Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.
Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.
Pramoonjago
P
P
Department of Bacteriology, Osaka University Medical School, Japan.
Kaneko
M
M
Kinoshita
T
T
Ohtsubo
E
E
Takeda
J
J
Hong
K S
KS
Inagi
R
R
Inoue
K
K
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D001770:Blood Bactericidal Activity; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003183:Complement C6 / Q000502:physiology; D004926:Escherichia coli / Q000276:immunology* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006461:Hemolysis; D008969:Molecular Sequence Data; D011815:R Factors
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D003176:Complement C3; D003183:Complement C6; D029968:Escherichia coli Proteins; C036297:TraT protein, E coli; D050577:Complement C3-C5 Convertases