scholarly journals Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1014
Author(s):  
Fabio Antenucci ◽  
Armen Ovsepian ◽  
Agnieszka Wrobel ◽  
Hanne Cecilie Winther-Larsen ◽  
Anders Miki Bojesen
Keyword(s):  
Outer Membrane ◽  
Acyl Carrier Protein ◽  
Actinobacillus Pleuropneumoniae ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Bacterial Strains ◽  
Spectrometry Analysis ◽  
Fast Screening ◽  
Chimeric Construct ◽  
Fluorescent Tag

Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In Actinobacillus pleuropneumoniae, a Gram-negative porcine pathogen responsible for extensive economic losses worldwide, we identified a conserved general secretion pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA (ApfA stem: ApfAs). ApfAs was used as an outer membrane anchor, to which potential immunogens can be attached. To enable confirmation of correct positioning, ApfAs, was cloned in combination with the modified acyl carrier protein (ACP) fluorescent tag ACP mini (ACPm) and the putative immunogen VacJ. The chimeric construct was inserted in the pMK-express vector, subsequently transformed into A. pleuropneumoniae for expression. Flow cytometry, fluorescence imaging and mass spectrometry analysis were employed to demonstrate that the outer membrane of the transformed strain was enriched with the chimeric ApfAs-ACPm-VacJ antigen. Our results confirmed correct positioning of the chimeric ApfAs-ACPm-VacJ antigen and supported this system’s potential as platform technology enabling antigenic enrichment of the outer membrane of A. pleuropneumoniae.

scholarly journals Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1531-1540 ◽  
Author(s):  
Rahul Shrivastava ◽  
Bhakti Basu ◽  
Ashwini Godbole ◽  
M. K. Mathew ◽  
Shree K. Apte ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Organic Acids ◽  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Aromatic Compounds ◽  
Mass Spectrometry Analysis ◽  
Binding Studies ◽  
Spectrometry Analysis ◽  
Glucose Binding

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [14C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain’s ability to utilize aromatics and organic acids over glucose.

scholarly journals LPS Remodeling Triggers Formation of Outer Membrane Vesicles inSalmonella

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Wael Elhenawy ◽  
Michael Bording-Jorgensen ◽  
Ezequiel Valguarnera ◽  
M. Florencia Haurat ◽  
Eytan Wine ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Membrane Remodeling ◽  
Macrophage Infection ◽  
Spectrometry Analysis

ABSTRACTOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employingSalmonella entericaserovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in apagLmutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.IMPORTANCEThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis inS. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. AnS. Typhimurium ΔpagLstrain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

scholarly journals Extracellular Secretion of the Borrelia burgdorferi Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein

2004 ◽  
Vol 72 (11) ◽  
pp. 6279-6286 ◽  
Author(s):  
Robert G. Cluss ◽  
Damon A. Silverman ◽  
Thomas R. Stafford
Keyword(s):  
Borrelia Burgdorferi ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Regulatory Networks ◽  
Binding Protein ◽  
Mass Spectrometry Analysis ◽  
Logarithmic Phase ◽  
Spectrometry Analysis ◽  
Rpmi Medium ◽  
Extracellular Secretion

ABSTRACT Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 107 to 109 cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.

scholarly journals The N -linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

Open Biology ◽  
2017 ◽  
Vol 7 (1) ◽  
pp. 160212 ◽  
Author(s):  
Jon Cuccui ◽  
Vanessa S. Terra ◽  
Janine T. Bossé ◽  
Andreas Naegeli ◽  
Sherif Abouelhadid ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Mass Spectrometry Analysis ◽  
Respiratory Pathogen ◽  
Transcriptional Unit ◽  
Biotechnological Application ◽  
Spectrometry Analysis ◽  
Porcine Pleuropneumonia ◽  
Repeat Units ◽  
Potential Use

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N -linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO , ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

scholarly journals Efficacy of the Aqueous Extract of Azadirachta indica Against the Marine Parasitic Leech and Its Phytochemical Profiling

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1908
Author(s):  
Balu Alagar Venmathi Maran ◽  
Dawglas Josmeh ◽  
Jen Kit Tan ◽  
Yoong Soon Yong ◽  
Muhammad Dawood Shah
Keyword(s):  
Mass Spectrometry ◽  
Aqueous Extract ◽  
Azadirachta Indica ◽  
Total Mortality ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Dependent Manner ◽  
Spectrometry Analysis ◽  
Orbitrap Mass Spectrometry ◽  
Q Exactive

