Abstract
Purpose :
Recently, our lab discovered that the eye harbors a resident ocular microbiome that includes Corynebacterium mastitidis (C. mast), which can stimulate local immunity to protect the eye from infection. We know that C. mast remains associated with the ocular surface indefinitely and can stimulat protective immunity. Our studies are now focused on better understanding the microbial factors that govern colonization of the ocular mucosa and stimulation of the host immune response. To achieve the study goals, we performed transposon mutagenesis on C. mast to generate a library of mutants for the purpose of identifying microbial genes critical for colonization and immunogenicity of C. mast.
Methods :
We created a transposon mutant library containing ~1,500 candidates with distinct insertion sites and varying phenotypes. To do this, we used the EZ-Tn5 kit (Lucigen) to generate transposomes that contained genes encoding the fluorescent protein, mCherry, and a kanamycin-resistance cassette. Tn mutants were initially selected on kamamycin-treated plates. Functional mutants were then screened for desired phenotypes.
Results :
We found several mutant candidates of interest. First, a mutant constitutively expressed mCherry in vitro and in vivo when associated with the ocular mucosa. This candidate colonized the eye and induced immunity similar to that observed with the parental C. mast strain. A separate candidate lost the ability to colonize the ocular mucosa and failed to elicit an immune response in vivo; however, it still induced the production of cytokines in vitro. A final candidate corresponded with a reduced immune response both in vitro and in vivo compared to the parental strain.
Conclusions :
We have demonstrated that an ocular commensal, C. mast, can be mutated. Using in vivo imaging and Tn-C. mast mutants, we can conclude that C. mast only colonizes the conjunctiva and does not associate with the cornea. Furthermore, we can conclude from our library of transposon mutants that simple exposure to C. mast does not induce a measurable immune response, but colonization of the ocular surface is required for in vivo immunity. These studies are the first steps towards understanding what makes an ocular commensal bacterium what it is, and from this, we can begin to engineer ocular bacteria to promote ocular health to limit disease and pathogenesis.
This is a 2021 ARVO Annual Meeting abstract.