Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome

Abstract

Purpose Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Methods Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Results Fluorescence-activated cell sorting (FACS) of mCherry + bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, Urease KO . Although uric acid is an antimicrobial factor produced in tears, Urease KO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast . Conclusions In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.

Ocular Bacterial Infections: A Ten-Year Survey and Review of Causative Organisms Based on the Oklahoma Experience

“…St. Leger and Caspi [ 44 ] isolated Corynebacterium mastitidis ( C. mastitidis ) from the conjunctiva of a group of C57BL/6 mice housed at the National Institutes of Health and demonstrated that C. mastitidis is able to form a stable ocular colonines in mouse eyes. This species has been used in mouse models to study how the ocular microbiome affects the ocular surface immune homeostasis [ 45 ]. To our knowledge, this organism has not been isolated from human ocular infection.…”