Analysis of Two-Component Systems in Group B Streptococcus Shows That RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Abstract

Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

The Sensor Histidine Kinase RgfC Affects Group B Streptococcal Virulence Factor Expression Independent of Its Response Regulator RgfA

“…Control of these genes suggests that RgfC regulation of gene expression is altered in the absence of a functional RgfA and that sensor kinase has a broader role in GBS virulence than previously appreciated. Support for this observation also is provided by the recent findings of Faralla et al (21). The authors reported that the deletion of RgfC in the GBS serotype V strain CJB11, which encodes a functional RgfA, resulted in increased mortality of mice without significant differences in bacterial dissemination (21).…”

“…The same point mutation in rgfA also was identified in other GBS clinical strains, such as H36B (9) (Fig. 1B) and 61 other GBS strains (21). To determine if RgfC had a significant role in gene regulation and virulence of GBS in strains lacking a functional RgfA, we constructed a strain deficient in rgfC expression from the wild-type (WT) strain A909.…”

“…However, in certain GBS clinical isolates that were obtained from infected newborns (e.g., A909 and H36B), a point mutation in rgfA results in the premature truncation of the response regulator (9; also see the schematic in Fig. 1A and B); this feature was observed in another 61 GBS strains (21). The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and regulation of gene expression.…”

“…Like other prokaryotic organisms, signaling in GBS is achieved primarily by two-component signaling systems (TCS) that regulate gene expression in response to external signals (1,3,4). The GBS genome sequence indicates the presence of 18 to 20 TCS (9), and the roles of a few TCS have been well characterized and include CovR/CovS (10)(11)(12)(13)(14)(15), DltR/DltS (16), CiaH/CiaR (17), SAK_0188/0189 (18), RgfC/A (19,20), and FpsR/S (21). Previous studies have indicated that GBS deficient for RgfC/A exhibit reduced adherence to extracellular matrix components due to altered expression of the fibrinogen binding proteins, such as FbsA and FbsB (19,20).…”

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD

“…Quantitative RT-PCR Analysis-RNA extracts were prepared as described (24). Bacteria were harvested at two time points, at A 600 ϭ 0.4 (log phase) and A 600 ϭ 1.7 (early stationary phase).…”