Disease relevance of oprF

• The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb HincII kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene [1].
• Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene [2].
• Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa [3].
• In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus [4].
• A parallel study performed with the whole oprF gene showed a lack of specificity of this probe: indeed, the probe hybridized not only with the 42 Pseudomonas aeruginosa strains but also with Escherichia coli and Salmonella minnesota [5].

High impact information on oprF

• One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector [6].
• Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process [7].
• The outer membrane of G49 was shown to lack OprF, suggesting that loss of this protein may be involved in the multiple antibiotic resistance phenotype; however, when G49 was transformed with a plasmid encoding oprF (pRW5), expression of oprF was shown to have no effect upon the phenotype [8].
• To develop the oprF gene as a carrier for foreign epitopes, linker insertion mutagenesis has been performed to introduce 12 nucleotide inserts marked by a unique PstI site [9].

Regulatory relationships of oprF

• Challenge with P. aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide-KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone [4].

Analytical, diagnostic and therapeutic context of oprF

• The oprF gene was cloned into plasmid vector pVR1020, and the plasmid vaccines were delivered to mice by biolistic (gene gun) intradermal inoculation [10].
• The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe [2].
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it [3].
• For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes [11].

References