Questo lavoro è stato supportato da sovvenzioni da parte del National Institutes of Health e dalla Fondazione Fibrosi Cistica.

1. Preparazione di batteri per l&#39;estrazione LPS <ol><li> Avviare una cultura pernottamento in 5 ml di Luria Broth (LB), integrato con antibiotici se necessario. Grow coltura durante la notte (12-18 ore) in un incubatore agitazione a 37 ° C e 200 rpm. </li><li> Diluire il 01:10 cultura con LB e prendere un 600 lettura OD in uno spettrofotometro. Sulla base della lettura 600 OD, una sospensione 1,5 mL dei batteri ad una OD 600 di 0,5. </li><li> Pellet i batteri in una microcentrifuga a 10.600 xg per 10 minuti. Rimuovere e scartare il surnatante. Il pellet può essere conservato a -20 ° C, se LPS non sta per essere estratto immediatamente. </li></ol> 2. Estrazione di LPS <ol><li> In primo luogo, preparare 2x tampone SDS. Effettuare una soluzione di 50 mL del 4% β-mercaptoetanolo (BME), 4% SDS e 20% glicerolo in 0,1 M Tris-HCl, pH 6,8. Aggiungere un pizzico di blu di bromofenolo per tingere la soluzione. Fai una SDS-buffer 1x magazzino diluendo 2X stock 1:1 in sterile distillata H 2 O. Questo può essere conservato a temperatura ambiente. </li><li> Fare tre distinti a 10 mg / mL soluzioni di DNasi, RNasi e proteinasi K in sterile distillata H 2 O. </li><li> Risospendere i batteri pellet a partire dal punto 1,3 in 200 microlitri di 1x SDS-buffer. Assicurarsi che il pellet è completamente risospeso pipettando la soluzione su e giù lentamente. Non vortice. </li><li> Lessare i batteri in sospensione in un bagnomaria per 15 minuti. Lasciare raffreddare la soluzione a temperatura ambiente per 15 minuti. </li><li> Aggiungere 5 microlitri di entrambi I DNasi RNasi e soluzioni preparate in 2.2. Incubare i campioni a 37 ° C per 30 minuti. * Questo passaggio è facoltativo, c&#39;è minima differenza nella qualità di LPS tra campioni nucleasi trattati e non trattati. </li><li> Aggiungere 10 pl di soluzione di proteinasi K preparata in 2,2. Incubare i campioni a 59 ° C per 3 ore. Questa fase può essere eseguita durante la notte, se ci sono vincoli di tempo. </li><li> Per ogni annuncio campione,d 200 pl di ghiacciato Tris-fenolo saturo (acqua-fenolo saturo può essere utilizzato come sostituto se Tris-fenolo saturo non è disponibile). Assicurarsi che i tappi sui tubi sono chiuse ermeticamente, e vortex ogni campione per circa 5 a 10 secondi. </li><li> Incubare i campioni a 65 ° C per 15 minuti, vortex occasionalmente. Dopo incubazione raffreddare a temperatura ambiente, poi aggiungere 1 ml di temperatura ambiente etere dietilico per ciascun campione