Infection-associated gene regulation of L-tartrate metabolism in Salmonella enterica serovar Typhimurium.

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for the sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, the regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, i.e., substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes the utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection. IMPORTANCE: Bacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms of how Salmonella sense metabolites in the gut to activate or repress its virulence program; however, our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

Gene regulation of infection-associated L-tartrate metabolism in Salmonella enterica serovar Typhimurium.

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection.

Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy.

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4-5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL.

Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy.

Currently, there is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and its analog mundulone acetate (MA) as inhibitors of in vitro intracellular Ca 2+ -regulated myometrial contractility. In this study, we probed the tocolytic and therapeutic potential of these small molecules using myometrial cells and tissues obtained from patients receiving cesarean deliveries, as well as a mouse model of PL resulting in preterm birth. In a phenotypic assay, mundulone displayed greater efficacy in the inhibition of intracellular-Ca 2+ from myometrial cells; however, MA showed greater potency and uterine-selectivity, based IC 50 and E max values between myometrial cells compared to aorta vascular smooth muscle cells, a major maternal off-target site of current tocolytics. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted concentration-dependent inhibition of ex vivo myometrial contractions and that neither mundulone or MA affected vasoreactivity of ductus arteriosus, a major fetal off-target of current tocolytics. A high-throughput combination screen of in vitro intracellular Ca 2+ -mobilization identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these synergistic combinations, mundulone + atosiban demonstrated a favorable in vitro therapeutic index (TI)=10, a substantial improvement compared to TI=0.8 for mundulone alone. The ex vivo and in vivo synergism of mundulone and atosiban was substantiated, yielding greater tocolytic efficacy and potency on isolated mouse and human myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone 5hrs after mifepristone administration (and PL induction) dose-dependently delayed the timing of delivery. Importantly, mundulone in combination with atosiban (FR 3.7:1, 6.5mg/kg + 1.75mg/kg) permitted long-term management of PL after induction with 30 µg mifepristone, allowing 71% dams to deliver viable pups at term (> day 19, 4-5 days post-mifepristone exposure) without any visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the future development of mundulone as a stand-alone single- and/or combination-tocolytic therapy for management of PL.

Selective Polyprotein Processing Determines Norovirus Sensitivity to Trim7.

Noroviruses are a leading cause of gastroenteritis worldwide, yet the molecular mechanisms of how host antiviral factors restrict norovirus infection are poorly understood. Here, we present a CRISPR activation screen that identifies mouse genes which inhibit murine norovirus (MNV) replication. Detailed analysis of the major hit Trim7 demonstrates a potent inhibition of the early stages of MNV replication. Leveraging in vitro evolution, we identified MNV mutants that escape Trim7 restriction by altering the cleavage of the viral NS6-7 polyprotein precursor. NS6, but not the NS6-7 precursor, directly binds the substrate-binding domain of Trim7. Surprisingly, the selective polyprotein processing that enables Trim7 evasion inflicts a significant evolutionary burden, as viruses with decreased NS6-7 cleavage are strongly attenuated in viral replication and pathogenesis. Our data provide an unappreciated mechanism of viral evasion of cellular antiviral factors through selective polyprotein processing and highlight the evolutionary tradeoffs in acquiring resistance to host restriction factors. IMPORTANCE To maximize a limited genetic capacity, viruses encode polyproteins that can be subsequently separated into individual components by viral proteases. While classically viewed as a means of economy, recent findings have indicated that polyprotein processing can spatially and temporally coordinate the distinct phases of the viral life cycle. Here, we present a function for alternative polyprotein processing centered on immune defense. We discovered that selective polyprotein processing of the murine norovirus polyprotein shields MNV from restriction by the host antiviral protein Trim7. Trim7 can bind the viral protein NS6 but not the viral precursor protein NS6-7. Our findings provide insight into the evolutionary pressures that define patterns of viral polyprotein processing and uncover a trade-off between viral replication and immune evasion.

Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness.

BACKGROUND: Indomethacin treatment for patent ductus arteriosus (PDA) is associated with acute kidney injury (AKI). Fenoldopam, a dopamine (DA) DA1-like receptor agonist dilates the renal vasculature and may preserve renal function during indomethacin treatment. However, limited information exists on DA receptor-mediated signaling in the ductus and fenoldopam may prevent ductus closure given its vasodilatory nature. METHODS: DA receptor expression in CD-1 mouse vessels was analyzed by qPCR and immunohistochemistry. Concentration-response curves were established using pressure myography. Pretreatment with SCH23390 (DA1-like receptor antagonist), phentolamine (α -adrenergic receptor antagonist) or indomethacin addressed mechanisms for DA-induced changes. Fenoldopam's effects on postnatal ductus closure were evaluated in vivo. RESULTS: DA1 receptors were expressed equally in ductus and aorta. High-dose DA induced modest vasoconstriction under newborn O2 conditions. Phentolamine inhibited DA-induced constriction, while SCH23390 augmented constriction, consistent with a vasodilatory role for DA1 receptors. Despite this, fenoldopam had little effect on ductus tone nor indomethacin- or O2-induced constriction and did not impair postnatal closure in vivo. CONCLUSION(S): DA receptors are present in the ductus but have limited physiologic effects. DA-induced ductus vasoconstriction is mediated via α-adrenergic pathways. The absence of DA1-mediated impairment of ductus closure supports the study of potential role for fenoldopam during PDA treatment.

Molecular and mechanical factors contributing to ductus arteriosus patency and closure.

Regulation of the ductus arteriosus, an essential fetal vessel connecting the pulmonary artery and aorta, is complex. Failure of this vessel to close after birth may result in a persistent left-to-right shunt through the patent ductus arteriosus, a condition associated with significant morbidities. Numerous factors contribute to the shift from fetal ductus patency to postnatal closure, requiring precise coordination of molecular cues with biomechanical forces and underlying genetic influences. Despite significant advances, questions remain regarding signaling dynamics and the natural time course of ductus closure, particularly in preterm neonates. This review highlights the contributions of early investigators and more recent clinician scientists to our understanding of the molecular and mechanical factors that mediate ductus patency and closure.