Abstract
Purpose :
Bacterial keratitis is a vision threatening infection of the cornea. Serratia marcescens causes contact lens associated bacterial keratitis and experiences a hostile environment on the ocular surface. Mutation of the stress regulator gene gumB in S. marcescens strongly reduces the bacteria’s ability to induce corneal inflammation. The goal of this study was to gain insight into the mechanisms responsible for this conserved stress response system protein in regulating ocular microbial pathogenesis.
Methods :
Transcriptomic analysis of S. marcescens contact lens isolate K904 and an isogenic gumB mutant. RNA-sequencing, NanoString, and qRT-PCR were used to measure RNA from bacteria grown in lysogeny broth and in New Zealand White rabbit corneas. Proteomics on surface fraction from cultures grown in lysogeny broth was performed. Cytotoxicity was measured with PrestoBlue and Calcein AM staining. Genes for individual candidate cytotoxic factors were cloned and expressed from plasmids. Protease activity was measured from normalized culture filtrates using azocasein. Surface proteins and type I pili were assessed using PAGE and yeast agglutination analysis. Biofilm formation was assessed by the microplate-crystal violet assay.
Results :
Approximately 15% of the S. marcescens genome had significantly altered expression in the gumB mutant (p<0.05). Of interest, major changes include reduction of toxic and host-pathogen regulator genes including flagella, pili, hemolysins, and serralysin metalloproteases. The gumB mutant secretomes were defective in protease activity (down 65±5% verus WT, p<0.001) and cytotoxicity to ocular surface cells (down 50±10% versus WT, p<0.01). Expression of serralysin, but not the ShlA cytolysin, PhlA phospholipase, or FlhDC virulence regulator restored cytotoxicity to the gumB mutant indicating that serralysin protease expression could complement the gumB mutant defect. Biofilm formation by the gumB mutant was defective (down 87±7% versus WT, p<0.05) and data suggest that this was due to loss of pilus biosynthesis rather than extracellular polysaccharide levels.
Conclusions :
This study indicates that GumB is a major regulator of virulence genes and bacterial interactions with ocular cells through transcriptional control of secreted metalloproteases and biofilm promoting adhesins.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.