446 - CovR Regulation of GBS Virulence in the Newborn Gut

Publication Number: 2434.446

Alexia Pearah, USF Health Morsani College of Medicine, Tampa, FL, United States; Madison R. Huszar, University of South Florida, Tampa, FL, United States; April Lindon, University of South Florida, Tampa, FL, United States; Sophia Carlin, University of Pittsburgh, Pittsburgh, PA, United States; Nichol John-Lewis Edwards, University of South Florida, Tampa, FL, United States; Hannah Wapshott-Stehli, University of South Florida, Wesley Chapel, FL, United States; Thao Ho, University of South Florida, Tampa, FL, United States; Thomas A. Hooven, University of Pittsburgh, Pittsburgh, PA, United States; Tara M. Randis, University of South Florida, Tampa, FL, United States

Background: Late-onset Group B Streptococcus (GBS) disease (LOD) occurs when GBS colonizing the newborn intestine breaches epithelial barriers with subsequent systemic dissemination. CovR/S is a two-component signal transduction system in GBS composed of a surface-anchored sensor kinase (CovS) and a transcriptional response regulator (CovR) that represses a large array of virulence-associated genes. The role of CovR/S in LOD is unknown.

Objective: Our objective was to define the contribution of the GBS CovR/S regulon to intestinal colonization and invasion using immature human intestinal enteroid monolayers and an established mouse model of LOD.

Design/Methods: We used GBS COH-1 as the wild-type (WT) strain. A CovR deficient mutant was constructed via Cas12a genome editing which was then complemented in trans using the expression plasmid pGBScomp. Enteroid monolayers were cultivated on transwell plates and confluency was verified by measuring transepithelial electrical resistance (TEER). Monolayers were exposed to GBS strains for two hours. To assess GBS translocation, media from the basolateral compartment of the transwell plate was collected and bacteria enumerated by serial dilutions. GBS-exposed cells were washed, dissociated from the transwell inserts, and adherent GBS were enumerated to calculate percent adherence. To examine intracellular invasion, cells were exposed to antibiotic-containing media (gentamicin [50 µg/mL] X 1 hour) then washed, lysed, and intracellular GBS enumerated. Cell viability was assessed after GBS exposure using a lactate dehydrogenase cytotoxicity assay. To test strains in our mouse model of LOD, 10-day-old pups were infected with the WT or CovR-deficient strain (108 CFU X 2 doses) via oral gavage. After 4 days, tissues were collected to quantify GBS intestinal burden and dissemination to the blood, liver, and brain.

Results: CovR deficient GBS exhibited increased epithelial cell adhesion, invasion and translocation in HIE monolayers compared to WT and complemented mutant strains (Fig.1A). All GBS strains had a similar impact on cell viability with variable impacts on monolayer permeability (Fig.1B). A loss of CovR regulation resulted in decreased GBS burden in colonized mice and a trend toward increased systemic dissemination (Fig.1C).

Conclusion(s): CovR/S modulates GBS adhesion to and disruption of intestinal epithelial barriers-a critical step in LOD pathogenesis. This enhanced cellular engagement may be detrimental to persistent colonization. The CovR regulon balances the colonizing and invasive GBS phenotypes in the neonatal gut.

Figure 1. GBS-host dynamics across in vitro and in vivo models.
A. CovR regulates GBS adhesion, invasion and translocation across HIE monolayers (Kruskal-Wallis with Dunn's Multiple Comparisons). B. GBS-induced changes in HIE monolayer permeability (Kruskal-Wallis with Dunn's Multiple Comparisons), and cell viability (ANOVA, Tukay's Post test) C. CovR deficient GBS exhibits decreased colonization (Mann-Whitney) and a trend toward increased invasion in a mouse model of GBS LOD (Fisher's exact test). * p<0.05, **p < 0.005, ***p < 0.0001)