Group B Strep and Pregnancy: Difference between revisions

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<br>By Shawn Ruiz<br>
<br>By Shawn Ruiz<br>


<br>Group B Strep (GBS), also known as Streptococcus agalactiae, is a Gram-positive, beta-hemolytic, catalase-negative, facultative anaerobe that is a normal component of the gastrointestinal and genitourinary tracts<ref name=aa>[https://en.wikipedia.org/wiki/Streptococcus_agalactiae “Streptococcus Agalactiae.” Wikipedia, Wikimedia Foundation, 24 Mar. 2021, en.wikipedia.org/wiki/Streptococcus_agalactiae.].</ref>. In fact, GBS colonizes the gastrointestinal and genitourinary tracts of up to 50% of healthy adults<ref name=bb>[Johri, Atul Kumar, et al. “Group B Streptococcus: Global Incidence and Vaccine Development.” Nature Reviews Microbiology, vol. 4, no. 12, 2006, pp. 932–942., doi:10.1038/nrmicro1552.].</ref>. Most healthy adults who are colonized by GBS will not experience any symptoms or GBS-related infections. While the bacteria is usually harmless in healthy adults, it is a major cause of meningitis, pneumonia, and and sepsis in neonates<ref name=cc>[Dekker, Rebecca. “The Evidence on: Group B Strep.” Evidence Based Birth , Evidence Based Birth , 17 July 2017, evidencebasedbirth.com/groupbstrep/.].</ref>. Moreover, GBS is the leading infectious cause of neonatal mortality and morbidity in the United States; between four and six percent of babies who develop GBS disease die<ref name=dd>[Morgan, John A. “Group B Streptococcus And Pregnancy.” StatPearls [Internet]., U.S. National Library of Medicine, 29 Jan. 2021, www.ncbi.nlm.nih.gov/books/NBK482443/#:~:text=Preterm
<br>Group B Strep (GBS), also known as Streptococcus agalactiae, is a Gram-positive, beta-hemolytic, catalase-negative, facultative anaerobe that is a normal component of the gastrointestinal and genitourinary tracts<ref name=aa>[https://en.wikipedia.org/wiki/Streptococcus_agalactiae “Streptococcus Agalactiae.” Wikipedia, Wikimedia Foundation, 24 Mar. 2021, en.wikipedia.org/wiki/Streptococcus_agalactiae.].</ref>. In fact, GBS colonizes the gastrointestinal and genitourinary tracts of up to 50% of healthy adults<ref name=bb>[https://pubmed.ncbi.nlm.nih.gov/17088932/Johri, Atul Kumar, et al. “Group B Streptococcus: Global Incidence and Vaccine Development.” Nature Reviews Microbiology, vol. 4, no. 12, 2006, pp. 932–942., doi:10.1038/nrmicro1552.].</ref>. Most healthy adults who are colonized by GBS will not experience any symptoms or GBS-related infections. While the bacteria is usually harmless in healthy adults, it is a major cause of meningitis, pneumonia, and sepsis in neonates<ref name=cc>[https://evidencebasedbirth.com/groupbstrep/ Dekker, Rebecca. “The Evidence on: Group B Strep.” Evidence Based Birth , Evidence Based Birth , 17 July 2017, evidencebasedbirth.com/groupbstrep/.].</ref>. GBS is the leading infectious cause of neonatal mortality and morbidity in the United States; between four and six percent of babies who develop GBS disease die<ref name=dd>[https://www.ncbi.nlm.nih.gov/books/NBK482443/#:~:text=Preterm%20%20infants%20with%20%20early%2Donset,in%20term%20infants%20%5B2%5DMorgan John A. “Group B Streptococcus And Pregnancy.” StatPearls, U.S. National Library of Medicine, 29 Jan. 2021, www.ncbi.nlm.nih.gov/books/]</ref><ref name=ee>[https://www.cdc.gov/groupbstrep/about/fast-facts.html#references “Group B Strep: Fast Facts and Statistics.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 11 June 2020, www.cdc.gov/groupbstrep/about/fast-facts.html#references.]</ref>. GBS causes both early onset (<7 days old) and late onset (7-90 days old) infections in neonates<ref name=dd/>. The main risk factor for an early-onset GBS infection in neonates is colonization of a birthing person's genital tract with Group B strep during labor<ref name=dd/>. About one in four pregnant individuals carry GBS in their body<ref name=ee/>. If the bacteria is present in a pregnant person, it can be directly transferred to their neonate(s) in a multitude of ways. For example, GBS can travel from the vagina into the amniotic fluid where the neonate(s) can ingest it. The neonate(s) can also come into contact with the bacteria as they make their way down the birth canal<ref name=cc/>. In the early 1990s, the early GBS infection rate was 1.7 cases per 1,000 births<ref name=cc/>. In an effort to decrease this infection rate, the American Congress of Obstetricians and Gynecologists and the American Academy of Pediatrics recommended screening pregnant individuals for GBS before they go into labor<ref name=cc/>. It is now common practice to screen pregnant individuals for GBS at some point between 35 and 37 weeks of pregnancy<ref name=ee/>. Pregnant people who test positive for GBS are treated with intravenous antibiotics during labor<ref name=ee/>. Penicillin and ampicillin are the recommended antibiotics for intrapartum GBS prophylaxis<ref name=ff>[https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5910a1.htm Horsley, Elizabeth. “CDC Updates Guidelines for the Prevention of Perinatal GBS Disease.” American Family Physician, The American Academy of Family Physicians Foundation , 1 May 2011, www.aafp.org]</ref>. If a pregnant person tests positive for GBS and they are treated with antibiotics during labor, the risk of their neonate(s) developing a serious, life-threatening GBS infection drops by 80%  <ref name=cc/>. Early GBS infection rates in the United States have significantly dropped (0.25 cases per 1,000 births) since these preventative measures went into effect around 1995<ref name=cc/>. Intrapartum prophylaxis effectively prevents GBS transmission from the birthing individual to their neonate(s) during labor and delivery. This preventative measure, however, does not target in utero infections that occur earlier in pregnancy, and little is known about the mechanisms that result in the infection of the amniotic cavity<ref name=gg>[https://pubmed.ncbi.nlm.nih.gov/23712433/ Whidbey, Christopher, et al. “A Hemolytic Pigment of Group B Streptococcus Allows Bacterial Penetration of Human Placenta.” Journal of Experimental Medicine, vol. 210, no. 6, 2013, pp. 1265–1281., doi:10.1084/jem.20122753.]</ref>. In utero GBS infections have devastating effects, including preterm birth and mortality in both the pregnant person and their neonate(s)<ref name=gg/>. That said, it is critical that researchers and public health officials work toward understanding exactly how GBS infects the amniotic cavity.
%20infants%20with%20
early%2Donset,in%20term%20infants
%5B2%5D.]</ref><ref name=ee>[“Group B Strep: Fast Facts and Statistics.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 11 June 2020, www.cdc.gov/groupbstrep/about/fast-facts.html#references.]</ref>. GBS causes both early onset (<7 days old) and late onset (7-90 days old) infections in neonates<ref name=dd/>. The main risk factor for an early-onset GBS infection in a neonate is colonization of a birthing person's genital tract with Group B strep during labor<ref name=dd/>. About one in four pregnant individuals carry GBS in their body<ref name=ee/>. If the bacteria is present in a pregnant person, it can be directly transferred to their baby in a multitude of ways. For example, GBS can travel from the vagina into the amniotic fluid where the baby can ingest it. The baby can also come into contact with the bacteria as they make their way down the birth canal<ref name=cc/>. In the early 1990s, the early GBS infection rate was 1.7 cases per 1,000 births<ref name=cc/>. In an effort to decrease this infection rate, the American Congress of Obstetricians and Gynecologists and the American Academy of Pediatrics recommended screening pregnant individuals for GBS before they go into labor<ref name=cc/>. As a result, it is now common practice to screen pregnant individuals for GBS at some point between 35 and 37 weeks of pregnancy<ref name=ee/>. Pregnant people who test positive for GBS are treated with intravenous antibiotics during labor<ref name=ee/>. Penicillin and ampicillin are the recommended antibiotics for intrapartum GBS prophylaxis<ref name=ff>[Horsley, Elizabeth. “CDC Updates Guidelines for the Prevention of Perinatal GBS Disease.” American Family Physician, The American Academy of Family Physicians Foundation , 1 May 2011, www.aafp.org/afp/2011/0501/p1106.html#:~:text=The%20
recommended%20antibiotic%20for%20intrapartum,units%20
intravenously%20
every%20four%20hours.]</ref>. If a pregnant person tests positive for GBS and they are treated with antibiotics during labor, the risk of their neonate developing a serious, life-threatening GBS infection drops by 80%  <ref name=cc/>. Early GBS infection rates in the United States have significantly dropped (0.25 cases per 1,000 births) since these preventative measures went into effect around 1995<ref name=cc/>. While intrapartum prophylaxis to prevent GBS transmission from the birthing individual to their neonate during labor and delivery has proven to be effective, this preventative measure does not target in utero infections that occur earlier in pregnancy, and little is known about the mechanisms that result in the infection of the amniotic cavity<ref name=gg>[Whidbey, Christopher, et al. “A Hemolytic Pigment of Group B Streptococcus Allows Bacterial Penetration of Human Placenta.” Journal of Experimental Medicine, vol. 210, no. 6, 2013, pp. 1265–1281., doi:10.1084/jem.20122753.]</ref>. In utero GBS infections have devastating effects, including preterm birth and mortality in both the pregnant person and their baby<ref name=gg/>. That said, the it is critical that researchers and public health officials work toward understanding exactly how GBS infects the amniotic cavity.


==Section 1==
==The Hemolytic Pigment of GBS==
[[Image:Screen Shot 2021-04-06 at 12.35.47 AM.png|thumb|300px|left|Figure 2. Primary hAECs were isolated from chorioamniotic membranes and the percent adherence of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig1.jpg].]]
[[Image:Screen Shot 2021-04-06 at 12.35.47 AM.png|thumb|300px|left|Figure 2. Primary hAECs were isolated from chorioamniotic membranes, and the percent adherence of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig1.jpg].]]


[[Image:Screen_Shot_2021-04-06_at_12.47.20_AM.png |thumb|300px|left|Figure 3. Primary hAECs were isolated from chorioamniotic the percent invasion of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared<ref name=gg/>. [https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig1.jpg].]]
[[Image:Screen_Shot_2021-04-06_at_12.47.20_AM.png |thumb|300px|left|Figure 3. Primary hAECs were isolated from chorioamniotic membranes, and the percent invasion of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared<ref name=gg/>. [https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig1.jpg].]]


