Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201ϕ2-1 and PVP-SE1

Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections

A novel use for sugarcane bagasse hemicellulosic fraction: Xylitol enzymatic production

The excess of sugarcane bagasse (SCB) from the sugar-alcohol industry is considered a by-product with great potential for many bioproducts production. This work had as objective to verify the performance of sugarcane bagasse hemicellulosic hydrolysate (SCBHH) as source of sugars for enzymatic or in vitro xylitol production. For this purpose, xylitol enzymatic production was evaluated using different concentrations of treated SCBHH in the commercial reaction media. The weak acid hydrolysis of SCB provided a hydrolysate with 18 g L(-1) and 6 g L(-1) of xylose and glucose, respectively. Considering the reactions, changes at xylose xylitol conversion efficiency and volumetric productivity in xylitol were not observed for the control experiment and using 20 and 40% v.v (1) of SCBHH in the reaction media. The conversion efficiency achieved 100% in all the experiments tested. The results showed that treated SCBHH is suitable as xylose and glucose source for the enzymatic xylitol production and that this process has potential as an alternative for traditional xylitol production ways. (C) 2011 Published by Elsevier Ltd.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[2005/0280-4]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[2005/02866-9

Schematic diagram of the domain organization of OBPgp279, 201φ2-1gp229 and PVP-SE1gp146.

<p>For each endolysin, the predicted N-terminal PG binding domains (PBD) are marked in light grey, whereas the C-terminal catalytic domains (lysozyme-like or goose-like lysozyme domains) are in dark grey. E-values for OBP_127–327 (predicted by HHpred), PBD₁ and PBD₂ of OBPgp279 (both HHpred), PVP_82–235 (Blastp), PBD of PVP-SE1gp146 (Pfam), 201φ2-1_75–210 (HHpred) and PBD of 201φ2-1gp229 (Pfam) are all indicated beneath the respective domains. The names of the different constructs used in this study are also marked above each predicted structure.</p

Apparent association and dissociation kinetic data for the binding of PVP_1–63-EGFP to immobilized <i>S.</i> Typhimurium LT2 cells.

<p>Two different concentrations (5 and 10 µM) were measured in three-fold and the corresponding average is represented. The association rate (k_a1), dissociation rate (k_d1), forward rate (k_a2) and backward rate (k_d2) constants are calculated according to a two-state model that describes a 1∶1 binding (AB) of the fusion protein analyte (A) to the immobilized ligand (B), followed by a conformational change in the complex (AB→AB^..).</p

Thermoresistance of OBPgp279, PVP-SE1gp146 and 201φ2-1gp229.

<p>(A) The residual muralytic activity of OBPgp279 (1 µM, light grey bars), PVP-SE1gp146 (5 µM, intermediate grey bars) and 201φ2-1gp229 (3 µM, dark grey bars) on OM permeabilized <i>P. aeruginosa</i> PAO1 cell substrate after 1, 2, 3 and 4 h heat treatment at 50°C is shown. (B) For PVP-SE1gp146 (5 µM), the residual activity on OM permeabilized <i>P. aeruginosa</i> PAO1 after incubation for 0 (white bars), 20 (light grey bars), 40 (dark grey bars) and 60 (black bars) min on different temperatures between 50 and 100°C was determined. For each curve, averages and standard deviations of three repeated and independent experiments are given.</p

Peptidoglycan binding capacity of fusion proteins OBP_1–117-EGFP, OBP_7–54-EGFP, OBP_57–117-EGFP, PVP_1–63-EGFP and 201φ2-1_8–63-EGFP.

<p>(A) The specific fluorescence (i.e. the fluorescence of the treated cells subtracted by the fluorescence of untreated cells and cell buffer) of 2 µM of each fusion protein measured after incubation with OM permeabilized <i>P. aeruginosa</i> PAO1 (dark grey bars) or <i>S.</i> Typhimurium LT2 cells (light grey bars) is shown here for the different EGFP fusion constructs. Obtained values were compared with the specific fluorescence of the unbound wt EGFP. 2 µM of KZ-EGFP, the fusion protein of the PBD of KZ144 and EGFP, was used as positive control. Average and standard deviation values of three independent experiments are shown. (B) Epifluorescence microscopy of OM permeabilized <i>P. aeruginosa</i> PAO1 cells treated with OBP_1–117-EGFP (left) and PVP_1–63-EGFP (right). Cells were incubated with 2 µM of each fusion protein for 5 min. Cell pellets were washed twice and visualized using epifluorescence microscopy (with 500× magnification, size bar is indicated). Both fusion proteins are visualized in green targeted to the bacterial cell wall.</p

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical IndustryFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p&lt;0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p&lt;0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status