OMIG, Abstract 8
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cAMP-phosphodiesterase activity mediates bacterial biofilm formation
Robert M. Q. Shanks,Eric J. Kalivoda, Nicholas A. Stella, James E. Fender
Campbell Laboratory, Department of Ophthalmology, University of Pittsburgh
Purpose: Serratia marcescens is an opportunistic pathogen that is emerging as a causative agent of keratitis. The formation of biofilms on soft contact lenses and lens cases likely plays a role in keratitis. Furthermore, biofilm formation is one way that bacteria resist antibiotics and the immune system. Previous studies have shown that Serratia marcescens regulates biofilm formation through positive regulators of cAMP production and the cAMP receptor protein, CRP. The purpose of this study is to test the hypothesis that cAMP-phosphodiesterase (cAMP-PDE) activity would regulate biofilm formation through degradation of cAMP.
Methods:The cpdA gene from E. coli, which codes for a known cAMP-PDE and uncharacterized homologs from S. marcescens (SM3506) and P. aeruginosa (PA4969)were cloned on a multicopy plasmid under transcriptional control of the Plac promoter. Site directed mutagenesis of the S. marcescens cpdA homolog and addition of C-terminal histidine tags were performed by yeast in vivo recombination. Recombinant proteins were purified from E. coli using metal-affinity chromatography. Phosphodiesterase activity from crude lysates and purified proteins were performed using bis-pNPP and cAMP as substrates. Biofilms were formed under high sheer conditions in LB broth and were stained with 0.1% crystal violet for visualization.
Results:Multicopy expression of a bona fide cAMP-PDE (cpdA) from E. coli in S. marcescens lead to a dramatic increase in S. marcescens biofilm formation, whereas mutant versions of cpdA that lack PDE activity did not support hyper-biofilm production. Novel cpdA homologs from both S. marcescens and P. aeruginosa conferred similar increases in biofilm formation. Purified CpdA from S. marcescens and P. aeruginosa exhibited phosphodiesterase activity. The mechanism for elevated biofilm formation was determined to be an increase in surface fimbriae coded by the fimABCD genes.
Conclusions:These data suggest a novel role for cAMP-PDE activity in biofilm formation. Previously uncharacterized genes from S. marcescens and P. aeruginosa were shown to code for phosphodiesterases that can impact biofilm formation in S. marcescens. Together these data suggest that cAMP-PDE inhibitors have potential in preventing biofilm formation.
Supported by the Campbell Laboratory and NEI core grant EY08098
Disclosure code: N