Lectin blot studies on proteins of Myxobolus cerebralis, the causative agent of whirling disease

Abstract

It is known that Myxobolus cerebralis antigens, both surficial and secreted, are key modulators for, or targets of, host immune system compounds. We undertook SDS-PAGE glycoprotein characterisation of M. cerebralis developmental stages isolated from infected rainbow trout and Western blot analyses using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and anti-triactinomyxon polyclonal antibodies. Glycoproteins were isolated with lectin-affinity chromatography, and prominent bands were characterised by matrix-assisted laser desorption/ionisationmass spectrometry (MALDI/MS). We identified glycoproteins of M. cerebralis myxospores that contained carbohydrate motifs reactive with Phaseolus vulgaris erythroagglutinin (proteins 20 to 209 kDa, PHA-E), Sophora japonica agglutinin (proteins 7 to 70 kDa, SJA), Griffonia simplicifolia Agglutinin I (proteins 10 to 209 kDa, GSA-I) and G. simplicifolia Agglutinin II (proteins 5 to 40 kDa, GSA-II). Mcgp33, a glycoprotein isolated by lectin-affinity chromatography, was reactive with SJA (about 33 kDa). Antiserum produced against M. cerebralis triactinomyxons was found to have differences in the antigenicity of isolated glycoproteins from both M. cerebralis myxospores and actinospores. We also demonstrated modified antigen expression, especially involving the glycoprotein Mcgp33, in different developmental stages of M. cerebralis. KEY WORDS: Myxobolus cerebralis · Glycoprotein · Lectin · MALDI/MS · Lectin blotting · Oncorynchus mykiss · Myxozoa · Interaction Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [227][228][229][230][231][232][233][234][235] 2005 ments, both in host-tissue and in free-spore stages. The survival strategies of these parasites frequently involve the participation of glycoconjugates, which can be used to build protective structures and to facilitate hostparasite interactions. Yet given the wide variety of environmental conditions faced and host adaptation over time to ward off parasite attack, the proteins and glycoconjugates of the parasite must also adapt over time if the organism is to survive. Fish immune systems are mainly based on non-specific immune responses (Ingram 1980) and frequently involve lectin activity in microbial defence (Hosono et al. 1999, Muramoto et al. 1999, Honda et al. 2000. The immune system has to be able to effectively recognise parasite glycan epitopes, in order to provide some resistance to infection. To counter the host response, protozoans rely on antigen mimicry (Damian 1987, Inal 2004) and antigen variation (Borst et al. 1996, Rudenko et al. 1998, processes which involve surface glycoproteins such as glycoepitopes of N-glycosides (Burghaus et al. 1999, Yang et al. 1999 and O-glycosides (Dieckmann-Schuppert et al. 1993, Khan et al. 1997. In Trypanosoma brucei (Borst et al. 1996, Ferguson 1997, 1999 and Giardia lamblia (Gillin et al. 1990, Gillin & Reiner 1996, Nash 1992, a variant surface glycoprotein (VSG) is expressed sequentially to evade the ...

Detection of carbohydrate terminals in the enteric parasite Enteromyxum scophthalmi (Myxozoa) and possible interactions with its fish host Psetta maxima

“…Pre-esporogonic and extra-esporogonic stages of T. bryosalmonae were also stained by BSI-I at LM (Marín de Mateo et al 1996;Morris and Adams 2004), though only some vacuolar structures of P cell were scarcely labelled at TEM (Morris and Adams 2004). Using blot studies, Knaus and El-Matbouli (2005b) identified glycoproteins that contained carbohydrate motifs reactive with the same lectin in Myxobolus cerebralis stages as well.…”

Antigenic characterization of Enteromyxum leei (Myxozoa: Myxosporea)

“…Such terminal sugar re sidues are believed to play a paramount role in host− parasite interactions and may form a protective sheath for the parasites that helps them to evade host recognition (Hicks et al 2000, Theodoropoulos et al 2001. Therefore, some parasites, such as Trypanosoma cruzi (Buscaglia et al 2006), employ an antigen variation strategy through modification of N-and Oglycosides of glycoepitopes in their surface glycoproteins (Gagneux & Varki 1999, Knaus & El-Matbouli 2005. In fish, as in all vertebrates, the lectin pathway of the complement system is an ancient first line defense mechanism of the innate immune system that relies on recognition of pathogen-associated glycan epitopes (Sunyer & Lambris 1998, Nakao et al 2006, Kania et al 2010.…”

“…In fish, as in all vertebrates, the lectin pathway of the complement system is an ancient first line defense mechanism of the innate immune system that relies on recognition of pathogen-associated glycan epitopes (Sunyer & Lambris 1998, Nakao et al 2006, Kania et al 2010. In previous studies, the glycoproteins of some piscine parasites have been studied by lectin blotting (Feng & Woo 1998b, Kim et al 1999, Muñoz et al 2000a, Knaus & El-Matbouli 2005.…”

“…Another approach to studying the antigenicity of myxozoans is the use of polyclonal antibodies (Pabs) (Bartholomew et al 1989, Saulnier & deKinkelin 1996, Muñoz et al 1999b, 2000a, Lu et al 2002, Knaus & El-Matbouli 2005, Zhang et al 2010). In the present study, the antigenic glycoproteins of Enteromyxum leei were analysed using 2 Pabs; one raised against E. leei and another raised against the polar filament of Myxobolus pendula (Ringuette et al 2011).…”

Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio

“…However, this approach can be time-and laborconsuming, and detection and identification is mainly based on the mature spore stages, with the early parasite stages likely to be neglected (Grabner et al 2012, Mahony et al 2015. Immunological methods based on antibodies or lectins (Knaus & El-Matbouli 2005, Estensoro et al 2013) have also been developed. Nevertheless, these methods are generally restricted by cross-reactivity between different species (Lu et al 2002, Estensoro et al 2014, Jia et al 2016.…”