Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor

Abstract

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

Author Summary

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. These worms infect the liver and can survive for many years in its animal or human host because they supress the host’s immune system that is important in clearing worm infection. Worms are similar to humans in that they are made of proteins, fats and sugars, and while there are many studies on worm proteins, few studies have examined the sugars. We are interested in the sugars because we believe that they help the parasite survive for many years within its host. To examine this, we have used a technique called mass spectrometric analysis to characterise the sugars present in F. hepatica. We also have developed systems in the laboratory to test if these sugars can suppress the host’s immune system. We conclude that F. hepatica sugars are crucial in suppressing its host’s immune system; however, the exact way the sugars can do this requires further studies. These studies are important for the development of worm vaccines or therapies.