Characterization of the Group B Streptococcus Hemolysin and its Role in Intrauterine Infection

Thesis (Ph.D.)--University of Washington, 2015Intrauterine 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

Kinase Inhibitors that Increase the Sensitivity of Methicillin Resistant Staphylococcus aureus to β-Lactam Antibiotics

Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermore, these inhibitors lacked alerting structures commonly associated with toxic effects, and toxicity was not observed with ST085384 or ST085405 in vivo in a murine model. These results suggest that kinase inhibitors may be useful in therapeutic strategies against MRSA infections

Bacterial Hyaluronidase Promotes Ascending GBS Infection and Preterm Birth

Preterm birth increases the risk of adverse birth outcomes and is the leading cause of neonatal mortality. A significant cause of preterm birth is in utero infection with vaginal microorganisms. These vaginal microorganisms are often recovered from the amniotic fluid of preterm birth cases. A vaginal microorganism frequently associated with preterm birth is group B streptococcus (GBS), or Streptococcus agalactiae. However, the molecular mechanisms underlying GBS ascension are poorly understood. Here, we describe the role of the GBS hyaluronidase in ascending infection and preterm birth. We show that clinical GBS strains associated with preterm labor or neonatal infections have increased hyaluronidase activity compared to commensal strains obtained from rectovaginal swabs of healthy women. Using a murine model of ascending infection, we show that hyaluronidase activity was associated with increased ascending GBS infection, preterm birth, and fetal demise. Interestingly, hyaluronidase activity reduced uterine inflammation but did not impact placental or fetal inflammation. Our study shows that hyaluronidase activity enables GBS to subvert uterine immune responses, leading to increased rates of ascending infection and preterm birth. These findings have important implications for the development of therapies to prevent in utero infection and preterm birth

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber

Abstract While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome’s functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon “Musculibacterium intestinale”, which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations

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

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