Characterization of the Group B Streptococcus Hemolysin and its Role in Intrauterine Infection
Whidbey, Christopher Mychael
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Intrauterine infection and inflammation are a major cause of perinatal morbidity, including preterm birth. A pathogen associated with intrauterine infection, preterm birth, and perinatal disease is Streptococcus agalactiae, or Group B streptococcus (GBS). Despite its importance to public health, little is known about the immune response during intrauterine GBS infection and the role of specific GBS virulence factors in this process. One major GBS virulence factor is the β-hemolysin which allows GBS to lyse host cells; however, the hemolysin had never been purified and its molecular nature was undefined. Additionally, the mechanism of hemolysin-mediated cytotoxicity, the host cellular immune response to the hemolysin, and the role of the hemolysin during intrauterine infection were unknown. Gaining insight into the molecular nature of the β-hemolysin and the role it plays in GBS pathogenesis is important to develop new therapeutic strategies for GBS associated disease. This dissertation describes our efforts to purify and characterize the GBS hemolysin, understand the mechanisms of pigment-mediated host cell lysis, and establish the role of this toxin during intrauterine infection that lead to fetal injury. The molecule responsible for GBS hemolytic activity had not been identified at the onset of this project. Using genetic and biochemical approaches, we demonstrated that the GBS hemolysin is not a protein toxin, but rather an ornithine rhamnolipid pigment produced by the bacterial cyl operon. The GBS pigment is toxic to both red blood cells and amniotic epithelial cells. Overproduction of this pigment allows GBS to traverse human placental membranes. Hyperpigmented GBS strains were identified among clinical isolates from women in preterm birth, supporting the hypothesis that the pigment is important during intrauterine infection. With purification of this major virulence factor, we were able to investigate how the GBS pigment causes membrane disruption and identify the immune pathways it activates. We found that the GBS pigment induces membrane perturbations in lipid membranes, which leads to ion flux. In red blood cells, this ion flux results in colloidal-osmotic lysis. In macrophages, membrane disruption triggers activation of the NLRP3 inflammasome, leading to activation of caspase 1. Caspase 1 activation by both whole cell GBS and the purified pigment is NLRP3 dependent, and results in secretion of IL-1β and the programmed, proinflammatory cell death known as pyroptosis. We developed a murine model of intrauterine infection to test the role of the GBS pigment in vivo. Infection with hyperpigmented/hyperhemolytic bacteria resulted in intrauterine fetal injury and preterm birth. Intrauterine fetal death in NLRP3 deficient mice was decreased compared to wild-type mice, demonstrating a role for pigment-mediated activation of NLRP3 during intrauterine infection. Interestingly, even in NLRP3-deficient mice, hyperpigmented GBS caused more fetal death that nonpigmented GBS. These results suggest that both NLRP3 dependent and NLRP3-independent pathways contribute to pathogenesis during intrauterine infection. Together, these data demonstrate that the GBS hemolysin, an ornithine rhamnolipid toxin, plays a key role during intrauterine infection.
- Pathobiology