The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene

Group B Streptococcus (GBS) is a b-hemolytic, Gram-positive bacterium that commonly colonizes the female lower genital tract and is associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A major factor promoting GBS virulence is the b-hemolysin/cytolysin, which is cytotoxic to several host cells. We recently showed that the ornithine rhamnolipid pigment, Granadaene, produced by the gene products of the cyl operon, is hemolytic. Here, we demonstrate that heterologous expression of the GBS cyl operon conferred hemolysis, pigmentation, and cytoxicity to Lactococcus lactis, a model non-hemolytic Gram-positive bacterium. Similarly, pigment purified from L. lactis is hemolytic, cytolytic, and identical in structure to Granadaene extracted from GBS, indicating the cyl operon is sufficient for Granadaene production in a heterologous host. Using a systematic survey of phyletic patterns and contextual associations of the cyl genes, we identify homologs of the cyl operon in physiologically diverse Gram-positive bacteria and propose undescribed functions of cyl gene products. Together, these findings bring greater understanding to the biosynthesis and evolutionary foundations of a key GBS virulence factor and suggest that such potentially toxic lipids may be encoded by other bacteria.This work was supported by funding from the National Institutes of Health grants R01AI112619, R01AI33976, R01AI00989, and R21AI125907 to LR. The NIH training grant T32AI07509 (PI: Lee Ann Campbell) supported CW. LI and LA were supported by intramural funds from NLM, NIH

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Elucidation of Group B Streptococcus hemolytic pigment and hyaluronidase during infection

Thesis (Ph.D.)--University of Washington, 2020Group 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 (also known as granadaene) 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

GBS hyaluronidase mediates immune suppression in a TLR2/4- and IL-10-dependent manner during pregnancy-associated infection

ABSTRACT Bacterial infections remain a significant cause of adverse pregnancy outcomes. Ascending infection of group B streptococcus (GBS) or Streptococcus agalactiae from the lower genital tract to the amniotic cavity leads to fetal injury, preterm births, or stillbirths. Factors increasing the invasive potential of bacteria at the maternal-fetal interface are poorly understood. Previous studies have indicated that the GBS hyaluronidase (HylB) can enhance systemic infection by breaking down host hyaluronan into disaccharides that dampen protective TLR2 and TLR4 signaling. Here, we examined the importance of hyaluronan receptors such as TLR2, TLR4, and CD44 in defense against GBS infections during pregnancy. While HylB promoted ascending GBS infection in wild-type (WT) and CD44-deficient mice, surprisingly, mice lacking both TLR2 and TLR4 (TLR2/4) were able to curtail these infections. Interleukin-10 (IL-10) and IL-10-expressing macrophages were significantly increased in the uterine tissues of WT mice during infection with HylB-proficient GBS compared with those of TLR2/4-deficient mice, and this likely promotes immune suppression and GBS dissemination. Consistent with these observations, pregnant IL-10-deficient mice exhibited diminished GBS ascension and dissemination. Similarly, the administration of a blocking antibody against the IL-10 receptor (IL-10R) in WT mice diminished ascending GBS infection. Collectively, these observations indicate that HylB promotes immune suppression in a TLR2/4- and IL-10-dependent manner to enhance the invasive potential of GBS during pregnancy-associated infections. IMPORTANCE Bacteria such as GBS can cause infections during pregnancy leading to preterm births, stillbirths, and neonatal infections. The interaction between host and bacterial factors during infections in the placenta is not fully understood. GBS secretes a hyaluronidase enzyme that is thought to digest host hyaluronan into immunosuppressive disaccharides that dampen TLR2/4 signaling, leading to increased bacterial dissemination and adverse outcomes. In this study, we show that GBS HylB mediates immune suppression and promotes bacterial infection during pregnancy that requires TLR2, TLR4, and IL-10. Understanding the interaction between host and bacterial factors can inform future therapeutic strategies to mitigate GBS infections

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

Zika virus and the nonmicrocephalic fetus: why we should still worry

Zika virus is a mosquito-transmitted flavivirus and was first linked to congenital microcephaly caused by a large outbreak in northeastern Brazil. Although the Zika virus epidemic is now in decline, pregnancies in large parts of the Americas remain at risk because of ongoing transmission and the potential for new outbreaks. This review presents why Zika virus is still a complex and worrisome public health problem with an expanding spectrum of birth defects and how Zika virus and related viruses evade the immune response to injure the fetus. Recent reports indicate that the spectrum of fetal brain and other anomalies associated with Zika virus exposure is broader and more complex than microcephaly alone and includes subtle fetal brain and ocular injuries; thus, the ability to prenatally diagnose fetal injury associated with Zika virus infection remains limited. New studies indicate that Zika virus imparts disproportionate effects on fetal growth with an unusual femur-sparing profile, potentially providing a new approach to identify viral injury to the fetus. Studies to determine the limitations of prenatal and postnatal testing for detection of Zika virus–associated birth defects and long-term neurocognitive deficits are needed to better guide women with a possible infectious exposure. It is also imperative that we investigate why the Zika virus is so adept at infecting the placenta and the fetal brain to better predict other viruses with similar capabilities that may give rise to new epidemics. The efficiency with which the Zika virus evades the early immune response to enable infection of the mother, placenta, and fetus is likely critical for understanding why the infection may either be fulminant or limited. Furthermore, studies suggest that several emerging and related viruses may also cause birth defects, including West Nile virus, which is endemic in many parts of the United States. With mosquito-borne diseases increasing worldwide, there remains an urgent need to better understand the pathogenesis of the Zika virus and related viruses to protect pregnancies and child health