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2008 OMIG, Abstract 9

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A genetic screen identified novel regulators of Serratia marcescens virulence factor production.
Eric J. Kalivoda, Nicholas A. Stella, Robert M. Q. Shanks,
Campbell Laboratory, Department of Ophthalmology, University of Pittsburgh

Purpose:The major metalloprotease and a pore forming hemolysin are the major known virulence factors of S. marcescens, however little is known about their regulation.  A genetic approach was taken to identify genes required for production of virulence factors by a model Gram-negative opportunistic pathogen that causes microbial keratitis
Methods:Random transposon mutagenesis of a pigmented laboratory strain and a human clinical isolate of Serratia marcescens was performed using a mariner-based transposon.  Bacteria were plated on protease indicator plates and bacteria unable to produce zones of proteolysis were selected for further analysis.  Proteolysis was further characterized with zymogram analysis using casein as a substrate, and by immunoblots with antibodies that detect a major metalloprotease.  Hemolysis was determined using blood agar plates.  Pigment production was determined by extraction with acidified ethanol and quantification by absorbance at 534 nm.  Transposons were mapped using arbitrary PCR.  Directed mutagenesis was utilized to verify the importance of candidate genes in proteolysis.  Cytopathogenic studies were done using Chang conjunctival cells and primary rabbit corneal epithelial cells.
Results: Protease negative mutants mapped to a predicted major metalloprotease gene and to secretion system components known to be required for protease secretion verifying the screen.  Other mutations mapped to both genes in a predicted two-component transcriptional regulator system.  Mutation of these genes also conferred a loss of hemolysis and pigment production.  These uncharacterized genes were named eepR and eepS for exoenzyme and pigment regulator and sensor, respectively.   Site directed mutagenesis of eepR confirms its role in exoenzyme production and pigment production.  Mutation of eepR attenuates cytopathogenic effects of secreted fractions on mammalian conjunctival and corneal cells. 
Conclusions:These data suggest that EepR and EepS are novel regulators of virulence factor production by S. marcescens.

Supported by the Campbell Laboratory and NEI core grant EY08098
Disclosure code: N

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