Ocular Microbiology and Immunology Group
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2020 OMIG Abstract

The Rcs Stress Response System Modulates Serratia Marcescens Induced Inflammation and Bacterial Proliferation in a Rabbit Keratitis Model

Robert M.Q. Shanks, PhD, Eric G. Romanowski, MS, Nicholas A. Stella, John E. Romanowski,
Kathleen A. Yates, BS, Deepinder K. Dhaliwal, MD, LAc
The Charles T. Campbell Ophthalmic Microbiology Laboratory, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA

Purpose: In this study, we tested the hypothesis that the conserved bacterial Rcs stress response system mediates corneal pathogenesis associated with Serratia marcescens ocular infections. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known.

Methods:: This was accomplished by modifying Rcs activity using mutant strains. These include a mutant that has a hyper-active Rcs system due to deletion of the IgaA family gene, gumB, and a gumB rcsC double mutant that is defective for Rcs signaling. Intrastromal injection of ~1000 CFU of bacteria into rabbit corneas was performed, and corneal inflammation was analyzed by a modified MacDonald-Shadduck scoring system at 24-48 h and CFU were determined at 48 h. ELISAs and NanoString technology was used to assess inflammatory markers.

Results: Here we observed that the Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h, and demonstrated a notable reduction in inflammation based on inflammatory signs and pro-inflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid and partially complemented by restoration of shlA cytolysin expression and elimination of capsular polysaccharide production. We observed that inactivation of the Rcs stress response system completely restored corneal virulence to the gumB mutant. Transcriptional analysis of bacterial genes expressed during microbial keratitis demonstrated expression of gumB, rcsB, shlA, and three metalloprotease genes.

Conclusion: Together, these data indicate that GumB regulates virulence factor production through the Rcs system and this overall stress response system is a key mediator of a bacterium’s ability to induce vision-threatening keratitis.

Disclosure: N

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