A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter

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

Gold nanocluster-DNase 1 hybrid materials for DNA contamination sensing

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Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, nikR and arsS single mutants as well as a nikR/arsS double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct ‘cross-talk’ between HpArsRS and HpNikR at neutral p

Role of the −5 bp in <i>sodB</i> regulation.

<p>WT G27, WT 26695, and the “−5 bp swap” strain were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>, and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using <i>sodB</i> and <i>pfr</i> riboprobes. Data from <i>sodB</i> RPAs are presented in Panel A, and data from <i>pfr</i> RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. ^*p-value of 0.0001. ^#p-value of 0.006.</p

Direct Comparison of <i>sodB</i> Regulation in <i>H. pylori</i> Strains G27 and 26695.

<p>WT and Δ<i>fur</i> strains of G27 and 26695 were grown to exponential (A) and stationary (B) phase in iron replete and iron-limited (growth) media (60 µM dpp). After growth overnight, one-half of the exponential phase, iron replete culture was removed for RNA isolation. 200 µM dpp (final concentration) was added to create an iron-depletion shock condition to the remaining half of the iron replete cultures, and those cultures were grown for an additional hour prior to RNA isolation. The same procedure was applied the following day to the iron replete, stationary phase culture. After overnight growth, one-half of the iron-limited growth culture was removed for RNA isolation in exponential phase while the remaining half was allowed to grow into stationary phase, and RNA was isolated the following day. RNase Protection Assays (RPAs) were performed on RNA isolated from these strains using <i>sodB</i>, <i>pfr</i>, and <i>amiE</i> riboprobes. Data for Exponential phase cultures are shown in Panel A, and data for Stationary phase cultures are shown in Panel B. Fold-changes are indicated below each pair and were calculated by comparing either the relative amount of protected riboprobe in the iron-depletion shock environment (S) or the relative amount of protected riboprobe in the iron limited growth environment (G) to the iron replete lane (N). These data are representative of multiple independent experiments.</p

Fur binding to the <i>sodB</i> promoters.

<p>EMSAs were performed by incubating various concentrations of purified Fur with radiolabeled fragments of the WT G27, “−5 bp swap,” and WT 26695 <i>sodB</i> promoters as well as the negative control promoter, <i>rpoB</i>, as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. In the first four lanes, the Fur concentrations are indicated by the triangle from highest to lowest and range from 1.07 µg/mL to 0.026 µg/mL. A no protein control for each promoter is found in the fifth lanes. The last lane shows the 100× cold (unlabeled) competition control for each promoter fragment, which were each performed with the highest concentration of Fur (1.07 µg/mL). Fur exhibits specific interaction with each of the <i>sodB</i> promoters, and no interaction with the <i>rpoB</i> promoter except for very little non-specific binding at the highest Fur concentration. These data are representative of multiple independent EMSA experiments.</p

Competitive Binding Studies.

<p>To assess the relative affinity of Fur for each of the <i>sodB</i> promoter fragments (WT G27, “−5 bp swap,” and WT 26695), Fur was incubated with each radiolabeled promoter and 5×, 10×, or 25× the amount of homologous or heterologous unlabeled <i>sodB</i> promoter fragments as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. For each labeled promoter, lane one contains a no competition control. Lanes two to four, five to seven, and eight to ten contain the competition EMSAs with unlabeled WT G27, “−5 bp swap,” and WT 26695 <i>sodB</i> fragments, respectively. The percent of labeled promoter that is outcompeted and remains unbound in each lane is given below each image. These data are representative of multiple independent experiments.</p

Flow Cytometry analysis of <i>sodB</i> and <i>pfr</i> GFP reporters.

<p>Strains bearing <i>sodB::gfpmut3</i> or <i>pfr::gfpmut3</i> promoter fusions were grown overnight in either iron replete or iron depleted media. Changes in fluorescence were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. Results for the <i>sodB</i> promoter fusions are displayed in Panel 1A, and results for the <i>pfr</i> promoter fusions are displayed in Panel 1B. For both A and B, solid lines indicate the plasmid in WT <i>H. pylori</i> G27 grown in iron replete conditions, dotted lines indicate the plasmid in WT bacteria grown in iron deplete conditions, and dashed lines indicate the plasmid in Δ<i>fur</i> bacteria grown in iron replete conditions. Fluorescence is measured in relative units, and the data are representative of multiple independent flow analyses.</p