Genetic modulation of Glycoconjugate/Lectin interactions in immune responses : how co-evolution of the microbes and their hosts modulates individual susceptibility to a given microbe ?
Histo-blood group antigens (HBGAs) constitute the terminal portion of glycans (either O- or N-linked on glycoproteins or glycolipids) that present an intra-species or inter-species polymorphism. Their biosynthesis proceeds by stepwise addition of monosaccharide units to 6 possible precursors, each step requiring a specific glycosyltransferase. Our work between 2010-2015 was the follow up of a project initiated over 10 years ago. We had shown that several members of the Caliciviridae family (RHDV, Human norovirus) use HBGAs for attachment to host epithelial cells, which is the first step of the infection process. Members of the norovirus genus (NoVs) are responsible for the vast majority of gastroenteritis outbreaks in humans of all ages and recent studies indicate that they cause the death of over 200.000 young children in developing countries (Glass et al N Engl J Med 361: 1776, 2009; Patel et al Emerg Infect Dis 14: 1224, 2008).
We previously showed that the prototype strain of NoV binds to the fucosylated motif Fucα2Galβ3GlcNAc that is expressed on human small intestine surface epithelial cells. The fucosyltransferase responsible for the synthesis of this ligand is coded by the FUT2 gene. 20% of the European or North American populations is FUT2-/- and therefore is completely resistant to many NoV strains. Our work and data from others show that various NoV strains recognize distinct HBGAs, indicating that the polymorphism at the ABO, FUT2 and FUT3 loci dictates the host spectrum of each strain in humans (reviewed in Tan and Jiang Trends Microbiol 19: 382, 2011). As a result of the human HBGA diversity and NoV variable carbohydrate specificity, a single strain infects only a subgroup of the population, although collectively, NoV strains can infect all of us. This situation is most likely the result of a long host-pathogen co-evolution where the polymorphism of glycosyltransferases genes allows the population to always be partly protected from an outbreak: the viral pressure maintaining the polymorphism to which the virus would have adapted by a diversification of the strains carbohydrate specificity. This hypothesis generates explanations for the existence of HBGAs polymorphism as well as for high diversity and moderate virulence of the NoV strains (and rotavirus, see below). HBGAs polymorphism would thus correspond to a mechanism of protection (or immunity) at the population level that we named « Herd Innate Protection »(Ruvoën-Clouet et al Rev Med Virol 23: 355, 2013).
Another calicivirus, RHDV, rabbit hemorrhagic disease virus (lagovirus genus), offered the opportunity to monitor an ongoing host-pathogen co-evolution and to validate the Herd Innate Protection model. RHDV is an extremely virulent pathogen that appeared some 30 years ago. It kills rabbits within 12-48h post-infection and eliminated up to (decimated) 90% wild rabbits populations in outbreaks throughout Europe (Abrantes et al. Vet Res 2012). Our work conducted with the ONCFS (Office de la Chasse et de la Faune Sauvage, who survey wild rabbit populations in France) indicates that animals weakly expressing the RHDV ligand in association with some alleles of the Sec1 gene (an α2fucosyltransferase gene of the GT11 family) were significantly over-represented among survivors to a devastating outbreak. This suggests selection at the GT11 family locus. We then observed that rabbit Sec1 acts as a dominant-negative on rabbit Fut1, controlling the level of α2-fucosylated RHDV ligands. Thus in rabbits, Sec1 evolved a new function with a dominant-negative effect on Fut1, contributing to fucosylated glycan diversity, allowing herd protection from pathogens such as RHDV (Nyström et al, PLoS Pathogens, 2015). Analysis of 6 RHDV strains, representative of the virus evolution over the last 25 years, documented shifts in HBGAs specificity and ability to recognize duodenal epithelial cells of rabbits with distinct HBGAs phenotypes during this period. The virus selectively killed animals based on the presence of HBGA corresponding to the circulating strain specificity. Later strains adapted by a shift in HBGA specificity allowing infection of animals spared by earlier strains. This indicated that the virus induced a selection pressure on glycosyltransferases genes to generate host glycan diversity and showed a parallel diversification of RHDV strains with respect to their HBGA specificity in order to cope with the host polymorphism, as expected from the model (Nyström et al. PLoS Pathogens 7: e1002188, 2011; Le Pendu et al Curr Opin Virol 7: 88, 2014).
1- How the co-evolution of host receptor to pathogens and pathogen surface glycoconjugates trigger differences in the host microbiota composition?
