The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes
ocular infections in healthy individuals. Secreted
protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of
protease-positive strains was observed among
keratitis isolates than among
conjunctivitis isolates. A positive correlation between
protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical
keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted
protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted
protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for
serralysin-like
proteases noted here as slpB,
slpC, and slpD. Induced expression of prtS and slpB, but not
slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung
carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted
protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated
calcium-dependent and AprI-inhibited
protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the
type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic
protease from S. marcescens.