Zeylanicobdella arugamensis (Hirudinea), a marine parasitic leech, not only resulted in the mortality of the host fish (Groupers) but also caused economic losses. The current study aimed to elucidate the antiparasitic efficacy of the aqueous extract of the Azadirachta indica leaves against Z. arugamensis and to profile the composition via LC-Q Exactive HF Orbitrap mass spectrometry. Different concentrations (25, 50 and 100 mg/mL) of A. indica extract were prepared and tested on the parasitic leeches. The total mortality of leeches was noticed with an exposure to the A. indica aqueous extract. The average times required for the aqueous extract at concentrations of 25, 50 and 100 mg/mL to kill the leeches were 42.65 ± 9.20, 11.69 ± 1.11 and 6.45 ± 0.45 min, respectively, in a dose-dependent manner. The Orbitrap mass spectrometry analysis indicated the presence of five flavonoids (myricetin 3-O-galactoside, trifolin, isorhamnetin, quercetin and kaempferol), four aromatics (4-methoxy benzaldehyde, scopoletin, indole-3-acrylic acid and 2,4-quinolinediol), three phenolics (p-coumaric acid, ferulic acid and phloretin) and two terpenoids (pulegone and caryophyllene oxide). Thus, our study indicates that A. indica aqueous extract is a good source of metabolites with the potential to act as a biocontrol agent against the marine parasitic leech in aquaculture.

scholarly journals Diversity of 4-Chloro-2-nitrophenol-Degrading Bacteria in a Waste Water Sample

2016 ◽  
Vol 2016 ◽  
pp. 1-5 ◽  
Author(s):  
Pankaj Kumar Arora ◽  
Alok Srivastava ◽  
Vijay Pal Singh
Keyword(s):  
Waste Water ◽  
Water Sample ◽  
Mass Spectrometry Analysis ◽  
Contaminated Site ◽  
Gas Chromatography Mass Spectrometry ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Spectrometry Analysis ◽  
Waste Water Sample ◽  
Degrading Bacteria

Eighteen bacterial strains, isolated from a waste water sample collected from a chemically contaminated site, Patancheru (17°32′N 78°16′E/17.53°N 78.27°E), India, were able to decolorize 4-chloro-2-nitrophenol (4C2NP) in the presence of an additional carbon source. These eighteen 4C2NP-decolorizing strains have been identified as members of four different genera, includingBacillus,Paenibacillus,Pseudomonas, andLeuconostocbased on the 16S rRNA gene sequencing and phylogenetic analysis. Most of the bacteria (10) belonged to the genusBacillusand contributed 56% of the total 4C2NP-degrading bacteria, whereas the members of generaPaenibacillusandPseudomonasrepresented 22% and 17%, respectively, of total 4C2NP-degrading isolates. There was only one species ofLeuconostoccapable of degrading 4C2NP. This is the first report of the diversity of 4C2NP-decolorizing bacteria in a waste water sample. Furthermore, one bacterium,Bacillus aryabhattaistrain PC-7, was able to decolorize 4C2NP up to a concentration of 2.0 mM. Gas chromatography-mass spectrometry analysis identified 5-chloro-2-methylbenzoxazole as the final product of 4C2NP decolorization in strain PC-7.

scholarly journals Binding of Actinobacillus pleuropneumoniae to Phosphatidylethanolamine

2003 ◽  
Vol 71 (8) ◽  
pp. 4657-4663 ◽  
Author(s):  
Marie-Eve Jeannotte ◽  
Maan Abul-Milh ◽  
J. Daniel Dubreuil ◽  
Mario Jacques
Keyword(s):  
Bacterial Colonization ◽  
Actinobacillus Pleuropneumoniae ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Respiratory Pathogens ◽  
Swine Industry ◽  
Molybdenum Blue ◽  
O Antigen ◽  
Serotype 1

ABSTRACT The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that A. pleuropneumoniae serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of A. pleuropneumoniae in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against A. pleuropneumoniae serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of A. pleuropneumoniae serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for A. pleuropneumoniae serotype 1. A. pleuropneumoniae was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of A. pleuropneumoniae binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.

scholarly journals Strigolactone Deficiency Confers Resistance in Tomato Line SL-ORT1 to the Parasitic Weeds Phelipanche and Orobanche spp.

