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

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Mechanisms of Group B Streptococcus Colonization and Ascending Infection

Thesis (Ph.D.)--University of Washington, 2017-06Group B Streptococcus (GBS), also known as Streptococcus agalactiae, are bacteria that commonly reside in the vagina of healthy women. GBS is not a major cause of infection in normal adults. However, newborns can acquire GBS from colonized mothers during birth leading to neonatal infections. Alternatively, GBS can migrate from the vagina to the uterus, during pregnancy leading to in utero infection and significant adverse pregnancy outcomes such as preterm birth and stillbirth. To successfully establish an in utero infection, GBS must bypass the host immune response to traffic from the vagina into the pregnant uterus in a process known as ascending infection. Despite the significant impact on perinatal and neonatal health, the mechanisms of GBS vaginal colonization and ascending infection are not completely understood. An increased understanding of these processes will enable the development of novel therapeutic strategies and interventions and are critical for the reduction of the burden of GBS disease. This dissertation summarizes our efforts to describe the interactions between GBS and the host during vaginal colonization, ascending in utero infection and preterm birth. First, we aimed to explore the host-pathogen interactions that mediate vaginal colonization. While significant work has been done to identify GBS virulence factors involved in vaginal colonization, little attention has been given to the host response during colonization. We demonstrate that the vaginal resident immune cells known as mast cells are critical for preventing colonization of hyper-virulent GBS, which are highly associated with invasive infections. GBS virulence is largely mediated by its unique hemolytic toxin which is a pigmented rhamnolipid (hereafter-called hemolytic pigment); yet hyper-pigmented GBS strains are rarely isolated from vagina. We found that the hemolytic pigment toxin activates a subset of host immune cells known as mast cells, leading to its clearance from the vagina. Interestingly, removal of the GBS hemolytic pigment from the bacteria, which renders it avirulent, also leads to inefficient vaginal colonization. Once GBS has ascended from the vagina to infect the placenta, the hemolytic pigment is required for resistance of the host immune response, and hyper-pigmented strains are more resistant to killing by host immune defenses. These data indicate that a delicate balance of hemolytic pigment expression, and therefore regulation of the host immune response, is necessary for successful colonization and ascending infection. Next, we sought to determine the mechanism by which GBS ascends from the vagina to the uterus. We show that GBS interactions with vaginal epithelial cells play an integral role in permitting ascending infection. GBS stimulate a process known as epithelial exfoliation that is critical for ascending infection. Epithelial exfoliation is a process wherein epithelial cells lose their junction properties and detach from the epithelial surface and basement membranes. To induce epithelial exfoliation, GBS actives a class of proteins called integrins, which leads to β-catenin signaling and epithelial-to-mesenchymal transition (EMT), resulting in a migratory cell phenotype. EMT drives loss of junctional and adherent properties, and permits bacterial dissemination into vaginal tissues for ascending infection. Reduction of integrin activation results in less epithelial exfoliation and a reduction in ascending infection and adverse pregnancy outcomes. This work is the first to outline a mechanism of GBS ascending infection. Finally, we endeavored to define the mechanism by which GBS establishes in utero infection. We observed that GBS isolated from cases of preterm birth had significantly higher levels of hyaluronidase activity than those isolated from commensal settings. The GBS hyaluronidase, HylB, is a secreted enzyme that cleaves the host extra-cellular matrix molecule hyaluronic acid into is disaccharide moiety. Hyaluronic acid disaccharides have the ability to block toll-like receptors, which are critical for the detection of pathogenic bacteria, and thus prevent immune recognition of GBS. Using a murine model of ascending infection, we show that GBS deficient for hylB have reduced ability to establish in utero infections due to increased immune recognition of the bacteria. The reduction in bacterial load in the uterus reduces bacterial invasion of placental and fetal tissues, leading to improved pregnancy outcomes. Together, the work in this dissertation describes how GBS successfully colonizes the vagina, ascends from the vagina into the pregnant uterus, and blunts the host immune response in the uterus, leading to placental and fetal infection and adverse pregnancy outcomes

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

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection.

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality, and hospitalization costs. Kp must acquire nutrients from the host for successful infection; however, the host is able to prevent bacterial nutrient acquisition through multiple systems. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing transposon mutant frequencies between mouse genotypes, we identified the Kp citrate synthase, GltA, as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo. Accordingly, addition of glutamate family amino acids to Lcn2+/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2+/+ organ lysates was sufficient to rescue the loss of gltA. Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection

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