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A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.

Whidbey C, Vornhagen J, Gendrin C, Boldenow E, Samson JM, Doering K, Ngo L, Ezekwe EA, Gundlach JH, Elovitz MA, Liggitt D, Duncan JA, Adams Waldorf KM, Rajagopal L - EMBO Mol Med (2015)

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.

View Article: PubMed Central - PubMed

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.

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The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanismsFemale pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106–7 CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown.In utero fetal death in wild-type CD-1 mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylE mice and is indicated as NS; P = 0.99.
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fig07: The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanismsFemale pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106–7 CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown.In utero fetal death in wild-type CD-1 mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylE mice and is indicated as NS; P = 0.99.

Mentions: Previous work from our group demonstrated that hyperhemolytic GBS strains with mutations in CovR/S were more proficient in penetration of human placenta and were also isolated from chorioamniotic membranes and amniotic fluid from women in preterm labor (Whidbey et al, 2013). To determine if hyperhemolytic GBS strains induce fetal injury and preterm birth, we adapted a murine model of E. coli-induced in utero infection (Elovitz et al, 2003) to GBS. We chose the intrauterine model of inoculation rather than a vaginal model of inoculation to minimize discrepancies that can be attributed to differences in mouse vaginal persistence (Patras et al, 2013). To this end, on day E14.5 of pregnancy, either WT GBS, the hyperpigmented ΔcovR, or the non-hemolytic ΔcovRΔcylE was infused into the right horn of the uterus between the first (P1) and second (P2) fetal sacs most proximal to the cervix as described (Hirsch et al, 1995; Elovitz et al, 2003) (also see Fig7A). Mice were monitored for 72 h for signs of preterm birth (i.e., at least 1 pup in cage). At either 72 h or at the onset of delivery (whichever occurred first), fetal survival rates were determined and tissue was collected to assess bacterial load. Preterm birth (i.e., at least 1 pup in cage) was observed in three of six mice infected with ΔcovR, in one of six infected with WT GBS, and in none of the six mice infected with the non-hemolytic ΔcovRΔcylE strain (preterm delivery rates were 50, 16, and 0%, respectively). We also observed significantly higher fetal death in mice infected with hemolytic GBS, that is, WT or hyperpigmented ΔcovR compared to the non-hemolytic ΔcovRΔcylE strain (Fig7B). Bacteria were recovered from all fetuses present in the uterus, and there was no significant difference in bacterial load between fetuses of mice infected with the three GBS strains (data not shown). H&E staining of infected uterine tissue showed that while only a few mononuclear cells are present in the ΔcovRΔcylE sample, increased presence of inflammatory cells and necrotic debris is seen in the ΔcovR sample (Fig7C). The frequency of fetal death between hemolytic strains of GBS such as WT and ΔcovR was not significantly different and may likely be due to decreased repression of hemolysin/pigment biosynthetic genes by CovR/S in utero as suggested previously (Santi et al, 2009; Sitkiewicz et al, 2009).


A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.

Whidbey C, Vornhagen J, Gendrin C, Boldenow E, Samson JM, Doering K, Ngo L, Ezekwe EA, Gundlach JH, Elovitz MA, Liggitt D, Duncan JA, Adams Waldorf KM, Rajagopal L - EMBO Mol Med (2015)

The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanismsFemale pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106–7 CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown.In utero fetal death in wild-type CD-1 mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylE mice and is indicated as NS; P = 0.99.
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fig07: The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanismsFemale pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106–7 CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown.In utero fetal death in wild-type CD-1 mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. ‘n’ indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylE mice and is indicated as NS; P = 0.99.
Mentions: Previous work from our group demonstrated that hyperhemolytic GBS strains with mutations in CovR/S were more proficient in penetration of human placenta and were also isolated from chorioamniotic membranes and amniotic fluid from women in preterm labor (Whidbey et al, 2013). To determine if hyperhemolytic GBS strains induce fetal injury and preterm birth, we adapted a murine model of E. coli-induced in utero infection (Elovitz et al, 2003) to GBS. We chose the intrauterine model of inoculation rather than a vaginal model of inoculation to minimize discrepancies that can be attributed to differences in mouse vaginal persistence (Patras et al, 2013). To this end, on day E14.5 of pregnancy, either WT GBS, the hyperpigmented ΔcovR, or the non-hemolytic ΔcovRΔcylE was infused into the right horn of the uterus between the first (P1) and second (P2) fetal sacs most proximal to the cervix as described (Hirsch et al, 1995; Elovitz et al, 2003) (also see Fig7A). Mice were monitored for 72 h for signs of preterm birth (i.e., at least 1 pup in cage). At either 72 h or at the onset of delivery (whichever occurred first), fetal survival rates were determined and tissue was collected to assess bacterial load. Preterm birth (i.e., at least 1 pup in cage) was observed in three of six mice infected with ΔcovR, in one of six infected with WT GBS, and in none of the six mice infected with the non-hemolytic ΔcovRΔcylE strain (preterm delivery rates were 50, 16, and 0%, respectively). We also observed significantly higher fetal death in mice infected with hemolytic GBS, that is, WT or hyperpigmented ΔcovR compared to the non-hemolytic ΔcovRΔcylE strain (Fig7B). Bacteria were recovered from all fetuses present in the uterus, and there was no significant difference in bacterial load between fetuses of mice infected with the three GBS strains (data not shown). H&E staining of infected uterine tissue showed that while only a few mononuclear cells are present in the ΔcovRΔcylE sample, increased presence of inflammatory cells and necrotic debris is seen in the ΔcovR sample (Fig7C). The frequency of fetal death between hemolytic strains of GBS such as WT and ΔcovR was not significantly different and may likely be due to decreased repression of hemolysin/pigment biosynthetic genes by CovR/S in utero as suggested previously (Santi et al, 2009; Sitkiewicz et al, 2009).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus