Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells
Aufaugh Emam 1, William G Carter 2, Clifford Lingwood 1, 3, *
1 Molecular Structure and Function, The Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
2 Fred Hutchinson Cancer Research Centre, Seattle Wa. USA Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
3 Departments of Laboratory Medicine & Pathobiology, and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed
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* Address correspondence to this author at the Molecular Structure and Function, The Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Tel: 416 813 5998; Fax: 416 813 5993; E-mail: firstname.lastname@example.org