Abstract
Purpose :
To identify bacterial regulators of virulence. Bacterial keratitis is a potentially blinding disease that is exacerbated by bacterial virulence factors and bystander damage caused by the immune system. The Gram-negative bacterium Serratia marcescens produces several cell damaging products including a biosurfactant, serratamolide, phospholipase A, and the ShlA cytolysin. The transcriptional regulation of these virulence determinants is incompletely understood. In this study, the smrF gene was identified in genetic screens for mutants unable to produce serratamolide and the autophagy-inducing pigment, prodigiosin.
Methods :
The smrF gene was mutated using transposon mutagenesis and alleleic replacement. Bacteria were tested for cytotoxicity to human corneal limbal epithelial cells (HCLE) using Calcein AM and PrestoBlue. Complementation and luminescent reporter constructs were built by recombineering. Motility assays were performed on low percentage agar plates, and mass spectrometry was performed to measure serratamolide. NanoString was performed on synchronized cultures at OD600=1.0.
Results :
Bioinformatic analysis indicates the smrF gene is predicted to code for an uncharacterized predicted GntR/HutC family transcription factor. Biochemical analysis indicates that SmrF is a positive regulator of prodigiosin and serratamolide. By contrast, NanoString and transcriptional reporter analysis suggests SmrF regulates ShlA, which is a pore-forming toxin. The smrF mutant had increased cytotoxicity to a human corneal cell line that was shlBA dependent.
Conclusions :
These data indicate a role for SmrF as a novel regulator of secreted virulence factors.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.