Mast cell degranulation by a hemolytic lipid toxin decreases GBS colonization and infection

Abstract

Ascending infection of microbes from the lower genital tract into the amniotic cavity increases the risk of preterm birth, stillbirth, and newborn infections. Host defenses that are critical for preventing ascending microbial infection are not completely understood. Group B Streptococcus (GBS) are Gram-positive bacteria that frequently colonize the lower genital tract of healthy women but cause severe infections during pregnancy, leading to preterm birth, stillbirth, or early-onset newborn infections. We recently described that the GBS pigment is hemolytic, and increased pigment expression promotes GBS penetration of human placenta. Here, we show that the GBS hemolytic pigment/lipid toxin and hyperpigmented GBS strains induce mast cell degranulation, leading to the release of preformed and proinflammatory mediators. Mast cell-deficient mice exhibit enhanced bacterial burden, decreased neutrophil mobilization, and decreased immune responses during systemic GBS infection. In a vaginal colonization model, hyperpigmented GBS strains showed increased persistence in mast cell-deficient mice compared to mast cell-proficient mice. Consistent with these observations, fewer rectovaginal GBS isolates from women in their third trimester of pregnancy were hyperpigmented/hyperhemolytic. Our work represents the first example of a bacterial hemolytic lipid that induces mast cell degranulation and emphasizes the role of mast cells in limiting genital colonization by hyperpigmented GBS.

Fig. 1. The hemolytic pigment of GBS triggers the release of preformed mediators from mast cells.

(A and B) About 10⁵ BMCMCs (A) or PCMCs (B) were treated with varying amounts of the GBS pigment (0.625 to 7.5 μM). As controls, equal amounts of extract from the nonpigmented ΔcylE strain or DTS buffer were included. The Ca²⁺ ionophore A23187 (5 μM) was included as a positive control for mast cell degranulation. β-Hex release was measured 1 hour after treatment. Data shown were obtained from three independent experiments performed in duplicate with three independent batches of purified pigment [n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Bonferroni’s multiple comparison test following analysis of variance (ANOVA); error bars, ±SEM]. (C and D) BMCMCs (C) or PCMCs (D) were exposed to either wild-type (WT) GBS A909, hyperhemolytic ΔcovR, or nonhemolytic ΔcovRΔcylE or ΔcylE strains. Uninfected mast cells (UI) and mast cells treated with the Ca²⁺ ionophore A23187 (5 μM) were included as controls. β-Hex release was measured 1 hour after infection. Data shown were obtained from three independent experiments performed in duplicate (n = 3; **P < 0.01, ****P < 0.0001, Bonferroni’s multiple comparison test following ANOVA; error bars, ±SEM). (E and F) PCMCs were exposed to either 0.625 μM pigment or controls (ΔcylE extract or DTS buffer) or the GBS strains indicated earlier for a period of 30 min. Release of PGD₂ and LTC₄ was measured. Data shown were obtained from four independent experiments (n = 4; *P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni’s multiple comparison test following ANOVA; error bars, ±SEM).

Fig. 2. Mast cell degranulation by the GBS pigment requires Ca2⁺ influx.

(A) PCMCs were pretreated with the calcium indicator Fluo-4-AM, and calcium influx was recorded by flow cytometry. At 60 s, either 5 μM A23187 (top panel), 0.5 μM GBS pigment (bottom panel), or an equivalent amount of control ΔcylE extract (middle panel) was added. Mean fluorescence intensities of mast cells before treatment (red) and after treatment (green) are shown. Time is given in seconds. A representative image from one of three independent experiments is shown. (B) PCMCs were pretreated with either EGTA (4 mM) or LY294002 (100 μM) for 30 min or with pertussis toxin (PT; 200 ng/ml) for 2 hours. Untreated PCMCs were included as controls for both pretreatment conditions. Subsequently, the mast cells were exposed to either 2.5 μM pigment or an equivalent amount of ΔcylE extract or 5 μM A23187 for 1 hour. Release of β-hex was then quantified in the mast cell supernatants. Data shown were obtained from three independent experiments performed in duplicate and compared to the respective untreated mast cells (n = 3; **P = 0.002, Dunnett’s multiple comparison test following ANOVA; error bars, ±SEM).

Fig. 3. Mast cell degranulation by the GBS pigment contributes to cytotoxicity.

(A) PCMCs were pretreated with the membrane impermeable dye PI and then exposed to either 5 μM A23187 (top panel), 2.5 μM GBS pigment (bottom panel), or an equivalent amount of ΔcylE extract (middle panel). PI influx was recorded by flow cytometry, and time is given in seconds. Mean fluorescence intensities of mast cells before treatment (red) and after treatment (green) are shown. A representative image from one of two independent experiments is shown. (B) Scanning electron micrographs showing mast cells that were briefly exposed to 0.5 μM pigment or controls (cell culture medium, ΔcylE extract, or 1.66 μM A23187). A representative image from two independent experiments is shown. A minimum of 30 cells were examined in a blinded fashion. (C) PCMCs were pretreated with either EGTA (4 mM) or LY294002 (100 μM) for 30 min or with pertussis toxin (PT; 200 ng/ml) for 2 hours. Untreated PCMCs were included as controls for both pretreatments. Subsequently, the mast cells were exposed to either 2.5 μM pigment or an equivalent amount of ΔcylE extract or 5 μM A23187 for 1 hour. Release of the cytosolic enzyme lactate dehydrogenase (LDH) was measured in the mast cell supernatants. Data shown were obtained from three independent experiments performed in duplicate (n = 3; **P =0.006, Tukey’s multiple comparison test following ANOVA; error bars, ±SEM).

