Exotoxin A, one of the principal virulence factors produced by Pseudomonas aeruginosa, is an ADP-ribosyl transferase. An opportunistic pathogen, P. aeruginosa causes severe and often fatal infections in patients with cystic fibrosis, severe bums, and leukemia or lymphomas. In preliminary studies, several putative exotoxin A regulatory mutants were characterized. Our hypothesis is that they contain mutations in uncharacterized genes which are part of a regulatory cascade controlling expression of exotoxin A (toxA). Such regulatory mechanisms allow pathogenic microorganisms to respond rapidly environmental changes such as the shift from the external environment to the host organism they are invading. In addition, the promoter of the toxA gene is especially unusual because of its high guanosine plus cytosine content and its extreme bias for pyrimidines on one strand. Thus, we hypothesize that description of toxA occurs by a novel and previously unrecognized mechanism. An exotoxin A regulatory gene, designated vfr, has been cloned by complementation of the mutations in P. aeruginosa PA103-15, PA103-16, and PA103-19. These mutants are deficient in production of exotoxin A, protease, and several extracellular proteins and are altered in expression of a positive regulator of toxA expression. The nucleotide sequence of the vfr gene will be determined. Isogenic mutants will be constructed by insertional mutagenesis and gene replacement techniques. The effect of the in-vitro constructed vfr mutation on expression of toxA and other virulence factors will be evaluated. Possible roles for the vfr gene in regulation of toxA aeruginosa expression which will be investigated are: as an alternative sigma factor, as a transcriptional activating factor required to initiate transcription from the exceedingly GC rich toxA and regA promoters, as a stone-like protein, or as a member of a two-component regulatory system which responds to specific mental signals. The regulation of vfr by various environmental stimuli will also be examined using v promoter-lacZ reporter plasmid. The proposed research will provide the .vfr information about e regulation of P. aeruginosa virulence factors, will provide basic knowledge about gene expression in P. aeruginosa, and may provide insight as to the global mechanisms involved in sensing and adapting to specific growth conditions. In addition, the information gained from these studies could lead to the development of novel therapeutic approaches against P. aeruginosa.
