Innate Interactions of Bacillus S-Layer During Endophthalmitis

Abstract

Purpose : To explore the mechanisms by which Bacillus S-layer protein (SLP) contributes to intraocular inflammation during endophthalmitis.

Methods : We compared phenotypes of Wild-type (WT) and SLP-deficient (△slpA) Bacillus thuringiensis by analyzing bacterial adherence to and phagocytosis by human retinal Muller cells and phagocytosis by human and mouse neutrophils. Innate immune receptor activation by the Bacillus envelope and purified SLP was analyzed using TLR2 and TLR4 reporter cell lines. A synthetic TLR2/4 inhibitor was used as a control for this receptor activation. To induce endophthalmitis, mice were injected intravitreally with 100 CFU WT or △slpA B. thuringiensis. At 4 hours postinfection, a group of WT-infected mice was treated intravitreally with the TLR2/4 inhibitor. At 10 hours postinfection, infected eyes were analyzed for bacterial counts, histology, inflammation, and retinal function.

Results : B. thuringiensis SLP contributed to adherence to retinal cells, and protected this pathogen from Muller cell- and neutrophil-mediated phagocytosis. The B. thuringiensis envelope activated TLR2 and, surprisingly, TLR4, suggesting the presence of a surface-associated TLR4 agonist in Bacillus. Purified SLP from B. thuringiensis activated TLR4, as well as TLR2, in vitro. Growth of WT B. thuringiensis was significantly greater and caused more inflammation in untreated eyes than in infected eyes treated with the TLR2/4 inhibitor. Retinal function analysis also showed greater retention of A-wave and B-wave function in infected eyes treated with the TLR2/4 inhibitor. The TLR2/4 inhibitor was not antibacterial in vitro and did not cause inflammation when injected into uninfected eyes.

Conclusions : Taken together, these results suggest a potential role for Bacillus SLP in host innate-bacterial interactions, as well as in endophthalmitis pathogenesis via TLR2- and TLR4-mediated pathways. This is also the first study to demonstrate an anti-inflammatory therapeutic effect from targeting TLRs during bacterial endophthalmitis.

This is a 2020 ARVO Annual Meeting abstract.