Inhibition of Haemonchus contortus larval development by fungal lectins.
BACKGROUND
Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus.


METHODS
The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin - AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin - Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage.


RESULTS
Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5 μg/ml caused >95 % inhibition of larval development while 40 μg/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae's gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults.


CONCLUSIONS
The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes.
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, 8057, Zurich, Switzerland. christianheim@access.uzh.ch.
Christian
C
Heim
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, 8057, Zurich, Switzerland. Hubertus.Hertzberg@uzh.ch.
Hubertus
H
Hertzberg
Malcisbo AG, Wagistrasse 27a, 8952, Schlieren, Switzerland. alex.butschi@malcisbo.com.
Alex
A
Butschi
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, 8093, Zürich, Switzerland. silvis.mr@gmail.com.
Silvia
S
Bleuler-Martinez
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, 8093, Zürich, Switzerland. markus.aebi@micro.biol.ethz.ch.
Markus
M
Aebi
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, 8057, Zurich, Switzerland. deplazesp@access.uzh.ch.
Peter
P
Deplazes
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, 8093, Zürich, Switzerland. markus.kuenzler@micro.biol.ethz.ch.
Markus
M
Künzler
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, 8057, Zurich, Switzerland. sasa.stefanic@uzh.ch.
Saša
S
Štefanić
D000363:Agaricales / Q000737:chemistry*; D000818:Animals; D000871:Anthelmintics / Q000302:isolation & purification / Q000494:pharmacology*; D001203:Ascomycota / Q000737:chemistry*; D041981:Gastrointestinal Tract / Q000737:chemistry; D006189:Haemonchus / Q000187:drug effects* / Q000254:growth & development*; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D037102:Lectins / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding
D000871:Anthelmintics; D037102:Lectins