Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment

Abstract

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.

Keywords: Coagulation; Group B streptococcus; Hemolysis; Inflammation; Pigment; Streptococcus agalactiae.

Fig. 1

Pigmented LUMC16 and STSS/NF-HH GBS strains cause hyperhemolysis. a Patient diagnosed with STSS, DIC, and purpura fulminans. b Pigmentation of the indicated strains after 16-h growth in THY medium. c, d Hemolytic activity of the indicated GBS strains shown by a clearance zone around the colonies on sheep blood agar plates (c) and by hemoglobin release assay with human blood (d). Each symbol represents one independent experiment. Bars denote median values (n = 4). e Relative mRNA expression of the indicated genes encoding for pigment biosynthesis from stationary-phase bacterial cultures (n = 4). DIC, disseminated intravascular coagulation; GBS, group B streptococcus; HH, hyperhemolytic; LH, low hemolytic; NF, necrotizing fasciitis; NSTI, necrotizing soft tissue infection; STSS, streptococcal toxic shock syndrome.

Fig. 2

Whole genome sequencing analyses. a Neighborhood joining tree calculated via genome-genome distance calculator and inferred from Streptococcus agalactiae assembled genomes. b Base differences per 5.0 kb based on the comparison of the whole genomes of the indicated strains with the annotated genome of the 2603VR strain. c Single nucleotide polymorphism (SNP; left panel) and insertion/deletion (INDEL; right panel) variants found within the whole genome of the case's LUMC16 GBS strain. GBS, group B streptococcus; HH, hyperhemolytic; LH, low hemolytic; NF, necrotizing fasciitis; STSS, streptococcal toxic shock syndrome.

Fig. 3

Hyperhemolytic LUMC16 and STSS/NF-HH strains induce a toxic shock syndrome-associated proinflammatory response in human PBMCs and monocytes. a Human PBMCs (left panel) and monocytes (right panel) were stimulated with viable bacteria for 6 h and cytotoxic effects were assessed. Each symbol represents primary cells from one donor. Bars denote median values (n = 4). b–e TNF (b), IL-1β (c), IL-6 (d), and IL-8 (e) release by human primary PBMCs and/or monocytes in response to bacterial stimulation. Each symbol represents stimulation of PBMCs/monocytes from one healthy volunteer. Horizontal lines denote median values (n = 4). The level of significance was determined using the Kruskal-Wallis test with Dunnett's multiple comparison (** p < 0.01, *** p < 0.001). HH, hyperhemolytic; LH, low hemolytic; NF, necrotizing fasciitis; PBMCs, peripheral blood mononuclear cells; STSS, streptococcal toxic shock syndrome; Unstim., unstimulated.

Fig. 4

Hemolytic and cytolytic activities of the pigment. a Hemolytic activity of the isolated pigment. Pigment was added to 10% human blood at the indicated dilutions. Equivalent amounts of sample buffer were used as controls (ctrl.). The data shown are the mean and range from 4 independent pigment preparations (n = 4). b Human PBMCs (left panel) and monocytes (right panel) were stimulated with different concentrations of the pigment for 6 h, and cytotoxic effects were assessed. Each symbol represents primary cells from one donor. Bars denote median values (n ≥ 4). PBMCs, peripheral blood mononuclear cells.

Fig. 5

Pigment-induced cytokine release by human PBMCs and monocytes. TNF (a), IL-1β (b), IL-6 (c), and IL-8 (d) release by human primary PBMCs and/or monocytes in response to pigment stimulation. Each symbol represents stimulation of PBMCs/monocytes from one healthy volunteer. Horizontal lines denote median values (n ≥ 4). The level of significance was determined using the Kruskal-Wallis test with Dunnett's multiple comparison (* p < 0.05, ** p < 0.01, *** p < 0.001). PBMCs, peripheral blood mononuclear cells.

Fig. 6

Interference of GBS strains with the coagulation system. Bacteria were incubated in human plasma for 30 min at 37°C and removed via centrifugation. aPTT (a) and PT (b) of the resulting supernatants were determined in a coagulometer. Plasma incubated with buffer alone served as a control (Ctrl.). Each symbol represents one independent experiment. Bars denote median values and range from 3 and more independent experiments (n ≥ 3). The level of significance was determined using the Kruskal-Wallis test with Dunnett's multiple comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). c Blood clotting after incubation with the indicated GBS strains. Bacteria were added to the same volume of blood. Buffer alone served as a control. After incubation for 1, 2, and 4 h at 37°C, the recalcification clotting times were measured. Each symbol represents one independent experiment. Bars denote median values and range from 3 independent experiments (n = 3). The level of significance between the plasma controls and STSS strain samples was determined using Welch's t test (*** p < 0.001). d Activation of FXII/PK on the bacterial surface of the indicated GBS strains. Bacteria were incubated in HEPES buffer (control buffer [Pos. ctrl.]) or normal human plasma and washed, and chromogenic substrate S-2303 was added to the reaction. The FXII/PK activity was determined by measuring the absorbance (A) at 405 nm. Data represent mean values ± SD from 4 independent experiments (n = 4). Statistical significance between the controls and GBS strains was calculated using Welch's t test (*** p < 0.001). aPTT, activated partial thromboplastin time; FXII, factor XII; GBS, group B streptococcus; HH, hyperhemolytic; LH, low hemolytic; NF, necrotizing fasciitis; PT, prothrombin time; STSS, streptococcal toxic shock syndrome.

Fig. 7

Interference of the GBS pigment with the coagulation system. The indicated concentrations of the pigment were incubated in human plasma for 30 min at 37°C and removed via centrifugation. aPTT (a) and PT (b) of the resulting supernatants were determined in a coagulometer. Plasma incubated with the same amount of elution buffer of the pigment served as a control. Each symbol represents one independent experiment. Bars denote median values and range from 3 independent experiments (n = 3). The level of significance was determined using Welch's t test (** p < 0.01). c Plasma clotting after incubation with the indicated dilutions of the GBS pigment. Elution buffer of the pigment served as a control. After incubation for 1 h at 37°C, the recalcification clotting times were measured. Each symbol represents one independent experiment. Bars denote median values and range from 3 independent experiments (n = 3). The level of significance between the controls and pigment samples was determined using Welch's t test (** p < 0.01, *** p < 0.001, **** p < 0.0001). d Activation of FXII/PK GBS pigment. Pigment or elution buffer (ctrl. buffer) were incubated in normal human plasma and washed, and chromogenic substrate S-2303 was added to the reaction. The FXII/PK activity was determined by measuring the absorbance (A) at 405 nm. Data represent mean values and range from 3 independent experiments (n = 3). Statistical significance between the controls and pigment was calculated using Welch's t test (* p < 0.05, *** p < 0.001). aPTT, activated partial thromboplastin time; FXII, factor XII; GBS, group B streptococcus; PT, prothrombin time.