2011 ◽  
Vol 101 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Evgenia Dor ◽  
Koichi Yoneyama ◽  
Smadar Wininger ◽  
Yoram Kapulnik ◽  
Kaori Yoneyama ◽  
...  
Keyword(s):  
Seed Germination ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Toxic Activity ◽  
Parasitic Weeds ◽  
Spectrometry Analysis ◽  
Wild Tomato Species ◽  
Effective Strategies ◽  
Tomato Species ◽  
Dicotyledonous Plants

The parasitic flowering plants of the genera Orobanche and Phelipanche (broomrape species) are obligatory chlorophyll-lacking root-parasitic weeds that infect dicotyledonous plants and cause heavy economic losses in a wide variety of plant species in warm-temperate and subtropical regions. One of the most effective strategies for broomrape control is crop breeding for broomrape resistance. Previous efforts to find natural broomrape-resistant tomato (Solanum lycopersicon) genotypes were unsuccessful, and no broomrape resistance was found in any wild tomato species. Recently, however, the fast-neutron-mutagenized tomato mutant SL-ORT1 was found to be highly resistant to various Phelipanche and Orobanche spp. Nevertheless, SL-ORT1 plants were parasitized by Phelipanche aegyptiaca if grown in pots together with the susceptible tomato cv. M-82. In the present study, no toxic activity or inhibition of Phelipanche seed germination could be detected in the SL-ORT1 root extracts. SL-ORT1 roots did not induce Phelipanche seed germination in pots but they were parasitized, at the same level as M-82, after application of the synthetic germination stimulant GR24 to the rhizosphere. Whereas liquid chromatography coupled to tandem mass spectrometry analysis of root exudates of M-82 revealed the presence of the strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer, these compounds were not found in the exudates of SL-ORT1. It can be concluded that SL-ORT1 resistance results from its inability to produce and secrete natural germination stimulants to the rhizosphere.

scholarly journals Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage

2004 ◽  
Vol 70 (3) ◽  
pp. 1487-1493 ◽  
Author(s):  
Jonathan D. Van Hamme ◽  
Phillip M. Fedorak ◽  
Julia M. Foght ◽  
Murray R. Gray ◽  
Heather D. Dettman
Keyword(s):  
Bond Cleavage ◽  
Organosulfur Compound ◽  
Viscosity Reduction ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Sulfur Source ◽  
Bacterial Strains ◽  
Spectrometry Analysis ◽  
Alkyl Chains ◽  
Degrading Bacteria

ABSTRACT The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C—S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C—S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound, bis-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C—S bonds. A unique bacterial strain, Rhodococcus sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C—S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including Rhodococcus sp. strain IGTS8, were all unable to cleave the C—S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C—S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.

scholarly journals Kinetic Study of the Biodegradation of Acephate by Indigenous Soil Bacterial Isolates in the Presence of Humic Acid and Metal Ions

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 433 ◽  
Author(s):  
Simranjeet Singh ◽  
Vijay Kumar ◽  
Sourav Singla ◽  
Minaxi Sharma ◽  
Dhananjaya P. Singh ◽  
...  
Keyword(s):  
Humic Acid ◽  
Metal Ions ◽  
Contaminated Soils ◽  
Mass Spectrometry Analysis ◽  
Bacterial Strains ◽  
Spectrometry Analysis ◽  
First Order ◽  
First Order Kinetics ◽  
Degradation Rates ◽  
Uv Visible

Many bacteria have the potential to use specific pesticides as a source of carbon, phosphorous, nitrogen and sulphur. Acephate degradation by microbes is considered to be a safe and effective method. The overall aim of the present study was to identify acephate biodegrading microorganisms and to investigate the degradation rates of acephate under the stress of humic acid and most common metal ions Fe(III) and copper Cu(II). Pseudomonas azotoformanss strain ACP1, Pseudomonas aeruginosa strain ACP2, and Pseudomonas putida ACP3 were isolated from acephate contaminated soils. Acephate of concentration 100 ppm was incubated with separate strain inoculums and periodic samples were drawn for UV—visible, FTIR (Fourier-transform infrared spectroscopy) and MS (Mass Spectrometry) analysis. Methamidophos, S-methyl O-hydrogen phosphorothioamidate, phosphenothioic S-acid, and phosphenamide were the major metabolites formed during the degradation of acephate. The rate of degradation was applied using pseudo-first-order kinetics to calculate the half-life (t1/2) values, which were 14.33–16.72 d−1 (strain(s) + acephate), 18.81–21.50 d−1 (strain(s) + acephate + Cu(II)), 20.06 –23.15 d−1 (strain(s) + acephate + Fe(II)), and 15.05–17.70 d−1 (strains + acephate + HA). The biodegradation efficiency of the three bacterial strains can be ordered as P. aeruginosa > P. putida > P. azotoformans. The present study illustrated the decomposition mechanism of acephate under different conditions, and the same may be applied to the removal of other xenobiotic compounds.

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