Influence of the Alternative Sigma Factor RpoN on Global Gene Expression and Carbon Catabolism in Enterococcus faecalis V583

The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection

SARS-CoV-2 Mac1 is an essential virulence factor

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.National Institutes of Health (NIH) grant P20GM103648 (RC) National Institutes of Health (NIH) grant 2P01AI060699 (LE) National Institutes of Health (NIH) grant P20GM113117 (ARF) National Institutes of Health (NIH) grant K22AI134993 (ARF) National Institutes of Health (NIH) grant R35GM138029 (ARF) National Science Foundation (NSF) grant 2135167 (RLU) University of Kansas General Research Fund (GRF) and Start-up funds (ARF) NIH Graduate Training at the Biology-Chemistry Interface grant T32GM132061 (CMK) University of Kansas College of Liberal Arts and Sciences Graduate Research Fellowship (CMK) Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE) LE European Commission (H2020-SC1-2019, ISOLDA Project nº 848166-2) LEN

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas. 2302083120/-/DCSupplemental.Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.Research reported in this publication was made possible in part by the services of the KU Genome Sequencing Core which is supported by the NIH under award number P30GM145499. We thank the Life Alliance Organ Recovery Agency from the University of Miami, Miami, FL, LifeCenter Northwest from Bellevue, Washington, Nevada Donor Network from Las Vegas, NV, and Midwest Transplant Network from Kansas City, KS, for provid- ing the lungs. NIH grant P20GM103648 (R.C.) NIH grant 2P01AI060699 (L.E.) NIH grant P20GM113117 (A.R.F.) NIH grant K22AI134993 (A.R.F.) NIH grant R35GM138029 (A.R.F.) NIH grant R01HL139365 (M.S.) NIH grant R01HL133240 (M.S.) NIH grant R01HL157942 (M.S.) Cystic Fibrosis Foundation grant SALATH18I0 (M.S.) NSF grant 2135167 (R.L.U.) University of Kansas General Research Fund and Start-up funds (A.R.F.) NIH Graduate Training at the Biology- Chemistry Interface grant T32GM132061 (C.M.K.) University of Kansas College of Liberal Arts and Sciences Graduate Research Fellowship (C.M.K.) Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE) (L.E.) European Commission (H2020-SC1-2019, ISOLDA Project no. 848166-2) (L.E.).Peer reviewe

PARP14: A key ADP-ribosylating protein in host–virus interactions?

Over 300 posttranslational modifications (PTMs) are known to modify the functions of proteins by affecting processes ranging from activation, degradation, localization, secretion, recognition, and regulation [1]. One such PTM, ADP-ribosylation, can be defined as the transfer of a single ADP-ribose (Mono-ADP-ribosylation (MAR)) or multiple ADP-ribose (Poly-ADP-ribosylation (PAR)) units to target proteins utilizing nicotinamide adenine dinucleotide (NAD+) as the substrate. PARP14 is a MARylating enzyme that is implicated in a range of processes from tumorigenesis to DNA repair. Most notably, PARP14 is well known in the literature for promoting the anti-inflammatory interleukin (IL)-4–mediated signaling pathway by activating STAT6-dependent gene expression and inhibiting STAT-1–dependent gene expression. However, PARP14 expression is also induced by interferon (IFN), and it enhances host IFN responses to lipopolysaccharide (LPS), poly(I:C), and viral infection, indicating a role for PARP14 in restricting viral and bacterial infections. Despite these results, data supporting a significant role for PARP14 in the antiviral response are limited. More studies are needed to identify specific roles for PARP14 during viral infections, determine its targets following infection, and elucidate the mechanisms by which PARP14 modulates inflammatory pathways

Distributed Esd Protection Network For Millimetre-Wave Rf Applications

ESD protection is critical to RF (Radio Frequency) circuits. However, linearity and parasitic capacitance that ESD protection devices present adversely affect the performance of such circuits. To address challenges in millimeter-wave RF I/O ESD protection, a robust ESD solution with adjustable working frequency is proposed. The design methodology is demonstrated through an example design. S-parameters characterization and ESD test using TLP electrical stress are presented to verify the proposed design

Esd Protection Structure With Reduced Capacitance And Overshoot Voltage For High Speed Interface Applications

Dual diodes with embedded silicon controlled rectifier (DD-SCR) for high-speed applications are presented. A new DD-SCR topography is shown to exhibit a high failure current (It2), small on-state resistance (RON), low voltage overshoot and low parasitic capacitance. This is a preferred device option for broadband high-speed data converter applications in advanced 28 nm CMOS processes. A comprehensive device characterization demonstrates the design tradeoffs and the superior ESD performance in relation to the devices\u27 variations capacitance in the sub 40 fF range

Embedded Shunt Diode Pair To Suppress Overshoot Voltage

An EMC (electromagnetic compatibility) bidirectional blocking voltage protection clamp is introduced. This device addresses shortcomings in existing solutions for interface applications with narrow design window. To suppress the voltage overshoot during transient stress conditions, embedded avalanche diodes accelerate the transition of the clamp to the on-state. By embedding the avalanche-driven triggering mechanism, this device provides a fast turn-on speed without compromising its high current handling capability. The device physics insight is demonstrated via electro-Thermal numerical simulations

Compact And Fast-Response Voltage Clamp For Bi-Directional Signal Swing Interface Applications

A compact bi-directional blocking voltage protection clamp with low overshoot voltage and high current handling capability is proposed. Under a high-stress 15-A very fast transmission line pulse, the initial overshoot voltage of the proposed structure is reduced to \u3c80 V. The voltage is clamped below 20 V (safe operation voltage) within 2 ns. This allows for a fast turn-on speed to protect interface circuits in system-on-a-chip applications with a narrow electrostatic discharge design window

Snapback And Postsnapback Saturation Of Pseudomorphic High-Electron Mobility Transistor Subject To Transient Overstress

The snapback and postsnapback saturation characteristics in a pseudomorphic high-electron mobility transistor (PHEMT) subject to electrostatic discharge (ESD) transient overstress are studied. This is undertaken, for the first time, via transmission line pulsing (TLP)-like 2-D device simulations and benchmarked against TLP measurements. Physical mechanisms underlying the postsnapback behavior and ESD-induced failure are identified and discussed by analyzing TLP-like simulation results rather than extrapolating dc-like numerical simulation data. © 2010 IEEE