The role of Vfr and adenylate cyclase in regulating exotoxin a production by pseudomonas aeruginosa

Exotoxin A (ETA) production by Pseudomonas aeruginosa depends on the virulence factor regulator Vfr, a homologue of the Escherichia coli catabolite repressor protein CRP. In E. coli, cyclic AMP activates CRP. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS enhances ETA production through the ETA regulatory gene regA. In this study, we examined the relationship between vfr, pvdS, and regA in regulating ETA production by P. aeruginosa. This was done using mutants defective in vfr, pvdS, regA, or the adenylate cyclase genes cyaA and cyaB (cyaAB); plasmids that individually overexpress these genes; and lacZ transcriptional/translational fusion plasmids. Throughout the growth cycle of PAOÄvfr, ETA concentration and regA expression were significantly reduced but pvdS expression was not affected. The presence of a multicopy vfr plasmid significantly increased ETA production in PAO::pvdS and PAOÄregA, yet overexpression of either regA or pvdS did not enhance ETA production in PAOÄvfr. RT-PCR analysis showed that iron had no effect on the accumulation of vfr mRNA in PAO1. Compared with its parent strain PAK, ETA production by PAKÄcyaAB was significantly reduced. This defect was complemented by a plasmid overexpressing cyaB. Neither regA overexpression nor pvdS overexpression enhanced ETA production by PAKÄcyaAB. However, cyaB overexpression enhanced ETA production in PAOÄpvdS and PAOÄregA. These results suggest that: (1) Vfr may regulate ETA production in P. aeruginosa independently of regA and pvdS; (2) Vfr does not regulate pvdS expression; (3) vfr expression is not regulated by iron; and (4) Vfr activation, by cyclic AMP through CyaB, is important for ETA production.