Ocular Microbiology and Immunology Group
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2016 OMIG Abstract 14

Serratia marcescens Induced Corneal Epithelial Cell Blebs are Mediated by
a Type V Secretion System
Robert MQ Shanks, JM Bachinsky, KM Brothers, NA Stella, RP Kowalski, KM Hunt, JD Callaghan

Purpose: The objectives of this study were to analyze the interactions between ocular surface cells and contact-lens associated bacterial pathogens. Specifically, we analyzed how the bacterium Serratia marcescens damages corneal cells.

Methods: Ocular clinical and other isolates were exposed to human corneal epithelial cells and other ocular cells at a multiplicity of infection of 10-200, stained with Calcein AM to assess viability and analyzed by confocal laser scanning microscopy and differential interference microscopy to assess morphological changes.  Presto Blue was used as a second approach to analyze viability. Scanning electron microscopy was used with porcine corneal organ culture exposed to contact lenses colonized by S. marcescens.  Mutagenesis was performed using a mariner-based transposon, or gene deletion by allelic replacement.  Genes were cloned using yeast-based recombineering for complementation analysis.

Results: Membrane blebs were observed on porcine ocular surface cells and primary human corneal epithelial cells exposed to S. marcescens and Proteus mirabilis. 100% (n=34) of isolates of S. marcescens induced massive membrane blebs in corneal epithelial and airway epithelial cell lines. Membrane blebs correlated with a loss of cell viability. Mutations in a type V secretion system rendered the bacteria unable to cause blebs and damage the epithelial cells. Complementation analysis confirmed the role of this type V secretion system. Mutation of an analogous type V secretion system in keratitis isolates of P. mirabilis also rendered the bacterium unable to produce membrane blebs.

Conclusions: Little is known about why contact lens is a risk factor for microbial keratitis or what causes sterile infiltrates. This study demonstrates damage to ocular surface cells by a bacterial type V secretion system mediated mechanism. This phenomenon may allow contact lens associated pathogens to bypass the epithelium and cause keratitis or lead to infiltrates.

S= Eye and Ear Institute of Pittsburgh, NIH grant EY08098, NIH grant AI085570

2016 Agenda and Abstracts | < Previous | Next >