Interactions between rotaviruses (RVAs) and histo-blood group antigens
Group A rotaviruses (RVAs) are the major cause of severe gastroenteritis in young children constituting the first cause of hospitalization among children below 5 years of age. In low-income countries they are associated with an important mortality, with an estimated number of deaths of about 450 000 in 2008. Thanks to the availability of vaccines, this number has now been divided by half. Initial cell attachment of RVAs is mediated by VP8*, the distal domain of the VP4 protein through glycan recognition. Recent developments concerning the ligands for attachment viral protein VP8* of human RVA strains uncovered two distinct types of glycans. Acidic glycolipids, called gangliosides and fucosylated HBGAs. Until recently it was considered that VP8* binds to either terminal, sialidase-sensitive sialic acid residues, or to internal, sialidase-insensitive sialic acid residues. However, recent analyses of the carbohydrate binding properties of VP8* led to a paradigm shift as VP8* from various human strains revealed specific recognition of fucosylated neutral oligosaccharides of the histo-blood group family (HBGAs). Most VP8* variants tested so far bind to either the H type 1, Lewis b or A antigens that have in common an α1,2-linked fucose residue (Hu et al Nature 485:256, 2012; Jiang et al J Virol 86: 4833, 2012; Ramani et al J Virol 87: 7255, 2013). In a retrospective study, we showed that FUT2-deficient individuals were not found among children hospitalized in Nantes for severe gastroenteritis due to RVA of the P genotype, indicating that such individuals are genetically resistant to RVA-gastroenteritis, similar to what has been previously observed for norovirus-mediated gastroenteritis (Imbert-Marcille et al J Infect Dis 209: 1227, 2013).
Common polymorphisms of fucosyltransferases such as FUT3 and FUT2, as well as the ABO polymorphisms generate diversity of the epithelial glycan cover. P strains recognize the difucosylated Lewis b tetrasaccharide which synthesis requires both FUT2 and FUT3. It is present in about 75% of the French population, the remaining 25% lacking either one or both of these enzymes. In this country, P strains are overly dominant (~90% of all strains). In some countries where combined FUT2/FUT3-deficient phenotypes are more frequent (up to 50% in some African countries and 40% in some South American countries) these strains are less often encountered (Desselberger Virus Res 190: 75, 2014).
Two live vaccines are presently licensed. In both instances, their VP8* corresponds to a P variant. Since the carbohydrate specificity of VP8* determines resistance or sensitivity to severe RVA gastroenteritis, we hypothesize that FUT2- and/or FUT3-deficient individuals should not be infected (or only poorly) by the vaccine strains. This would contribute to explain the lack of efficacy of the vaccines in a minor fraction of children from high-income countries and in a very significant fraction of children from several African and South-American countries (Glass et al J Infection 68: S9, 2014).
One of our goals is to put this hypothesis to the test through studies of genetic epidemiology from both France and from tropical areas as well as through the outcome of vaccine according to childrens’ HBGA status. If our work can support the hypothesis, it would point to the necessity of generating new vaccines for these countries that contain adapted VP8* in the case of live attenuated vaccines, or to a shift toward non live vaccines.
Another goal is to understand where in the infection process by human RVAs the HBGA intervene using appropriate in vitro experimental models.
Relationship with the Microbiote
We have seen above that in human, terminal sugar units of epithelial glycans are polymorphic, defining HBGAs. We suggested that this polymorphism is the result of a co-evolution between our species and enteric pathogens. It is likely that the microbiote had to adapt to this terminal glycosylation diversity. Binding to the polymorphic HBGAs through specific adhesins has been documented for various pathogenic bacteria such as Campilobacter jejuni, Escherichia coli, Salmonella enterica and Helicobacter pylori strains (Cooling, Clin Microbiol Rev 28: 801, 2015). In order to strive in a host gut, nonpathogenic bacteria of the microbiota, like pathogenic bacteria, likely use adhesins that may show HBGA specificities such as the recently described HBGA-specific adhesin-like protein from a strain of Lactobacillus (Watanabe et al J Appl Microbiol 109: 927, 2010). They also use glycosidases that permit degradation of the glycan cover into monosaccharides used as carbon sources (Koropatkin Nat Rev Microbiol 10: 323, 2012). Distinct sets of glycosidases should be required to degrade host glycans depending on the HBGA polymorphism. The human gut microbiota composition strongly influences the development of immunity. Intriguing associations were recently reported between FUT2 and ABO polymorphisms and the composition of Bifidobacteria or Clostridium bacteria, respectively in the human gut (Mäkivuoko BMC Microbiol 12: 94, 2012). On the other hand the susceptibility or resistance to type 1 diabetes or Crohn’s disease is strongly influenced by FUT2 (Rausch et al PNAS 108: 19030, 2011; Smyth et al Diabetes 60: 3081, 2011). This suggests that HBGAs polymorphisms might influence the composition of the gut microbiota, which in turn affects susceptibility to inflammatory diseases. In other words, our working hypothesis is that pathogens such as enteric viruses, and possibly some pathogenic bacteria, contributed to select and maintain common HBGA polymorphisms that provide innate protection at the species level. The microbiota may then have adapted to this polymorphism of epithelial glycans, by generating variable composition associated with distinct susceptibility to inflammatory diseases.
We therefore aim at uncovering a connection between the gut microbiota, the combined genetic polymorphisms at the ABO, FUT2 and FUT3 loci and the presence of DP8α regulatory T cells.