Fig. 4. Hyperpigmented GBS strains induce rapid mast cell degranulation in vivo.

WT C57BL6/J mice (n = 6 per group) were infected intraperitoneally with 10⁷ CFU of either WT GBS or isogenic ΔcovR or ΔcovRΔcylE strains. Peritoneal fluid and blood were collected 2 hours after infection. Data shown are representative of two independent experiments. (A and B) Peritoneal cells were cytocentrifuged and stained with May-Grünwald-Giemsa. A representative image showing intact (PBS) and activated mast cells (ΔcovR) is shown. Activated mast cells in peritoneal fluid were scored in a blinded fashion, and percent activated mast cells was calculated as the number of activated mast cells divided by the total number of mast cells in randomly selected fields × 100 (n = 30 cells per group; *P < 0.05, Bonferroni’s multiple comparison test following ANOVA; medians are shown). (C) Histamine levels were measured in the plasma isolated from the blood of mice infected with the GBS strains indicated earlier (n = 6 per group; *P < 0.05, Bonferroni’s multiple comparison test following ANOVA; medians are shown).

Fig. 5. Mast cell–deficient mice exhibit impaired bacterial clearance and reduced levels of proinflammatory cytokines and neutrophils during systemic GBS infection.

Mast cell–deficient mice and mast cell–proficient littermate controls were infected intraperitoneally with hyperhemolytic/hyperpigmented GBSΔcovR and control nonhemolytic/nonpigmented ΔcovRΔcylE. At 24 hours after infection, bacterial burden, inflammatory cytokines, and neutrophil levels were evaluated. Data shown are from a representative experiment of two independent experiments containing seven animals per group. The Mann-Whitney test was used for comparison between two groups, and Bonferroni’s multiple comparison test following ANOVA was used for multiple comparisons. Medians are indicated. (A) Bacterial burden in the spleens of mast cell–proficient and mast cell–deficient mice infected with GBSΔcovR and ΔcovRΔcylE [*P < 0.05; n.s. (not significant), P > 0.4]. (B to D) Cytokine TNF, IL-6, and KC levels in the spleens of mast cell–proficient and mast cell–deficient mice infected with GBSΔcovR and ΔcovRΔcylE (*P < 0.05; **P < 0.01; ***P < 0.005; n.s., P >0.1). (E) Percent neutrophils (Ly6G⁺ CD11b⁺ cells) in the spleens of mast cell–proficient and mast cell–deficient mice infected with GBSΔcovR (*P < 0.05). (F) Histamine levels in the plasma isolated from mast cell–proficient and mast cell–deficient mice infected with GBSΔcovR and ΔcovRΔcylE (*P < 0.05).

Fig. 6. Mast cell activation promotes clearance of hyperhemolytic GBS from the lower genital tract.

Mast cell–deficient mice or heterozygous littermate controls were intravaginally inoculated with ~10⁸ CFU of GBS ΔcovR or ΔcovRΔcylE. At 4 days after inoculation, bacterial persistence and dissemination were evaluated in the lower genital tract and both uterine horns. (A) Data shown are from an experiment containing eight animals per group. Barnard’s test was used to estimate differences in percent clearance/persistence. (B to D) The Mann-Whitney test was used for comparison between two groups, or Bonferroni’s multiple comparison test following ANOVA was used for multiple comparisons. (A) Negative or positive bacterial cultures obtained from the lower genital tract and both uterine horns of mast cell–proficient mice and mast cell–deficient mice that were inoculated with either GBSΔcovR or ΔcovRΔcylE. Data are represented as percent clearance compared to persistence (n = 8 per group; *P = 0.028, **P = 0.007, Barnard’s test). (B) Bacterial burden in the uterine horns and lower genital tract of mast cell–proficient and mast cell–deficient mice infected with GBS ΔcovR or ΔcovRΔcylE (n = 8 per group; *P < 0.05). In the mast cell–proficient group inoculated with GBSΔcovR, the same mouse had bacterial CFU in both the lower genital tract and uterine horns (denoted as a partially filled symbol). (C) Histamine levels in the genital tract of mast cell–proficient and mast cell–deficient mice infected with GBSΔcovR or ΔcovRΔcylE (n = 8 per group; *P < 0.05).

Fig. 7. Histological sections of the genital tracts of female mast cell–proficient and mast cell–deficient mice infected with hyperpigmented and nonpigmented GBS strains.

Histology of mouse genital tracts at 4 days after inoculation with GBS (ΔcovR or ΔcovRΔcylE) or control PBS. (A) Toluidine blue–stained sections. Mast cells were not observed in the lower genital tracts of mast cell–deficient (MC^−/−) mice (panel iv to vi). (B) H&E-stained sections. (A) Arrows and boxed area indicate nondegranulated mast cells in mast cell–proficient (MC⁺) mice treated with control PBS or GBSΔcovRΔcylE (panels i and iii and magnified insets). In mast cell–proficient mice treated with GBSΔcovR, arrowheads indicate degranulated mast cells (panel ii and magnified inset). (B) H&E-stained sections of mouse genital tracts reveals the presence of inflammatory foci in mast cell–proficient mice infected with GBSΔcovR (arrowhead in panel ii) in contrast to mast cell–proficient mice treated with PBS or GBSΔcovRΔcylE (panels i and iii). Inflammatory foci are also absent in mast cell–deficient (MC^−/−) mice treated with PBS, GBSΔcovR, or GBSΔcovRΔcylE (panels iv to vi). Scale bars, 100 μm.