e vortex per 5 a 10 secondi. Assicurarsi di eseguire etere manico in una cappa aspirante, in quanto è volatile. </li><li> Centrifugare i campioni a 20600 x g per 10 minuti. Rimuovere con cautela i campioni dalla centrifuga ed estrarre lo strato di fondo blu. Essere sicuri di evitare la parte superiore, strato trasparente. Lasciando una piccola quantità dello strato blu è preferibile contaminazione con lo strato superiore. </li><li> Ri-estrarre i campioni ripetendo i punti da 2,7-2,9. Due estrazioni sono di solito sufficienti. Se i campioni appaiono nuvoloso, più estrazioni può essere Effettuared. Aggiungere 200 pl di 2x SDS-buffer per ciascuno dei campioni estratti prima separazione mediante SDS-PAGE. I campioni possono essere eseguiti su 8% -15% SDS-poliacrilammide gel. Cinque a quindici microlitri di LPS preparate utilizzando questo metodo è di solito sufficiente per la visualizzazione. </li></ol> 3. Risultati rappresentativi LPS campioni preparati come sopra può essere visualizzato mediante colorazione diretta dopo la separazione su SDS-PAGE usando uno standard di argento o di un protocollo di colorazione disponibile in commercio kit di colorazione LPS. In alternativa, LPS separati su un gel di poliacrilammide può essere trasferita ad una membrana di nitrocellulosa e sottoposto a Western immunoblotting usando LPS-specifici anti-sieri. Per questo protocollo, abbiamo usato il Pro-Q Emerald 300 Kit Gel Stain Lipopolisaccaride (Molecular Probes), e seguito le istruzioni del produttore. In figura 1 è una Pro-Q Emerald 300 tinto 12% SDS-gel di poliacrilammide. Ciascuna corsia contiene 15 pl di LPS preparati da DIFdifferenti ceppi di Burkholderia dolosa isolati da campioni di espettorato di pazienti affetti da fibrosi cistica. Differenti modelli di LPS banding scala, riflettenti di numeri diversi O-antigene unità ripetitive attaccato al centro oligosaccaride, sono evidenti con questo metodo, ad esempio, campioni in corsia 1 e 6 sono simili schemi bande tra loro (scatolato in rosso). <img alt="Figura 1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Figura 1. Pro-Q Emerald 300 LPS macchiate da Burkholderia dolosa isolati clinici. LPS di sette B. ceppi dolosa di un focolaio in pazienti affetti da fibrosi cistica sono stati isolati come descritto in questo protocollo. Quindici pl sono stati separati su un 12% SDS-gel di poliacrilammide, e colorate con Pro-Q Emerald 300, secondo le istruzioni del produttore. LPS nucleo è indicata una freccia, e LPS che mostrano modelli simili bande di O-antigene ripetizioni sono boxed in rosso. M = marcatore di peso molecolare.

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

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Abbiamo descritto un metodo di purificazione LPS lontano dagli altri componenti cellulari, tra cui gli acidi nucleici e proteine. Questo metodo consente di alta qualità LPS che possono essere utilizzati in un certo numero di metodi di visualizzazione differenti, compresa la colorazione carboidrati di SDS-PAGE gel, come mostrato nella Figura 1. Questo metodo può essere utilizzato per sierotipo LPS da una varietà di ceppi, utilizzando specifici anti-sieri, o per mostrare connessione tra isolati tramite visualizzazione ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> DNasi ricombinante, RNasi-free </td><td> Roche </td><td> 04716728001 </td></tr><tr><td> RNasi A </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> Proteinasi K </td><td> Pescatore </td><td> BP1700 </td></tr><tr><td> Tris-saturi Fenolo </td><td> Pescatore </td><td> BP1750-100 </td></tr><tr><td> Etere etilico </td><td> Thomas Scientific </td><td> C313K31 </td></tr><tr><td> Pro-Q Emerald 300 Gel Kit Lipopolisaccaride Stain </td><td> Molecular Probes </td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

Lipopolisaccaride (LPS) è un componente principale delle membrane batteriche Gram-negativi esterni. Si tratta di una molecola composta dal lipide A, che è incorporato nella membrana esterna, un oligosaccaride e ripetibile nucleo O-antigene unità che si estendono verso l&#39;esterno dalla superficie della cella 1, 2. LPS è una molecola immunodominante che è importante per la virulenza e patogenesi di molte specie batteriche, tra Pseudomonas aeruginosa, Salmonella spp, Escherichia coli e 3-5, e le differenze di LPS O-antigene composizione forma la base per la sierotipizzazione di ceppi. LPS è coinvolto in attaccamento alle cellule ospiti all&#39;inizio della infezione e fornisce protezione da complemento-mediata uccisione; ceppi privi LPS può essere attenuato per virulenza 6-8. Per queste ragioni, è importante visualizzare LPS, in particolare da isolati clinici. LPS visualizzando bande modelli e il riconoscimento da uno specificontibodies possono essere strumenti utili per identificare i lignaggi di deformazione e caratterizzare i vari mutanti. In questa relazione, si descrive un hot-fenolo acquoso metodo per l&#39;isolamento e la purificazione di LPS da cellule batteriche gram-negativi. Questo protocollo consente l&#39;estrazione di LPS lontano da acidi nucleici e proteine ​​che possono interferire con visualizzazione di LPS che si verifica con brevi e metodi di estrazione meno intensi 9. LPS preparato in questo modo possono essere separati da sodio dodecil solfato (SDS)-poliacrilammide gel elettroforesi (PAGE) e direttamente colorati usando carboidrati / glicoproteina macchie o normali metodi di colorazione d&#39;argento. Molti anti-sieri di LPS contenere anticorpi che cross-reagiscono con le proteine ​​della membrana esterna o altri bersagli antigenici che possono ostacolare la reattività osservati dopo immunoblot occidentale SDS-PAGE separate da lisati cellulari grezzi. Proteasi trattamento di lisati cellulari grezzi da sola non è sempre un modo efficace per rimuovere questosfondo di utilizzare questo o altri metodi di visualizzazione. Inoltre, il trattamento della proteasi esteso nel tentativo di eliminare questo sfondo può portare a LPS scarsa qualità che non è ben risolto da uno qualsiasi dei metodi summenzionati. Per queste ragioni, riteniamo che il seguente protocollo, adattato da Westpahl Jann e 10, è l&#39;ideale per l&#39;estrazione LPS.

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Descriviamo una modificato caldo acquosa-fenolo metodo di estrazione per purificare lipopolisaccaride (LPS) da batteri Gram-negativi. Una volta estratto, le LPS può essere successivamente analizzati mediante SDS-PAGE e visualizzati tramite colorazione diretta o immunoblot occidentale.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

purification-visualization-lipopolysaccharide-from-gram-negative

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

35.0K Views.  University of Virginia Health System. Descriviamo una modificato caldo acquosa-fenolo metodo di estrazione per purificare lipopolisaccaride (LPS) da batteri Gram-negativi. Una volta estratto, le LPS può essere successivamente analizzati mediante SDS-PAGE e visualizzati tramite colorazione diretta o immunoblot occidentale.

Video: Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Isolamento e Caratterizzazione chimica del lipide A da batteri Gram-negativi

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Ricerca

Chimica

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

Isolamento e preparazione delle pareti cellulari batteriche per l'analisi compositia mediante cromatografia liquida ad alte prestazioni

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Estrazione e l&#39;analisi delle microbica fosfolipidi acidi grassi nei terreni

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Ambiente

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Au-Interazione di SLP1 Polimeri e monostrato da<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, ICP-MS e AFM come strumenti di studi Biomolecule metallo

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Isolamento delle particelle di lipoproteina dal tuorlo di uovo di pollo per lo studio dell'incorporazione di acido acido grasso patogeno batterico in fosfolipidi di membrana

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Biochimica

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Analisi semi-quantitativa del peptidoglicano mediante cromatografia liquida spettrometria di massa e bioinformatica

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Estrazione, purificazione e profilatura del ganglioside

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Arricchimento delle lipoproteine batteriche e preparazione del N-terminale lipopeptidi per determinazione strutturale mediante spettrometria di massa

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Immunologia e infezioni

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

Separazione dell'involucro cellulare per i batteri Gram-negativi in frazioni di membrana interne ed esterne con regolazioni tecniche per Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Analisi della composizione lipidica dei micobatteri mediante cromatografia a strato sottile

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

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