The CiaR response regulator in group B Streptococcus promotes intracellular survival and resistance to innate immune defenses

Abstract

Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS DeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and DeltaciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

FIG. 1.

The GBS CiaR/H system contributes to AMP resistance. (A) Schematic representation of the GBS ciaR/ciaH region. The position of the Tn917 insertion in original mutants is indicated by arrows. (B) Killing kinetics of WT COH1 and the COH1ΔciaR::pHY304 and COH1ΔciaH::pHY304 mutants after exposure to 18 μM mCRAMP. (C) Alignment of the amino acid sequence of the ciaR gene product in GBS COH1 with those of its homologues in Streptococcus pneumoniae (SPN) TIGR4 and Streptococcus pyogenes (group A Streptococcus [GAS]) M1. Stars indicate residues that are identical in all sequences; colons indicate conserved substitutions; and periods indicate semiconserved substitutions.

FIG. 2.

GBS ciaR enhances intracellular survival in hBMEC and J774 macrophages, as well as resistance to killing by human neutrophils. (A) Killing kinetics of WT and ΔciaR mutant GBS upon exposure to human neutrophils. (B) Survival in J774 macrophages. (C) Survival in hBMEC. (D) Adherence to and invasion of hBMEC by WT and ΔciaR GBS (multiplicity of infection, 1). *, P < 0.05; **, P < 0.01. All experiments were performed at least three times with similar results. Results of representative experiments are shown.

FIG. 3.

CiaR contributes to GBS resistance to killing by antimicrobial effectors. (A and B) Killing kinetics of WT GBS, the ΔciaR mutant, and the ΔciaR mutant complemented with pciaR upon exposure to 16 μM mCRAMP (A) or 64 μM polymyxin B (B). (C to E) Survival of WT GBS, the ΔciaR mutant, and the complemented strain after exposure to 0.03% H₂O₂ (C), 0.01% hypochlorite (D), or 100 ng/ml of lysozyme (E). The survival index was calculated as total CFU recovered/CFU in the starting inoculum. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed at least three times with similar results. Results of representative experiments are shown.

FIG. 4.

GBS CiaR is essential for bacterial fitness and overall virulence. Shown are analyses of bacterial counts in blood 24 h after intravenous injection with equal amounts of WT and ΔciaR mutant GBS strains (A) and in blood, spleen, and brain tissues at the experimental end point of 72 h (B). CFU were enumerated on THA plates or on THA plates supplemented with Cm to distinguish between WT and mutant bacteria. An overall ratio of 1 indicates equal numbers of WT and ΔciaR GBS CFU. *, P < 0.05; **, P < 0.01; ***, P < 0.001.