Characterizing the two-component regulatory system (TCS) LytSR in Group B Streptococcus

Abstract

Abstract Streptococcus agalactiae or Group B Streptococcus (GBS) is a commensal bacterium of the female urogenital tract and the leading cause of neonatal meningitis worldwide. GBS has two-component systems (TCSs) that detect and respond to environmental changes during colonization or invasive disease contributing to bacterial survival and virulence. In this thesis, I investigated the role of the LytST two-component system in GBS pathogenesis, with a specific focus on the sensory component lytS, using in vitro and in vivo approaches along with the host transcriptomics analysis. An isogenic lytS deletion mutant (ΔlytS) was generated in a hypervirulent serotype III, CC17 GBS strain. ΔlytS and wild-type (WT) strains were compared in vitro for growth dynamics in different conditions, autolysis (Triton X-100 induced and natural), biofilm formation, β-hemolysin/cytolysin activity, and morphology via scanning electron microscope. Adhesion and invasion assays in human brain microvascular endothelial cells (hCMEC/D3) were also performed to evaluate the role of lytS and the contribution of extracellular DNA (eDNA) to these phenotypic properties. Blood-brain barrier (BBB) transmigration and trans-endothelial electrical resistance (TEER) were evaluated using a Transwell model. Time-kill assays with penicillin assessed antibiotic susceptibility. A murine intravenous infection model was used to measure survival, bacterial loads, cytokine responses, and BBB integrity. A vaginal colonization model examined the role of lytS in bacterial persistence and the contribution of eDNA to maintaining carriage. Finally, RNA sequencing of hCMEC/D3 cells at 3 h and 6 h post-infection was performed to characterize host transcriptional responses to WT and ΔlytS infection. ΔlytS exhibited increased autolysis, elevated eDNA release, and enhanced biofilm formation. Scanning electron microscope revealed dense clumping in ΔlytS compared to WT. ΔlytS showed greater adhesion but reduced invasion and transmigration across the BBB, DNase treatment abolished the adhesion difference highlighting the influence of eDNA in adhesion by the ΔlytS. Hemolytic activity was lower in ΔlytS and the mutant strain was more susceptible to penicillin at most of the time points tested. In the in vivo experiments, mice infected with ΔlytS showed better survival, reduced brain bacterial loads, and lower proinflammatory cytokines compared to WT. The mutant also preserved BBB integrity following infection. Vaginal colonization by ΔlytS was prolonged, with significantly higher eDNA levels over time, compared to WT. RNA-seq analysis showed that cells infected with the ΔlytS mutant had impaired activation of key immune pathways, vesicular-transport pathways, and pathways involved in the modulation of neuronal signaling, compared with cells infected with the WT strain. Our data indicates that the two-component system LytST domain, through lytS, plays an important role in regulating GBS phenotypic properties, such as autolysis, biofilm formation, adherence and invasion to brain endothelial cells. It also regulates overall virulence in vivo along with altered host transcriptomic profile. These findings are important for understanding the pathogenesis of GBS-induced meningitis in neonates and may aid in the development of targeted therapeutics and vaccines in future.