Determination of the role of quorum sensing response regulator LuxR on flavobacterium covae biofilm formation and virulence in channel catfish (Ictalurus punctatus)

Authors: CHURCHMAN, EMILY - Auburn University; WHITE, RACHEL - Auburn University; Lange, Miles; LAFRENTZ, STACEY - Auburn University; Lafrentz, Benjamin; LILES, MARK - Auburn University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/3/2023
Publication Date: 6/15/2023
Citation: Churchman, E.M., White, R., Lange, M.D., Lafrentz, S., Lafrentz, B.R., Liles, M.R. 2023. Determination of the role of quorum sensing response regulator LuxR on flavobacterium covae biofilm formation and virulence in channel catfish (Ictalurus punctatus) [ABSTRACT]. American Society of Microbiology Microbe Conference, Houston, TX, June 15-19, 2023.

Interpretive Summary:

Technical Abstract: Columnaris disease leads to massive losses in freshwater fish production due to the bacterial pathogen Flavobacterium covae, which causes disease by forming biofilms on skin and gill surfaces. The regulation of biofilm formation in F. covae may involve quorum sensing. Previous research has shown that vertebrate mucus significantly upregulates the quorum sensing response regulator LuxR in F. covae biofilm cells1. Since quorum sensing is known to regulate biofilm production and virulence in other aquatic pathogens, we hypothesized that LuxR has a similar role in F. covae. A luxR deletion mutant was developed in F. covae C#2 using an allelic exchange method. Microtiter biofilm formation was analyzed for F. covae C#2 and F. covae C#2 luxR. Briefly, overnight cultures were diluted 1:100, seeded into a 96-well plate (n=8) and grown under static conditions at 28 ºC for 24 hours. Plates were washed with sterile water, stained with 1% crystal violet (wt/vol), and washed. After drying overnight, stained biofilms were solubilized with 30% acetic acid (vol/vol), and the OD550 was recorded. A significant increase of biofilm production was observed in F. covae C#2 luxR compared to the wild-type strain. Interestingly, when supplemented with 10 mM mannose, the wild-type strain exhibited an increase in biofilm formation while the luxR mutant remained unaffected. These results suggest a phenotypic difference in biofilm formation due to the luxR deletion. Recently, the F. covae C#2 luxR has been complemented with a plasmid containing the luxR gene and these biofilm studies will be repeated with the wild-type, mutant and complemented mutant. Future research includes evaluating the transcriptome of these strains to evaluate differentially expressed genes regulated by LuxR. Lastly, a disease challenge in channel catfish is planned that will include three tanks of fish (n=25) challenged with either wild-type, luxR, or the complemented mutant with approximately 5.0 x 107 colony forming units/mL as conducted previously. A fourth tank will be a mock challenge with sterile bacteriological medium. Mortality rates will be monitored twice daily for 14 days post-challenge, and F. covae will be reisolated from morts. Collectively, these data will yield greater insights into the role of quorum sensing and carbohydrate-mediated interactions in F. covae pathogenesis.