Abstract
Purpose :
Corynebacterium mastitidis (C. mast) can stably colonize the ocular surface while inducing protective IL-17 from γδ T cells through T cell receptor (TCR) engagement and IL-1β from antigen presenting cells. This immune response results in neutrophil recruitment, anti-microbial production, and protection from infection by Candida albicans and Pseudomonas aeruginosa. Despite knowledge of this causal relationship, the microbial factors governing C. mast colonization and immunogenicity are still unknown. Knowing microbial factors that permit eye-colonization will reveal critical information on how commensal or pathogenic bacteria may affect ocular surface homeostasis and disease.
Methods :
Using transposon mutagenesis, we generated a library of C. mast mutants. We used a biofilm formation assay as a method for high throughput screening C. mast mutants to identify candidates that may lack an ability to colonize the eye. Normal culture methods were used to expand and quantify C. mast mutants. In vivo colonization experiments determined whether mutants could colonize the eye. Finally, in vitro stimulations that combined gd T cells, bone marrow-derived dendritic cells, and bacteria allowed us to assess—by flow cytometry—how immune cells recognized C. mast mutants in vitro.
Results :
After high throughput screening our transposon mutant library for biofilm formation, we identified a mutant (BiofilmKO) deficient in biofilm formation with a transposon insertion interrupting a sortase gene. This mutant grew normally in culture, but it could not colonize the ocular surface (P<0.0001). Because of this, we observed no induction of T or B cell immunity. Notably, BiofilmKO C. mast maintained its ability to induce IL-17 production from gd T cells in vitro (P=0.8796). Conversely, C. mast-specific antibodies could not recognize the BiofilmKO C. mast.
Conclusions :
Here, we identify a microbial gene—sortase—that is critical for C. mast to colonize the eye. Because this gene is not required for in vitro immunity, we conclude that colonization with a commensal is required to generate protective immunity at the ocular surface. Further, sortase is required for the proper expression of B cell epitopes on the surface of C. mast, which has clear implications for the generation of humoral immunity against commensal bacteria at the ocular surface.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.