Name: high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability
Uploaded: 2014-11-21T14:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

This research was supported by the Department of Veterans Affairs Career Development Award 1IK2BX001701 and the CTSA award UL1TR000445 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. Scanning electron microscopy experiments were performed in part through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126.

1. H. pylori crescita in condizioni particolari di ferro Disponibilità e co-coltura con cellule umane epiteliali gastriche <ol><li> Seleziona H. pylori ceppo PMSS1 per questi studi perché ha un intatto patogenicità cag isola ed esprime un tipo di funzionamento del sistema di secrezione IV. Utilizzare anche una gabbia mutante isogenico (PMSSI Δ Cage) come controllo negativo. Grow batteri su piastre di TSA addizionato con 5% di sangue di pecora (piastre di agar sangue) per 24 ore a 37 ° C in presenza del 5% di CO 2. NOTA: Preparare i reagenti e assemblare materiali come indicato in materiali e attrezzature della tabella. Le concentrazioni finali per ciascun reagente è elencato tra parentesi. </li><li> Raschiare i batteri dalla piastra utilizzando un applicatore con punta di cotone sterili e inoculare in brodo di Brucella modificato preparata dai componenti (vedi Materiali e attrezzature Table) e integrato con il colesterolo. Incubare le culture per una notte a 37° C in aria ambiente integrato con il 5% di CO 2 con agitazione a 200 giri al minuto (rpm). NOTA: colesterolo viene utilizzato al posto di siero fetale bovino a causa del fatto che il siero è complesso; contenente numerose fonti di ferro nutrienti come eme che provoca variabilità nei risultati. </li><li> Il giorno della coltura batterica, seminare le cellule epiteliali gastriche umane AGS (ATCC CRL-1739) su poli-D-lisina coprioggetto trattati in piastre da 12 pozzetti (circa 1 x 10 5 cellule per pozzetto). </li><li> Il giorno seguente, diluire colture batteriche ad una OD600 di 0,3 in solo o integrato con 100 mM FeCl 3, 200 mM dipiridile (un chelante del ferro sintetico), o 200 micron dipiridile più 250 micron FeCl 3 brodo brucella modificato. Incubare le colture per 4 ore a 37 ° C in aria ambiente supplementato con 5% di CO 2 con agitazione a 200 rpm. </li><li> Batteri centrifugare a 1000 xg per raccogliere le cellule e rimuovere surnatanti prima di risospendere in un volume uguale di fresco Brucella modificato brodo integrata con colesterolo. Misura OD600 della cultura (OD600 = 1,0 = 5,5 x 10 8 cellule / ml) e aggiungere batteri alle cellule epiteliali in una molteplicità di infezione (MOI) di 20: 1. Effettuare diluizioni seriali e le cellule batteriche piastra su piastre di agar sangue per valutare la vitalità cellulare batterica. </li><li> Incubare H. cellule epiteliali gastriche umane pylori e AGS co-coltura per 4 ore a 37 ° C in presenza del 5% di CO 2 in condizioni statiche prima di eseguire la microscopia elettronica a scansione (SEM) preparazione del campione. </li></ol> 2. SEM Preparazione del campione per visualizzare H. pylori Cag-T4SS Pili <ol><li> Rimuovere H. pylori e co-colture AGS dal surnatante incubatore e decantare la cultura (salvo per IL-8 ELISA). Lavare delicatamente tre volte con sodio 0,05 M tampone cacodilato (pH 7,4). Fix campioni per 2-4 ore a temperatura ambiente in una soluzione di 2,0% paraformaldeide, 2.5% Glutaraldeide, e tampone cacodilato di sodio 0,05 M. Dopo fissazione primaria, lavare campioni tre volte con tampone cacodilato di sodio 0,05 M. </li><li> Eseguire ancoraggio secondario utilizzando 0,1% tetrossido di osmio in 0,05 M di sodio tampone cacodilato in due fasi sequenziali 10 min di fissaggio. Dopo la fissazione secondaria, lavare campioni tre volte con tampone cacodilato di sodio 0,05 M. </li><li> Dopo fissazione primaria e secondaria, disidratano campioni di lavaggio sequenziale con concentrazioni crescenti di etanolo come da