The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1^-/- more susceptible to P. aeruginosa adhesion while TRPV1^-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.