GBS has been isolated from the amniotic fluid of birthing people with intact chorioamniotic membranes, suggesting that GBS is capable of invading and breaching amniotic epithelium and chorioamnion<ref name=gg/><ref name=hh>[JR, Bobitt, and Ledger WJ. “Unrecognized Amnionitis and Prematurity: a Preliminary Report.” The Journal of Reproductive Medicine., vol. 19, no. 1, 30 June 1977, pp. 8–12., europepmc.org/article/med/874942.]</ref><ref name=ii>[Naeye , RL, and EC Peters. “Amniotic Fluid Infections with Intact Membranes Leading to Perinatal Death: a Prospective Study.” Pediatrics, U.S. National Library of Medicine, Feb. 1978, pubmed.ncbi.nlm.nih.gov/634667/.]</ref><ref name=jj>[Winram, Scott B., et al. “Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro.” Infection and Immunity, vol. 66, no. 10, 1998, pp. 4932–4941., doi:10.1128/iai.66.10.4932-4941.1998.]</ref><ref name=kk>[Goldenberg, Robert L., et al. “Intrauterine Infection and Preterm Delivery.” New England Journal of Medicine, vol. 342, no. 20, 2000, pp. 1500–1507., doi:10.1056/nejm200005183422007.]</ref>. This led Whidbey et al. to hypothesize that “intra-amniotic GBS infections in patients with intact placenta or chorioamniotic membranes may be due to elevated virulence factor expression”<ref name=gg/><ref name=hh/><ref name=ii/><ref name=jj/><ref name=kk/>.Previous studies showed that the expression of GBS virulence genes is regulated by a two-component regulatory system: COVR/S<ref name=gg/><ref name=ll>[Lamy, Marie-Cécile, et al. “CovS/CovR of Group B Streptococcus: a Two-Component Global Regulatory System Involved in Virulence.” Molecular Microbiology, vol. 54, no. 5, 2004, pp. 1250–1268., doi:10.1111/j.1365-2958.2004.04365.x.]</ref><ref name=mm>[Jiang, Sheng-Mei, et al. “Variation in the Group B Streptococcus CsrRS Regulon and Effects on Pathogenicity.” Journal of Bacteriology, vol. 190, no. 6, 2008, pp. 1956–1965., doi:10.1128/jb.01677-07.]</ref><ref name=nn>[Rajagopal, Lakshmi, et al. “Regulation of Cytotoxin Expression by Converging Eukaryotic-Type and Two-Component Signalling Mechanisms in Streptococcus Agalactiae.” Molecular Microbiology, vol. 62, no. 4, 2006, pp. 941–957., doi:10.1111/j.1365-2958.2006.05431.x.]</ref>. More specifically, COVR/S was described to repress a multitude of GBS virulence genes, including the cyl operon, which contains the cylE gene that is that is important for the production of the pluripotent toxin, hemolysin <ref name=gg/><ref name=ll/><ref name=mm/><ref name=nn/>. In order to test whether or not increased expression of GBS virulence genes promotes the invasion of amniotic epithelium, Whidbey et al. compared the abilities of wild-type (WT) GBS and hyper-hemolytic GBS (ΔcovR) GBS to adhere to and invade human amniotic epithelial cells (hAEC)<ref name=gg/>.The hAECs were isolated and cultured from “normal, term placentas obtained immediately after cesarean delivery from” birthing people without labor<ref name=gg/>. Non-hemolytic GBS strains (ΔcovRΔcylE and ΔcylE) were also included in the experiment to investigate hemolysin’s role in GBS strains’ ability to adhere to and invade hAECs<ref name=gg/><ref name=oo>[Pritzlaff, Craig A., et al. “Genetic Basis for the Beta-Haemolytic/Cytolytic Activity of Group B Streptococcus.” Molecular Microbiology, vol. 39, no. 2, 2001, pp. 236–248., doi:10.1046/j.1365-2958.2001.02211.x.]</ref>. The results suggest that all GBS strains adhered to hAECs, as seen in Figure 2.Notably, as shown in Figure 3, WT GBS invaded hAECs (~4% invasion), non-hemolytic GBS showed a significantly decreased invasion when compared to the WT (~0.3% invasion), and the hyper-hemolytic strain (ΔcovR) was significantly more invasive when compared to the WT (~80% invasion)<ref name=gg/>.In sum, Whidbey et al. concluded that hemolysin promotes GBS invasion of hAECs.
GBS has been isolated from the amniotic fluid of birthing people with intact chorioamniotic membranes, suggesting that GBS is capable of invading and breaching amniotic epithelium and chorioamnion<ref name=gg/><ref name=hh>[https://europepmc.org/article/med/874942 Bobitt, JR, and WJ Ledger. “Unrecognized Amnionitis and Prematurity: a Preliminary Report.” The Journal of Reproductive Medicine., vol. 19, no. 1, 30 June 1977, pp. 8–12., europepmc.org/article/med/874942.]</ref><ref name=ii>[https://pubmed.ncbi.nlm.nih.gov/634667/ Naeye , RL, and EC Peters. “Amniotic Fluid Infections with Intact Membranes Leading to Perinatal Death: a Prospective Study.” Pediatrics, U.S. National Library of Medicine, Feb. 1978, pubmed.ncbi.nlm.nih.gov/634667/.]</ref><ref name=jj>[https://europepmc.org/article/med/9746599 Winram, Scott B., et al. “Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro.” Infection and Immunity, vol. 66, no. 10, 1998, pp. 4932–4941., doi:10.1128/iai.66.10.4932-4941.1998.]</ref><ref name=kk>[https://pubmed.ncbi.nlm.nih.gov/10816189/ Goldenberg, Robert L., et al. “Intrauterine Infection and Preterm Delivery.” New England Journal of Medicine, vol. 342, no. 20, 2000, pp. 1500–1507., doi:10.1056/nejm200005183422007.]</ref>. Whidbey et al. hypothesized that “intra-amniotic GBS infections in patients with intact placenta or chorioamniotic membranes may be due to elevated virulence factor expression”<ref name=gg/><ref name=hh/><ref name=ii/><ref name=jj/><ref name=kk/>. Previous studies showed that the expression of GBS virulence genes is regulated by a two-component regulatory system: COVR/S<ref name=gg/><ref name=ll>[https://pubmed.ncbi.nlm.nih.gov/15554966/ Lamy, Marie-Cécile, et al. “CovS/CovR of Group B Streptococcus: a Two-Component Global Regulatory System Involved in Virulence.” Molecular Microbiology, vol. 54, no. 5, 2004, pp. 1250–1268., doi:10.1111/j.1365-2958.2004.04365.x.]</ref><ref name=mm>[https://pubmed.ncbi.nlm.nih.gov/18203834/ Jiang, Sheng-Mei, et al. “Variation in the Group B Streptococcus CsrRS Regulon and Effects on Pathogenicity.” Journal of Bacteriology, vol. 190, no. 6, 2008, pp. 1956–1965., doi:10.1128/jb.01677-07.]</ref><ref name=nn>[https://pubmed.ncbi.nlm.nih.gov/17005013/ Rajagopal, Lakshmi, et al. “Regulation of Cytotoxin Expression by Converging Eukaryotic-Type and Two-Component Signalling Mechanisms in Streptococcus Agalactiae.” Molecular Microbiology, vol. 62, no. 4, 2006, pp. 941–957., doi:10.1111/j.1365-2958.2006.05431.x.]</ref>. More specifically, COVR/S was described to repress a multitude of GBS virulence genes, including the cyl operon, which contains the cylE gene that is important for the production of the pluripotent toxin, hemolysin <ref name=gg/><ref name=ll/><ref name=mm/><ref name=nn/>. In order to test whether or not increased expression of GBS virulence genes promotes the invasion of amniotic epithelium, Whidbey et al. compared the abilities of wild-type (WT) GBS and hyper-hemolytic GBS (ΔcovR) to adhere to and invade human amniotic epithelial cells (hAECs) that were isolated and cultured from “normal, term placentas obtained immediately after cesarean delivery from” birthing people without labor<ref name=gg/>. Non-hemolytic GBS strains (ΔcovRΔcylE and ΔcylE) were also included in the experiment to investigate hemolysin’s role in GBS strains’ ability to adhere to and invade hAECs<ref name=gg/><ref name=oo>[https://pubmed.ncbi.nlm.nih.gov/11136446/ Pritzlaff, Craig A., et al. “Genetic Basis for the Beta-Haemolytic/Cytolytic Activity of Group B Streptococcus.” Molecular Microbiology, vol. 39, no. 2, 2001, pp. 236–248., doi:10.1046/j.1365-2958.2001.02211.x.]</ref>. The results, shown in Figures 2 and 3, show that all GBS strains adhered to hAECs. WT GBS invaded hAECs (~4% invasion), non-hemolytic GBS showed a significantly decreased invasion when compared to the WT (~0.3% invasion), and the hyper-hemolytic GBS strain (ΔcovR) was significantly more invasive when compared to the WT strain (~80% invasion)<ref name=gg/>. In sum, Whidbey et al. concluded that hemolysin promotes GBS invasion of hAECs.




Next, Whidbey et al. investigated if the increased expression of hemolysin in the hyper-hemolytic GBS strain (ΔcovR) activates an inflammatory response<ref name=gg/>. Whidbey et al. isolated RNA from GBS-infected hAECs four hours after infection, and they used qRT-PCR to examine any changes in the expression of inflammatory genes<ref name=gg/><ref name=pp>[Lembo, Annalisa, et al. “Regulation of CovR Expression in Group B Streptococcus Impacts Blood-Brain Barrier Penetration.” Molecular Microbiology, vol. 77, no. 2, 2010, pp. 431–443., doi:10.1111/j.1365-2958.2010.07215.x.]</ref>. The results, depicted in Figure 4, suggest that infection with ΔcovR caused a significant increase in the transcription of cytokines (e.g., IL-6 and IL-8) in hAECs<ref name=gg/>The increase in the expression of inflammatory genes in hAECS infected with ΔcovR was abolished in hAECs infected with the non-hemolytic strain, ΔcovRΔcylE. In sum, hAECs that were infected with the hyper-hemolytic GBS strain, ΔcovR, did show an inflammatory response. 
Next, Whidbey et al. investigated if the increased expression of hemolysin in the hyper-hemolytic GBS strain (ΔcovR) activates an inflammatory response<ref name=gg/>. Whidbey et al. isolated RNA from GBS-infected hAECs four hours after infection, and they used qRT-PCR to examine any changes in the expression of inflammatory genes in hAECs<ref name=gg/><ref name=pp>[https://pubmed.ncbi.nlm.nih.gov/20497331/ Lembo, Annalisa, et al. “Regulation of CovR Expression in Group B Streptococcus Impacts Blood-Brain Barrier Penetration.” Molecular Microbiology, vol. 77, no. 2, 2010, pp. 431–443., doi:10.1111/j.1365-2958.2010.07215.x.]</ref>. The results, depicted in Figure 4, suggest that infection with ΔcovR caused a significant increase in the transcription of cytokines (e.g., IL-6 and IL-8) in hAECs<ref name=gg/>. In sum, the results suggest that hAECs that were infected with the hyper-hemolytic GBS strain (ΔcovR) did show an inflammatory response. 
[[Image:Screen_Shot_2021-04-06_at_12.59.39_AM.png|thumb|300px|right|Figure 4. Whidbey et al. isolated RNA from hAECs infected with either WT GBS COH1 or isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants at 4 h after infection. qRT-PCR was performed on the RNA, and to examine the expression of cytokines/chemokines. "Data shown are the mean and SD obtained from hAECs that were isolated from three independent placentas, performed in triplicate (n = 3; **, P = 0.007; *, P = 0.03, Student’s t test, error bars ± SD)<ref name=gg/>."[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig2.jpg].]]
[[Image:Screen_Shot_2021-04-06_at_12.59.39_AM.png|thumb|300px|right|Figure 4. Whidbey et al. isolated RNA from hAECs infected with WT GBS COH1, isogenic ΔcovR, ΔcovRΔcylE, or ΔcylE mutants at 4 h after infection. qRT-PCR was performed on the RNA to examine the expression of cytokines/chemokines. "Data shown are the mean and SD obtained from hAECs that were isolated from three independent placentas, performed in triplicate (n = 3; **, P = 0.007; *, P = 0.03, Student’s t test, error bars ± SD)<ref name=gg/>."[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig2.jpg].]]




