A covR promoter mutation enhances Group B Streptococcus virulence by upregulating pgaC to reinforce capsular surface...

Group B Streptococcus (GBS) is a major zoonotic pathogen responsible for neonatal sepsis and cerebrospinal meningitis. A single adenine (T) insertion in the covR promoter, identified in a clinical isolate, has been shown to enhance pathogenicity. However, the mechanistic basis remains unclear. In this study, we demonstrated that this insertion downregulates covR expression and enhances hemolysin production. Unexpectedly, we also found that it leads to a thicker and more compact capsule based on dot blot and flow cytometry analyses. Transcriptome analysis performed on the covR promoter mutant revealed a significant upregulation in biofilm-related genes, including pgaC. Electrophoretic mobility shift assays confirmed the direct binding of CovR to the pgaC promoter, thereby identifying it as a transcriptional repressor involved in capsule regulation. Deletion of pgaC (ΔpgaC) severely compromised capsular surface anchoring, as validated by Western blot and transmission electron microscopy. The ΔpgaC mutant also exhibited impaired abilities in host cell adhesion, invasion, intracellular survival, and stress resistance. In a tilapia infection model, ΔpgaC caused significantly lower mortality, reduced bacterial loads in tissues, along with a marked attenuation of pro-inflammatory cytokine production relative to the wild type strain. Collectively, our results reveal a novel mechanism by which an insertion of a single nucleotide within the covR promoter enhances GBS virulence not only by increasing hemolysin production but also by reinforcing capsule surface anchoring via the direct upregulation of pgaC.