1. Group B Streptococcus: Not just a mother’s problem Group B Streptococcus induces trophoblast death. Kaplan, Amber; et al. Microbial Pathogenesis. Sept. 2008; 45(3): 231-235. By: Cassie Saikin, Microbial Pathogenesis

2. Group B Streptococcus (GBS) Streptococcus agalactiae Gram positive Encapsulated Commensal in vagina, GI tract Strong adherence to epithelial cells Leading cause of neonatal infection ◦ pneumonia, prenatal delivery, septicemia, and meningitis

3. Neonatal Infection EARLY ONSET Ascending infection 6-8 hours to 7 days after birth Characterized by: ◦ Pneumonia, respiratory failure, septicemia LATE ONSET Up to 7 months after birth Characterized by: ◦ Septicemia, neonatant meningitis GBS virulence: Adhesion  penetration  immune evasion  inflammation Penetration of host cell barriers is indication of pathogenicity

4. GBS Epithelial Adhesion Serotype III (capsule characteristic) – most capable of adhering to neonate epithelial cells Neonates colonized with GBS – increased expression of attachment receptors ◦ Lipoteichoic Acid (LTA) receptors ◦ Interact with extracellular matrix proteins – fibronectin, fibrinogen, laminin

5. Hypothesis GBS induces apoptosis of trophoblast cells by β- hemolysis, conducted by the bacterial protein β- hemolysin/cytolysin. Trophoblast death is responsible for neonatal infection. β-hemolysin/cytolysin is responsible for GBS toxicity.

6. β-hemolysin/cytolysin Pore-forming toxin – punctures holes in epithelial surfaces ◦ Weakens cell-barrier integrity (within placenta – trophoblasts) – allow for invasion Contributes to GBS pathogenicity/toxicity: ◦ Direct tissue damage ◦ Triggering apoptosis ◦ Promotion of intracellular invasion ◦ Immune evasion ◦ Activation of host inflammatory response

7. β-hemolysis – cytolytic damage and trophoblast death Leads to bacterial destruction and invasion of trophoblasts (placental epithelial tissue) Proving the hypothesis ◦ Exposed human JEG3 trophoblast cells and primary placental trophoblasts and fibroblasts to varying levels of purified β- hemolysin/cytolysin or GBS isolates with varying degrees of hemolytic activity  NCTC10/84 – high hemolytic activity (wildtype)  COH1 strain – low hemolytic activity  Mutant NCTC10/84 – no hemolytic activity (null) ◦ Measured cell death by colorometric lactate dehydrogenase (LDH) assay – measured the supernatant levels of active LDH ◦ Experiments performed in triplicate

8. Results of study Exposure to high levels of β-hemolysin/cytolysin from wild- type strain (NCTC10/84) induces trophoblast death in comparison with low levels (COH1 strain) and mutant strain (mutant NCTC10/84) Cell death is dose-dependent in relation to levels of β- hemolysin/cytolysin ◦ Higher levels results in increased death

9. Figure 5. Exposure to varying β- hemolysin/cytolysin levels

10. Chorioamnionitis Destruction of placental cells by GBS leads to amniotic fluid infections and chorioamnionitis (inflammation of placental amniotic membranes) – raises risk for neonatal infection Chorioamnion – bilayer of cells ◦ Monolayer of chorion epithelial cells – maternal side of placenta ◦ Monolayer of amnion epithelial cells – embryonic side of placenta GBS can bind to both chorion cells and amnion cells, but only invades and survive within chorion cells. ◦ Weakens the placental barrier, invade and transcytose through the bilayer of epithelial cells into the placenta ◦ Invasion of the placenta gives GBS access to the fetus in the amniotic cavity

11. Immune Evasion and Host Inflammatory Response Phagocytic evasion ◦ β-hemolysin/cytolysin – cytolytic damage and apoptosis of phagocytes ◦ GBS can survive within immature phagocytes – evade oxidative stress CiaR response ◦ AMP-resistance ◦ Survive in epithelial cells and blood stream Activation of host-inflammatory responses – sepsis, multiorgan failure ◦ Release of inflammatory mediators ◦ Amount of inflammatory mediators proportional to severity of placental infection

12. Neonatal Meningitis β-hemolysin/cytolysin ◦ Direct cytolytic damage to brain microvascular endothelial cells (BMEC), which allows GBC to breach the blood brain barrier (BBB) GBS is able to infect the lungs, where β- hemolysin/cytolysin causes destruction of the lung epithelia and weakening the tensile strength of the epithelial barrier = easy access to blood stream Survival in blood stream – travel to brain and transcytose through BBB ◦ β-hemolysin/cytolysin destroys BMEC ◦ GBS takes control of CNS

13. For the Future Research into the complications of bacterial prophylaxis by antibiotics Vaccine development against neonate GBS infection. ◦ Vaccines targeted against GBS serotype proteins of the streptococcal capsule are currently in phase III clinical trials. ◦ Investigation into currently-known bacterial factors as well as potential virulence factors could lead to new insights into a vaccine to protect neonates from this frightfully dangerous organism. Genomic research has discovered other protein factors that contribute to GBS virulence, including LTA, serine protease, fibrinogen receptor, and C5a peptidase. Further research into new virulence factors such as transcriptional factors, especially those that are homologous to other Streptococcus strains, could aid in the discovery of targets for prophylaxis and prevention as well as a common ancestor of the streptococcal species.

14. References [1] Berner, R. Significance, management, and prevention of Streptococcus agalactiae infection during the perinatal period. Expert Rev Anti Infect Ther. 2004 Jun;2(3):427-37. [2] De Paepe, Monique E, et al. The Histological Fetoplacental Inflammatory Response in Fatal Perinatal Group B-Streptococcus Infection. Journal of Perinatology. 2004; 24:441-445. [3] Doran, Kelly S., Victor Nizet. Molecular pathogenesis of neonatal group B streptococcal infection: no longer in its infancy. Molecular Microbiology. 2004; 54(1):23-31. [4] Kaplan, Amber, et al. Group B streptococcus induces trophoblast death. Microbial Pathogenesis. 2008; 45(3): 231-235. [5] Quach, Darin, et al. The CiaR Response Regulator in Group B Streptococcus Promotes Intracellular Survival and Resistance to Innate Immune Defences. Journal of Bacteriology. 2009; 191(7):2023-2032. [6] Teti, Giuseppe, et al. Adherence of Group B Steptococci to Adult and Neonatal Epithelial Cells Mediated by Lipoteichoic Acid. Infection and Immunity. 1987; 55(12):3057-3064. [7] Winram, Scott B, et al. Characterization of Croup B Streptococcal Invasion of Hman Chorion and Amnion Epithelial Cells In Vitro. Infection and Immunity. 1998; 66(10):4932-4941.