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R. M. Shanks, E. J. Kalivoda, N. A. Stella; cAMP-Phosphodiesterase Activity Mediates Bacterial Biofilm Formation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):847.
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Pseudomonas aeruginosa (PA) and Serratia marcescens (SM) are opportunistic pathogens that cause keratitis. The formation of biofilms on 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 SM 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 bacterial biofilm formation through degradation of cAMP.
The cpdA gene from Escherichia coli (EC), which codes for a known cAMP-PDE and uncharacterized homologs from SM (SMA3506) and PA (PA4969) were cloned on a multicopy plasmid under transcriptional control of the Plac promoter. Site directed mutagenesis of the SM cpdA homolog and addition of C-terminal histidine tags generated with yeast recombineering methods. Recombinant proteins were purified from EC using metal-affinity chromatography. PDE 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 crystal violet. IBMX was used as a PDE inhibitor.
Purified CpdA homologs from SM and PA exhibited cAMP-PDE activity. Multicopy expression of novel cpdA homologs from SM and PA, and the known EC CpdA conferred dramatic increases in SM biofilm formation. The mechanism for elevated biofilm formation was determined to be an increase in surface adhesins coded by the fimABCD genes for SM. Similar, type IV pili-dependent biofilm stimulation results were observed with PA. Levels of PDE activity of purified proteins could be inhibited by IBMX in a dose dependent manner in vitro. IBMX exhibited inhibition of biofilm formation.
These data suggest a novel role for cAMP-PDE activity in biofilm formation. Previously uncharacterized genes from SM and PA were shown to code for cAMP-PDEs that can impact biofilm formation by ocular pathogens. PDE inhibitors have potential in preventing biofilm formation.
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