It is known that microbial toxins use the nuclear transcription factor, NF-𝜅B, which is recruited from the cytoplasm to the nucleus, to activate inflammatory signaling pathways<ref name=qq>[Gonzalez, M. R., et al. “Bacterial Pore-Forming Toxins: The (w)Hole Story?” Cellular and Molecular Life Sciences, vol. 65, no. 3, 2007, pp. 493–507., doi:10.1007/s00018-007-7434-y. ]</ref>. Given that hAECs that were infected with ΔcovR showed an inflammatory response, Whidbey et al. investigated whether or not the increase in the expression of pro-inflammatory genes was associated with the nuclear recruitment/localization of NF-𝜅B<ref name=gg/>.Whidbey et al. isolated total nuclear and cytoplasmic proteins from infected and uninfected hAECs, and they resolved them on 10% SDS-PAGE and Western blots<ref name=gg/>. As shown in Figure 5,hAECs that were infected with the hyper-hemolytic GBS strain (ΔcovR) showed a 2.5 fold increase in the nuclear recruitment of  NF-𝜅B<ref name=gg/>.[[Image:Screen Shot 2021-04-06 at 1.21.29 AM.png|thumb|300px|right|Figure 5. Whidbey et al. performed Western Blots of nuclear and cytoplasmic proteins in GBS-infected hAECs(NF-𝜅B antibody). N and C stand for nuclear and cytoplasmic<ref name=gg/>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig2/?report=objectonly].]]  
It is known that microbial toxins use the nuclear transcription factor, NF-𝜅B, which is recruited from the cytoplasm to the nucleus, to activate inflammatory signaling pathways<ref name=qq>[https://pubmed.ncbi.nlm.nih.gov/17989920/ Gonzalez, M. R., et al. “Bacterial Pore-Forming Toxins: The (w)Hole Story?” Cellular and Molecular Life Sciences, vol. 65, no. 3, 2007, pp. 493–507., doi:10.1007/s00018-007-7434-y. ]</ref>. Given that hAECs that were infected with ΔcovR showed an inflammatory response, Whidbey et al. investigated if the increase in the expression of pro-inflammatory genes was associated with the nuclear recruitment/localization of NF-𝜅B<ref name=gg/>. Whidbey et al. isolated total nuclear and cytoplasmic proteins from infected and uninfected hAECs, and they resolved them on 10% SDS-PAGE and Western blots<ref name=gg/>. As shown in Figure 5, hAECs that were infected with the hyper-hemolytic GBS strain (ΔcovR) showed a 2.5 fold increase in the nuclear recruitment of  NF-𝜅B compared with WT GBS-infected or uninfected controls. These results suggest that the increase in the expression of inflammatory genes seen in ΔcovR-infected hAECs "is associated with the nuclear recruitment of NF-𝜅B"<ref name=gg/>.[[Image:Screen Shot 2021-04-06 at 1.21.29 AM.png|thumb|300px|right|Figure 5. Whidbey et al. performed Western Blots of nuclear and cytoplasmic proteins in GBS-infected hAECs(NF-𝜅B antibody). N and C stand for nuclear and cytoplasmic<ref name=gg/>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig2/?report=objectonly].]]  




Despite finding that hemolysin is associated with an increase in the expression of pro-inflammatory genes, as well as the nuclear recruitment of NF-𝜅B, Whidbey et al. still did not know whether or not the presence of hemolysin accelerates the failure of the human amniotic epithelial barrier<ref name=gg/>. To examine this, Whidbey et al. monitored the changes in transepithelial electrical resistance across hAEC using electric cell-substrate impedance sensing<ref name=gg/><ref name=rr>[Giaever, I., and C. R. Keese. “Micromotion of Mammalian Cells Measured Electrically.” Proceedings of the National Academy of Sciences, vol. 88, no. 17, 1991, pp. 7896–7900., doi:10.1073/pnas.88.17.7896.]</ref>. Figure six shows that hAECs that were infected with the hyper-hemolytic strain (ΔcovR) showed an accelerated decrease in their barrier resistance compared to hAECs that were infected with WT GBS<ref name=gg/>. In sum, increased hemolysin expression decreased the barrier function of the amniotic epithelium, enabling GBS to breach the epithelial barrier<ref name=gg/>.[[Image:Screen_Shot_2021-04-06_at_1.37.40_AM.png|thumb|300px|left|Figure 6. Whidbey et al. used Electric Cell-substrate Impedance Sensing (ECIS) to monitor the barrier resistance of hAECs in real time.[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig2/?report=objectonly].]]
Despite finding that hemolysin is associated with an increase in the expression of pro-inflammatory genes, as well as the nuclear recruitment of NF-𝜅B, Whidbey et al. still did not know if the presence of hemolysin accelerates the failure of the human amniotic epithelial barrier<ref name=gg/>. To examine this, Whidbey et al. monitored the changes in transepithelial electrical resistance across hAEC using electric cell-substrate impedance sensing<ref name=gg/><ref name=rr>[https://www.pnas.org/content/88/17/7896 Giaever, I., and C. R. Keese. “Micromotion of Mammalian Cells Measured Electrically.” Proceedings of the National Academy of Sciences, vol. 88, no. 17, 1991, pp. 7896–7900., doi:10.1073/pnas.88.17.7896.]</ref>. Figure 6 shows that hAECs that were infected with the hyper-hemolytic strain (ΔcovR) showed an accelerated decrease in their barrier resistance compared to hAECs that were infected with WT GBS<ref name=gg/>. In sum, increased hemolysin expression decreased the barrier function of the amniotic epithelium, enabling GBS to breach the epithelial barrier<ref name=gg/>.[[Image:Screen_Shot_2021-04-06_at_1.37.40_AM.png|thumb|300px|left|Figure 6. Whidbey et al. used Electric Cell-Substrate Impedance Sensing (ECIS) to monitor the barrier resistance of hAECs in real time.[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig2/?report=objectonly].]]




Knowing that an increase in hemolysin expression enabled hyper-hemolytic GBS to breach the hAEC epithelium, Whidbey et al. investigated whether or not GBS are capable of penetrating intact chorioamniotic membranes<ref name=gg/>.  Whidbey et al. mounted, maintained, and, after 48 hours of stabilization, infected chorioamniotic membranes with various GBS strains (WT, ΔcovR, ΔcovRΔcylE). Figure 7 shows that only ΔcovR penetrated and invaded the chorioamnion, including the amniotic epithelium.This suggests that an increase in hemolysin expression can “facilitate bacterial penetration of chorioamniotic membranes and the amniotic cavity”<ref name=gg/>.[[Image:Screen Shot 2021-04-06 at 1.49.39 AM.png|thumb|300px|left|Figure 7. GBS penetration of the chorion, amnion, and chorioamniotic membranes (n = 6; NS, P > 0.3; *, P = 0.02, Mann Whitney test, error bars ± SD)<ref name=gg/>."[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig3/?report=objectonly].]]
Knowing that an increase in hemolysin expression enabled hyper-hemolytic GBS to breach the hAEC epithelium, Whidbey et al. investigated if GBS are capable of penetrating intact chorioamniotic membranes<ref name=gg/>.  Whidbey et al. mounted, maintained, and, after 48 hours of stabilization, infected chorioamniotic membranes with various GBS strains (WT, ΔcovR, ΔcovRΔcylE). Figure 7 shows that only ΔcovR penetrated and invaded the chorioamnion, including the amniotic epithelium. This suggests that an increase in hemolysin expression can “facilitate bacterial penetration of chorioamniotic membranes and the amniotic cavity”<ref name=gg/>.[[Image:Screen Shot 2021-04-06 at 1.49.39 AM.png|thumb|300px|left|Figure 7. GBS penetration of the chorion, amnion, and chorioamniotic membranes (n = 6; NS, P > 0.3; *, P = 0.02, Mann Whitney test, error bars ± SD)<ref name=gg/>."[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674703/figure/fig3/?report=objectonly].]]




Considering that hyper-hemolytic GBS is capable of penetrating the chorioamniotic membranes and the amniotic cavity, resulting in an in-utero infection and preterm labor, Whidbey et al. hypothesized that increased hemolytic activity could be observed in GBS isolated from birthing people in preterm labor<ref name=gg/>. To test this hypothesis, they collected clinical isolates from amniotic fluid and chorioamnion from six birthing people in preterm labor<ref name=gg/>. These isolates were examined for potential mutations in the covR/S locus, as well as hemolytic properties. Eight GBS strains were obtained: most of them demonstrated increased hemolytic activity, and six of them had a mutation in the covR/S loci<ref name=gg/>. Notably, two GBS isolates did not have a mutation in the covR/S loci, but they did demonstrate increased hemolytic activity. That said, it is likely that hemolytic activity is regulated by multiple regulatory mechanisms. Site-directed mutations of the COVR/S mutations that were collected from clinical samples confirmed that the increase in hemolytic activity was in part attributable to these mutations<ref name=gg/>.
Considering that hyper-hemolytic GBS is capable of penetrating the chorioamniotic membranes and the amniotic cavity, which may result in an in-utero infection and/or preterm labor, Whidbey et al. hypothesized that increased hemolytic activity could be observed in GBS isolated from birthing people in preterm labor<ref name=gg/>. To test this hypothesis, they collected clinical isolates from amniotic fluid and chorioamnion from six birthing people in preterm labor<ref name=gg/>. These isolates were examined for potential mutations in the covR/S locus, as well as hemolytic properties. Eight GBS strains were obtained: most of them demonstrated increased hemolytic activity, and six of them had a mutation in the covR/S loci<ref name=gg/>. Notably, two GBS isolates did not have a mutation in the covR/S loci, but they did demonstrate increased hemolytic activity. That said, it is likely that hemolytic activity is regulated by multiple regulatory mechanisms. Site-directed mutations of the COVR/S mutations that were collected from clinical samples confirmed that the increase in hemolytic activity was in part attributable to these mutations<ref name=gg/>. Thus, hyper-hemolytic GBS can be associated with birthing people in preterm labor.




