Immune Inhibitor A Metalloproteases Contribute to Virulence inBacillusEndophthalmitis

Abstract

<jats:title>Abstract</jats:title><jats:p>Bacterial endophthalmitis is a devastating infection that can cause blindness following the introduction of organisms into the posterior segment of the eye. Over half of <jats:italic>Bacillus</jats:italic> endophthalmitis cases result in significant loss of useful vision. Often, these eyes have to be enucleated. <jats:italic>Bacillus</jats:italic> produces many virulence factors in the eye that may contribute to retinal damage and robust inflammation. This study analyzed <jats:italic>Bacillus</jats:italic> immune inhibitor A (InhA) metalloproteases, which digest extracellular matrix, tight junction proteins, and antimicrobial proteins. We hypothesized that InhAs contribute to <jats:italic>Bacillus</jats:italic> intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild type (WT), InhA1-deficient (Δ<jats:italic>inhA1</jats:italic>), InhA2-deficient (Δ<jats:italic>inhA2</jats:italic>), or InhA1, A2, and A3-deficient (Δ<jats:italic>inhA1-3</jats:italic>) Bacillus thuringiensis. <jats:italic>In vitro</jats:italic> analysis of growth, proteolysis, and cytotoxicity were compared between <jats:italic>B. thuringiensis</jats:italic> strains. WT and InhA mutants were similarly cytotoxic to retinal cells. Mutant Δ<jats:italic>inhA1</jats:italic> and Δ<jats:italic>inhA2</jats:italic> entered log phase growth earlier than WT. Proteolysis of the Δ<jats:italic>inhA1-3</jats:italic> mutant was decreased, but this strain grew similar to WT <jats:italic>in vitro</jats:italic>. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of <jats:italic>B. thuringiensis</jats:italic> WT or InhA mutants. Intraocular <jats:italic>Bacillus</jats:italic> and retinal function loss were quantified. Intraocular myeloperoxidase concentrations were quantified and histology was analyzed. Eyes infected with Δ<jats:italic>inhA1</jats:italic> or Δ<jats:italic>inhA2</jats:italic> strains 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 Δ<jats:italic>inhA1-3</jats:italic> cleared 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 in <jats:italic>Bacillus</jats:italic> endophthalmitis.</jats:p><jats:sec><jats:title>Author summary</jats:title><jats:p>Bacterial 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 as <jats:italic>Bacillus</jats:italic> cause ocular damage via the activities of an array of virulence factors, including proteases. A class of proteases that are expressed by <jats:italic>Bacillus</jats:italic> during ocular infection are the immune inhibitor A metalloproteases. Here, we used a mouse model of endophthalmitis to test mutant <jats:italic>Bacillus</jats:italic> that 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, <jats:italic>Bacillus</jats:italic> could not cause severe infection. Our study provides new insights into the virulence of <jats:italic>Bacillus</jats:italic> in the eye, and the contribution of its InhA metalloproteases to establishing infection.</jats:p></jats:sec>

Bacterial Burden Declines But Neutrophil Infiltration and Ocular Tissue Damage Persist in Experimental Staphylococcus epidermidis Endophthalmitis

“…Because different pathogens cause experimental endophthalmitis at certain infectious doses (Gupta et al, 2019;Francis et al, 2020;Kumar et al, 2020;Livingston et al, 2021), we performed a doseresponse study beginning with an intravitreal injection of 5,000 cfu of S. epidermidis, a dose required for S. aureus endophthalmitis (Singh et al, 2021). However, this inoculum was rapidly cleared from the mouse eyes resulting in transient inflammation (data not shown).…”

DataSheet_1.docx

“…Because different pathogens cause experimental endophthalmitis at certain infectious doses (Gupta et al, 2019;Francis et al, 2020;Kumar et al, 2020;Livingston et al, 2021), we performed a doseresponse study beginning with an intravitreal injection of 5,000 cfu of S. epidermidis, a dose required for S. aureus endophthalmitis (Singh et al, 2021). However, this inoculum was rapidly cleared from the mouse eyes resulting in transient inflammation (data not shown).…”