title: A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.
abstract: We sought to develop a practical and representative model to study the interactions of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) with human intestinal tissue. For this purpose, human intestinal epithelial HCT-8 cells were cultured under low-shear microgravity conditions in a rotating cell culture system. After 10 days, layered cell aggregates, or 'organoids', developed. Three lines of evidence indicated that these organoids exhibited traits characteristic of normal tissue. First, the organoids expressed normal intestinal tissue markers in patterns that suggested greater cellular differentiation in the organoids than conventionally grown monolayers. Second, the organoids produced higher levels of intestinally expressed disaccharidases and alkaline phosphatase on a cell basis than did conventionally cultured monolayers. Third, HCT-8 organoid tissue developed microvilli and desmosomes characteristic of normal tissue, as revealed by electron microscopy. Because the low-shear microgravity condition is proposed by modelling studies to more closely approximate conditions in the intestinal microvilli, we also tested the impact of microgravity of bacterial growth and virulence gene expression. No influence on growth rates was observed but intimin expression by EHEC was elevated during culture in microgravity as compared with normal gravity. That the responses of HCT-8 organoids to infection with wild-type EPEC or EHEC under microgravitational conditions approximated infection of normal tissue was demonstrated by the classical appearance of the resultant attaching and effacing lesions. We concluded that the low shear microgravity environment promoted growth of intestinal cell organoids with greater differentiation than was seen in HCT-8 cells maintained in conventional tissue culture and provided a reduced gravity environment for study of bacterial-host cell interactions.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
authors.forename: Humberto M
authors.initials: HM
authors.lastname: Carvalho
authors.forename: Louise D
authors.initials: LD
authors.lastname: Teel
authors.forename: Gertrud
authors.initials: G
authors.lastname: Goping
authors.forename: Alison D
authors.initials: AD
authors.lastname: O'Brien
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism; D001422:Bacterial Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor / Q000382:microbiology / Q000648:ultrastructure; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics; D006801:Humans; D018472:Hypogravity; D008854:Microscopy, Electron; D009154:Mutation; D014774:Virulence / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; C104901:BfpA protein, E coli; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli; D036781:Fimbriae Proteins