Tabella 1. </li><li> Dopo etanolo disidratazione, effettuare tre lavaggi sequenziali di anidride carbonica liquida. Poi i campioni a secco in un punto critico con una macchina asciugatrice punto critico ad una temperatura di 31,1 ° C e pressione di 1073 PSI. </li><li> Monte coprioggetto sul stub campione alluminio SEM e dipingere con una linea sottile di argento colloidale sul bordo del campione per ottenere adeguata messa a terra ed evitare di ricarica durante l&#39;imaging SEM. </li><li> Campioni cappotto con 5 nm dioro-palladio utilizzando una macchina cappotto polverizzazione di aumentare campione di segnale degli elettroni secondari e l&#39;effetto bordo. </li></ol> 3. I parametri di imaging per analisi ad alta risoluzione SEM <ol><li> Vedi i campioni con un campo-Emission Gun microscopio elettronico a scansione (SEM-FEG). </li><li> Immagine in una modalità di aspirazione ad alta ad una distanza di lavoro di 5-10 mm. </li><li> Impostare la tensione di accelerazione di 5 kV. </li><li> Impostare dimensione del punto di 2-2,5. </li><li> Inclinare il campione tra i 15-25 ° per ottenere una migliore visualizzazione dell&#39;interfaccia ospite-patogeno. </li><li> Priorità di imaging batteri aderenti ai bordi delle cellule epiteliali per la visualizzazione ad alto ingrandimento, in quanto queste aree di interazione ospite-patogeno sono arricchiti per i batteri-Pili formando. </li></ol> 4. Analisi statistica per valutare Pili Quantificazione <ol><li> Analizzare H. pylori Cag-T4SS pili utilizzando il software ImageJ per quantificare il numero di pili / cellule e per cento delle cellule esibendo il piliated phenotype secondo le istruzioni riportate di seguito. </li><li> Aprire i file microfotografia nel software ImageJ. Identificare pili come strutture formate tra la cellula batterica e cellula ospite, con larghezza uniforme (10-13 nm) e la lunghezza (60-150 nm). NOTA: La struttura pilus è presente su ceppi batterici WT, ma assente in ceppi isogeni quali Δ Cage ceppi mutanti, che ospitano l&#39;inattivazione del gene che codifica per il principale ATPasi che l&#39;assemblaggio tipo poteri secrezione IV pilus. </li><li> Calibrare lo strumento di misurazione tracciando una linea con lo strumento linea retta e quindi facendo clic sulla scheda &quot;Analizza&quot;. Selezionare &quot;Imposta Scala&quot; dal menu a discesa e immettere il valore bar di ingrandimento nella casella &quot;Noto Distanza&quot; e impostare l&#39;unità di misura per l&#39;impostazione appropriata (nm). Fare clic su &quot;OK&quot;. </li><li> Misurare Cag-T4SS pili utilizzando lo strumento linea retta per disegnare su tutta la lunghezza o la larghezza del pilus e poi premere &quot;Ctrl-M&quot; per misurare ogni pilus. Osservare una finestra di dialogo con i valori misurati. Registrare questi valori o copiare e incollare in un programma di foglio di calcolo. </li><li> Utilizzare la lunghezza e la larghezza dei parametri stabiliti in precedenza 24, caratteristiche non tengono conto che non si adattano al profilo di Cag-T4SS. Poi quantificare il numero di pili in base ai valori che rientrano nelle cut-off 10-13 nm di larghezza e 60-150 nm di lunghezza. </li><li> Analizzare la quantificazione dei pili statisticamente con il test t di Student a due code utilizzando il software GraphPad Prism. </li></ol> 5. Opzionale: Valutazione della secrezione di IL-8 in correlazione con Pilus produzione <ol><li> Utilizzando surnatanti dal punto 2.1, eseguire un IL-8 ELISA secondo le istruzioni del produttore con il kit (vedi Materiali e attrezzature Tabella). </li><li> Analizzare l&#39;IL-8 secreto dalle cellule ospiti e calcolare la percentuale di IL-8 induzione rispetto al WT PMSS1 coltivate in terreno da solo. Eseguire l&#39;analisi statistica con due code il test t di Student con GraphPad Prism cosìftware. </li></ol>

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The authors declare that they have no competing financial interests.

