Ingenium 2020
Genetically engineering ocular probiotics to manipulate ocular immunity and disease Yannis Rigasa, b, Benjamin Treatb, Anthony St. Legerb, c Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, PA, USA b Department of Ophthalmology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA c Department of Immunology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA a
Yannis Rigas is a current bioengineering major on the track of cellular engineering. His current research interests include microbiology, immunology, genetic and microbial engineering. Yannis plans to attend graduate school upon graduating from the University of Pittsburgh. Yannis Rigas
Dr. Anthony St. Leger
Dr. St. Leger is an ocular immunologist in the Department of Ophthalmology at the University of Pittsburgh. Since joining Pitt in late 2017, his lab has focused on understanding the relationships between the microbiome and ocular immune system during health and disease. Specifically, his lab currently investigates how the microbiome may be manipulated to alleviate ocular surface disease.
Significance Statement
Every year eye-related diseases account for a huge financial and medical burden. For example, in the U.S., approximately 1 million doctor visits and $175 million are spent annually on treating keratitis and contact lens disorders [1]. In an effort to characterize and manipulate the interactions between ocular immunity and microbiome, our lab is developing a model to characterize and manipulate the relationship between the microbiome and ocular immunity. To study this, we are developing a model that allows the study of commensal bacterium, Corynebacterium mastitidis, and its interactions with ocular immunity. The long-term aim of these studies is the development of a vehicle for long-term therapy delivery to the ocular surface in order to help treat ocular diseases.
Category: Methods
Keywords: Ocular immunity, genetic/microbial engineering, probiotic, and microbiology Abbreviations: C. mast (Corynebacterium mastitidis), IL-17 (Interleukin 17), γδTC (gamma delta T-cells), WT (Wild Type)
Abstract
Recently, our lab discovered that the eye harbors a microbiome that includes Corynebacterium mastitidis, which can stimulate local immunity to protect the eye from more serious infections. From our previous work, we know that C. mast can remain on the ocular surface indefinitely. Therefore, the goal of this project is to genetically engineer C. mast, so that it can act as a natural vehicle to deliver therapeutics locally to alleviate or prevent ocular surface diseases. Here, we took initial steps towards this goal by genetically modifying the C. mast genome so that a fluorescent protein is selected for by utilizing an antibiotic resistance cassette. This fluorescence will allow for real-time in vivo detection of genetically modified C. mast. In this study we have discovered four possible mutants that are resistant to the antibiotic, kanamycin, and fluoresce with varying degrees intensity in the red channel. We hypothesized that these mutants would retain the ability to colonize the eye and induce immunity similar to wild type C. mast. Indeed, we observed that all four mutants were able to colonize the eyes of mice and elicit immune responses similar to wild type C. mast. We further demonstrated that genetically engineered strains of C. mast can effectively colonize the ocular mucosa and elicit an immune response similar to WT C. mast.
1. Introduction
Previously, research on the ocular surface has shown that Corynebacterium mastitidis is stably present on the conjunctiva, an ocular mucosal immune tissue, while displaying commensal properties by eliciting an immune response from γδT cells. The presence of this commensal bacterium, and its accompanying immune response were previously shown to protect the eye from pathogenic eye infections caused by Pseudomonas aeruginosa and Candida albicans. [2]. Through a series of experiments, we were able to link ocular colonization of C. mast with an induced antimicrobial immune response [2]. Due to the ability of this microbe to asymptomatically thrive at the ocular surface for indefinite periods of time, C. mast is an attractive candidate to engineer as a long-term drug delivery vehicle for inflammatory diseases like keratitis, Dry Eye Disease, and Sjogren’s Syndrome. A similar technique was used to deliver the immune regulating cytokine, interleukin (IL)-10, in mice with inflammatory bowel syndrome (IBS). More specifically, when Lactococcus lactis was engineered to express IL-10, disease associated with colitis was reduced compared to controls in two separate mouse models [3]. In this current study, we successfully genetically engineered C. mast by electroporation with a novel plasmid, and showed that C. mast can stably remain on the ocular surface and elicit an immune response similar to the WT strain. This discovery will pave the way for future modifications that will one day allow for a longterm delivery of therapeutics to the ocular surface.
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