GBS hyaluronidase mediates immune suppression in a TLR2/4- and IL-10-dependent manner during pregnancy-associated infection.

IMPORTANCE: Bacteria such as GBS can cause infections during pregnancy leading to preterm births, stillbirths, and neonatal infections. The interaction between host and bacterial factors during infections in the placenta is not fully understood. GBS secretes a hyaluronidase enzyme that is thought to digest host hyaluronan into immunosuppressive disaccharides that dampen TLR2/4 signaling, leading to increased bacterial dissemination and adverse outcomes. In this study, we show that GBS HylB mediates immune suppression and promotes bacterial infection during pregnancy that requires TLR2, TLR4, and IL-10. Understanding the interaction between host and bacterial factors can inform future therapeutic strategies to mitigate GBS infections.

CD1 and iNKT cells mediate immune responses against the GBS hemolytic lipid toxin induced by a non-toxic analog.

Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known as R-P4 had reduced bacterial dissemination during systemic infection. However, mechanisms important for R-P4 mediated immune protection was not understood. Here, we show that immune serum from R-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naïve mice from GBS infection. Further, CD4+ T cells isolated from R-P4-immunized mice proliferated in response to R-P4 stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations, R-P4 immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells from R-P4 vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins.

Mast cell-derived factor XIIIA contributes to sexual dimorphic defense against group B streptococcal infections.

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.

Group B Streptococcal Hemolytic Pigment Impairs Platelet Function in a Two-Step Process.

Group B streptococci (GBS) cause a range of invasive maternal-fetal diseases during pregnancy and post-partum. However, invasive infections in non-pregnant adults are constantly increasing. These include sepsis and streptococcal toxic shock syndrome, which are often complicated by systemic coagulation and thrombocytopenia. GBS express a hyper-hemolytic ornithine rhamnolipid pigment toxin with cytolytic and coagulatory activity. Here, we investigated the effects of GBS pigment on human platelets. Infections of platelets with pigmented GBS resulted initially in platelet activation, followed by necrotic cell death. Thus, this study shows that GBS pigment kills human platelets.

A Recombinant Alpha-Like Protein Subunit Vaccine (GBS-NN) Provides Protection in Murine Models of Group B Streptococcus Infection.

BACKGROUND: Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes. METHODS: Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination. RESULTS: Vaccinated mice had higher GBS-NN-specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge. CONCLUSIONS: These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates.

Hyaluronidase Impairs Neutrophil Function and Promotes Group B Streptococcus Invasion and Preterm Labor in Nonhuman Primates.

Invasive bacterial infections during pregnancy are a major risk factor for preterm birth, stillbirth, and fetal injury. Group B streptococci (GBS) are Gram-positive bacteria that asymptomatically colonize the lower genital tract but infect the amniotic fluid and induce preterm birth or stillbirth. Experimental models that closely emulate human pregnancy are pivotal for the development of successful strategies to prevent these adverse pregnancy outcomes. Using a unique nonhuman primate model that mimics human pregnancy and informs temporal events surrounding amniotic cavity invasion and preterm labor, we show that the animals inoculated with hyaluronidase (HylB)-expressing GBS consistently exhibited microbial invasion into the amniotic cavity, fetal bacteremia, and preterm labor. Although delayed cytokine responses were observed at the maternal-fetal interface, increased prostaglandin and matrix metalloproteinase levels in these animals likely mediated preterm labor. HylB-proficient GBS dampened reactive oxygen species production and exhibited increased resistance to neutrophils compared to an isogenic mutant. Together, these findings demonstrate how a bacterial enzyme promotes GBS amniotic cavity invasion and preterm labor in a model that closely resembles human pregnancy.IMPORTANCE Group B streptococci (GBS) are bacteria that commonly reside in the female lower genital tract as asymptomatic members of the microbiota. However, during pregnancy, GBS can infect tissues at the maternal-fetal interface, leading to preterm birth, stillbirth, or fetal injury. Understanding how GBS evade host defenses during pregnancy is key to developing improved preventive therapies for these adverse outcomes. In this study, we used a unique nonhuman primate model to show that an enzyme secreted by GBS, hyaluronidase (HylB) promotes bacterial invasion into the amniotic cavity and fetus. Although delayed immune responses were seen at the maternal-fetal interface, animals infected with hyaluronidase-expressing GBS exhibited premature cervical ripening and preterm labor. These observations reveal that HylB is a crucial GBS virulence factor that promotes bacterial invasion and preterm labor in a pregnancy model that closely emulates human pregnancy. Therefore, hyaluronidase inhibitors may be useful in therapeutic strategies against ascending GBS infection.

