June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Identification and characterization of a novel microbial virulence-associated transcription factor and its role in ocular infection
Author Affiliations & Notes
  • Robert M Q Shanks
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Nicholas A Stella
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Kimberly M Brothers
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Kristin M Hunt
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Xinu Liu
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Eric G Romanowski
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Regis P Kowalski
    Chemistry, University of Pittsburgh, Pittsburgh, PA
  • Footnotes
    Commercial Relationships Robert Shanks, None; Nicholas Stella, None; Kimberly Brothers, None; Kristin Hunt, None; Xinu Liu, None; Eric Romanowski, None; Regis Kowalski, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4844. doi:https://doi.org/
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      Robert M Q Shanks, Nicholas A Stella, Kimberly M Brothers, Kristin M Hunt, Xinu Liu, Eric G Romanowski, Regis P Kowalski; Identification and characterization of a novel microbial virulence-associated transcription factor and its role in ocular infection. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4844. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To identify bacterial regulators necessary for microbial keratitis. Bacterial keratitis progresses rapidly and can cause vision loss even with effective antibiotics. An unknown gene was isolated in genetic screens. Its role in controlling virulence factor production, cytotoxicity to human corneal cells, corneal wound healing, and keratitis was determined.

Methods: Molecular genetic, biochemical, RNA-Seq, proteomic, and metabolomic approaches were used to characterize an eepR mutant in a keratitis isolate of the bacteria Serratia marcescens. HCLE cells were used in cytotoxicity assays and cell migration assays. Pig eyes were used for corneal organ culture, and NZW rabbits were used for keratitis studies.

Results: Transposon mutations were isolated in an uncharacterized gene in a screen for genes involved in secretion of protease and hemolysin activity using S. marcescens. Targeted deletion of eepR in clinical and laboratory strains and complementation analysis confirmed the importance of EepR in transcription of metalloprotease and hemolysin genes. EMSA analysis confirmed a direct binding of EepR to the hemolysin gene promoter, but not the protease gene promoter. Proteomic analysis supported the importance of EepR in protease secretion. Mass-spec and metabolomic analysis confirmed loss of hemolysin production in the eepR mutant. Bacteria lacking the eepR gene lost cytotoxicity to HCLE cells. This defect could be restored by expression of the metalloprotease gene in the eepR mutant. The eepR mutant was unable to inhibit corneal cell migration and wound healing. In a rabbit keratitis model, EepR was necessary for proliferation of the bacteria with a significant reduction in CFU recovered from corneas infected with the eepR mutant compared to the parental strain.

Conclusions: These results indicate EepR is a novel bacterial transcription factor required for expression of secreted protease and hemolysin activity from ocular clinical bacterial isolates. The EepR protein regulates cytotoxicity through protease regulation and host-pathogen interactions through a yet unknown mechanism. EepR is required for bacterial proliferation in mammalian corneas. Further studies are required to determine the mechanism behind EepR’s role in proliferation and corneal wound healing inhibition.

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