Sortase F from C. mastitidis targets novel adhesion proteins in a unique way to colonize the eye

Abstract

Purpose : Historically, the investigation of the ocular microbiome has been difficult due to the paucibacterial nature and inhospitable environment of the ocular surface. Despite this, Corynebacterium mastitidis (C. mast) is capable of colonizing the murine ocular mucosa. After colonization, C. mast stimulates protective immunity that enhances the eye’s ability to resist infection. Here, using clinical isolates of C. mast, we show that few Corynebacteria possess an ability to colonize the eye. Therefore, we sought to understand the unique bacterial machinery that C. mast may use to colonize the ocular surface.

Methods : We used transposon mutagenesis to create mutant libraries of C. mast, so that we can identify genes responsible for colonizing the ocular surface. In vivo colonization studies allowed us to identify a single mutant that lacked the ability to colonize the eye. Whole Genome Sequencing revealed the location of mutations within the C. mast genome. Mass spectrometry was used to identify proteins that are differentially expressed between WT and mutant C. mast. Complementation of the gene knock out was delivered via plasmid into the mutant bacteria to confirm our results.

Results : We identified a Tn mutant that was completely unable to colonize the eye (p<0.0001), and WGS revealed a disruption in the sortase F gene, which anchors proteins to the cell wall of C. mast and governs biofilm formation and other microbial activities. After “shaving” off extracellular proteins from WT C. mast and Sortase F^KO C. mast, MS revealed that two adhesin proteins were significantly reduced in Sortase F^KO bacteria compared to WT. Notably, bacteria lacking one of the adhesin proteins had a significantly reduced ability to colonize the ocular surface (p<0.001).

Conclusions : We conclude from these data that Sortase F is responsible for appropriately decorating the bacteria surface of C. mast with two adhesin proteins. Without Sortase F, those adhesin proteins are not appropriately represented on the bacteria cell wall, which results in an inability to colonize the ocular surface. These data reveal a unique protein, Sortase F, as a critical factor in ocular colonization, and the presence of this protein in other bacteria may indicate colonizers from non-colonizers.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.