Intestinal adverse outcomes in COVID-19: current evidence and uncertainties using the Adverse Outcome Pathway framework

Presentation to the European Society of Toxicology in Vitro (ESTIV) November 2022 Search for CCTE records in EPA’s Science Inventory by typing in the title at this link. https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Biochemical analysis of RimK.

<p><b>3A.</b> ATPase activity of RimK_Pf incubated with RpsF_Pf and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (open circles). Addition of RpsF_Pf (triangles), glutamate (filled circles) or both (square) increases the <i>V</i>_<i>max</i> of RimK_Pf ATPase activity. <b>3B.</b> Glutamation assays with <i>E</i>. <i>coli</i> and SBW25 RimK and RpsF. The contents of each assay are indicated underneath the relevant lanes. Independent preparations of RimK_Pf and RpsF_Pf were used in the two panels, which were run separately and are shown side by side for comparative purposes only. Running positions of RimK, RpsF and glutamated-RpsF (RpsF**) are marked with arrows. <b>3C.</b> Glutamation assays with RimK_Pf and RpsF_Pf. The contents of each assay are indicated, with 0.2, 2.0 and 20 mM glutamate added to the test samples as shown. Running positions of RimK, RpsF and glutamated-RpsF (RpsF*) are marked. <b>3D.</b> Glutamation assays with RimK_Pf, RpsF_Pf and U-¹⁴C- glutamate. The contents of each reaction is indicated underneath the relevant lane. Control samples were incubated overnight, while time-course samples show 5, 10, 30, 60, 180 minutes, and overnight incubation. The left hand panel shows an overlay of Coomassie stained and radiolabel visualizations of a single gel. The right hand panel shows radiolabel incorporation into RpsF alone.</p

RimK interacting proteins.

<p>Numbers denote unique peptides detected in each sample.</p

A model for RimABK function in <i>Pseudomonas</i> plant interactions.

<p>During early stage colonisation/initial infection (top row), increased RimK activity leads to increased RpsF glutamation. This leads to increased Hfq levels, with the resulting translational repression promoting phenotypes important for niche colonisation and the establishment of infection, including motility and virulence [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005837#pgen.1005837.ref004" target="_blank">4</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005837#pgen.1005837.ref044" target="_blank">44</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005837#pgen.1005837.ref051" target="_blank">51</a>]. In the established rhizosphere/plant infection environment (bottom row), <i>rimK</i> transcription decreases. Less RpsF is glutamated, leading to altered ribosomal protein abundance and ribosome function, and lower Hfq levels. Release of Hfq translational repression leads to increased production of amino-acid transporters (ABC), oxidative stress response pathways (SodA), NRPSs and attachment factors. These changes promote resource acquisition, stress resistance and root attachment, and prioritize long-term adaptation to the plant environment. RimK activity is further regulated by direct interaction with the RimA/B proteins and the signalling molecule cdG. ‘+/-‘ denotes cases where the nature of protein/dinucleotide control is currently undefined.</p

RimA, RimB and cdG impacts on RimK activity.

<p><b>4A.</b> ATPase activity of RimK_Pf incubated with RimB, BSA and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (red, circles). Addition of RimB (purple, up-triangles) increases the RimK_Pf <i>V</i>_<i>max</i>, while BSA (brown, down-triangles) does not. RimB alone displays no ATPase activity (green, squares). <b>4B.</b> ATPase activity of RimK_Pf incubated with RimA and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (purple, squares). Addition of RimA (green, up-triangles) increases the RimK_Pf <i>V</i>_<i>max</i>, while RimA alone displays no ATPase activity (yellow, down-triangles). <b>4C.</b> Thin layer chromatography of α-³²P-labeled cdG incubated with BSA, YhjH or RimA for the time periods shown. The product of cdG hydrolysis; pGpG, migrates further than cdG but less than α-³²P-GTP. <b>4D.</b> Biotinylated-cdG pulldown for RimK_Pf. <i>E</i>. <i>coli</i> overexpression cell lysate (RimK sample) is loaded alongside the washed cdG-bead sample (b-cdG pulldown). RimK and streptavidin are indicated with arrows. <b>4E.</b> SPR sensorgram and affinity data for RimK_Pf binding to biotinylated cdG. A range of RimK_Pf concentrations was used (0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 μM) and concentration replicates included as appropriate together with buffer only controls. Protein binding and dissociation phases are shown. For the affinity fit, binding responses were measured 4s before the end of the injection and <i>K</i>_<i>d</i> values for each protein calculated using BiaEvaluation software and confirmed by GraphPad. <b>4F.</b> The effect of cdG addition on glutamation of RpsF_Pf by RimK_Pf. The contents of each reaction is indicated underneath the relevant lanes. Control samples were incubated overnight, while time-course samples show 5, 10, 30, 60, 180 minutes, and overnight incubation. The panel shows an overlay of Coomassie staining and radiolabel visualization (red) of the same gel, as with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005837#pgen.1005837.g003" target="_blank">Fig 3C</a>.</p

Up-regulated proteins common to both Δ<i>hfq</i> and Δ<i>rimK</i> mutants.