Il ferro è un micronutriente essenziale per la maggior parte delle forme di vita, compresi i batteri patogeni. Nel tentativo di limitare la validità di microrganismi invasori, ospiti vertebrati sequestrano il ferro di nutrienti in un processo noto come &quot;immunità nutrizionale&quot; 18. In risposta a questo, batteri patogeni sono evoluti per utilizzare il ferro come molecola di segnalazione globale di percepire l&#39;ambiente circostante e regolano l&#39;elaborazione delle caratteristiche di virulenza c...

H. pylori infection is a significant risk factor for gastric cancer1. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet11. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression12. Acute H. pylori infection is also associated with hypochlorhydria which impairs the host&#8217;s ability to absorb nutrient iron, and ultimately leads to changes in iron homeostasis13. These clinical findings suggest that iron availability within the gastic niche could be an important factor in disease outcome. In fact, animal models of H. pylori infection have demonstrated that low dietary iron consumption exacerbates gastric disease14. The reduced iron levels in these animals necessitate that H. pylori induce an iron-acquisition response in order to obtain the iron needed for bacterial replication. H. pylori has the capacity to perturb iron trafficking within host cells to facilitate bacterial replication in a CagA-dependent fashion15. Interestingly, the cag-pathogenicity island has been shown to be regulated by the iron-responsive transcription factor Fur16,17. Furthermore, Cag+ strains are associated with increased inflammation and gastric diseases such as cancer1. These findings support a model whereby H. pylori alters Cag-T4SS expression in an effort to obtain iron from host cells that reside in an iron deplete environment resulting in exacerbated disease outcomes. 
Two factors that increase inflammation and morbidity are Cag expression and low dietary iron intake. These facts support the hypothesis that reduced iron availability increases the production of Cag-T4SS pili at the host pathogen interface resulting in worse gastric disease11-14. The goal of the method provided in this manuscript is to establish the role of the micronutrient iron in the regulation of the Cag-T4SS pilus biogenesis. In previous work, we utilized two approaches to observe an iron-dependent increase in Cag-T4SS expression. First, output strains from animals maintained on high and low iron diets were analyzed and revealed that low-iron diet output strains produced more Cag-T4SS pili than high-iron diet strains14. Second, growing the H. pylori 7.13 strain in vitro in iron replete conditions resulted in reduced pili formation while cells grown in the presence of an iron chelator produced significantly more pili. 
We have continued to investigate the iron-dependent regulation of Cag-T4SS pili phenotype and offer the following optimized protocol and representative results performed with an additional Helicobacter pylori strain, PMSS1. The rationale behind the development of this technique was to correlate increased Cag-T4SS activity in conditions of iron-limitation with increased Cag-T4SS pilus formation. The broader implication and use of this technique will provide optimized culture conditions that result in elevated production of the Cag-T4SS pili. This assay will be useful to researchers seeking to determine the composition and architecture of the Cag-T4SS by enriching for this important bacterial surface feature. The sample preparation and visualization by field-emission gun electron microscopy has numerous advantages over alternative techniques such as light-microscopy methods to visualize the Cag-T4SS and will be appropriate to investigators interested in studying the regulation of this organelle10.

