Adherence to and invasion of Vero cells by recombinant Escherichia coli expressing the outer membrane protein rOmpB of Rickettsia japonica.
Recombinant Escherichia coli expressing the outer membrane protein rOmpB of rickettsiae on the surface were generated. The DNA corresponding to the open reading frame of the ompB gene of a spotted fever group rickettsia, Rickettsia japonica, was amplified by polymerase chain reaction. The amplified fragment was inserted between the Sal I and the Xho I sites of the expression vector pET-22b(+). E. coli BL21(DE3) was transformed by the constructed plasmid. The recombinant bacteria expressed a recombinant protein with a molecular size of 165 kilodaltons on the surface. The size was consistent with that of the precursor of rOmpB. The protein was reactive with monoclonal antibodies to heat-labile epitopes of the rOmpB. This result suggested a rather native conformation of the recombinant protein. Immunofluorescence of the recombinant bacteria demonstrated the surface expression of the protein. The recombinant bacteria acquired properties to enter Vero cells. The morphological change was observed by means of transmission electron microscopy and scanning electron microscopy. Adherence triggered the generation of abundant microvilli and membrane ruffling for the cells to engulf the bacteria The manner of entry of the recombinant bacteria was similar to that of rickettsiae. Thus it is suggested that the rOmpB plays an important role in the adherence to and invasion of host cells by rickettsiae. Moreover, since even the recombinant rOmpB precursor protein expressed on the surface of the bacteria promotes adherence and invasion, the conformation of the functional domain may be similar to that of the processed mature rOmpB.
Uchiyama
Tsuneo
T
Department of Virology, University of Tokushima Graduate School of Medicine, Kuramoto 3-18-15, Tokushima 770-8503, Japan. uchiyama@basic.med.tokushima-u.ac.jp
D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012281:Rickettsia / Q000472:pathogenicity / Q000502:physiology* / Q000648:ultrastructure; D014709:Vero Cells
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D011994:Recombinant Proteins