Identification of a Putative Transcription Factor From Serratia marcescens That Regulates Metalloprotease and Hemolysis Activity

Abstract

Purpose: : Serratia marcescens

Methods: : Random mutagenesis of human clinical isolates of S. marcescens was performed using a mariner-based transposon. Mutants defective in extracellular proteolysis production were tested for hemolysin production on blood agar plates. Proteolysis was further characterized with zymogram analysis using casein as a substrate, and by immunoblots with antibodies that detect a serralysin. Transposons were mapped using arbitrary PCR. Directed mutagenesis was performed using two-step allelic replacement. Bacterial keratitis studies were performed using New Zealand White rabbits. Clinical signs were graded in a masked fashion by an ophthalmologist using a slit-lamp, and colony counts were determined from corneas. Transcription of eepR was determined using a chromosomal lacZ fusion. The CRP protein was purified using IMAC, and EMSA assays were performed using a commercial kit and biotin-labeled PCR-primers.

Results: : Multiple genes were found that altered protease/hemolysis production. Several mutant strains were screened in an animal model of keratitis (n=2-3 eyes per mutant strain). One mutant defective in extracellular protease/hemolysis production and attenuated in the animal keratitis experiment mapped to a novel putative two-component transcriptional regulatory protein. This uncharacterized gene was named eepR for exoenzyme and pigment regulator. Site directed mutagenesis of eepR in several clinical keratitis isolates confirms its role in serralysin production and secreted hemolysis activity. A CRP-binding site was found upstream of the eepR gene. Transcriptional analysis and EMSA studies indicate that eepR is regulated by the global transcription factor CRP. Preliminary pathogenesis studies support EepR as required for bacterial keratitis.

Conclusions: : EepR is the first identified regulator of serralysin, and may be a novel regulator of S. marcescens virulence.

Keywords: microbial pathogenesis: experimental studies • microbial pathogenesis: clinical studies • transcription factors