Abstract
The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene-dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.
Highlights
The successful generation of human mini-guts cultured ex vivo [1], known as human intestinal enteroids (HIEs) or organoids, has boosted a large number of studies of physiology and Research Center, NIAID, National Institutes of Health, Bethesda, Maryland, USA
Exploiting this significant advancement, we have conducted a study of lipid and sphingolipid compositions of seven HIE cultures and characterized the interaction between their lipid membranes and human norovirus (HuNoV) virus-like particles (VLPs)
HIE lines were phenotyped by ELISAs and genotyped by PCR amplification of ABO, FUT2, and FUT3 genes followed by DNA sequencing to determine their histo-blood group antigen (HBGA) status
Summary
The successful generation of human mini-guts cultured ex vivo [1], known as human intestinal enteroids (HIEs) or organoids, has boosted a large number of studies of physiology and Research Center, NIAID, National Institutes of Health, Bethesda, Maryland, USA. One remarkable finding is the ability of these nontransformed human cultures to support replication and recapitulate unique aspects of infection of previously noncultivatable pathogens, such as human noroviruses [10], cryptosporidium [11, 12], and Salmonella Typhi [13] Exploiting this significant advancement, we have conducted a study of lipid and sphingolipid compositions of seven HIE cultures and characterized the interaction between their lipid membranes and human norovirus (HuNoV) virus-like particles (VLPs). These HIEs uniquely represent individuals with different ABO, Lewis, and secretor histo-blood group geno- and phenotypes with varying susceptibilities to human HuNoV infection [10, 14]. It is important to characterize the lipid composition of a series of HIEs and their individual interactions with HuNoV VLPs as a model to compare the differences between susceptible and resistant enteroid cultures
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