One study suggested that the cylE gene, which is a part of the cyl operon, encoded and produced hemolysin<ref name=oo/>. Researchers proved, however, that while CylE is necessary for the production of hemolysin, it is not sufficient for GBS hemolysis. Notably, studies have shown that the hemolytic phenotype of GBS is pigmented and the non-hemolytic phenotype is not; this pigment is described as an ornithine rhamnolipid (i.e., granadaene)<ref name=oo/><ref name=ss>[Nizet, V, et al. “Group B Streptococcal Beta-Hemolysin Expression Is Associated with Injury of Lung Epithelial Cells.” Infection and Immunity, vol. 64, no. 9, 1996, pp. 3818–3826., doi:10.1128/iai.64.9.3818-3826.1996.]</ref><ref name=tt>[Spellerberg, Barbara, et al. “Thecylgenes OfStreptococcus Agalactiaeare Involved in the Production of Pigment.” FEMS Microbiology Letters, vol. 188, no. 2, 2000, pp. 125–128., doi:10.1111/j.1574-6968.2000.tb09182.x.]</ref>.  Like hemolytic activity, pigment biosynthesis in GBS also requires the cyl open Whidbey et al. conducted a sequence profile analysis of CylE, which they used in combination with predicted homology profiles, to propose a pathway (depicted in Figure 9) for GBS pigment biosynthesis that is dependent on the majority of genes in the cyl operon<ref name=gg/>.  
One study suggested that the cylE gene, which is a part of the cyl operon, encoded and produced hemolysin<ref name=oo/>. Whidbey et al. proved, however, that, while CylE is necessary for the production of hemolysin, it is not sufficient for GBS hemolysis. Notably, studies have shown that the hemolytic phenotype of GBS is pigmented and the non-hemolytic phenotype is not; this pigment is described as an ornithine rhamnolipid (i.e., granadaene)<ref name=oo/><ref name=ss>[https://pubmed.ncbi.nlm.nih.gov/8751934/ Nizet, V, et al. “Group B Streptococcal Beta-Hemolysin Expression Is Associated with Injury of Lung Epithelial Cells.” Infection and Immunity, vol. 64, no. 9, 1996, pp. 3818–3826., doi:10.1128/iai.64.9.3818-3826.1996.]</ref><ref name=tt>[https://academic.oup.com/femsle/article/188/2/125/614898 Spellerberg, Barbara, et al. “Thecylgenes OfStreptococcus Agalactiaeare Involved in the Production of Pigment.” FEMS Microbiology Letters, vol. 188, no. 2, 2000, pp. 125–128., doi:10.1111/j.1574-6968.2000.tb09182.x.]</ref>.  Like hemolytic activity, pigment biosynthesis in GBS also requires the cyl open. Whidbey et al. conducted a sequence profile analysis of CylE, which they used in combination with predicted homology profiles, to propose a pathway for GBS pigment biosynthesis that is dependent on the majority of genes in the cyl operon, which is depicted in Figure 8<ref name=gg/>.  




Whidbey et al. propose that the ornithine rhamnolipid pigment, rather than the CylE protein, is the cause of the hemolytic activity in GBS. To test this, they extracted and purified pigment from WT GBS and examined the hemolytic activity of the pigment. Lysis of red blood cells occurred in the presence of the pigment, and brief (8 min) exposure to the pigment dramatically altered the membrane morphology of the disc-shaped red blood cells, as shown in Figure 10. Researchers confirmed that the hemolytic activity observed in the lysis of red blood cells could not be attributed to a protein toxin by demonstrating that the purified pigment was not affected by proteinase K. Other conformational experiments, such as SDS-PAGE analysis of the purified pigment, were also performed<ref name=gg/>.[[Image:Screen_Shot_2021-04-06_at_2.26.22_AM.png|thumb|300px|right|Figure 9.Proposed pathway for the biosynthesis of the GBS pigment, granadaene<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753R_Fig6.jpg.].]][[Image:Screen_Shot_2021-04-06_at_2.49.27_AM.png |thumb|300px|right|Figure 10.Scanning electron micrographs of hRBCs 8 minutes after exposure to GBS pigment (12.5 µM), buffer or ΔcylE extract.<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig7.jpg].]]
That said, Whidbey et al. propose that the ornithine rhamnolipid pigment, rather than the CylE protein, is the cause of the hemolytic activity in GBS. To test this, they extracted and purified pigment from WT GBS and examined the hemolytic activity of the pigment. Lysis of red blood cells occurred in the presence of the pigment, and brief (8 min) exposure to the pigment dramatically altered the membrane morphology of the disc-shaped red blood cells, as shown in Figure 9. Researchers confirmed that the hemolytic activity observed in the lysis of red blood cells could not be attributed to a protein toxin by demonstrating that the purified pigment was not affected by proteinase K. Other experiments, such as SDS-PAGE analysis of the purified pigment, also helped confirm this finding<ref name=gg/>.[[Image:Screen_Shot_2021-04-06_at_2.26.22_AM.png|thumb|300px|right|Figure 8. Proposed pathway for the biosynthesis of the GBS pigment, granadaene<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753R_Fig6.jpg.].]][[Image:Screen_Shot_2021-04-06_at_2.49.27_AM.png |thumb|300px|right|Figure 9. Scanning electron micrographs of hRBCs 8 minutes after exposure to GBS pigment (12.5 µM), buffer, or ΔcylE extract.<ref name=gg/>.[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3674703_JEM_20122753_Fig7.jpg].]]


==Section 3==
==The Lytic Mechanisms and Cytolytic Abilities of the GBS Hemolytic Pigment==
Whidbey et al. investigated how the GBS pigment induces cell lysis by measuring the kinetics of both K+ and hemoglobin (Hb) release from red blood cells treated with 400nM of the pigment<ref name=ww>[Whidbey, Christopher, et al. “A Streptococcal Lipid Toxin Induces Membrane Permeabilization and Pyroptosis Leading to Fetal&nbsp;Injury.” EMBO Molecular Medicine, vol. 7, no. 4, 2015, pp. 488–505., doi:10.15252/emmm.201404883.]</ref>. These results suggest that membrane permeabilization enables K+ and Hb to efflux, respectively. This delay in Hb suggests that the GBS pigment induces a colloidal osmotic mechanism of lysis rather than direct lysis<ref name=ww/>. Protection assays revealed that small osmoprotectants (e.g., PEG200) are incapable of protecting RBC from pigment-mediated hemolysis, whereas larger osmoprotectants (e.g, PEG1500) completely protect RBC from pigment-mediated hemolysis<ref name=ww/>. Interestingly, pigment-mediate membrane permeability is independent of cellular response, and it does not conform to a pore-forming protein toxin nor the induction of instant lysis<ref name=ww/>. 
Whidbey et al. investigated how the GBS pigment induces cell lysis by measuring the kinetics of both K<sup>+</sup> and hemoglobin (Hb) release from red blood cells treated with 400nM of the pigment<ref name=ww>[https://pubmed.ncbi.nlm.nih.gov/25750210/ Whidbey, Christopher, et al. “A Streptococcal Lipid Toxin Induces Membrane Permeabilization and Pyroptosis Leading to Fetal&nbsp;Injury.” EMBO Molecular Medicine, vol. 7, no. 4, 2015, pp. 488–505., doi:10.15252/emmm.201404883.]</ref>. These results suggest that membrane permeabilization enables K<sup>+</sup> and Hb to efflux, respectively. This delay in Hb suggests that the GBS pigment induces a colloidal osmotic mechanism of lysis rather than direct lysis<ref name=ww/>. Protection assays revealed that small osmoprotectants (e.g., PEG200) are incapable of protecting RBC from pigment-mediated hemolysis, whereas larger osmoprotectants (e.g, PEG1500) completely protect RBC from pigment-mediated hemolysis<ref name=ww/>. Interestingly, pigment-mediate membrane permeability is independent of cellular response, and it does not conform to a pore-forming protein toxin nor the induction of instant lysis<ref name=ww/>. 


To further investigate the cytolytic abilities of GBS, researchers treated macrophages with various GBS strains (WT, ΔcovR, ΔcylE, and ΔcovRΔcylE) for four hours; cytotoxicity was measured by the release of lactate dehydrogenase<ref name=ww/>. Macrophage death and IL-1β release in cells that were infected with hyper-hemolytic GBS was significantly higher compared to those infected with non-hemolytic strains<ref name=ww/>. This, combined with further experiments that confirmed these findings and revealed an increase in IL-18 levels in macrophages treated with hyper-hemolytic GBS strains, suggest that the purified hemolytic GBS pigments is pro-inflammatory and cytotoxic<ref name=ww/>. 


To investigate the cytolytic abilities of GBS, researchers treated macrophages with various GBS strains (WT, ΔcovR, ΔcylE, and ΔcovRΔcylE) for four hours; cytotoxicity was measured by the release of lactate dehydrogenase<ref name=ww/>. Macrophage death and IL-1β and IL-18 release in cells that were infected with hyper-hemolytic GBS was significantly higher compared to those infected with non-hemolytic strains<ref name=ww/>. This, combined with further experiments that confirmed these findings and revealed an increase in IL-1β and IL-18 levels in macrophages treated with hyper-hemolytic GBS strains, suggest that the purified hemolytic GBS pigments is pro-inflammatory and cytotoxic<ref name=ww/>. 


