Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

Abstract

ABSTRACT The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H 2 O 2 ) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H 2 O 2 . Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H 2 O 2 , whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H 2 O 2 . The katB mutant, lacking the H 2 O 2 -inducible catalase KatB, was similar to the wild-type strain with respect to H 2 O 2 resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H 2 O 2 , while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H 2 O 2 . Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H 2 O 2 , suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB :: lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H 2 O 2 , while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H 2 O 2 , particularly at high H 2 O 2 concentrations; KatB is induced in both planktonic and biofilm cells in response to H 2 O 2 insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H 2 O 2 are sublethal.