A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.
Bottom Line: Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells.Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage.These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.
Affiliation: Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA.Show MeSH
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Mentions: A known activator of the NLRP3 inflammasome is the efflux of intracellular potassium that occurs upon membrane permeabilization (Munoz-Planillo et al, 2013). Given that the GBS pigment is able to induce membrane disruptions in RBC and even in artificial lipid bilayers (Fig1C and D), we hypothesized that intercalation of the GBS pigment into host cells such as human macrophages should trigger membrane permeabilization and the efflux of intracellular potassium, irrespective of the presence or absence of the inflammasome. To test this hypothesis, we measured membrane disruption and quantified intracellular potassium levels in NLRP3-proficient and NLRP3-deficient macrophages that were treated with the GBS pigment. To examine membrane permeabilization, we exposed NLRP3-proficient and NLRP3-deficient macrophages to GBS pigment (1 μM) or controls for 20 min and measured uptake of a membrane impermeable dye, propidium iodide (see Materials and Methods for details). The results shown in Fig5A and B indicate that increased fluorescence is seen in both NLRP3-proficient and NLRP3-deficient macrophages treated with GBS pigment (1 μM) when compared to controls (ΔcylE extract). These data indicate that the GBS pigment induces membrane permeability in host cells independent of the inflammasome. To further confirm this, we utilized ion-coupled plasma atomic emission spectroscopy (ICP-AES) as described (Munoz-Planillo et al, 2013) to measure levels of intracellular potassium in NLRP3-proficient and NLRP3-deficient macrophages that were exposed to GBS pigment or to controls over time (0, 30, 60, 120, 180, and 240 min). These results demonstrate that intracellular potassium levels dramatically decreased within 30 min in NLRP3-proficient macrophages (THP-1/scrambled, Fig5C) and in NLRP3-deficient macrophages (THP-1/shNLRP3, Fig5D) that were exposed to the GBS pigment (1 μM), demonstrating that both pigment-mediated membrane disruption and potassium efflux occur independently of NLRP3 inflammasome activation and even cell death. Notably, NLRP3-deficient macrophages appear to be able to recover from the initial K+ efflux (see 120, 180, and 240 min in Fig5D). In contrast, cells proficient for NLRP3 undergo cell death and do not significantly recover (Fig5C). To determine if K+ efflux was important for pigment-induced cell death, we performed cytolysis assays of THP-1 cells exposed to the GBS pigment in the presence of excess extracellular potassium (final concentration 50 mM) compared to culture media containing 5 mM potassium. The results shown in Fig5E indicate that addition of extracellular potassium partially protected the cells from GBS pigment-mediated cytolysis, further supporting a role of K+ efflux and NLRP3 activation in cytolysis.
Affiliation: Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA.