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&#160;Growth in Various Conditions of Iron Availability and Co-culture with Human Gastric Epithelial Cells
<ol>
	<li>Select H. pylori strain PMSS1 for these studies because it has an intact cag pathogenicity island and expresses a functioning type IV secretion system. Also utilize an isogenic cagE mutant (PMSSI &#916;cagE) as a negative control. Grow bacteria on TSA plates supplemented with 5% sheep blood (blood agar plates) for 24 hr&#160;at 37 &#176;C in the presence of 5% CO2. 
	NOTE: Prepare reagents and assemble materials as outlined in the Materials and Equipment Table. Final concentrations for each reagent is listed in parentheses.</li>
	<li>Scrape bacteria from the plate using a sterile cotton-tipped applicator and inoculate into modified brucella broth prepared from components (see Materials and Equipment Table) and supplemented with cholesterol. Incubate cultures overnight at 37 &#176;C in room air supplemented with 5% CO2 with shaking at 200 rotations per minute (rpm). 
	NOTE: Cholesterol is used in place of fetal bovine serum due to the fact that serum is complex; containing numerous sources of nutrient iron such as heme which causes variability in results.</li>
	<li>On the day of bacterial culturing, seed AGS human gastric epithelial cells (ATCC CRL-1739) onto poly-D-lysine treated coverslips in 12-well plates (about 1 x 105 cells per well).</li>
	<li>The following day, dilute bacterial cultures to an OD600 of 0.3 in modified brucella broth alone or supplemented with 100 &#181;M FeCl3, 200 &#181;M dipyridyl (a synthetic iron chelator), or 200 &#181;M dipyridyl plus 250 &#181;M FeCl3. Incubate these cultures for 4 hours at 37 &#176;C in room air supplemented with 5% CO2 with shaking at 200 rpm.</li>
	<li>Centrifuge bacteria at 1,000 x g to collect cells and remove supernatants before resuspending in an equal volume of fresh modified brucella broth supplemented with cholesterol. Measure OD600 of the culture (OD600 = 1.0 = 5.5 x 108 cells/ml) and add bacteria to the epithelial cells in a multiplicity of infection (MOI) of 20:1. Perform serial dilutions and plate bacterial cells onto blood agar plates to assess bacterial cell viability.</li>
	<li>Incubate H. pylori and AGS human gastric epithelial cells co-cultures for 4 hr&#160;at 37 &#176;C in the presence of 5% CO2 in static conditions before performing scanning electron microscopy (SEM) sample preparation.</li>
</ol>
2. SEM Sample Preparation to Visualize H. pylori Cag-T4SS Pili
<ol>
	<li>Remove H. pylori and AGS co-cultures from the incubator and decant culture supernatant (save for IL-8 ELISA). Wash gently three times with 0.05 M sodium cacodylate buffer (pH 7.4). Fix samples for 2-4 hours at room temperature in a solution of 2.0% paraformaldehyde, 2.5% gluteraldehyde, and 0.05 M sodium cacodylate buffer. After primary fixation, wash samples three times with 0.05 M sodium cacodylate buffer.</li>
	<li>Perform secondary fixation using 0.1% osmium tetroxide in 0.05 M sodium cacodylate buffer in two sequential 10 min&#160;fixation steps. After secondary fixation, wash samples three times with 0.05 M sodium cacodylate buffer.</li>
	<li>After primary and secondary fixation, dehydrate samples by sequential washing with increasing concentrations of ethanol as per Table 1.</li>
	<li>After ethanol dehydration, perform three sequential washes of liquid carbon dioxide. Then dry samples at a critical point using a critical point dryer machine at a temperature of 31.1 &#186;C and pressure of 1073 PSI.</li>
	<li>Mount coverslips onto aluminum SEM sample stubs and paint with a thin line of colloidal silver at the sample edge to achieve appropriate grounding and avoid charging during SEM imaging.</li>
	<li>Coat samples with 5 nm of gold-palladium using a sputter coat machine to increase sample secondary electron signal and edge effect.</li>
</ol>
3. Imaging Parameters for High Resolution SEM Analyses
<ol>
	<li>View samples with a Field-Emission Gun Scanning Electron Microscope (FEG-SEM).</li>
	<li>Image in a high vacuum mode at a working distance of 5-10 mm.</li>
	<li>Set the accelerating voltage to 5 kV.</li>
	<li>Set spot size to 2-2.5.</li>
	<li>Tilt the sample between 15-25 degrees to achieve a better view of the host-pathogen interface.</li>
	<li>Prioritize imaging bacteria adherent to the edges of the epithelial cells for high-magnification viewing, as these areas of host-pathogen interaction are enriched for pili-forming bacteria.</li>
</ol>
4. Statistical Analyses to Evaluate Pili Quantifications
<ol>
	<li>Analyze H. pylori Cag-T4SS pili using ImageJ software to quantify number of pili/cell as well as percent of cells exhibiting the piliated phenotype per instructions below.</li>
	<li>Open micrograph files in ImageJ software. Identify pili as structures formed between the bacterial cell and host cell, with uniform width (10-13 nm) and length (60-150 nm). 
	NOTE: The pilus structure is present on WT bacterial strains, but absent on isogenic strains such as &#916;cagE mutant strains, which harbor an inactivation in the gene encoding the major ATPase that powers type IV secretion pilus assembly.</li>
	<li>Calibrate the measurement tool by drawing a line with the straight line tool and then clicking on the &#8220;Analyze&#8221; tab. Select &#8220;Set Scale&#8221; from the dropdown menu and enter the magnification bar value into the box labeled &#8220;Known Distance&#8221; and set the unit of length to the appropriate setting (nm). Click &#8220;OK&#8221;.</li>
	<li>Measure Cag-T4SS pili using the straight line tool to draw over the length or width of the pilus and then press &#8220;Ctrl-M&#8221; to measure each pilus. Observe a dialogue box with the measured values. Record these values or copy and paste into a spreadsheet program.</li>
	<li>Use the length and width parameters previously established24, disregard features that do not fit the profile of Cag-T4SS. Then quantify the number of pili based on values that are within the 10-13 nm width and 60-150 nm length cutoffs.</li>
	<li>Analyze the quantification of pili statistically by two-tailed Student&#8217;s t test using GraphPad Prism software.</li>
</ol>
5. Optional: Evaluation of IL-8 Secretion to Correlate with Pilus Production
<ol>
	<li>Using supernatants from Step 2.1, perform an IL-8 ELISA per manufacturer&#8217;s instructions with the kit (See Materials and Equipment Table).</li>
	<li>Analyze the IL-8 secreted by host cells and calculate the percent IL-8 induction with respect to WT PMSS1 grown in medium alone. Perform statistical analysis using two-tailed Student&#8217;s t test using GraphPad Prism software.</li>
</ol>

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The authors declare that they have no competing financial interests.

Iron is an essential micronutrient for most forms of life, including bacterial pathogens. In an effort to restrict the viability of invading microorganisms, vertebrate hosts sequester nutrient iron in a process known as &#8220;nutritional immunity&#8221;18. In response to this, bacterial pathogens have evolved to use iron as a global signaling molecule to sense their surroundings and regulate the elaboration of virulence features such as iron acquisition systems, toxins, and toxin secretion machinery19,20.
H. pylori requires micronutrients such as iron, zinc, and magnesium to grow22 and can use alternate iron sources such as heme, hemoglobin, transferrin, and lactoferrin23. H. pylori, like most successful pathogens, has evolved a wide repertoire of virulence factors to help it scavenge nutrient iron from the host21. One virulence factor, CagA, the effector molecule of the Cag-T4SS, is implicated in rerouting transferrin from the basolateral side of polarized cells to the apical surface where it can be readily used by the bacterial cells as a nutrient iron source and promote replication15. Thus, it seems an intuitive evolutionary strategy to promote the secretion of CagA in conditions of low iron availability.
The H. pylori Cag-T4SS is an important bacterial organelle, due to the proinflammatory host cell responses it elicits. The effects of the Cag-T4SS have been well studied in both co-cultures and animal models of infection. However, studies of the macromolecular structure and composition of this toxin secretion system have been hindered by the lack of high-resolution imaging protocols. Our previously published reports studying the Cag-T4SS have reported an average of four Cag-T4SS pili per bacterial cell by high-resolution electron microscopy analyses24. Our improved method provides a robust imaging technique to visualize the Cag-T4SS by using a culture technique that promotes the production of this feature on the surface of the bacterial cell. Utilization of culture techniques in conjunction with high-resolution field-emission gun scanning electron microscope is critical for this imaging protocol. Two-fold enrichment of the Cag-T4SS surface feature is a significant increase for investigators interested in performing immunohistochemical analyses such as immunofluorescence or immunoprecipitation techniques to further characterize the subcellular location or composition of the Cag-T4SS. Once the high-resolution electron microscopy imaging of the Cag-T4SS has been mastered, it is also possible to develop other applications, such as immunogold-labeling, to determine the location of bacterial proteins with respect to the Cag-T4SS pilus25.
One limitation of our technique is the fact that the investigator is still restricted to using a co-culture system for visualization of the Cag-T4SS. In a single field, only a subset of H. pylori under normal culture conditions form these structures (about 50-70 percent, see Figure 3 panel B). So numerous cells in a field will be non-piliated. It is unknown why these subpopulations exist in the same location, but these cells are always accounted within both the quantifications of pili/cell and percent of cells with pili on the surface. Because the Cag-T4SS is only observed at the host-bacterial interface, the primary and secondary fixation steps within the SEM sample preparation are critical for the preservation of both proteinaceous structures and the membranes of the eukaryotic and prokaryotic cells. However, over-fixation can occur if fixation steps are carried out longer than outlined, or if the solutions are not freshly prepared. If over-fixation occurs, samples will have a stony appearance when viewed under SEM and the Cag-T4SS will be masked by this. To troubleshoot, repeat the SEM sample preparation using less concentrated fixatives and/or less time incubated in the presence of the fixative.
The co-culture system also renders biochemical characterization (such as mass spectrometry analyses) cumbersome due to the contamination of large quantities of host proteins. However, understanding the molecular mechanisms that regulate the production of this bacterial secretion system could ultimately lead to new techniques that will trigger Cag-T4SS pilus formation in the absence of host cells. This would greatly improve the isolation and purification techniques for biochemical assays.
In conclusion, we report that conditions of restricted iron availability result in increased production of H. pylori Cag-T4SS that can be visualized by high resolution scanning electron microscopy techniques. We also report the iron-dependent repression of this surface feature at the host-pathogen interface. This assay may be optimized and utilized for numerous bacterial pathogens that form iron-regulated toxin-secretions and is broadly applicable to a variety of host-microbe interactions.

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 this report, we have demonstrated that conditions of varying iron availability have the capacity to modulate H. pylori Cag-T4SS pilus biogenesis at the host pathogen interface. When cultured in medium alone, H. pylori forms an average of 3 pili/cell. When H. pylori is grown in iron deplete conditions (using the synthetic chelator dipyridyl) that are sub-inhibitory to bacterial growth (Figure 1), the bacteria produce numerous Cag-T4SS pili (~7 pili/cell) when co-cultured with human gastric cells (Figure 2). Conversely, when an exogenous source of nutrient iron is present, the formation of Cag-T4SS pili is repressed (less than 1 pilus/cell in FE sample, less than 1 pilus/cell in FE+DIP sample) (Figure 2). Quantification of the pili at the host-pathogen interface reveals that in conditions of iron restriction, H. pylori exhibits an 2-fold increase in Cag-T4SS pili (p&lt;0.0001) and the percent piliated cells increases by 11% (p&lt;0.05) compared to cells grown in medium alone (Figure 3). Interestingly, the activity of the Cag-T4SS, as measured by host IL-8 secretion, is enhanced 107% under conditions of iron restriction and repressed 49% under conditions of excess iron availability compared to medium alone (p=0.03, and p&lt;0.001, respectively) a result that supports the SEM data. However, pili dimensions remain consistent among all conditions of iron availability (Figure 4). Furthermore, iron availability does not change the biogenesis of pili on the surface of a cagE mutant, nor does it change this isogenic derivative’s ability to induce an IL-8 response from host cells, supporting the hypothesis that the structures demonstrated are, in fact, Cag-T4SS and not an additional unrelated surface feature.
<img alt="Bacterial growth comparison under varied culture conditions; bar chart; Cfu/mL; experimental results." fo:content-width="5in" src="/files/ftp_upload/52122/52122fig1highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Bacterial viability determined in various conditions of iron availability. H. pylori was cultured in modified brucella broth alone (Medium Alone), or supplemented with 100 µM FeCl3 (FE), 200 µM dipyridyl (DIP), or 200 µM dipyridyl plus 250 µM FeCl3 (FE+DIP). Bacteria were diluted serially and plated onto bacteriological medium prior to incubation for 2 days at 37 °C in the presence of 5% CO2. Colonies were counted and colony forming units/mL (Cfu/ml) were calculated. Bars indicate mean of three independent experiments +/- SEM. Treatment with DIP, FE, or FE+DIP does not significantly alter bacterial viability.
<img alt="Scanning electron micrographs of bacteria morphology; diagrams A-D show structural variations, scale bars." fo:content-width="5in" src="/files/ftp_upload/52122/52122fig2highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: High resolution scanning electron microscopy analyses of H. pylori Cag-T4SS pili. Bacteria were grown in A) medium alone, B) medium supplemented with 100 µM FeCl3, C) medium supplemented with 200 µM dipyridyl, or D) medium supplemented with 200 µM dipyridyl plus 250 µM FeCl3 prior to co-culture with AGS human gastric cells. Arrows indicate Cag-T4SS pili formed at the host-pathogen interface. Samples were analyzed by high-resolution scanning electron microscopy to evaluate Cag-T4SS biogenesis. Conditions of low iron availability increase the prevalence of Cag-T4SS pilus formation, while conditions of excess available nutrient iron repress Cag-T4SS pilus formation.
<img alt="Bacterial pilus quantification; charts compare pilus number and cell percentage under conditions." fo:content-width="5in" src="/files/ftp_upload/52122/52122fig3highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Quantification of pili/cell and percent piliated cells cultured in various conditions of iron availability. Bacteria were grown in modified brucella broth alone (Medium Alone), or medium supplemented with 100 µM FeCl3 (FE), 200 µM dipyridyl (DIP), or 200 µM dipyridyl plus 250 µM FeCl3 (FE+DIP) prior to co-culture with human gastric epithelial cells. Samples were analyzed by high-resolution scanning electron microscopy and pili were enumerated using ImageJ software. A) Scatterplot of pili per cell (*p&lt;0.0001 compared to medium alone) and B) Bar graph depicting percent of piliated cells (*p&lt;0.05 compared to medium alone) derived from 60-112 cells per culture condition derived from 3 separate biological experiments.
<img alt="Bar graph comparing pili width and length (nm) across culture conditions; static equilibrium analysis." fo:content-width="5in" src="/files/ftp_upload/52122/52122fig4highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Pilus dimensions measured from cells cultured in various conditions of iron availability. Bacteria were grown in modified brucella broth alone (Medium Alone), or medium supplemented with 100 µM FeCl3 (FE), 200 µM dipyridyl (DIP), or 200 µM dipyridyl plus 250 µM FeCl3 (FE+DIP) prior to co-culture with human gastric epithelial cells. Samples were analyzed by high-resolution scanning electron microscopy and pili dimensions were measured using ImageJ software. A) Pilus width is an average of 13.1+/- 2.4 nm among all conditions. B) Pilus length is an average of 75.8 +/- 16.0 nm among all conditions. No significant difference in pilus dimensions are seen in the various conditions of iron availability.
Supplemental Figure 1: High resolution scanning electron microscopy analyses of H. pylori cagE mutant in various conditions of iron availability. Bacteria were grown in A) medium supplemented with 100 µM FeCl3, B) medium supplemented with 200 µM dipyridyl (DIP) or C) medium supplemented with 200 µM dipyridyl (DIP) plus 250 µM FeCl3 prior to co-culture with AGS human gastric cells. Samples were analyzed by high-resolution scanning electron microscopy to evaluate Cag-T4SS biogenesis. Conditions of low or high iron do not modulate any Cag-independent pilus biogenesis, supporting the hypothesis that the structures visualized in Figure 2 are Cag-T4SS pili.
Supplemental Figure 2: ELISA analysis of host IL-8 secretion in response to bacteria grown in varying conditions of iron availability. Wild-type (white bars) or cagE mutant (grey bars) bacteria were grown in modified brucella broth alone (Medium Alone), or medium supplemented with 100 µM FeCl3 (FE), 200 µM dipyridyl (DIP), or 200 µM dipyridyl plus 250 uM FeCl3 (FE+DIP) prior to co-culture with human gastric epithelial cells. Iron restriction results in increased IL-8, a host response that is dependent upon Cag-T4SS activity. Bars indicate mean +/- SEM, derived from 3 separate biological experiments. (*p&lt;0.05 compared to PMSS1 grown in medium alone).

