Bacterial keratitis is a costly and global problem that results in vision loss and blindness, and Serratia marcescens is a leading agent of community-acquired Gram-negative bacterial keratitis. S. marcescens also causes many hospital acquired infections including pneumonia, endocarditis, bacteremia and urinary tract infections that are commonly resistant to current antibiotics and whose outcomes are associated with significant morbidity and mortality. There is a lack of studies that investigate how S. marcescens genes contribute to ocular infections using isogenic mutant strains. The broad long-term objective of this research is to prevent vision loss following corneal infections caused by this organism. A better understanding of the mechanisms by S. marcescens virulence factors are regulated will allow for a novel approach to reduce tissue damage and corneal opacification that results from the expression of these factors. This new knowledge about specific pathways can be used to custom design novel anti-infectives. Establishing new therapeutic targets is becoming ever more important as bacteria continue to develop resistance to existing classes of antibiotics. Our overall specific hypothesis to be tested is that the transcription factor EepR/S is a critical virulence factor that controls expression of tissue damaging metalloprotease and hemolysin activities. Our preliminary data supports that the mutation of EepR/S in S. marcescens severely attenuates the pathogenesis of S. marcescens in an in vivo ocular model of keratitis. Our central hypothesis will be tested by accomplishing the following aims:
Aim 1. Test the hypothesis that EepR/S, four metalloproteases and the ShlA hemolysin are required for S. marcescens cytotoxicity in vitro and ocular pathogenesis in vivo.
Aim 2. Test the hypothesis that EepR and EepS regulate transcription of metalloprotease and hemolysin genes, and that EepR/S is in a regulatory pathway with other transcription factors (crp, hexS and pigP).

Public Health Relevance

Ocular infections caused by bacteria are a common and costly problem in the United States and in both developed and developing nations abroad. Over 100,000 Americans live with vision loss due to corneal infections (keratitis) caused by dangerous bacteria. The bacterium Serratia marcescens is a frequent cause of serious and sometimes fatal hospital acquired infections and vision impairing community-acquired corneal infections. S. marcescens genes that facilitate vision-threatening corneal infections have not been studied to date. This study is designed to determine how S. marcescens is able to successfully infect and damage the cornea. Specifically, we will evaluate the role of secreted proteases, a pore-forming hemolysin, and a newly discovered genetic regulatory system. The answers to these questions will allow for the creation of new drugs to treat S. marcescens ocular infections and reduce infection-associated vision loss.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI085570-04
Application #
8460550
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Korpela, Jukka K
Project Start
2010-05-15
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
4
Fiscal Year
2013
Total Cost
$339,168
Indirect Cost
$106,518
Name
University of Pittsburgh
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Shanks, Robert M Q; Stella, Nicholas A; Brothers, Kimberly M et al. (2016) Exploitation of a "hockey-puck" phenotype to identify pilus and biofilm regulators in Serratia marcescens through genetic analysis. Can J Microbiol 62:83-93
Romanowski, Eric G; Stella, Nicholas A; Brothers, Kimberly M et al. (2016) Predatory bacteria are nontoxic to the rabbit ocular surface. Sci Rep 6:30987
Hover, Tal; Maya, Tal; Ron, Sapir et al. (2016) Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae. Appl Environ Microbiol 82:2585-94
Romanowski, Eric G; Kowalski, Tyler A; O'Connor, Katherine E et al. (2016) The In Vitro Evaluation of Tigecycline and the In Vivo Evaluation of RPX-978 (0.5% Tigecycline) as an Ocular Antibiotic. J Ocul Pharmacol Ther 32:119-26
Zambelli, Alison M; Brothers, Kimberly M; Hunt, Kristin M et al. (2015) Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses. Eye Contact Lens 41:277-80
Mukherjee, Somdatta; Brothers, Kimberly M; Shanks, Robert M Q et al. (2015) Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein. Appl Environ Microbiol 82:1653-61
Brothers, Kimberly M; Stella, Nicholas A; Romanowski, Eric G et al. (2015) EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model. Infect Immun 83:4373-82
Stella, Nicholas A; Lahr, Roni M; Brothers, Kimberly M et al. (2015) Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites. J Bacteriol 197:2468-78
Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M et al. (2015) Identification of SlpB, a Cytotoxic Protease from Serratia marcescens. Infect Immun 83:2907-16
Brothers, Kimberly M; Stella, Nicholas A; Hunt, Kristin M et al. (2015) Putting on the brakes: Bacterial impediment of wound healing. Sci Rep 5:14003

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