studied by lectin binding assays, which confirmed the abundance of fucose residues, especially in the cercarial glycocalyx, and revealed differences in lectin binding patterns among particular life stages [17–23,6]. The mechanism of glycocalyx shedding during the penetration of cercariae into the host is known only to a limited extent. Hypotheses based on indirect evidence suggest that peptidases or (phospho)lipases from cercarial penetration glands might be involved [24–26, 6]. Specific secretions from the glands are in charge for lysis of the surface epithelia and underlying tissues during invasion (...truncated)