Hemolytic Membrane Vesicles of Group B Streptococcus Promote Infection.

BACKGROUND: Group B streptococci (GBS) are ß-hemolytic, Gram-positive bacteria associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A key factor promoting GBS virulence is the ß-hemolysin/cytolysin, a pigmented ornithine rhamnolipid (also known as granadaene) associated with the bacterial surface. METHODS: A previous study indicated that GBS produce small structures known as membrane vesicles (MVs), which contain virulence-associated proteins. In this study, we show that GBS MVs are pigmented and hemolytic, indicating that granadaene is functionally active in MVs. RESULTS: In addition, MVs from hyperhemolytic GBS induced greater cell death of neutrophils, T cells, and B cells compared with MVs from isogenic nonhemolytic GBS, implicating MVs as a potential mechanism for granadaene-mediated virulence. Finally, hemolytic MVs reduced oxidative killing of GBS and aggravated morbidity and mortality of neonatal mice infected with GBS. CONCLUSIONS: These studies, taken together, reveal a novel mechanism by which GBS deploy a crucial virulence factor to promote bacterial dissemination and pathogenesis.

Lipid analogs reveal features critical for hemolysis and diminish granadaene mediated Group B Streptococcus infection.

Although certain microbial lipids are toxins, the structural features important for cytotoxicity remain unknown. Increased functional understanding is essential for developing therapeutics against toxic microbial lipids. Group B Streptococci (GBS) are bacteria associated with preterm births, stillbirths, and severe infections in neonates and adults. GBS produce a pigmented, cytotoxic lipid, known as granadaene. Despite its importance to all manifestations of GBS disease, studies towards understanding granadaene's toxic activity are hindered by its instability and insolubility in purified form. Here, we report the synthesis and screening of lipid derivatives inspired by granadaene, which reveal features central to toxin function, namely the polyene chain length. Furthermore, we show that vaccination with a non-toxic synthetic analog confers the production of antibodies that inhibit granadaene-mediated hemolysis ex vivo and diminish GBS infection in vivo. This work provides unique structural and functional insight into granadaene and a strategy to mitigate GBS infection, which will be relevant to other toxic lipids encoded by human pathogens.

Corrigendum: The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene.

[This corrects the article DOI: 10.3389/fmicb.2019.03123.].

The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene.

Group B Streptococcus (GBS) is a ß-hemolytic, Gram-positive bacterium that commonly colonizes the female lower genital tract and is associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A major factor promoting GBS virulence is the ß-hemolysin/cytolysin, which is cytotoxic to several host cells. We recently showed that the ornithine rhamnolipid pigment, Granadaene, produced by the gene products of the cyl operon, is hemolytic. Here, we demonstrate that heterologous expression of the GBS cyl operon conferred hemolysis, pigmentation, and cytoxicity to Lactococcus lactis, a model non-hemolytic Gram-positive bacterium. Similarly, pigment purified from L. lactis is hemolytic, cytolytic, and identical in structure to Granadaene extracted from GBS, indicating the cyl operon is sufficient for Granadaene production in a heterologous host. Using a systematic survey of phyletic patterns and contextual associations of the cyl genes, we identify homologs of the cyl operon in physiologically diverse Gram-positive bacteria and propose undescribed functions of cyl gene products. Together, these findings bring greater understanding to the biosynthesis and evolutionary foundations of a key GBS virulence factor and suggest that such potentially toxic lipids may be encoded by other bacteria.

Group B streptococcus exploits vaginal epithelial exfoliation for ascending infection.

Thirteen percent of pregnancies result in preterm birth or stillbirth, accounting for fifteen million preterm births and three and a half million deaths annually. A significant cause of these adverse pregnancy outcomes is in utero infection by vaginal microorganisms. To establish an in utero infection, vaginal microbes enter the uterus by ascending infection; however, the mechanisms by which this occurs are unknown. Using both in vitro and murine models of vaginal colonization and ascending infection, we demonstrate how a vaginal microbe, group B streptococcus (GBS), which is frequently associated with adverse pregnancy outcomes, uses vaginal exfoliation for ascending infection. GBS induces vaginal epithelial exfoliation by activation of integrin and ß-catenin signaling. However, exfoliation did not diminish GBS vaginal colonization as reported for other vaginal microbes. Rather, vaginal exfoliation increased bacterial dissemination and ascending GBS infection, and abrogation of exfoliation reduced ascending infection and improved pregnancy outcomes. Thus, for some vaginal bacteria, exfoliation promotes ascending infection rather than preventing colonization. Our study provides insight into mechanisms of ascending infection by vaginal microbes.

Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus.

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

Diminished Capsule Exacerbates Virulence, Blood-Brain Barrier Penetration, Intracellular Persistence, and Antibiotic Evasion of Hyperhemolytic Group B Streptococci.

Group B streptococci (GBS) are encapsulated, ß-hemolytic bacteria that are a common cause of infections in human newborns and certain adults. Two factors important for GBS virulence are the sialic acid capsular polysaccharide that promotes immune evasion and the hemolytic pigment that induces host cell cytotoxcity. These virulence factors are often oppositely regulated by the CovR/CovS two-component system. Clinical GBS strains exhibiting hyperhemolysis and low capsule due to pathoadaptive covR/S mutations have been isolated from patients. Given the importance of capsule to GBS virulence, we predicted that a decrease or loss of capsule would attenuate the virulence of covR/S mutants. Surprisingly, hyperhemolytic GBS with low or no capsule exhibit increased virulence, intracellular persistence, and blood-brain barrier penetration, which was independent of a Trojan horse mechanism of barrier penetration. Additionally, intracellular persistence enabled both hemolytic and hyperhemolytic GBS to evade antibiotics routinely used to treat these infections. The finding that diminished capsule expression promotes GBS virulence, intracellular persistence, and antibiotic evasion has important implications for sustained antibiotic therapy and efficacy of capsule-based vaccines.

A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence.

Group B streptococci (GBS) are Gram-positive bacteria that are a leading cause of neonatal infections. Most invasive isolates are ß-hemolytic, and hemolytic activity is critical for GBS virulence. Although nonhemolytic GBS strains are occasionally isolated, they are often thought to be virulence attenuated. In this study, we show that a nonhemolytic GBS strain (GB37) isolated from a septic neonate exhibits hypervirulence. Substitution of tryptophan to leucine (W297L) in the sensor histidine kinase CovS results in constitutive kinase signaling, leading to decreased hemolysis and increased activity of the GBS hyaluronidase, HylB. These results describe how nonpigmented and nonhemolytic GBS strains can exhibit hypervirulence.

Bacterial Hyaluronidase Promotes Ascending GBS Infection and Preterm Birth.

UNLABELLED: Preterm birth increases the risk of adverse birth outcomes and is the leading cause of neonatal mortality. A significant cause of preterm birth is in utero infection with vaginal microorganisms. These vaginal microorganisms are often recovered from the amniotic fluid of preterm birth cases. A vaginal microorganism frequently associated with preterm birth is group B streptococcus (GBS), or Streptococcus agalactiae However, the molecular mechanisms underlying GBS ascension are poorly understood. Here, we describe the role of the GBS hyaluronidase in ascending infection and preterm birth. We show that clinical GBS strains associated with preterm labor or neonatal infections have increased hyaluronidase activity compared to commensal strains obtained from rectovaginal swabs of healthy women. Using a murine model of ascending infection, we show that hyaluronidase activity was associated with increased ascending GBS infection, preterm birth, and fetal demise. Interestingly, hyaluronidase activity reduced uterine inflammation but did not impact placental or fetal inflammation. Our study shows that hyaluronidase activity enables GBS to subvert uterine immune responses, leading to increased rates of ascending infection and preterm birth. These findings have important implications for the development of therapies to prevent in utero infection and preterm birth. IMPORTANCE: GBS are a family of bacteria that frequently colonize the vagina of pregnant women. In some cases, GBS ascend from the vagina into the uterine space, leading to fetal injury and preterm birth. Unfortunately, little is known about the mechanisms underlying ascending GBS infection. In this study, we show that a GBS virulence factor, HylB, shows higher activity in strains isolated from cases of preterm birth than those isolates from rectovaginal swabs of healthy women. We discovered that GBS rely on HylB to avoid immune detection in uterine tissue, but not placental tissue, which leads to increased rates of fetal injury and preterm birth. These studies provide novel insight into the underlying mechanisms of ascending infection.