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.1K 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.

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

Biochemistry

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.

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

Bioengineering

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.

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

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.

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

Chemistry

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.

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.

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

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

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.

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

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 ...

Impact of Helicobacter pylori Infection on Retina and Choroid Using SS-OCT

Retinal and Choroidal Thickness Changes in Populations with Helicobacter pylori Infection by Swept-Source Optical Coherence Tomography

Around half of the world&#39;s population is infected with Helicobacter pylori (H. pylori), which is closely related to several ocular diseases. The study aims to evaluate the retinal and choroidal thickness changes in subjects with H. pylori infection by swept-source optical coherence tomography (SS-OCT). The ophthalmic examination and 13C-urea breath test (13C-UBT) were performed on all subjects participating in the cross-sectional study. The participants were divided into H. pylori (+) and H. pylori (&#8722;) groups depending on the 13C-UBT results. This study covered 2574 right eyes from 2574 subjects with H. pylori infection and 2574 right eyes from 2574 age- and sex-matched individuals without H. pylori infection. Out of the nine sectors of the early treatment diabetic retinopathy study (ETDRS) grid, the maximum retinal thickness was in the inner superior sector, while the minimum was in the center sector. The maximum choroidal thickness was in the inner superior sector, while the minimum was in the outer nasal sector. The choroid of each area of the ETDRS subfield in the H. pylori (+) group was significantly thicker than that in the H. pylori (&#8722;) group, but retinal thickness did not show any difference between the two groups. Increased choroidal thickness may be an early indicator of H. pylori-associated retinal or choroidal diseases.

Here, we compared macular retinal and choroidal thickness in populations with and without Helicobacter pylori infection using a swept-source optical coherence tomography (SS-OCT) device, which is one of the recent milestones in the development of retinal and choroidal visualization.

Around half of the world&#39;s population is infected with Helicobacter pylori (H. pylori), which is closely related to several ocular diseases. The study ...

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

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