Developing anti-adhesion therapies for microbial keratitis: a porcine model

Abstract

Microbial keratitis poses a severe threat to corneal health, particularly in developing nations, leading to potential blindness. Bacterial keratitis, primarily attributed to Staphylococcus aureus and Pseudomonas aeruginosa, can result in permanent corneal scarring and vision loss, especially with the escalating antibiotic resistance. To address this, we explored anti-adhesive treatments based on the host tetraspanin protein CD9 and secretory immunoglobulin A (sIgA). Tetraspanins, transmembrane proteins forming tetraspanin-enriched microdomains (TEM), play a crucial role in organizing cell surface molecules involved in bacterial adhesion. Short (16 aa) synthetic peptides derived from the large extracellular domain of CD9 disrupt TEM, and have previously been shown to inhibit bacterial adhesion to human skin cells. Using porcine intestinal epithelial cells (IPEC-J2 and primary porcine corneal epithelial cells (PCEpiC)), and primary human corneal epithelial cells (H6621), we investigated the effects of CD9- derived peptides (designated 800C) on S. aureus adhesion. Flow cytometry confirmed porcine CD9 expression, and synthetic peptides inhibited S. aureus adhesion to IPEC-J2 and H6621 cells. Notably, human CD9 peptide 800C significantly reduced S. aureus adhesion to human primary corneal cells. The involvement of CD9 in S. aureus adhesion to human and IPEC-J2 cells, mediated by heparan sulphate proteoglycans, was validated using soluble heparin. By contrast, no effect of the porcine 800C peptide or heparin was observed for primary porcine epithelial cells, indicating that CD9 is not involved in S. aureus adhesion to the porcine cornea. Furthermore, our study extended prior findings on sIgA from Nepalese cows' milk inhibiting S. aureus adhesion to human cells. sIgA from a local Sheffield dairy herd also inhibited S. aureus adhesion to porcine and human cells, correlating with specific binding to S. aureus in ELISA assays. However, limited reactivity was observed against P. aeruginosa. In contrast, Nepalese sIgA demonstrated good reactivity against P. aeruginosa, and inhibited bacterial adhesion indicating its potential in an ex-vivo human cadaveric model of keratitis.

In conclusion, our findings suggest that CD9-derived peptides and sIgA from local cows' milk may offer promising interventions for bacterial keratitis. However, further research is needed to explore their full potential, particularly regarding P. aeruginosa infections. This study contributes to the development of low-cost reagents to combat bacterial adhesion, crucial in the face of rising antibiotic resistance.