Temperature-specific adaptations and genetic requirements in a biofilm formed by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen often associated with nosocomial infections that are made more severe by this bacterium’s ability to form robust biofilms. A biofilm is a microbial community encompassing cells embedded within an extracellular polymeric substrate (EPS) matrix that is typically secreted by the encased microbial cells. Biofilm formation is influenced by several environmental cues, and temperature fluctuations are likely to be an important stimulus in the lifecycle of P. aeruginosa as it transitions between life in aquatic or soil environments to sites of infection in the human host. Previous work has demonstrated that human body temperature can induce a shift in the biofilm EPS relative to room temperature growth, resulting in an incorporation of a filamentous phage coat protein into the biofilm EPS. In this study, we sought to identify adaptations enabling biofilm formation at room temperature or temperatures mimicking the natural environment of P. aeruginosa (23°C and 30°C) relative to temperatures mimicking life in the human host (37°C and 40°C). We identified higher biofilm: biomass ratios at lower temperatures on certain substrates, which correlated with a higher relative abundance of apparent polysaccharide EPS content. However, the known genes for EPS polysaccharide production in P. aeruginosa PA14 did not appear to be specifically important for temperature-dependent biofilm adaptation, with the pelB gene appearing to be generally important and the algD gene being generally expendable in all conditions tested. Instead, we were able to identify two previously uncharacterized hypothetical proteins (PA14_50070 and PA14_67550) specifically required for biofilm formation at 23°C and/or 30°C relative to temperatures associated with the human host. These unstudied contributors to biofilm integrity may have been previously overlooked since most P. aeruginosa biofilm studies tend to use 37°C growth temperatures. Overall, our study demonstrates that temperature shifts can have dramatic impacts on biofilm structure and highlights the importance of studying environment-specific adaptations in biofilm physiology

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.Public Health Englan

An Overview of Antiviral Peptides and Rational Biodesign Considerations

Viral diseases have contributed significantly to worldwide morbidity and mortality throughout history. Despite the existence of therapeutic treatments for many viral infections, antiviral resistance and the threat posed by novel viruses highlight the need for an increased number of effective therapeutics. In addition to small molecule drugs and biologics, antimicrobial peptides (AMPs) represent an emerging class of potential antiviral therapeutics. While AMPs have traditionally been regarded in the context of their antibacterial activities, many AMPs are now known to be antiviral. These antiviral peptides (AVPs) have been shown to target and perturb viral membrane envelopes and inhibit various stages of the viral life cycle, from preattachment inhibition through viral release from infected host cells. Rational design of AMPs has also proven effective in identifying highly active and specific peptides and can aid in the discovery of lead peptides with high therapeutic selectivity. In this review, we highlight AVPs with strong antiviral activity largely curated from a publicly available AMP database. We then compile the sequences present in our AVP database to generate structural predictions of generic AVP motifs. Finally, we cover the rational design approaches available for AVPs taking into account approaches currently used for the rational design of AMPs

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes

Acknowledgements: The authors would like to thank Dr Michael Oade, Rene Vigeveno, and Sarah van Leeuwen for discussions and reagents. Portions of the work reported in this paper were performed using the Princeton Research Computing resources at Princeton University, which is a consortium of groups led by the Princeton Institute for Computational Science and Engineering (PICSciE) and Office of Information Technology’s Research Computing. CVR was supported by a studentship from Public Health England. K.R.S. was supported by NIH Grant Nos. R01 GM140032 and R01 AI170520. K.B. was supported by NIH Grant No. DP2 AI175474. A.T.V. was supported by NIH Grant Nos. R21 AI147172, DP2 AI175474, and R01 AI170520, and Wellcome Trust and Royal Society Grant No. 206579/17/Z.Funder: Public Health England; DOI: https://doi.org/10.13039/501100002141RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes

Acknowledgements: The authors would like to thank Dr Michael Oade, Rene Vigeveno, and Sarah van Leeuwen for discussions and reagents. Portions of the work reported in this paper were performed using the Princeton Research Computing resources at Princeton University, which is a consortium of groups led by the Princeton Institute for Computational Science and Engineering (PICSciE) and Office of Information Technology’s Research Computing. CVR was supported by a studentship from Public Health England. K.R.S. was supported by NIH Grant Nos. R01 GM140032 and R01 AI170520. K.B. was supported by NIH Grant No. DP2 AI175474. A.T.V. was supported by NIH Grant Nos. R21 AI147172, DP2 AI175474, and R01 AI170520, and Wellcome Trust and Royal Society Grant No. 206579/17/Z.Funder: Public Health England; DOI: https://doi.org/10.13039/501100002141RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes

Acknowledgements: The authors would like to thank Dr Michael Oade, Rene Vigeveno, and Sarah van Leeuwen for discussions and reagents. Portions of the work reported in this paper were performed using the Princeton Research Computing resources at Princeton University, which is a consortium of groups led by the Princeton Institute for Computational Science and Engineering (PICSciE) and Office of Information Technology’s Research Computing. CVR was supported by a studentship from Public Health England. K.R.S. was supported by NIH Grant Nos. R01 GM140032 and R01 AI170520. K.B. was supported by NIH Grant No. DP2 AI175474. A.T.V. was supported by NIH Grant Nos. R21 AI147172, DP2 AI175474, and R01 AI170520, and Wellcome Trust and Royal Society Grant No. 206579/17/Z.Funder: Public Health England; DOI: https://doi.org/10.13039/501100002141RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes

Acknowledgements: The authors would like to thank Dr Michael Oade, Rene Vigeveno, and Sarah van Leeuwen for discussions and reagents. Portions of the work reported in this paper were performed using the Princeton Research Computing resources at Princeton University, which is a consortium of groups led by the Princeton Institute for Computational Science and Engineering (PICSciE) and Office of Information Technology’s Research Computing. CVR was supported by a studentship from Public Health England. K.R.S. was supported by NIH Grant Nos. R01 GM140032 and R01 AI170520. K.B. was supported by NIH Grant No. DP2 AI175474. A.T.V. was supported by NIH Grant Nos. R21 AI147172, DP2 AI175474, and R01 AI170520, and Wellcome Trust and Royal Society Grant No. 206579/17/Z.Funder: Public Health England; DOI: https://doi.org/10.13039/501100002141RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population

Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes

Acknowledgements: The authors would like to thank Dr Michael Oade, Rene Vigeveno, and Sarah van Leeuwen for discussions and reagents. Portions of the work reported in this paper were performed using the Princeton Research Computing resources at Princeton University, which is a consortium of groups led by the Princeton Institute for Computational Science and Engineering (PICSciE) and Office of Information Technology’s Research Computing. CVR was supported by a studentship from Public Health England. K.R.S. was supported by NIH Grant Nos. R01 GM140032 and R01 AI170520. K.B. was supported by NIH Grant No. DP2 AI175474. A.T.V. was supported by NIH Grant Nos. R21 AI147172, DP2 AI175474, and R01 AI170520, and Wellcome Trust and Royal Society Grant No. 206579/17/Z.Funder: Public Health England; DOI: https://doi.org/10.13039/501100002141RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population