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2014 Agenda and Abstracts | < Previous | Next >

2014 OMIG Abstract 2

Identification of a Novel Cytotoxic Protease and Characterization of Secreted
Proteases by Keratitis Isolates of Serratia marcescens

RM Shanks, NA Stella, KM Hunt, KM Brothers, P Thibodeau
The Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, PA

Purpose: To characterize secreted cytotoxic protease production by ocular isolates of S. marcescensSerratia species are the most frequently isolated genus of the Enterobacteriaceae that cause ocular infections. Systematic characterization of protease production by ocular isolates has not been reported, and only one secreted metalloprotease gene, the serralysin gene, has been cloned. 

Methods: S. marcescens from conjunctivitis (n=7), endophthalmitis (n=2), and keratitis (n=35) were obtained from the Campbell Laboratory and tested for secreted protease activity with milk agar plates. Protease activity and cytotoxicity of secreted fractions to a human corneal limbal epithelial cell line (HCLE) was measured using azocasein and resazurin. The K904 keratitis isolate genome was sequenced. Candidate protease genes were cloned using a yeast-based recombineering system. Quantitative real time reverse transcriptase PCR was performed using standard techniques and SYBR Green.

Results: The majority (93%) of S. marcescens ocular isolates produced protease activity on milk plates, and there was a significantly (p<0.05) higher occurrence of protease activity associated with keratitis isolates compared to conjunctivitis isolates. Most clinical isolates produced high levels of protease and there was a positive correlation between secreted protease activity and cytotoxicity to HCLEs. Genomic analysis revealed the presence of 4 putative metalloprotease genes. Induction of the known serralysin gene and one of the putative protease genes in a laboratory strain produced toxicity to epithelial cells in vitro and measurable secreted protease activity. This novel protease gene was notated here as slpB for serralysin-like protease B. Mutation of slpB in strain K904 reduced cytotoxicity to HCLE monolayers in vitro.

Conclusions: We provide novel data on protease production by ocular isolates of S. marcescens and identify a new cytotoxic protease that may contribute to keratitis.

Disclosure: S= NIH grants: AI085570, EY08098, EY017271, Eye and Ear Institute of Pittsburgh,
Research to Prevent Blindness

2014 Agenda and Abstracts | < Previous | Next >