Elucidation of Group B Streptococcus hemolytic pigment and hyaluronidase during infection Blair Armistead A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2020 Reading Committee: Lakshmi Rajagopal, Chair Jairam Lingappa Chetan Seshadri Program Authorized to Offer Degree: Pathobiology © Copyright 2020 Blair Armistead University of Washington Abstract Elucidation of Group B Streptococcus hemolytic pigment and hyaluronidase during infection Blair E. Armistead Chair of the Supervisory Committee: Lakshmi Rajagopal, Ph.D. Department of Pediatrics Department of Global Health Group B Streptococci (GBS) are β-hemolytic, Gram-positive bacteria that colonize the lower genital tract of approximately 18% of women worldwide as asymptomatic components of the gastrointestinal and/or vaginal flora. If established in other host niches, however, GBS are highly pathogenic. During pregnancy, ascending GBS infection from the vagina to the intrauterine space is associated with preterm birth, stillbirth, and fetal injury. In addition, vertical transmission of GBS during birth results in life-threatening neonatal infections, including pneumonia, sepsis, and meningitis. Notably, invasive GBS infections in non-pregnant adults are on the rise. Novel strategies to prevent GBS colonization and disease are an urgent global health priority and require a better understanding of the complex interactions between GBS virulence factors and host defenses. This dissertation summarizes efforts to uncover fundamental insights into two GBS virulence factors during infection, namely the surface-associated hemolytic pigment toxin and the extracellular enzyme hyaluronidase. Over 100 years ago, GBS isolates of human origin were first described as β-hemolytic, which was invariantly linked to orange/red pigmentation. In the century that followed, research uncovered the importance of hemolysis to GBS colonization and disease. Non-hemolytic/non- pigmented strains were shown to be deficient vaginal colonizers and less able to disseminate into vulnerable host niches in in vivo models. Moreover, hyper-hemolytic/hyper-pigmented strains were isolated from people with invasive infections and shown to more readily breach host barriers in vitro and in vivo. Despite the strong genetic and phenotypic link between hemolysis and pigment, these factors were thought to be independent. In 2013, the GBS hemolysin and pigment were shown to be one and the same, initiating a paradigm shift in understanding this virulence factor. The GBS hemolytic pigment, also known as granadaene, is a cell-surface-associated ornithine rhamnolipid consisting of a 12-alkene chain and is dissimilar to many other commonly studied Gram-positive pore-forming toxins, which are proteinaceous in nature. Here, we describe the genetic elements sufficient for granadaene production using a heterologous bacterial host (Lactococcus lactis) and trace the evolutionary origins of this toxin before the divergence of Gram- positive bacteria. In addition, we show that GBS release granadaene in membrane vesicles (MVs), which are cytotoxic to host immune cells and promote GBS infection in neonatal mice. Using organic chemical synthesis for the generation of granadaene analogs, we demonstrate the importance of the polyene chain length and terminal head groups to hemolytic activity. And lastly, we identify a non-toxic analog that, unlike granadaene, is well tolerated by cells of the adaptive immune system and confers protection against infection with hyper-hemolytic GBS. Collectively, the studies on the GBS hemolytic pigment presented in this dissertation provide novel biosynthetic, evolutionary, structural, and functional insight into this crucial GBS virulence factor and offer proof-of-concept for a targeted vaccine approach to attenuate its activity during infection. Next, we explore the role of another key virulence factor, hyaluronidase, in promoting GBS dissemination during pregnancy using a non-human primate model. Hyaluronidase, or HylB, has endoglycosidase activity, which is correlated to GBS’s invasive potential. Using a pregnant non- human primate model, we show that a non-pigmented/non-hemolytic GBS strain invaded the amniotic cavity and induced fetal injury and preterm labor in a HylB-dependent manner. Despite increased recruitment of neutrophils at the maternal-fetal interface, HylB dampened ROS production and neutrophil-mediated killing of GBS. Our studies show how this unique bacterial enzyme enables GBS to circumvent host defenses, leading to the rapid induction of infection of the amniotic cavity and preterm labor. Together, the work presented in this dissertation elucidates novel mechanisms of host-GBS interactions through the study of two key virulence factors, the hemolytic pigment and hyaluronidase. The findings here offer basic insights that may alleviate challenges associated with the development of improved therapeutic strategies against GBS. TABLE OF CONTENTS List of Figures .............................................................................................................................. viii List of Tables ................................................................................................................................. ix Chapter 1. Introduction ................................................................................................................. 12 1.1 Epidemiology of Group B Streptococcus colonization and disease ............................. 13 1.2 An introduction to GBS factors discussed in this dissertation ...................................... 14 1.2.1 Hemolytic pigment, granadaene ............................................................................... 15 1.2.2 Hyaluronidase ........................................................................................................... 17 1.3 Host factors that mediate GBS disease ......................................................................... 18 1.3.1 Humoral immunity .................................................................................................... 18 1.3.2 Immunity at the maternal-fetal interface................................................................... 19 1.4 Dissertation Summary ................................................................................................... 19 1.5 Figures........................................................................................................................... 21 Chapter 2. The cyl genes reveal the biosynthetic and evolutionary origins of the GBS hemolytic lipid, granadaene ........................................................................................................................... 22 2.1 Abstract ......................................................................................................................... 23 2.2 Introduction ................................................................................................................... 23 2.3 Results and Discussion ................................................................................................. 24 2.3.1 Heterologous expression of the GBS cyl operon in Lactococcus lactis confers hemolysis, pigmentation, and cytotoxicity. .......................................................................... 24 2.3.2 Pigment isolated from L. lactis pcylX-K is identical to granadaene from GBS. ....... 26 i 2.3.3 Phyletic analysis suggests the cyl operon evolved prior to the diversification of Gram-positive bacteria. ......................................................................................................... 27 2.3.4 The evolution of CylE and CylF conferred critical functions in Granadaene biosynthesis to the cyl operon. .............................................................................................. 29 2.4 Materials and Methods .................................................................................................. 31 2.4.1 Ethics statement ........................................................................................................ 31 2.4.2 Bacterial strains ......................................................................................................... 31 2.4.3 Heterologous expression of the cyl operon in Lactococcus lactis ............................ 31 2.4.4 Isolation and purification of pigment from GBS and L. lactis ................................. 32 2.4.5 Hemolytic assays ...................................................................................................... 33 2.4.6 Isolation of neutrophils from adult human blood ..................................................... 33 2.4.7 Testing bacterial strains and pigment extracts for neutrophil cytotoxicity ............... 34 2.4.8 Phyletic analysis........................................................................................................ 35 2.4.9 Statistical analysis ..................................................................................................... 35 2.5 Acknowledgements ....................................................................................................... 35 2.5.1 Conflict of interest .................................................................................................... 35 2.5.2 Author contributions ................................................................................................. 36 2.5.3 Funding ....................................................................................................................