Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies.
TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies. In contrast, OprF derivatives with predicted fusion joints at amino acids 180, 204, 289, and 299 reacted with one to five of the monoclonal antibodies. Four other immunoreactive OprF derivatives were created by subcloning and encoded amino acids 1 to 187, 188 to 326, 1 to 273 and 1 to 170 plus 301 to 326. On the basis of reactivity with the TnphoA-truncated derivatives and subclones of oprF, the epitopes for all 10 monoclonal antibodies were localized, in part, to specific regions of OprF. Nine of the 10 monoclonal antibodies, 8 of which recognize surface-exposed epitopes, mapped within the carboxy-terminal region of OprF that is homologous to the Escherichia coli outer membrane protein OmpA. Thus, we concluded that parts of the carboxy terminus of OprF are exposed on the external face of the outer membrane. In addition, a clone containing only the first two cysteine residues of OprF demonstrated reactivity with monoclonal antibodies MA4-4 and MA7-8 that was destroyed by 2-mercaptoethanol treatment, as was reactivity with intact OprF. Thus, we conclude that this first pair of cysteines at residues 176 and 185 of mature OprF form a disulfide bond.
Finnen
R L
RL
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Martin
N L
NL
Siehnel
R J
RJ
Woodruff
W A
WA
Rosok
M
M
Hancock
R E
RE
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D004251:DNA Transposable Elements; D000939:Epitopes / Q000032:analysis; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D000939:Epitopes