To determine if hyper-hemolytic GBS strains induce fetal injury or preterm birth, Whidbey et al. used an intrauterine model of inoculation to introduce GBS strains--WT, hyperpigmented (ΔcovR), or non-hemolytic (ΔcovRΔcylE)--into the right horn of the uteruses of pregnant mice<ref name=ww/><ref name=xx>[Patras, Kathryn A., et al. “Group B Streptococcus CovR Regulation Modulates Host Immune Signalling Pathways to Promote Vaginal Colonization.” Cellular Microbiology, vol. 15, no. 7, 2013, pp. 1154–1167., doi:10.1111/cmi.12105. ]</ref><ref name=yy>[Hirsch, Emmet, et al. “A Model of Intrauterine Infection and Preterm Delivery in Mice.” American Journal of Obstetrics and Gynecology, vol. 172, no. 5, 1995, pp. 1598–1603., doi:10.1016/0002-9378(95)90503-0. ]</ref><ref name=zz>[Elovitz, Michal A., et al. “A New Model for Inflammation-Induced Preterm Birth.” The American Journal of Pathology, vol. 163, no. 5, 2003, pp. 2103–2111., doi:10.1016/s0002-9440(10)63567-5.]</ref>. The inoculated mice were monitored for 72 hour for signs of preterm, which Whidbey et al. defined as the birth of at least one pup in a mouse’s cage<ref name=ww/>. One of six mice infected with WT GBS and three of six mice infected with ΔcovR GBS experienced preterm birth<ref name=ww/>. Moreover, Whidbey et al. also observed significantly higher fetal deaths in mice that were infected with WT GBS and ΔcovR GBS (hemolytic strains)<ref name=ww/>. Whidbey et al. reported that there was not a statistically significant difference between the frequency of fetal deaths between the two hemolytic strains, and they explained that this may be due to a decrease in the repression of hemolysin/pigment by COVR/S<ref name=ww/><ref name=santi>[Santi, Isabella, et al. “CsrRS Regulates Group B Streptococcus Virulence Gene Expression in Response to Environmental PH: a New Perspective on Vaccine Development.” Journal of Bacteriology, vol. 191, no. 17, 2009, pp. 5387–5397., doi:10.1128/jb.00370-09.]</ref><ref name=sit>[Sitkiewicz, Izabela, et al. “Transcriptome Adaptation of Group B Streptococcus to Growth in Human Amniotic Fluid.” PLoS ONE, vol. 4, no. 7, 2009, doi:10.1371/journal.pone.0006114.]</ref>. 


==Section 4==
The increase in the secretion of IL-1β and IL-18 in macrophages treated with the GBS pigment suggests that the pigment is capable of triggering activation of the inflammasome, which is a cytosolic complex that is responsible for activating inflammatory responses to injury and/or illness<ref name=ww/>. Whidbey et al. write, "one major inflammasome comprises the NLR (nucleotide binding, leucine-rich repeat containing) protein known as NLRP3, which associates with the adaptor ASC (apoptosis-associated speck-like protein containing the caspase recruitment domain, CARD)"<ref name=ww/><ref name=infl>[https://pubmed.ncbi.nlm.nih.gov/20638636/ Taxman, Debra J., et al. “Inflammasome Inhibition as a Pathogenic Stealth Mechanism.” Cell Host &amp; Microbe, vol. 8, no. 1, 2010, pp. 7–11., doi:10.1016/j.chom.2010.06.005. ]</ref>. Whideby et al. conducted several experiments to investigate if the GBS pigment activates NLRP3, and they concluded that GBS induced hemolysin-dependent cell death in macrophages that contain the NLRP3 inflammasome<ref name=ww/>.
Unlike the mice discussed in the previous paragraph, pregnant people do not get infected with GBS via intrauterine inoculation. In order for in utero infection to occur in humans, vaginal microbes must enter the uterus by ascending infection. The mechanisms behind ascending infection are not clear, but Vornhagen et al. propose a convincing mechanism that GBS uses to cause in utero infections<ref name=vorn>[Vornhagen, Jay, et al. “Group B Streptococcus Exploits Vaginal Epithelial Exfoliation for Ascending Infection.” Journal of Clinical Investigation, vol. 128, no. 5, 2018, pp. 1985–1999., doi:10.1172/jci97043.]</ref>.Notably, not all pregnant people experience ascending microbial infections. Vornhagen et al. propose that one reason for this may be a breakdown in the host’s defenses that work to prevent ascending infection<ref name=vorn/>. Epithelial cells, for example, create a physical barrier that is able to recognize a pathogenic attack and respond to it appropriately<ref name=vorn/>. One of these defense mechanisms is called epithelial exfoliation (i.e., shedding), during which epithelial cells detach from their basement membrane, which prevent pathogens from harming the host<ref name=vorn/>. Vaginal epithelial cells use exfoliation to protect their host<ref name=vorn/>. Interestingly, vaginal GBS colonization increases a pregnant person’s risks of experiencing an ascending infection and, in turn, adverse pregnancy outcomes<ref name=vorn/>. Given this, Vornhagen et al. hypothesized that vaginal epithelial exfoliation may regulate GBS colonization<ref name=vorn/>. 


Vaginal exfoliation is characterized by vaginal epithelial cells losing their adhesive properties and detaching from their base membrane in response to a pathological threat<ref name=vorn/>. To test if GBS may trigger the loss of barier function and cellular detachment of vaginal epithelial cell, which will eventually result in exfoliation, Vornhagen et al. infected immortalized human vaginal epithelial cells (hVECs) with the GBS WT strain COH1<ref name=vorn/>. Vonhegan et al. noticed a significant amount of cellular detachment, as well as a significant difference in cell structure and shape, and a loss of barrier function in hVECS infected with WT GBS<ref name=vorn/>. In other words, these results suggest that WT GBS infection of hVECS stimulates vaginal epithelial cell exfoliation<ref name=vorn/>. Next, Vonhegan et al. measured “the ability of GBS to migrate across hVEC monolayers in the Transwell assay<ref name=vorn/>". Vonhegan et al. observed that a significant amount of GSB crossed the epithelial barrier. Notably, these crossing times were associated with vaginal epithelial cell exfoliation time points, suggesting that GBS induces vaginal epithelial cell exfoliation, and it allows for an increase in GBS cell migration across vaginal epithelia<ref name=vorn/>. 
==The GBS Hemolytic Pigment is the Critical Component of GBS-Related Fetal Injuries==
[[Image:Screen Shot 2021-04-06 at 11.25.40 AM.png|thumb|300px|left|Figure 10. In utero fetal deaths in GBS-infected (GBS WT, hyperhemolytic ΔcovR, or non-hemolytic ΔcovRΔcylE) mice. Fetal deaths were measured by the number of dead fetuses obtained from a total of six parent mice.****P < 0.0001, Fisher's exact test.<ref name=vorn/>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403049/figure/fig07/?report=objectonly].]]
 
To determine if hyper-hemolytic GBS strains induce fetal injury or preterm birth, Whidbey et al. used an intrauterine model of inoculation to introduce GBS strains (WT, hyperpigmented (ΔcovR), or non-hemolytic (ΔcovRΔcylE)) into the right horn of the uteruses of pregnant mice<ref name=ww/><ref name=xx>[https://pubmed.ncbi.nlm.nih.gov/23298320/ Patras, Kathryn A., et al. “Group B Streptococcus CovR Regulation Modulates Host Immune Signalling Pathways to Promote Vaginal Colonization.” Cellular Microbiology, vol. 15, no. 7, 2013, pp. 1154–1167., doi:10.1111/cmi.12105. ]</ref><ref name=yy>[https://www.ajog.org/article/0002-9378(95)90503-0/abstract Hirsch, Emmet, et al. “A Model of Intrauterine Infection and Preterm Delivery in Mice.” American Journal of Obstetrics and Gynecology, vol. 172, no. 5, 1995, pp. 1598–1603., doi:10.1016/0002-9378(95)90503-0. ]</ref><ref name=zz>[https://www.sciencedirect.com/science/article/pii/S0002944010635675 Elovitz, Michal A., et al. “A New Model for Inflammation-Induced Preterm Birth.” The American Journal of Pathology, vol. 163, no. 5, 2003, pp. 2103–2111., doi:10.1016/s0002-9440(10)63567-5.]</ref>. The inoculated mice were monitored for 72 hours for signs of preterm, which Whidbey et al. defined as the birth of at least one pup in a mouse’s cage<ref name=ww/>. One of six mice infected with WT GBS and three of six mice infected with ΔcovR GBS experienced preterm birth compared with the controls groups<ref name=ww/>. Moreover, Figure 10 shows that there were significantly higher fetal deaths in mice that were infected with WT GBS and ΔcovR GBS (hemolytic strains)<ref name=ww/>. Whidbey et al. reported that there was not a statistically significant difference between the frequency of fetal deaths between the two hemolytic strains, and they explained that this may be due to a decrease in the repression of hemolysin/pigment by COVR/S in vivo<ref name=ww/><ref name=santi>[https://pubmed.ncbi.nlm.nih.gov/19542277/ Santi, Isabella, et al. “CsrRS Regulates Group B Streptococcus Virulence Gene Expression in Response to Environmental PH: a New Perspective on Vaccine Development.” Journal of Bacteriology, vol. 191, no. 17, 2009, pp. 5387–5397., doi:10.1128/jb.00370-09.]</ref><ref name=sit>[https://pubmed.ncbi.nlm.nih.gov/19568429/ Sitkiewicz, Izabela, et al. “Transcriptome Adaptation of Group B Streptococcus to Growth in Human Amniotic Fluid.” PLoS ONE, vol. 4, no. 7, 2009, doi:10.1371/journal.pone.0006114.]</ref>.
 
 
Whidbey et al. also investigated if the activation of NLRP3 inflammasome is important for fetal injuries that are the caused by hyper-hemolytic GBS strains<ref name=ww/>. Notably, Whidbey et al. found that the production of the hemolytic pigment is the critical component of GBS fetal injury, as it contributes to fetal injuries that are associated with GBS infections in ways that are independent and dependent on the NLRP3 inflammasome<ref name=ww/>.
 
==GBS Exploits Vaginal Epithelial Exfoliation for Ascending Infection==
[[Image:Screen Shot 2021-04-06 at 11.54.30 AM.png|thumb|300px|left|Figure 11. Proposed mechanism that explains how GBS exploits vaginal epithelial exfoliation for ascending infection<ref name=vorn/>[https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5919824_jci-128-97043-g007.jpg].]]
In order for in utero infections to occur in humans, vaginal microbes must enter the uterus by ascending infection. The mechanisms behind ascending infection are not clear, but Vornhagen et al. propose a convincing mechanism, which is depicted in Figure 11, that GBS may use to cause in utero infections.<ref name=vorn>[https://pubmed.ncbi.nlm.nih.gov/29629904/ Vornhagen, Jay, et al. “Group B Streptococcus Exploits Vaginal Epithelial Exfoliation for Ascending Infection.” Journal of Clinical Investigation, vol. 128, no. 5, 2018, pp. 1985–1999., doi:10.1172/jci97043.]</ref>. Notably, not all pregnant people experience ascending microbial infections. Vornhagen et al. propose that one reason for this may be a breakdown in the host’s defenses that work to prevent ascending infection<ref name=vorn/>. Epithelial cells, for example, create a physical barrier that is able to recognize a pathogenic attack and respond to it appropriately<ref name=vorn/>. One of these defense mechanisms is called epithelial exfoliation (i.e., shedding), during which epithelial cells detach from their basement membrane, which prevents pathogens from harming the host<ref name=vorn/>. Vaginal epithelial cells use exfoliation to protect their host<ref name=vorn/>. Interestingly, vaginal GBS colonization increases a pregnant person’s risks of experiencing an ascending infection and, in turn, adverse pregnancy outcomes<ref name=vorn/>. Given this, Vornhagen et al. hypothesized that vaginal epithelial exfoliation may regulate GBS colonization<ref name=vorn/>. 
 
