Abstract
Purpose: :
Serratia marcescens is an opportunistic bacterium capable of causing sight-threatening keratitis and other eye infections. Biofilm formation and exoenzyme production are major components in S. marcescens pathogenesis, and these processes are highly influenced by carbon source availability. This study describes the role of glucose and the cyclic nucleotide cAMP in regulation of S. marcescens biofilm formation and exoenzyme production.
Methods: :
Directed mutations were made in the chromosome of S. marcescens by homologous recombination. Biofilms from mutant and wild-type strains were formed under high sheer conditions and levels were determined using crystal violet (CV) staining and confocal laser scanning microscopy. Transmission electron microscopy was used to assess surface adhesin production. Quantitative RT-PCR (qPCR) was used to assay transcription. Cytotoxicity was measured by mixing spent culture supernatant with either Saccharomyces cerevisiae or primary rabbit corneal epithelial cells. Viability was determined by colony counts with yeast cells or with vital stains for epithelial cells. Intracellular levels of cAMP were determined by ELISA assay. Proteolysis and hemolysis were measured by agar plate assays using casein or sheep red blood cells as indicators.
Results: :
Growth in glucose-rich medium or mutations that either reduce cAMP levels (cyaA or crr) or the ability to respond to cAMP (crp) dramatically increased the biofilm capacity of S. marcescens. These data contrast with previous reports on E. coli and other enteric bacteria. The mechanism of increased biofilm formation was determined using a genetic screen. Suppressor mutations of the hyper-biofilm phenotype of crp mutants mapped to the fimABCD operon, which codes for a large surface adhesin required for attachment to corneal cells. Mutation of fimC eliminated the hyper-biofilm phenotype of cyaA, crr and crp mutants, demonstrating the requirement of the fimABCD encoded adhesin in biofilm formation. Electron microscopy and qPCR indicate that fimABCD is elevated in cyaA and crp mutants. Proteolysis, hemolysis, and cytotoxicity were elevated by mutation of cyaA and crp.
Conclusions: :
Our data supports a model in which cAMP controls multiple factors that may contribute to S. marcescens virulence. Mutation of the cyaA and crp genes led to elevated exoenzyme production and cytotoxicity. Ongoing studies will determine the role of cAMP-dependent regulation of S. marcescens pathogenesis using rabbit keratitis models.
Keywords: bacterial disease • contact lens • keratitis