Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces

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

EPEC strains used in this work.

<p>EPEC strains used in this work.</p

Functionality of T3SS of the EPEC effector mutants.

<p><b>A</b>. Analysis of secreted and bacterial proteins in the indicated EPEC strains after 4 h of growth in DMEM at 37°C. Top panel: Coomassie staining of proteins found in the extracellular medium labeling the translocators EspABD and the autotransporter EspC. Molecular standards are shown in kDa. Bottom panels: Western blots of bacterial whole-cell protein extracts incubated with polyclonal antibodies to detect EscC, EscJ, EscD injectisome proteins and EspB translocator protein. Detection of cytoplasmic GroEL was used as a loading control. Discontinuous lines indicate borders of independent gels. <b>B</b>. Immunofluorescence confocal microscopy of HeLa cells infected for 90 min with WT EPEC, EPEC2, EPEC1 and EPEC0 strains. EPEC bacteria are labeled with anti-intimin-280 polyclonal (green), actin is labeled with TRITC-phalloidin (red) and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath the adherent bacteria is observed in EPECwt, EPEC2 and EPEC1 strains. Scale bar 5 μm. <b>C.</b> Quantification of the number of HeLa cells with actin pedestals after infection with the indicated strains WT, EPEC2, EPEC1 and EPEC0. The data shown are the mean of two independent experiments with standard deviation (SD). In each experiment, one hundred cells per infection sample were counted. <b>D</b>. Protein translocation into HeLa cells of β-lactamase (Bla) fusion by EPEC effector mutants. HeLa cells were infected for 90 min with the indicated EPEC strains (WT EPEC, EPECΔ<i>escN</i>, EPEC2, EPEC1 EPEC0), expressing EspF_1-20-Bla fusion (WT EPEC, EPECΔ<i>escN</i>, EPEC2, EPEC1, EPEC0) or the control vector pCX340, and then incubated with the BLA substrate CCF2/AM for additional 1 h. Bla activity was quantified measuring the emission ratio of fluorescence at 450/520 nm. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **p<0.01 and ***<i>p</i> <0.001.</p

Deletion of effector genes in EPEC E2348/69.

<p><b>A</b>. Representation of the LEE pathogenicity island. Effector genes are labeled in red. The order of deletion is numbered: deletion 1, 2, 3, 4 are for <i>map</i>, <i>espG</i>, <i>espF</i> and <i>espH</i> respectively. The <i>espZ</i> and <i>tir</i> genes were deleted after the non-LEE effectors, with order 12 and 13 respectively. Scale of 5 kb is indicated at the bottom. <b>B</b>. Effector genes located outside the LEE are localized in integrative elements (IE) and prophages (PP). Effector genes are labeled in red. Pseudogenes are specified with asterisk. The red bars indicate the deletions carried out. The order of deletion is numbered. Scale of 5 kb is indicated at the bottom.</p

A/E lesions formation by EPEC deletion mutants in IE effectors.

<p>A/E lesions formation by EPEC deletion mutants in IE effectors.</p

A/E lesions in human intestinal biopsies infected with EPEC effector mutants.

<p>A/E lesions in human intestinal biopsies infected with EPEC effector mutants.</p

Induction of filopodia by EPEC effector mutants with integrated <i>map</i>.

<p>Immunofluorescence microscopy of Swiss 3T3 cells infected for 10 min with EPEC2, EPEC1 and EPEC0 and isogenic strains with <i>map</i> integrated in the chromosome. EPEC was detected with rabbit polyclonal anti-O127 (red) and actin was stained with Oregon-green phalloidin (green). Filopodia spikes are labelled with arrowheads. Actin accumulation beneath bacteria is indicated with arrows. Scale bar 2 μm.</p

Translocation of NleC by EPEC effectors mutants induces p65 degradation.

<p><b>A</b>. Western blot detecting NF-κB p65 protein in HeLa cells infected for 4 h (1 h+3 h gentamicin) with EPEC2, EPEC1 and EPEC0 and isogenic strains with <i>nleC</i> integrated in the chromosome. Uninfected (UI) cells and cells infected with WT EPEC are used as controls. Detection of α-tubulin was used as a loading control. <b>B</b>. Quantification of p65 in Hela cells infected with the indicated strains. Protein loading was normalized with α-tubulin. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **<i>p</i> <0.01; ***<i>p</i> <0.001.</p

Mild Hypercapnia or Normocapnia after Out-of-Hospital Cardiac Arrest

BACKGROUND Guidelines recommend normocapnia for adults with coma who are resuscitated after out-of-hospital cardiac arrest. However, mild hypercapnia increases cerebral blood flow and may improve neurologic outcomes. METHODS We randomly assigned adults with coma who had been resuscitated after out-of-hospital cardiac arrest of presumed cardiac or unknown cause and admitted to the intensive care unit (ICU) in a 1:1 ratio to either 24 hours of mild hypercapnia (target partial pressure of arterial carbon dioxide [Paco ], 50 to 55 mm Hg) or 2 normocapnia (target Paco2, 35 to 45 mm Hg). The primary outcome was a favorable neurologic outcome, defined as a score of 5 indicating lower moderate disability) or higher, as assessed with the use of the Glasgow Outcome Scale–Extended (range, 1 death] to 8, with higher scores indicating better neurologic outcome) at 6 months. Secondary outcomes included death within 6 months. RESULTS A total of 1700 patients from 63 ICUs in 17 countries were recruited, with 847 pa- tients assigned to targeted mild hypercapnia and 853 to targeted normocapnia. A favorable neurologic outcome at 6 months occurred in 332 of 764 patients (43.5%) in the mild hypercapnia group and in 350 of 784 (44.6%) in the normocapnia group (relative risk, 0.98; 95% confidence interval [CI], 0.87 to 1.11; P=0.76). Death within 6 months after randomization occurred in 393 of 816 patients (48.2%) in the mild hypercapnia group and in 382 of 832 (45.9%) in the normocapnia group (relative risk, 1.05; 95% CI, 0.94 to 1.16). The incidence of adverse events did not differ significantly between groups. CONCLUSIONS In patients with coma who were resuscitated after out-of-hospital cardiac arrest, targeted mild hypercapnia did not lead to better neurologic outcomes at 6 months than targeted normocapnia