[[Image:Screen Shot 2021-04-06 at 12.02.40 PM.png|thumb|300px|right|Figure 12. (A) hVECs were infected with WT GBS for 0, 16, or 24 hours and stained with 10% crystal violet for 30 minutes.(n = 3; **P < 0.005 and P = 0.08, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM. (B) Scanning electron microscopy images of hVEC's were infected with WT GBS for 24 hours. (C) ECIS was used to measure transepithelial resistance of hVEC monolayers (n = 3; data represent the mean). [https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5919824_jci-128-97043-g001.jpg].]]
 
 
To test if GBS may trigger the loss of barrier function in and cellular detachment of vaginal epithelial cells, which will eventually result in exfoliation, Vornhagen et al. infected immortalized human vaginal epithelial cells (hVECs) with the GBS WT strain COH1<ref name=vorn/>. Figure 12 shows that Vornhagen et al. noticed (A) a significant amount of cellular detachment in hVECs, (B) a significant difference in hVEC structure and shape, and (C) a loss of barrier function in hVECs infected with WT GBS<ref name=vorn/>. In other words, these results suggest that WT GBS infection of hVECs stimulates vaginal epithelial cell exfoliation<ref name=vorn/>.  
 
 
Next, Vornhagen et al. measured “the ability of GBS to migrate across hVEC monolayers in [a] Transwell assay<ref name=vorn/>." Vornhagen et al. observed that a significant amount of GBS crossed the epithelial barrier. Notably, these crossing times were associated with vaginal epithelial cell exfoliation time points, suggesting that GBS induces vaginal epithelial cell exfoliation, allowing for an increase in GBS cell migration across vaginal epithelia<ref name=vorn/>.
 
 
Vornhagen et al. vaginally inoculated female mice with WT GBS to see if GBS infection results in epithelial exfoliation<ref name=vorn/>. Interestingly, blinded quantification scoring of scanning electron microscopy images revealed that mice that were infected with GBS experienced significantly higher levels of vaginal exfoliation than the control group 72 and 96 hours post-inoculation<ref name=vorn/>. Vornhagen et al. probed the vaginal epithelial with fluorescent nanoparticles in vivo to confirm that GBS-induced epithelial exfoliation also resulted in a loss of epithelial barrier function in vivo<ref name=vorn/>. Notably, over time, more nanoparticles penetrated the epithelial barrier in GBS-inoculated mice than in the control sample, suggesting that in vivo GBS infections resulted in epithelial exfoliation, as well as a disruption in epithelial barrier function, which is associated with increased bacterial dissemination<ref name=vorn/>. 
 
 
It is known that the loss of barrier function and increased mobility of epithelial cells is associated with EMT, which is the process whereby nonmotile epithelial cells become mobile metastatic cells; this process is often characterized by a loss of the epithelial marker E-cadherin and a gain in the expression of the mesenchymal marker N-cadherin<ref name=vorn/><ref name=lam>[https://pubmed.ncbi.nlm.nih.gov/24556840/ Lamouille, Samy, et al. “Molecular Mechanisms of Epithelial–Mesenchymal Transition.” Nature Reviews Molecular Cell Biology, vol. 15, no. 3, 2014, pp. 178–196., doi:10.1038/nrm3758.]</ref>. Although, there is evidence of EMT occurring in cells when only the expression of E-cadherin was decreased without the increase in the expression of N-cadherin<ref name=costa>[https://pubmed.ncbi.nlm.nih.gov/26018309/ Costa, Liana Cristina, et al. “Expression of Epithelial-Mesenchymal Transition Markers at the Invasive Front of Oral Squamous Cell Carcinoma.” Journal of Applied Oral Science, vol. 23, no. 2, 2015, pp. 169–178., doi:10.1590/1678-775720140187.]</ref>. Vornhagen et al. infected hVECs with WT GBS, and they used flow cytometry to detect whether or not infected cells showed evidence of the EMT mesenchymal change. WT GBS-infected hVECs showed a decrease in the epithelial marker E-cadherin, and immunohistochemistry of in vivo murine samples of GBS-infected hVECS validated this finding<ref name=vorn/>. While a decrease of the epithelial marker E-cadherin was observed in murine vaginal samples, Vornhagen et al. did not observe a decrease in the expression of the mesenchymal marker N-cadherin in murine vaginal tissue, which may be the result of the difference in the expression levels of N-cadherin in different types of cells<ref name=vorn/>. That said, Vornhagen et al. concluded that GBS are capable of inducing EMT in vaginal epithelial cells, which may be the cause of epithelial exfoliation in vaginal epithelial cells<ref name=vorn/>.
 
 
[[Image:Screen Shot 2021-04-06 at 12.46.21 PM.png|thumb|300px|left|Figure 13. "Expression of β-catenin target genes in GBS-infected hVECs were compared with expression in mock-treated controls 24 hours after infection (n = 3; **P < 0.005 and ****P < 0.00005, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM)<ref name=vorn/>."[ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5919824_jci-128-97043-g004.jpg].]]
It is known that the β-catenin signaling pathway can regulate EMT<ref name=lam/>. To determine if the GBS can trigger the β-catenin signaling pathway to regulate EMT, Vornhagen et al. examined the gene expression of β-catenin regulated genes in GBS-infected hVECs<ref name=vorn/>. Interestingly, Figure 13 shows that β-catenin regulated genes were significantly upregulated in WT GBS-infected hVECs and vaginal tissues from GBS inoculated mice, suggesting that GBS may trigger the β-catenin signaling pathway. This finding, combined with the findings of several other molecular experiments, lead Vornhagen et al. to conclude that the data suggest that β-catenin signaling controls EMT in vaginal epithelial cells and that GBS may use this signaling mechanism to induce epithelial exfoliation<ref name=vorn/>. 
 
 
While interesting, these findings did not enable Vornhagen et al. to identify the extracellular signal that GBS may use to prompt β-catenin signaling<ref name=vorn/>. Vornhagen et al. eventually turned their attention to the integrin protein family, because they are known for their ability to control various cellular processes <ref name=int>[https://pubmed.ncbi.nlm.nih.gov/27687254/ Streuli, Charles H. “Integrins as Architects of Cell Behavior.” Molecular Biology of the Cell, vol. 27, no. 19, 2016, pp. 2885–2888., doi:10.1091/mbc.e15-06-0369.]</ref>. The molecular mechanisms of the integrin signaling pathway are described in detail in the Vornhagen et al. study<ref name=vorn/>. Vornhagen et al.’s knowledge of the molecular mechanisms of the integrin signaling pathway encouraged them to conduct several molecular experiments that lead to the suggestion that GBS are able to stimulate integrin signaling, which permits the nuclear translocation of β-catenin<ref name=vorn/>. This translocation potential leads to EMT, barrier disruption, and ultimately vaginal epithelial exfoliation<ref name=vorn/>. This mechanism is depicted in Figure 11. The data suggest that, while vaginal epithelial cell exfoliation serves as a defense mechanism against certain pathogens, GBS actually use this defense mechanism as a way to ascend further into the vagina<ref name=vorn/>.  


==Conclusion==
==Conclusion==
GBS infections are associated with adverse pregnancy outcomes, such as stillbirth. The molecular mechanisms of these outcomes are still unknown, but the two Whidbey et al. studies and the Vornhagen study discussed above significantly advanced the efforts to combat these pregnancy outcomes by expanding our knowledge about the molecular mechanisms of GBS infections and their negative effects on pregnant and birthing individuals and their neonate(s). These studies show that GBS hemolysin is not a protein, as it was previously described, but rather a toxic lipid pigment. Increased expression of this pigment has several consequences, including but not limited to bacterial (GBS) penetration of chorioamniotic membranes and the amniotic cavity, activation of the NLRP3 inflammasome, and higher rates of murine fetal death. The proposed mechanism of hemolysin biosynthesis and the proposed mechanism that explains how GBS exploits vaginal epithelial exfoliation for ascending infection (which were proposed by Whidbey et al. and Vornhagen et al., respectively) are promising starting points for future research that aims to discover and implement preventative measures against in utero GBS infections, which is important because the current intrapartum prophylaxis to prevent GBS transmission from the birthing individual to their neonate(s) does not target in utero GBS infections.


==References==
==References==
<br><br>Authored for BIOL 238 Microbiology, taught by [mailto:slonczewski@kenyon.edu Joan Slonczewski], 2021, [http://www.kenyon.edu/index.xml Kenyon College].
<br><br>Authored for BIOL 238 Microbiology, taught by [mailto:slonczewski@kenyon.edu Joan Slonczewski], 2021, [http://www.kenyon.edu/index.xml Kenyon College].

Latest revision as of 20:43, 7 April 2021

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Introduction

This artistic recreation, based on scanning electron microscopy (SEM), depicts a three-dimensional (3D), computer-generated image, of a group of Gram-positive, Streptococcus agalactiae (group B Streptococcus) bacteria. Photo Credit: Alissa Eckert, who is a medical illustrator at the CDC.


By Shawn Ruiz


Group B Strep (GBS), also known as Streptococcus agalactiae, is a Gram-positive, beta-hemolytic, catalase-negative, facultative anaerobe that is a normal component of the gastrointestinal and genitourinary tracts[1]. In fact, GBS colonizes the gastrointestinal and genitourinary tracts of up to 50% of healthy adults[2]. Most healthy adults who are colonized by GBS will not experience any symptoms or GBS-related infections. While the bacteria is usually harmless in healthy adults, it is a major cause of meningitis, pneumonia, and sepsis in neonates[3]. GBS is the leading infectious cause of neonatal mortality and morbidity in the United States; between four and six percent of babies who develop GBS disease die[4][5]. GBS causes both early onset (<7 days old) and late onset (7-90 days old) infections in neonates[4]. The main risk factor for an early-onset GBS infection in neonates is colonization of a birthing person's genital tract with Group B strep during labor[4]. About one in four pregnant individuals carry GBS in their body[5]. If the bacteria is present in a pregnant person, it can be directly transferred to their neonate(s) in a multitude of ways. For example, GBS can travel from the vagina into the amniotic fluid where the neonate(s) can ingest it. The neonate(s) can also come into contact with the bacteria as they make their way down the birth canal[3]. In the early 1990s, the early GBS infection rate was 1.7 cases per 1,000 births[3]. In an effort to decrease this infection rate, the American Congress of Obstetricians and Gynecologists and the American Academy of Pediatrics recommended screening pregnant individuals for GBS before they go into labor[3]. It is now common practice to screen pregnant individuals for GBS at some point between 35 and 37 weeks of pregnancy[5]. Pregnant people who test positive for GBS are treated with intravenous antibiotics during labor[5]. Penicillin and ampicillin are the recommended antibiotics for intrapartum GBS prophylaxis[6]. If a pregnant person tests positive for GBS and they are treated with antibiotics during labor, the risk of their neonate(s) developing a serious, life-threatening GBS infection drops by 80% [3]. Early GBS infection rates in the United States have significantly dropped (0.25 cases per 1,000 births) since these preventative measures went into effect around 1995[3]. Intrapartum prophylaxis effectively prevents GBS transmission from the birthing individual to their neonate(s) during labor and delivery. This preventative measure, however, does not target in utero infections that occur earlier in pregnancy, and little is known about the mechanisms that result in the infection of the amniotic cavity[7]. In utero GBS infections have devastating effects, including preterm birth and mortality in both the pregnant person and their neonate(s)[7]. That said, it is critical that researchers and public health officials work toward understanding exactly how GBS infects the amniotic cavity.