<p>Up-regulated proteins common to both Δ<i>hfq</i> and Δ<i>rimK</i> mutants.</p

RimK is important for <i>P</i>. <i>syringae</i> and <i>P</i>. <i>aeruginosa</i> plant infection.

<p><b>2A.</b> Swarming motility of <i>Pto</i> DC3000 and PA01 Δ<i>rimK</i> relative to their respective WT strains. <b>2B.</b> Congo Red binding of <i>Pto</i> DC3000 and PA01 Δ<i>rimK</i> compared with their respective WT strains. <b>2C.</b> Representative spray-infected <i>Arabidopsis</i> Col-0 plants 4 days post-infection with <i>Pto</i> DC3000 WT/Δ<i>rimK</i>. Disease symptoms are less marked with Δ<i>rimK</i> infection. <b>2D.</b> log (<i>P</i>. <i>syringae</i> CFU per cm² leaf tissue) recovered from <i>Arabidopsis</i> Col-0 plants infected with <i>Pto</i> DC3000 WT or Δ<i>rimK</i>, 2 and 3 days post-infection (dpi). The infection method in each case is stated beneath the graph. <b>2E.</b> Lettuce leaf infections with <i>P</i>. <i>aeruginosa</i> WT/Δ<i>rimK</i> strains. Lesions photographed after 5 days. <b>2F.</b> β-hemolysis by <i>P</i>. <i>aeruginosa</i> WT/Δ<i>rimK</i> strains after 24 h growth on horse blood agar.</p

Comparison of the <i>P</i>. <i>fluorescens</i> Δ<i>rimK</i>, Δ<i>hfq</i> and <i>rpsF-D139K</i> mutant strains.

<p><b>6A.</b> Swarming motility of Δ<i>hfq</i> relative to SBW25 WT. <b>6B.</b> Congo Red binding of Δ<i>hfq</i>, compared to SBW25 WT and Δ<i>rimK</i>. <b>6C.</b> Glutamation assays with <i>E</i>. <i>coli</i> and SBW25 RimK, and RpsF/RpsF-D139K. The contents of each assay are indicated underneath the relevant lanes. Running positions of RimK, RpsF and glutamated-RpsF (RpsF*) are marked with arrows. Incubation time is shown above the gel image; all controls were incubated overnight. <b>6D.</b> Wheat root attachment assay for Δ<i>rimK</i>, Tn<i>7</i>::<i>rimK</i> and <i>rpsF-D139K</i>, relative to WT SBW25. <b>6E.</b> Rhizosphere colonisation competition assays for Δ<i>hfq</i>, Δ<i>rimK</i>, <i>rpsF-D139K</i> and WT SBW25. The graph shows the ratio of mutant to WT-<i>lacZ</i> CFU recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents CFU recovered from an individual plant. <b>6F.</b> Western blot showing RpsF levels in mutant cell lysates. Statistically significant differences between WT SBW25 and mutant strains are indicated throughout (*** = p < 0.01).</p

RimABK is important for <i>P</i>. <i>fluorescens</i> rhizosphere colonisation.

<p><b>1A.</b> The SBW25 <i>rimABK</i> operon consists of three co-transcribed genes. Gene numbers and predicted translated proteins are shown in each case. <b>1B.</b> Wheat root attachment by the Δ<i>rimABK</i> and complementation strains relative to SBW25 WT. <b>1C.</b> Swarming motility of the Δ<i>rimABK</i> and complementation strains relative to SBW25 WT. <b>1D.</b> Rhizosphere colonisation competition assays. The graph shows the ratio of SBW25 WT or Δ<i>rimABK</i> to WT-<i>lacZ</i> colony forming units (CFU) recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents CFU recovered from an individual plant. Statistically significant differences between SBW25 and Δ<i>rimABK</i> strains are indicated (*** = p < 0.01, * = p < 0.05) in each case. <b>1E.</b> SBW25 <i>rimK</i> mRNA abundance determined by qRT-PCR, for wheat rhizospheres sampled at various intervals post-inoculation. Expression of <i>rimK</i> is shown relative to M9 0.4% pyruvate liquid-culture (LC).</p

Up- and Down-regulated proteins in the Δ<i>rimK</i> mutant background.

<p>Up- and Down-regulated proteins in the Δ<i>rimK</i> mutant background.</p