The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low-copy-number, high-affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high-affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor-binding domain (ColN-R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid- and solid-state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

Interactions of lipopolysaccharide with lipid membranes, raft models - a solid state NMR study.

Lipopolysaccharide (LPS) is a major component of the external leaflet of bacterial outer membranes, key pro-inflammatory factor and an important mediator of host-pathogen interactions. In host cells it activates the complement along with a pro-inflammatory response via a TLR4-mediated signalling cascade and shows preference for cholesterol-containing membranes. Here, we use solid state (13)C and (31)P MAS NMR to investigate the interactions of LPS from three bacterial species, Brucella melitensis, Klebsiella pneumoniae and Escherichia coli, with mixed lipid membranes, raft models. All endotoxin types are found to be pyrophosphorylated and Klebsiellar LPS is phosphonylated, as well. Carbon-13 MAS NMR indicates an increase in lipid order in the presence of LPS. Longitudinal (31)P relaxation, providing a direct probe of LPS molecular and segmental mobility, reveals a significant reduction in (31)P T1 times and lower molecular mobility in the presence of ternary lipid mixtures. Along with the ordering effect on membrane lipid, this suggests a preferential partitioning of LPS into ordered bilayer sphingomyelin/cholesterol-rich domains. We hypothesise that this is an important evolutionary drive for the selection of GPI-anchored raft-associated LPS-binding proteins as a first line of response to membrane-associated LPS.

Heteronuclear chemical shift correlation and J-resolved MAS NMR spectroscopy of lipid membranes.

Direct observation of J-couplings remains a challenge in high-resolution solid-state NMR. In some cases, it is possible to use Lee-Goldburg (LG) homonuclear decoupling during rare spin observation in MAS NMR correlation spectroscopy of lipid membranes to obtain J-resolved spectra in the direct dimension. In one simple implementation, a wide line separation-type (13)C-(1)H HETCOR can provide high-resolution (1)H/(13)C spectra, which are J-resolved in both dimensions. Coupling constants, (1)J(HC), obtained from (1)H doublets, can be compared with scaled (1)J(θ)(CH)-values obtained from the (13)C multiplets to assess the LG efficiency and scaling factor. The use of homonuclear decoupling during proton evolution, LG-HETCOR-LG, can provide J-values, at least in the rare spin dimension, and allows measurements in less mobile membrane environments. The LG-decoupled spectroscopic approach is demonstrated on pure dioleoylphosphatidylcholine (DOPC) membranes and used to investigate lipid mixtures of DOPC/cholesterol and DOPC/cholesterol/sphingomyelin.

Predictive Variables for Failure in Administration of Intrapleural Tissue Plasminogen Activator/Deoxyribonuclease in Patients With Complicated Parapneumonic Effusions/Empyema.

BACKGROUND: Combined intrapleural therapy with tissue plasminogen activator (tPA) and deoxyribonuclease (DNase) has been shown to reduce the need for surgical intervention for complicated pleural effusion/empyema (CPE/empyema). For patients in whom tPA/DNase is likely to fail, however, receipt of this therapy may simply delay the inevitable. The goal of this study was to identify risk factors for failure of combined intrapleural therapy. METHODS: We performed a retrospective chart review of patients who received intrapleural tPA/DNase for the treatment of CPE/empyema. Clinical variables included demographic data, radiographic parameters at time of diagnosis, and results from pleural fluid analysis. We used gradient boosted trees-an ensemble machine learning technique, with hyperparameter tuning to mitigate overfitting-to rank the importance of 19 candidate clinical variables with respect to their ability to predict failure of tPA/DNase therapy. RESULTS: We identified 84 patients who received intrapleural tPA/DNase for the treatment of complicated pleural effusions/empyema over a 5-year period. Resolution of CPE/empyema with intrapleural tPA/DNase was achieved in two-thirds of the patients (n = 57). Of the 19 candidate predictors of tPA/DNase failure, the presence of pleural thickening was found to be the most important (48% relative importance), followed by the presence of an abscess or necrotizing pneumonia (24%), the pleural protein level (6%), and the presence of loculated effusion (4%). CONCLUSIONS: Our analysis found that the presence of pleural thickening and the presence of an abscess/necrotizing pneumonia helps to triage patients in whom combined intrapleural therapy is likely to fail. The results warrant further study and validation in a prospective multicenter study.

Diagnostic Yield and Safety of Bronchoscopist-directed Moderate Sedation With a Bolus Dose Administration of Propofol During Endobronchial Ultrasound Bronchoscopy.

BACKGROUND: The propofol use for moderate sedation (MS) during endobronchial ultrasound (EBUS) bronchoscopy is primarily restricted for use by an anesthesiologist because of safety concerns. The goals of this study were to demonstrate the safety and the diagnostic yield of the use of propofol by bronchoscopists and trained endoscopy nurses during EBUS bronchoscopy without intubation. METHODS: We tested a bolus propofol administration protocol targeting MS for EBUS bronchoscopy. A fixed initial dose of 40 mg of propofol along with a fixed 50 mcg fentanyl dose were administered. Sedation assessment was performed every 2 minutes, and repeated bolus doses of propofol were given to maintain MS under the direction of the bronchoscopist. RESULTS: A total of 122 subjects underwent EBUS bronchoscopy with a goal of MS from August 2015 to April 2017. In total, 110 subjects who underwent convex EBUS bronchoscopy under MS with propofol were included in the analysis. Median procedure duration was 57 minutes (range, 15 to 97 min). Deep sedation and agitation-related delay were occurred in 14 and 21 subjects, respectively. Hemodynamic instability and hypoxemia occurred in 23 subjects. However, there was no need for vasopressors or artificial airway placement. Median of total propofol dose per case was 560 mg. Diagnostic yield for malignancy and granuloma was 68%, and a median of 4 lymph node stations were sampled per subject. All specimens with adenocarcinoma were sufficient for genetic marker analysis. There were no major sedation-related complications. CONCLUSION: A bolus administration of propofol during EBUS bronchoscopy provided excellent adequacy of sedation and well tolerance safety profile.

Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A (13)C MAS NMR Study.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state (13)C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol (13)C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.

High-resolution J-coupled 13C MAS NMR spectroscopy of lipid membranes.

We propose the use of Lee-Goldburg decoupling in high-resolution natural abundance 13C CP-MAS NMR spectroscopy to obtain J-resolved multiplets from membrane lipids, and the use of these in spectral assignment and to investigate changes in molecular and segmental dynamics within chemical shift-resolved lipid groups. Spectroscopic characteristics of hydrated DPPC bilayers are reported, including J1CH-couplings from the liquid crystalline and gel phases. The observed J1thetaCH values are scaled in the Lee-Goldburg experiment by a factor of approximately 3(-1/2) and corrected J1CH values on the order of 150 Hz compare well with indirect measurements. The J-resolved multiplets show J1thetaCH-couplings from the chain region to be approximately 20% lower than couplings determined from the headgroup, with backbone values falling between the two. Sensitivity of Lee-Goldburg decoupling to molecular motions reveals changes in hydrocarbon chain and backbone segmental dynamics across the main phospholipid transition and reduction in headgroup mobility below the pre-transition.