Role of lipid rafts and flagellin in invasion of colonic epithelial cells by Shiga-toxigenic Escherichia coli O113:H21.
Shiga-toxigenic Escherichia coli (STEC) O113:H21 strains that lack the locus of enterocyte effacement (LEE) efficiently invade eukaryotic cells in vitro, unlike LEE-positive O157:H7 strains. We used a fliC deletion mutant of the O113:H21 STEC strain 98NK2 (98NK2ΔfliC) to show that invasion of colonic epithelial (HCT-8) cells is heavily dependent on production of flagellin, even though adherence to the cells was actually enhanced in the mutant. Flagellin binds and signals through Toll-like receptor 5 (TLR5), but there was no evidence that either TLR5, the adaptor protein myeloid differentiation primary response gene 88 (MyD88), or the serine kinase interleukin-1 receptor-associated kinase (IRAK) were required for invasion of HCT-8 cells by strain 98NK2, as judged by transfection, RNA knockdown, or inhibitor studies. However, pretreatment of cells with anti-asialo-GM1 significantly decreased 98NK2 invasion (by 40.8%), while neuraminidase treatment (which cleaves terminal sialic acid residues, thus converting GM1 into asialo-GM1) significantly increased invasion (by 70.7%). Pretreatment of HCT-8 cells with either the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD) or the tyrosine kinase inhibitor genistein significantly decreased invasion by 98NK2, indicating a potential role for lipid rafts in the invasion mechanism. Confocal microscopy also showed invading 98NK2 colocalized with lipid raft markers caveolin-1 and GM1. Interestingly, anti-asialo-GM1, neuraminidase, MβCD, and genistein have similar effects on the vestigial level of STEC invasion seen for STEC strain 98NK2ΔfliC, indicating that lipid rafts mediate a common step in flagellin-dependent and flagellin-independent cellular invasion.
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
Trisha J
TJ
Rogers
Cheleste M
CM
Thorpe
Adrienne W
AW
Paton
James C
JC
Paton
D045744:Cell Line, Tumor; D003106:Colon / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology*; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006801:Humans; D021962:Membrane Microdomains / Q000502:physiology*; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051198:Toll-Like Receptor 5 / Q000378:metabolism
D034741:RNA, Small Interfering; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005408:Flagellin; D005677:G(M1) Ganglioside