<table><tbody><tr><td>Modified brucella broth</td><td></td><td></td><td></td></tr><tr><td>Peptone from casein (10 g/L)</td><td>Sigma</td><td>70172</td><td></td></tr><tr><td>Peptic digest of animal tissue (10 g/L)</td><td>Sigma</td><td>70174</td><td></td></tr><tr><td>Yeast extract (2 g/L)</td><td>Sigma</td><td>92144</td><td></td></tr><tr><td>Dextrose (1 g/L)</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Sodium chloride (5 g/L)</td><td>Thermo Fisher</td><td>S271-10</td><td></td></tr><tr><td>Cholesterol (250X) (4 ml/L)</td><td>Life Technologies</td><td>12531018</td><td></td></tr><tr><td>Ferric chloride (100 or 250 uM)</td><td>Sigma</td><td>157740-100G</td><td></td></tr><tr><td>Dipyridyl (200 uM)</td><td>Sigma</td><td>D216305-100G</td><td></td></tr><tr><td>Modified RPMI</td><td></td><td></td><td></td></tr><tr><td>RPMI+HEPES (1X)</td><td>Life Technologies</td><td>22400-121</td><td></td></tr><tr><td>Fetal bovine serum (100 ml/L)</td><td>Life Technologies</td><td>10438-026</td><td></td></tr><tr><td>Electron Microscopy Preparation</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (2.0% aqueous)</td><td>Electron Microscopy Sciences</td><td>15713</td><td></td></tr><tr><td>Gluteraldehyde (2.5% aqueous)</td><td>Electron Microscopy Sciences</td><td>16220</td><td></td></tr><tr><td>Sodium cacodylate (0.05 M)</td><td>Electron Microscopy Sciences</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide (0.1% aqueous)</td><td>Electron Microscopy Sciences</td><td>19150</td><td></td></tr><tr><td>Ethanol (absolute)</td><td>Sigma</td><td>E7023</td><td></td></tr><tr><td>Colloidal silver paint</td><td>Electron Microscopy Sciences</td><td>12630</td><td></td></tr><tr><td>SEM sample stubs</td><td>Electron Microscopy Sciences</td><td>75220</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>08-774-383</td><td></td></tr><tr><td>IL-8 Secretion Evaluation</td><td></td><td></td><td></td></tr><tr><td>Quantikine IL-8 ELISA kit</td><td>R&amp;amp;D Systems</td><td>D8000C</td><td></td></tr></tbody></table>

high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

In questa relazione, abbiamo dimostrato che le condizioni di diversa disponibilità di ferro hanno la capacità di modulare H. pylori Cag-T4SS pilus biogenesi all&#39;interfaccia patogeno host. Quando coltivate in terreno da solo, H. pylori fa una media di 3 pili / cell. Quando H. pylori è coltivato in ferro esaurire condizioni (utilizzando il chelante dipiridile sintetico) che sono sub-inibitorio per la crescita batterica (Figura 1), i batteri producono numerosi Cag-T4SS pil...

Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Ad alta risoluzione microscopia elettronica del<em&gt; Helicobacter pylori</em&gt; Cag secrezione di tipo IV Sistema Pili Prodotto in diverse condizioni di Ferro Disponibilità

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

high-resolution-electron-microscopy-helicobacter-pylori-cag-type-iv

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

15.0K Views.  Vanderbilt University School of Medicine. Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Video: High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

Analisi di particelle singole ad alta risoluzione da immagini elettroniche a cryo-microscopia usando SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Ricerca

Biochimica

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Ingegneria batteri aderenti mediante la creazione di un unico sintetico curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioingegneria

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus crescita con emoglobina umana come fonte di ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Immunologia e infezioni

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

L'assorbimento del metallo essenziale nei batteri gram-negativi: fluorescenza, radioisotopi, a raggi x e delle cellule di frazionamento

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Chimica

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis come organismo modello per la Valutazione Interazione di batteri anaerobici con cellule ospiti

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

Visualizzazione di Spasmi motilità e caratterizzazione del ruolo del<em&gt; Pell</em&gt; in<em&gt; Fastidiosa Xylella</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applicazione di Live Cell Imaging e tomografia Cryo-Electron a risolvere le caratteristiche spatiotemporali del sistema di secrezione Legionella pneumophila Dot/Icm

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Metal-Limited Crescita di Neisseria gonorrhoeae per la caratterizzazione dei geni metal-reattivi e l'acquisizione di metalli da Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

T3SS Activity in Yersinia pseudotuberculosis Under Iron Depletion & Anaerobic Stress

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 &#176;C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Imaging a super-risoluzione di proteine secrete da batteri utilizzando l'espansione del codice genetico

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

Ad alta risoluzione microscopia elettronica del

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