Immune Inhibitor A Metalloproteases Contribute to Virulence inBacillusEndophthalmitis
Authors: Livingston Erin T. , Mursalin Md Huzzatul , Coburn Phillip S. , Astley Roger , Miller Frederick C. , Amayem Omar , Lereclus Didier , Callegan Michelle C. 
Published: July 2020

AbstractBacterial endophthalmitis is a devastating infection that can cause blindness following the introduction of organisms into the posterior segment of the eye. Over half ofBacillusendophthalmitis cases result in significant loss of useful vision. Often, these eyes have to be enucleated.Bacillusproduces many virulence factors in the eye that may contribute to retinal damage and robust inflammation. This study analyzedBacillusimmune inhibitor A (InhA) metalloproteases, which digest extracellular matrix, tight junction proteins, and antimicrobial proteins. We hypothesized that InhAs contribute toBacillusintraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis.In vitroanalysis of growth, proteolysis, and cytotoxicity were compared betweenB. thuringiensisstrains. WT and InhA mutants were similarly cytotoxic to retinal cells. Mutant ΔinhA1and ΔinhA2entered log phase growth earlier than WT. Proteolysis of the ΔinhA1-3mutant was decreased, but this strain grew similar to WTin vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU ofB. thuringiensisWT or InhA mutants. IntraocularBacillusand retinal function loss were quantified. Intraocular myeloperoxidase concentrations were quantified and histology was analyzed. Eyes infected with ΔinhA1or ΔinhA2strains contained greater numbers of bacteria than eyes infected with WT throughout the course of infection. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with WT strain. Eyes infected with ΔinhA1-3cleared the infection, with less retinal function loss and inflammation compared to eyes infected with the WT strain. RT-PCR results suggested that single InhA mutant results may be explained by compensatory expression of the other InhAs in these mutants. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation inBacillusendophthalmitis.Author summaryBacterial endophthalmitis is an infection of the eye, which can follow accidental contamination of the posterior segment following ocular surgery (postoperative), a penetrating wound (post-traumatic), or during spread of bacteria into the eye from the bloodstream (endogenous). During bacterial endophthalmitis, virulent pathogens such asBacilluscause ocular damage via the activities of an array of virulence factors, including proteases. A class of proteases that are expressed byBacillusduring ocular infection are the immune inhibitor A metalloproteases. Here, we used a mouse model of endophthalmitis to test mutantBacillusthat lack single or multiple InhAs to determine if these metalloproteases contributed to the virulence during the disease. In the absence of the production of all InhAs,Bacilluscould not cause severe infection. Our study provides new insights into the virulence ofBacillusin the eye, and the contribution of its InhA metalloproteases to establishing infection.