Thursday, April 25, 2024
4/25/2024
Project Summary / Abstract The NIH funded Steroids for Corneal Ulcers Study found that keratitis caused by naturally occurring lasR mutants of the bacteria Pseudomonas aeruginosa resulted in significantly worse vision outcomes than keratitis caused by wild-type P. aeruginosa. All the isolates, from India, were obtainted between 2006-2010. Using the vast repository at the University of Pittsburgh Campbell Laboratory, which has isolates back to 1992, a concerning trend was observed that lasR mutants have dramatically increased among P. aeruginosa keratitis isolates in the United States. This is a potential public health problem that this study would help to communicate to ophthalmologists and optometrists. The long-term goal of this research is to develop approaches to prevent vision loss caused by infections. The overall objective of this application is to determine the mechanism by which lasR mutants of P. aeruginosa cause worse clinical outcomes. Our central hypothesis is that the LasR transcription factor inhibits expression of genes that influence P. aeruginosa survival on the harsh ocular surface, such that genes upregulated in the mutant confer an increased infectivity phenotype. The rationale for this study is that identifying the mechanisms by which LasR controls ocular virulence will provide a scientific basis to develop therapeutic strategies to combat vision loss. Three specific aims have been designed to interrogate our central hypothesis: 1) to characterize the genetic basis of lasR mutations among the clinical isolate collection at the Campbell Laboratory, and evaluate the extent to which the mutations confer dominant or recessive virulence phenotypes; 2) to test the importance of specific P. aeruginosa genes that have increased expression in lasR mutants for a role in ocular surface survival, establishing corneal infection in a rabbit contact lens infection model, and in inducing corneal inflammation, and 3) to test whether lasR mutants have elevated resistance to ocular surface innate defenses and ophthalmically relevant antibiotic therapy. This study will use a combination of advanced molecular genetics to manipulate bacterial genomes including clinical isolates, next generation sequencing, and well-established in vitro and in vivo models. This study is innovative because current diagnostic approaches do not differentiate between P. aeruginosa strains that cause keratitis, other than antibiotic susceptibility, and this study will introduce a new approach to identify highly vision threatening P. aeruginosa isolates. This study will also evaluate several candidate virulence factors that have not been tested with respect to the eye. The significance of this study is based on two items: A) its high potential to elucidate new pathogenic mechanisms that bacteria wield to establish blinding ocular infections, and the resulting knowledge can ultimately be used to develop approaches to prevent vision loss due to ocular infections, and B) introducing a feasible form of personalized medicine that can be used to alert health care workers to patients most at risk for vision loss.
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