AUTHOR=Mursalin Md Huzzatul , Coburn Phillip S. , Livingston Erin , Miller Frederick C. , Astley Roger , Flores-Mireles Ana L. , Callegan Michelle C. 

TITLE=Bacillus S-Layer-Mediated Innate Interactions During Endophthalmitis

JOURNAL=Frontiers in Immunology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00215

DOI=10.3389/fimmu.2020.00215

ISSN=1664-3224

ABSTRACT=<p><italic>Bacillus</italic> endophthalmitis is a severe intraocular infection. Hallmarks of <italic>Bacillus</italic> endophthalmitis include robust inflammation and rapid loss of vision. We reported that the absence of <italic>Bacillus</italic> surface layer protein (SLP) significantly blunted endophthalmitis severity. Here, we further investigated SLP in the context of <italic>Bacillus-</italic>retinal cell interactions and innate immune pathways to explore the mechanisms by which SLP contributes to intraocular inflammation. We compared phenotypes of Wild-type (WT) and SLP deficient <italic>(</italic>Δ<italic>slpA</italic>) <italic>Bacillus thuringiensis</italic> by analyzing bacterial adherence to and phagocytosis by human retinal Muller cells and phagocytosis by mouse neutrophils. Innate immune receptor activation by the <italic>Bacillus</italic> envelope and purified SLP was analyzed using TLR2/4 reporter cell lines. A synthetic TLR2/4 inhibitor was used as a control for this receptor activation. To induce endophthalmitis, mouse eyes were injected intravitreally with 100 CFU WT or Δ<italic>slpA B. thuringiensis</italic>. A group of WT infected mice was treated intravitreally with a TLR2/4 inhibitor at 4 h postinfection. At 10 h postinfection, infected eyes were analyzed for viable bacteria, inflammation, and retinal function. We observed that <italic>B. thuringiensis</italic> SLPs contributed to retinal Muller cell adherence, and protected this pathogen from Muller cell- and neutrophil-mediated phagocytosis. We found that <italic>B. thuringiensis</italic> envelope activated TLR2 and, surprisingly, TLR4, suggesting the presence of a surface-associated TLR4 agonist in <italic>Bacillus</italic>. Further investigation showed that purified SLP from <italic>B. thuringiensis</italic> activated TLR4, as well as TLR2 <italic>in vitro</italic>. Growth of WT <italic>B. thuringiensis</italic> was significantly higher and caused greater inflammation in untreated eyes than in 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 <italic>in vitro</italic>, and did not cause inflammation when injected into uninfected eyes. Taken together, these results suggest a potential role for <italic>Bacillus</italic> SLP in host-bacterial interactions, as well as in endophthalmitis pathogenesis via TLR2- and TLR4-mediated pathways.</p>