The Hemolytic Pigment of GBS

Figure 2. Primary hAECs were isolated from chorioamniotic membranes, and the percent adherence of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared[7].[1].
Figure 3. Primary hAECs were isolated from chorioamniotic membranes, and the percent invasion of GBS WT, isogenic ΔcovR, ΔcovRΔcylE, and ΔcylE mutants were compared[7]. [2].

GBS has been isolated from the amniotic fluid of birthing people with intact chorioamniotic membranes, suggesting that GBS is capable of invading and breaching amniotic epithelium and chorioamnion[7][8][9][10][11]. Whidbey et al. hypothesized that “intra-amniotic GBS infections in patients with intact placenta or chorioamniotic membranes may be due to elevated virulence factor expression”[7][8][9][10][11]. Previous studies showed that the expression of GBS virulence genes is regulated by a two-component regulatory system: COVR/S[7][12][13][14]. More specifically, COVR/S was described to repress a multitude of GBS virulence genes, including the cyl operon, which contains the cylE gene that is important for the production of the pluripotent toxin, hemolysin [7][12][13][14]. In order to test whether or not increased expression of GBS virulence genes promotes the invasion of amniotic epithelium, Whidbey et al. compared the abilities of wild-type (WT) GBS and hyper-hemolytic GBS (ΔcovR) to adhere to and invade human amniotic epithelial cells (hAECs) that were isolated and cultured from “normal, term placentas obtained immediately after cesarean delivery from” birthing people without labor[7]. Non-hemolytic GBS strains (ΔcovRΔcylE and ΔcylE) were also included in the experiment to investigate hemolysin’s role in GBS strains’ ability to adhere to and invade hAECs[7][15]. The results, shown in Figures 2 and 3, show that all GBS strains adhered to hAECs. WT GBS invaded hAECs (~4% invasion), non-hemolytic GBS showed a significantly decreased invasion when compared to the WT (~0.3% invasion), and the hyper-hemolytic GBS strain (ΔcovR) was significantly more invasive when compared to the WT strain (~80% invasion)[7]. In sum, Whidbey et al. concluded that hemolysin promotes GBS invasion of hAECs.


Next, Whidbey et al. investigated if the increased expression of hemolysin in the hyper-hemolytic GBS strain (ΔcovR) activates an inflammatory response[7]. Whidbey et al. isolated RNA from GBS-infected hAECs four hours after infection, and they used qRT-PCR to examine any changes in the expression of inflammatory genes in hAECs[7][16]. The results, depicted in Figure 4, suggest that infection with ΔcovR caused a significant increase in the transcription of cytokines (e.g., IL-6 and IL-8) in hAECs[7]. In sum, the results suggest that hAECs that were infected with the hyper-hemolytic GBS strain (ΔcovR) did show an inflammatory response. 

Figure 4. Whidbey et al. isolated RNA from hAECs infected with WT GBS COH1, isogenic ΔcovR, ΔcovRΔcylE, or ΔcylE mutants at 4 h after infection. qRT-PCR was performed on the RNA to examine the expression of cytokines/chemokines. "Data shown are the mean and SD obtained from hAECs that were isolated from three independent placentas, performed in triplicate (n = 3; **, P = 0.007; *, P = 0.03, Student’s t test, error bars ± SD)[7]."[3].


It is known that microbial toxins use the nuclear transcription factor, NF-𝜅B, which is recruited from the cytoplasm to the nucleus, to activate inflammatory signaling pathways[17]. Given that hAECs that were infected with ΔcovR showed an inflammatory response, Whidbey et al. investigated if the increase in the expression of pro-inflammatory genes was associated with the nuclear recruitment/localization of NF-𝜅B[7]. Whidbey et al. isolated total nuclear and cytoplasmic proteins from infected and uninfected hAECs, and they resolved them on 10% SDS-PAGE and Western blots[7]. As shown in Figure 5, hAECs that were infected with the hyper-hemolytic GBS strain (ΔcovR) showed a 2.5 fold increase in the nuclear recruitment of  NF-𝜅B compared with WT GBS-infected or uninfected controls. These results suggest that the increase in the expression of inflammatory genes seen in ΔcovR-infected hAECs "is associated with the nuclear recruitment of NF-𝜅B"[7].

Figure 5. Whidbey et al. performed Western Blots of nuclear and cytoplasmic proteins in GBS-infected hAECs(NF-𝜅B antibody). N and C stand for nuclear and cytoplasmic[7][4].


Despite finding that hemolysin is associated with an increase in the expression of pro-inflammatory genes, as well as the nuclear recruitment of NF-𝜅B, Whidbey et al. still did not know if the presence of hemolysin accelerates the failure of the human amniotic epithelial barrier[7]. To examine this, Whidbey et al. monitored the changes in transepithelial electrical resistance across hAEC using electric cell-substrate impedance sensing[7][18]. Figure 6 shows that hAECs that were infected with the hyper-hemolytic strain (ΔcovR) showed an accelerated decrease in their barrier resistance compared to hAECs that were infected with WT GBS[7]. In sum, increased hemolysin expression decreased the barrier function of the amniotic epithelium, enabling GBS to breach the epithelial barrier[7].

Figure 6. Whidbey et al. used Electric Cell-Substrate Impedance Sensing (ECIS) to monitor the barrier resistance of hAECs in real time.[5].


Knowing that an increase in hemolysin expression enabled hyper-hemolytic GBS to breach the hAEC epithelium, Whidbey et al. investigated if GBS are capable of penetrating intact chorioamniotic membranes[7]. Whidbey et al. mounted, maintained, and, after 48 hours of stabilization, infected chorioamniotic membranes with various GBS strains (WT, ΔcovR, ΔcovRΔcylE). Figure 7 shows that only ΔcovR penetrated and invaded the chorioamnion, including the amniotic epithelium. This suggests that an increase in hemolysin expression can “facilitate bacterial penetration of chorioamniotic membranes and the amniotic cavity”[7].

Figure 7. GBS penetration of the chorion, amnion, and chorioamniotic membranes (n = 6; NS, P > 0.3; *, P = 0.02, Mann Whitney test, error bars ± SD)[7]."[6].


Considering that hyper-hemolytic GBS is capable of penetrating the chorioamniotic membranes and the amniotic cavity, which may result in an in-utero infection and/or preterm labor, Whidbey et al. hypothesized that increased hemolytic activity could be observed in GBS isolated from birthing people in preterm labor[7]. To test this hypothesis, they collected clinical isolates from amniotic fluid and chorioamnion from six birthing people in preterm labor[7]. These isolates were examined for potential mutations in the covR/S locus, as well as hemolytic properties. Eight GBS strains were obtained: most of them demonstrated increased hemolytic activity, and six of them had a mutation in the covR/S loci[7]. Notably, two GBS isolates did not have a mutation in the covR/S loci, but they did demonstrate increased hemolytic activity. That said, it is likely that hemolytic activity is regulated by multiple regulatory mechanisms. Site-directed mutations of the COVR/S mutations that were collected from clinical samples confirmed that the increase in hemolytic activity was in part attributable to these mutations[7]. Thus, hyper-hemolytic GBS can be associated with birthing people in preterm labor.


One study suggested that the cylE gene, which is a part of the cyl operon, encoded and produced hemolysin[15]. Whidbey et al. proved, however, that, while CylE is necessary for the production of hemolysin, it is not sufficient for GBS hemolysis. Notably, studies have shown that the hemolytic phenotype of GBS is pigmented and the non-hemolytic phenotype is not; this pigment is described as an ornithine rhamnolipid (i.e., granadaene)[15][19][20]. Like hemolytic activity, pigment biosynthesis in GBS also requires the cyl open. Whidbey et al. conducted a sequence profile analysis of CylE, which they used in combination with predicted homology profiles, to propose a pathway for GBS pigment biosynthesis that is dependent on the majority of genes in the cyl operon, which is depicted in Figure 8[7].


That said, Whidbey et al. propose that the ornithine rhamnolipid pigment, rather than the CylE protein, is the cause of the hemolytic activity in GBS. To test this, they extracted and purified pigment from WT GBS and examined the hemolytic activity of the pigment. Lysis of red blood cells occurred in the presence of the pigment, and brief (8 min) exposure to the pigment dramatically altered the membrane morphology of the disc-shaped red blood cells, as shown in Figure 9. Researchers confirmed that the hemolytic activity observed in the lysis of red blood cells could not be attributed to a protein toxin by demonstrating that the purified pigment was not affected by proteinase K. Other experiments, such as SDS-PAGE analysis of the purified pigment, also helped confirm this finding[7].

Figure 8. Proposed pathway for the biosynthesis of the GBS pigment, granadaene[7].[7].
Figure 9. Scanning electron micrographs of hRBCs 8 minutes after exposure to GBS pigment (12.5 µM), buffer, or ΔcylE extract.[7].[8].

