Genetically Engineering ocular probiotics to manipulate ocular immunity and disease

Abstract

Recently, our lab discovered that the eye harbors a microbiome that includes Corynebacterium mastitidis, which induces the recruitment of gdT cells and the production of interleukin 17 (IL-17). This local immune response to C. mast can protect the eye from more serious infections, such as Candida albicans and Pseudomonas aeruginosa. Because of C. mast’s ability to stably colonize the ocular surface while inducing a beneficial immune response, we believe that this bacterium has the potential to act as a natural vehicle to deliver therapeutics locally at the ocular surface to alleviate ocular surface disease. Before this can become a reality, a reliable method to genetically modify C. mast is required. Therefore, we took the initial steps toward achieving this goal by genetically modifying C. mast to express genes of interest using transposon mutagenesis. This allowed us to create a mutant library with 1,000+ distinct candidates that expressed the fluorescent protein, mCherry, and kanamycin resistance. Using FACS, we were able to select the brightest C. mast mutants. The brightest four mutants were used to inoculate the eyes of C57BL/6 mice, so that we could: 1) track C. mast in vivo and 2) ensure that the promoters upstream of the genes of interest remain active when C. mast has colonized the ocular mucosa. We were able to image recombinant bacteria directly ex vivo and also confirmed that there was no decrease in bacterial fitness or the protective immune response elicited by C. mast. We concluded from these studies that C. mast can be engineered to express proteins of interest without a reduction in bacterial fitness or protective immunity.