Identification of SlpB, a Cytotoxic Protease from Serratia marcescens
The Gram-negative bacterium and opportunistic pathogenSerratia marcescenscauses 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 cellsin vitrowas determined. Deletion ofprtSin 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 theS. marcescensDb11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here asslpB,slpC, andslpD. Induced expression ofprtSandslpB, but notslpCandslpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, onlyprtSinduction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of bothprtSandslpBgenes 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 linesin 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 fromS. marcescens.