The Lytic Mechanisms and Cytolytic Abilities of the GBS Hemolytic Pigment

Whidbey et al. investigated how the GBS pigment induces cell lysis by measuring the kinetics of both K+ and hemoglobin (Hb) release from red blood cells treated with 400nM of the pigment[21]. These results suggest that membrane permeabilization enables K+ and Hb to efflux, respectively. This delay in Hb suggests that the GBS pigment induces a colloidal osmotic mechanism of lysis rather than direct lysis[21]. Protection assays revealed that small osmoprotectants (e.g., PEG200) are incapable of protecting RBC from pigment-mediated hemolysis, whereas larger osmoprotectants (e.g, PEG1500) completely protect RBC from pigment-mediated hemolysis[21]. Interestingly, pigment-mediate membrane permeability is independent of cellular response, and it does not conform to a pore-forming protein toxin nor the induction of instant lysis[21]


To investigate the cytolytic abilities of GBS, researchers treated macrophages with various GBS strains (WT, ΔcovR, ΔcylE, and ΔcovRΔcylE) for four hours; cytotoxicity was measured by the release of lactate dehydrogenase[21]. Macrophage death and IL-1β and IL-18 release in cells that were infected with hyper-hemolytic GBS was significantly higher compared to those infected with non-hemolytic strains[21]. This, combined with further experiments that confirmed these findings and revealed an increase in IL-1β and IL-18 levels in macrophages treated with hyper-hemolytic GBS strains, suggest that the purified hemolytic GBS pigments is pro-inflammatory and cytotoxic[21]


The increase in the secretion of IL-1β and IL-18 in macrophages treated with the GBS pigment suggests that the pigment is capable of triggering activation of the inflammasome, which is a cytosolic complex that is responsible for activating inflammatory responses to injury and/or illness[21]. Whidbey et al. write, "one major inflammasome comprises the NLR (nucleotide binding, leucine-rich repeat containing) protein known as NLRP3, which associates with the adaptor ASC (apoptosis-associated speck-like protein containing the caspase recruitment domain, CARD)"[21][22]. Whideby et al. conducted several experiments to investigate if the GBS pigment activates NLRP3, and they concluded that GBS induced hemolysin-dependent cell death in macrophages that contain the NLRP3 inflammasome[21].

The GBS Hemolytic Pigment is the Critical Component of GBS-Related Fetal Injuries

Figure 10. In utero fetal deaths in GBS-infected (GBS WT, hyperhemolytic ΔcovR, or non-hemolytic ΔcovRΔcylE) mice. Fetal deaths were measured by the number of dead fetuses obtained from a total of six parent mice.****P < 0.0001, Fisher's exact test.[23][9].

To determine if hyper-hemolytic GBS strains induce fetal injury or preterm birth, Whidbey et al. used an intrauterine model of inoculation to introduce GBS strains (WT, hyperpigmented (ΔcovR), or non-hemolytic (ΔcovRΔcylE)) into the right horn of the uteruses of pregnant mice[21][24][25][26]. The inoculated mice were monitored for 72 hours for signs of preterm, which Whidbey et al. defined as the birth of at least one pup in a mouse’s cage[21]. One of six mice infected with WT GBS and three of six mice infected with ΔcovR GBS experienced preterm birth compared with the controls groups[21]. Moreover, Figure 10 shows that there were significantly higher fetal deaths in mice that were infected with WT GBS and ΔcovR GBS (hemolytic strains)[21]. Whidbey et al. reported that there was not a statistically significant difference between the frequency of fetal deaths between the two hemolytic strains, and they explained that this may be due to a decrease in the repression of hemolysin/pigment by COVR/S in vivo[21][27][28].


Whidbey et al. also investigated if the activation of NLRP3 inflammasome is important for fetal injuries that are the caused by hyper-hemolytic GBS strains[21]. Notably, Whidbey et al. found that the production of the hemolytic pigment is the critical component of GBS fetal injury, as it contributes to fetal injuries that are associated with GBS infections in ways that are independent and dependent on the NLRP3 inflammasome[21].

GBS Exploits Vaginal Epithelial Exfoliation for Ascending Infection

Figure 11. Proposed mechanism that explains how GBS exploits vaginal epithelial exfoliation for ascending infection[23][10].

In order for in utero infections to occur in humans, vaginal microbes must enter the uterus by ascending infection. The mechanisms behind ascending infection are not clear, but Vornhagen et al. propose a convincing mechanism, which is depicted in Figure 11, that GBS may use to cause in utero infections.[23]. Notably, not all pregnant people experience ascending microbial infections. Vornhagen et al. propose that one reason for this may be a breakdown in the host’s defenses that work to prevent ascending infection[23]. Epithelial cells, for example, create a physical barrier that is able to recognize a pathogenic attack and respond to it appropriately[23]. One of these defense mechanisms is called epithelial exfoliation (i.e., shedding), during which epithelial cells detach from their basement membrane, which prevents pathogens from harming the host[23]. Vaginal epithelial cells use exfoliation to protect their host[23]. Interestingly, vaginal GBS colonization increases a pregnant person’s risks of experiencing an ascending infection and, in turn, adverse pregnancy outcomes[23]. Given this, Vornhagen et al. hypothesized that vaginal epithelial exfoliation may regulate GBS colonization[23]

Figure 12. (A) hVECs were infected with WT GBS for 0, 16, or 24 hours and stained with 10% crystal violet for 30 minutes.(n = 3; **P < 0.005 and P = 0.08, by ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM. (B) Scanning electron microscopy images of hVEC's were infected with WT GBS for 24 hours. (C) ECIS was used to measure transepithelial resistance of hVEC monolayers (n = 3; data represent the mean). [11].


To test if GBS may trigger the loss of barrier function in and cellular detachment of vaginal epithelial cells, which will eventually result in exfoliation, Vornhagen et al. infected immortalized human vaginal epithelial cells (hVECs) with the GBS WT strain COH1[23]. Figure 12 shows that Vornhagen et al. noticed (A) a significant amount of cellular detachment in hVECs, (B) a significant difference in hVEC structure and shape, and (C) a loss of barrier function in hVECs infected with WT GBS[23]. In other words, these results suggest that WT GBS infection of hVECs stimulates vaginal epithelial cell exfoliation[23].


Next, Vornhagen et al. measured “the ability of GBS to migrate across hVEC monolayers in [a] Transwell assay[23]." Vornhagen et al. observed that a significant amount of GBS crossed the epithelial barrier. Notably, these crossing times were associated with vaginal epithelial cell exfoliation time points, suggesting that GBS induces vaginal epithelial cell exfoliation, allowing for an increase in GBS cell migration across vaginal epithelia[23].


Vornhagen et al. vaginally inoculated female mice with WT GBS to see if GBS infection results in epithelial exfoliation[23]. Interestingly, blinded quantification scoring of scanning electron microscopy images revealed that mice that were infected with GBS experienced significantly higher levels of vaginal exfoliation than the control group 72 and 96 hours post-inoculation[23]. Vornhagen et al. probed the vaginal epithelial with fluorescent nanoparticles in vivo to confirm that GBS-induced epithelial exfoliation also resulted in a loss of epithelial barrier function in vivo[23]. Notably, over time, more nanoparticles penetrated the epithelial barrier in GBS-inoculated mice than in the control sample, suggesting that in vivo GBS infections resulted in epithelial exfoliation, as well as a disruption in epithelial barrier function, which is associated with increased bacterial dissemination[23]


It is known that the loss of barrier function and increased mobility of epithelial cells is associated with EMT, which is the process whereby nonmotile epithelial cells become mobile metastatic cells; this process is often characterized by a loss of the epithelial marker E-cadherin and a gain in the expression of the mesenchymal marker N-cadherin[23][29]. Although, there is evidence of EMT occurring in cells when only the expression of E-cadherin was decreased without the increase in the expression of N-cadherin[30]. Vornhagen et al. infected hVECs with WT GBS, and they used flow cytometry to detect whether or not infected cells showed evidence of the EMT mesenchymal change. WT GBS-infected hVECs showed a decrease in the epithelial marker E-cadherin, and immunohistochemistry of in vivo murine samples of GBS-infected hVECS validated this finding[23]. While a decrease of the epithelial marker E-cadherin was observed in murine vaginal samples, Vornhagen et al. did not observe a decrease in the expression of the mesenchymal marker N-cadherin in murine vaginal tissue, which may be the result of the difference in the expression levels of N-cadherin in different types of cells[23]. That said, Vornhagen et al. concluded that GBS are capable of inducing EMT in vaginal epithelial cells, which may be the cause of epithelial exfoliation in vaginal epithelial cells[23].


Figure 13. "Expression of β-catenin target genes in GBS-infected hVECs were compared with expression in mock-treated controls 24 hours after infection (n = 3; **P < 0.005 and ****P < 0.00005, by 1-way ANOVA followed by Sidak’s multiple comparisons test; data represent the mean ± SEM)[23]."[ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5919824_jci-128-97043-g004.jpg].

It is known that the β-catenin signaling pathway can regulate EMT[29]. To determine if the GBS can trigger the β-catenin signaling pathway to regulate EMT, Vornhagen et al. examined the gene expression of β-catenin regulated genes in GBS-infected hVECs[23]. Interestingly, Figure 13 shows that β-catenin regulated genes were significantly upregulated in WT GBS-infected hVECs and vaginal tissues from GBS inoculated mice, suggesting that GBS may trigger the β-catenin signaling pathway. This finding, combined with the findings of several other molecular experiments, lead Vornhagen et al. to conclude that the data suggest that β-catenin signaling controls EMT in vaginal epithelial cells and that GBS may use this signaling mechanism to induce epithelial exfoliation[23]


While interesting, these findings did not enable Vornhagen et al. to identify the extracellular signal that GBS may use to prompt β-catenin signaling[23]. Vornhagen et al. eventually turned their attention to the integrin protein family, because they are known for their ability to control various cellular processes [31]. The molecular mechanisms of the integrin signaling pathway are described in detail in the Vornhagen et al. study[23]. Vornhagen et al.’s knowledge of the molecular mechanisms of the integrin signaling pathway encouraged them to conduct several molecular experiments that lead to the suggestion that GBS are able to stimulate integrin signaling, which permits the nuclear translocation of β-catenin[23]. This translocation potential leads to EMT, barrier disruption, and ultimately vaginal epithelial exfoliation[23]. This mechanism is depicted in Figure 11. The data suggest that, while vaginal epithelial cell exfoliation serves as a defense mechanism against certain pathogens, GBS actually use this defense mechanism as a way to ascend further into the vagina[23].  

Conclusion

GBS infections are associated with adverse pregnancy outcomes, such as stillbirth. The molecular mechanisms of these outcomes are still unknown, but the two Whidbey et al. studies and the Vornhagen study discussed above significantly advanced the efforts to combat these pregnancy outcomes by expanding our knowledge about the molecular mechanisms of GBS infections and their negative effects on pregnant and birthing individuals and their neonate(s). These studies show that GBS hemolysin is not a protein, as it was previously described, but rather a toxic lipid pigment. Increased expression of this pigment has several consequences, including but not limited to bacterial (GBS) penetration of chorioamniotic membranes and the amniotic cavity, activation of the NLRP3 inflammasome, and higher rates of murine fetal death. The proposed mechanism of hemolysin biosynthesis and the proposed mechanism that explains how GBS exploits vaginal epithelial exfoliation for ascending infection (which were proposed by Whidbey et al. and Vornhagen et al., respectively) are promising starting points for future research that aims to discover and implement preventative measures against in utero GBS infections, which is important because the current intrapartum prophylaxis to prevent GBS transmission from the birthing individual to their neonate(s) does not target in utero GBS infections.

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Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2021, Kenyon College.

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