Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.
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.
The Charles T. Campbell Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA shanksrm@upmc.edu.
Robert M Q
RM
Shanks
The Charles T. Campbell Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Nicholas A
NA
Stella
The Charles T. Campbell Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Kristin M
KM
Hunt
The Charles T. Campbell Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Kimberly M
KM
Brothers
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Liang
L
Zhang
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Patrick H
PH
Thibodeau
D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects*; D015818:Eye Infections, Bacterial / Q000382:microbiology; D006801:Humans; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D016868:Serratia Infections / Q000382:microbiology; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D037521:Virulence Factors; D010447:Peptide Hydrolases