Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein.
In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.
Koga
Hisashi
H
Department of Tumor Genetics and Biology, Graduate School of Medical Science Kumamoto University, Kumamoto, Japan. hkoga@kazusa.or.jp
Izawa
Ichiro
I
Araki
Norie
N
Saya
Hideyuki
H
Mimori
Tatsuyuki
T
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003593:Cytoplasm / Q000737:chemistry; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D047468:Immunoprecipitation; D007456:Iodine Isotopes; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015011:Yersinia pseudotuberculosis / Q000235:genetics*
D018829:Adhesins, Bacterial; D007456:Iodine Isotopes; D011993:Recombinant Fusion Proteins; C053476:invasin, Yersinia