Uptake of Shiga-toxigenic Escherichia coli SubAB by HeLa cells requires an actin- and lipid raft-dependent pathway.
The novel cytotoxic factor subtilase cytotoxin (SubAB) is produced mainly by non-O157 Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves the molecular chaperone BiP/GRP78 in the endoplasmic reticulum (ER), leading to activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK), followed by caspase-dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB-induced BiP cleavage was inhibited by high concentrations of Dynasore, and methyl-β-cyclodextrin (mβCD) and Filipin III, but not suppressed in clathrin-, dynamin I/II-, caveolin1- and caveolin2-knockdown cells. We observed that SubAB treatment led to dramatic actin rearrangements, e.g. formation of plasma membrane blebs, with a significant increase in fluid uptake. Confocal microscopy analysis showed that SubAB uptake required actin cytoskeleton remodelling and lipid raft cholesterol. Furthermore, internalized SubAB in cells was found in the detergent-resistant domain (DRM) structure. Interestingly, IPA-3, an inhibitor of serine/threonine kinase p21-activated kinase (PAK1), an important protein of macropinocytosis, directly inhibited SubAB-mediated BiP cleavage and SubAB internalization. Thus, our findings suggest that SubAB uses lipid raft- and actin-dependent, but not clathrin-, caveolin- and dynamin-dependent pathways as its major endocytic translocation route.
Department of Legal Medicine, Graduate School of Medicine, Chiba University, Chiba, 260-8670, Japan.
Sayaka
S
Nagasawa
Kohei
K
Ogura
Hiroyasu
H
Tsutsuki
Hisako
H
Saitoh
Joel
J
Moss
Hirotaro
H
Iwase
Masatoshi
M
Noda
Kinnosuke
K
Yahiro
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology; D001692:Biological Transport; D022461:Caveolins / Q000235:genetics; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000235:genetics; D004220:Disulfides / Q000494:pharmacology; D034281:Dynamins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005372:Filipin / Q000494:pharmacology; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D021962:Membrane Microdomains / Q000378:metabolism*; D009284:Naphthols / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D054323:Shiga-Toxigenic Escherichia coli / Q000472:pathogenicity*; D013381:Subtilisins / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019892:eIF-2 Kinase / Q000378:metabolism; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors
D000998:Antiviral Agents; D022461:Caveolins; D002966:Clathrin; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D006835:Hydrazones; C541795:IPA-3 compound; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D009284:Naphthols; D034741:RNA, Small Interfering; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005372:Filipin; D002784:Cholesterol; C460640:EIF2AK3 protein, human; D019892:eIF-2 Kinase; D054462:p21-Activated Kinases; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli; D034281:Dynamins