The Streptococcus agalactiae LytSR two-component regulatory system promotes vaginal colonization and virulence in vivo

Abstract

Streptococcus agalactiae (or group B Streptococcus, GBS) is a leading cause of neonatal sepsis and meningitis globally. To sense and respond to variations in its environment, GBS possesses multiple two-component regulatory systems (TCSs), such as LytSR. Here, we aimed to investigate the role of LytSR in GBS pathogenicity. We generated an isogenic lytS knockout mutant in a clinical GBS isolate and used a combination of phenotypic in vitro assays and in vivo murine models to investigate the contribution of lytS to the colonization and invasive properties of GBS. Deletion of the lytS gene in the GBS chromosome resulted in significantly higher survival rates in mice during sepsis, accompanied by reduced bacterial loads in blood, lung, spleen, kidney, and brain tissues compared to infection with the wild-type strain. In a mouse model of GBS vaginal colonization, we also observed that the lytS knockout mutant was cleared more readily from the vaginal tract compared to its wild-type counterpart. Interestingly, lower levels of proinflammatory cytokines were found in the serum of mice infected with the lytS mutant. Our results demonstrate that the LytSR TCS plays a key role in GBS tissue invasion and pathogenesis, and persistence of mucosal colonization.IMPORTANCEStreptococcus agalactiae (group B Streptococcus, or GBS) is a common commensal of the female urogenital tract and one of WHO's priority pathogens. The bacterium has evolved mechanisms to adapt and survive in its host, many of which are regulated via two-component signal transduction systems (TCSs); however, the exact contributions of TCSs toward GBS pathogenicity remain largely obscure. We have constructed a TCS lytS-deficient mutant in a CC-17 hypervirulent GBS clinical isolate. Using murine models, we showed that LytSR regulatory system is essential for vaginal colonization via promoting biofilm production. We also observed that lytS deficiency led to significantly attenuated virulence properties and lower levels of proinflammatory cytokines in blood. Our findings are of significant importance in that they unveil a previously unreported role for LytSR in GBS and pave the way toward a better understanding of its ability to transition from an innocuous commensal to a deadly pathogen.

Keywords: CC-17; LytSR two-component regulatory system; Streptococcus agalactiae; group B Streptococcus; newborn infections; pathogenesis.

Fig 1

Phenotypic characterization of GBS wild-type and the isogenic ΔlytS strains. (A) Bacterial growth of GBS strain HQ199 WT and the isogenic ΔlytS mutant was determined in Todd-Hewitt broth at 37°C and 5% CO₂. Optical density at 600 nm (OD₆₀₀) was measured for 24 hours. Data represent the mean ± SD of three independent experiments. (B) Bacterial survival in blood. Bacterial strains were grown in human whole blood at 37°C for 48 hours, and viable bacteria were enumerated. Data represent the mean ± SD of three independent experiments. (C) Observation of β-hemolysis on sheep blood agar base medium. Bacteria were grown at 37°C overnight. (D) Hemolytic activity. Bacterial strains were incubated in fresh sheep red blood cells. After incubation for 30 min, samples were centrifuged, and the levels of hemoglobin released in the supernatant were determined by measuring the optical density at OD₄₂₀. Data represent the mean ± SD of three independent experiments. (E) Rates of bacterial autolysis. Bacterial strains were grown at an OD₆₀₀ = 1 prior to incubation in 0.01% Triton-X-100 at 37°C with shaking. After incubation for 120 min, OD₆₀₀ was measured and converted as the percent of the initial OD₆₀₀ reading. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed using the unpaired t test. *P < 0.05 and ***P < 0.001.

Fig 2

lytS deficiency impairs GBS colonization and persistence in the genital tract of mice. Female Balb/c mice (n = 30 per group, 5 mice per day) were infected intravaginally with 1 × 10⁶ CFU of GBS HQ199 WT or the isogenic ΔlytS strains. Levels of bacterial colonization in the vagina (A), cervix (B), and uterus (C) were quantified at days 1, 3, 7, 14, and 21 post infection. Each dot represents a single mouse, and error bars represent the mean ± SEM in each time point. Statistical analysis between the two groups in each day was performed using the Mann-Whitney U test. *P < 0.05 and **P < 0.01.

Fig 3

lytS deficiency reduces GBS virulence and dissemination in a mouse sepsis model. (A) Kaplan-Meier curve representing percentage of survival of female Balb/c mice (n = 10 per group) infected intravenously with 1 × 10⁸ CFU of GBS HQ199 wild-type or the isogenic ΔlytS strains. Statistical analysis was performed using the log-rank (Mantel-Cox) test. (B) Levels of bacterial loads in blood, lung, and brain tissues at time of death. In the WT group, animals were euthanized upon reaching humane endpoints at 18, 24, 30, and 36 hours post infection (n = 1, 7, 1, and 1, respectively). In the ΔlytS group, all tissues were collected at 72 hours post infection (n = 10). Each dot represents a single mouse, and error bars represent the mean ± SEM. Statistical analysis was performed using the Mann-Whitney U test. ***P < 0.001 and ****P < 0.0001.

Fig 4

lytS deficiency prevents bacterial dissemination and brain invasion in vivo in a mouse sepsis model. Female Balb/c mice (n = 20 per group, 5 mice per day) were intravenously infected with 1 × 10⁸ CFU of GBS HQ199 wild-type or the isogenic ΔlytS strains. Levels of bacterial loads in blood (A), lung (B), spleen (C), kidney (D), and brain (E) tissues were quantified at 0.5, 6, 24, and 32 hours post infection. Each dot represents a single mouse, and error bars represent the mean ± SEM in each time point. Statistical analysis between the two groups in each time point was performed using the Mann-Whitney U test. *P < 0.05 and **P < 0.01.

Fig 5

lytS deficiency impacts proinflammatory host responses during GBS sepsis. Female Balb/c mice (n = 20 per group, 5 mice per day) were intravenously infected with 1 × 10⁸ CFU of GBS HQ199 wild-type or the isogenic ΔlytS strains. Levels of proinflammatory cytokines IL-1β (A), IL-2 (B), IL-6 (C), IL-17A/F (D), MIP-3α (E), KC/GRO (F), and TNF-α (G) were quantified in the serum of mice at 0.5, 6, 24, and 32 hours post infection. Data represent the mean $\pm$ SD in each time point. Statistical analysis between the two groups in each time point was performed using the Mann-Whitney U test. *P < 0.05 and **P < 0.01.