This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1. Synthesis of 10 nm Gold Nanoparticles (Gold NPs)15
<ol>
	<li>Prepare solution A: add 2 ml 1% aqueous gold chloride into 160 ml Milli-Q, or double distilled, water.</li>
	<li>Prepare solution B: add 8 ml 1% tri-sodium citrate x 2 H2O and 160 &#181;l 1% Tannic acid into 32 ml Milli-Q, or double distilled, water.</li>
	<li>Warm up solution A and B to 60 &#176;C and mix them while stirring. Observe a dark blue color immediately. Observe red color after about 15 min. Then heat up to 95&#176;C, keep 5 min and cool the solution to RT.</li>
	<li>Add a drop of the NP suspension onto a carbon coated grid and allow to dry in air. Check the size and morphology of NPs by transmission electron microscopy.</li>
</ol>
2. Coating of Gold NPs with BSA and Labeling with Rhodamine N-hydroxysuccinimidyl Ester (NHS)16
<ol>
	<li>Add 900 &#956;l gold NPs into a 1.5 ml Eppendorf tube, centrifuge at 15,000 x g for 30 min. Optionally, prepare multiple tubes may at once.</li>
	<li>Discard the supernatant, re-suspend the pellet in 900 &#956;l sterilized Milli-Q water and add 100 &#956;l 2 mg/ml BSA/PBS, mix on a Vortex at 800 rpm for 30 min.</li>
	<li>To remove excess BSA, centrifuge the preparation at 15,000 x g for 60 min.</li>
	<li>Discard the supernatant, and re-suspend the gold-BSA NPs in 125 &#956;l PBS, add 12.5 &#181;l of 1 M bicarbonate.</li>
	<li>Immediately before use, prepare a 10 mg/ml solution of rhodamine NHS/DMSO, add 30 &#181;l of rhodamine NHS/DMSO solution to 1ml of the gold-BSA NPs suspension. Incubate the reaction for 2 hr (or O/N) at RT during 800 rpm mixing, avoiding exposure to light.</li>
	<li>Purify the gold-BSA-rhodamine NPs through dialysis against PBS at 4 &#186;C with 5 buffer changes over the period of 36 - 48 hr.</li>
	<li>To stabilize NPs, add 10 &#956;l of 200 mg/ml BSA/PBS into each 1 ml gold-BSA-rhodamine NPs. To remove the free or released BSA-rhodamine, centrifuge at 15,000 x g for 60 min. Resuspend the pellet in 2 mg/ml BSA/PBS, measure OD520, and store at 4 &#176;C, avoiding exposure to light.</li>
	<li>Dilute the NPs 10 times with Milli-Q or double distilled water, add a drop onto a carbon-coated grid, and allow to dry in air. Check size and morphology of NPs by transmission electron microscopy (TEM). 
	NOTE: All materials used for NP preparation were sterilized and the operation was conducted in a cell culture hood or on a bench beside a flame.</li>
</ol>
3. Culture of HeLa Cells
<ol>
	<li>HeLa cells permanently expressing LAMP1-GFP are cultured in DMEM with 10% fetal calf serum (FCS) and grown at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>Seed the cells at a density of 50,000 per well in an 8-well chamber slide (Ibidi) and incubate O/N.</li>
</ol>
4. Culture of Bacteria
<ol>
	<li>Use Salmonella enterica serovar Typhimurium NCTC 12023 wild-type (WT) strain. For comparison, use mutant strains &#916;ssaV defective in the SPI2-T3SS and &#916;sifA lacking key effector SifA for SIF. Use strains harboring plasmid pFPV25.1 for constitutive expression of enhanced GFP. 
	NOTE: Bacterial strains are routinely cultured in Luria-Bertani broth (LB) with addition of 50 &#181;g/ml carbenicillin (WT) and LB with addition of 50 &#181;g/ml carbenicillin and/or 50 &#181;g/ml kanamycin (&#916;ssaV, &#916;sifA) of required to maintain plasmids.</li>
	<li>Inoculate a single colony of bacteria in 3 ml LB with appropriate antibiotics and grow O/N&#160;at 37 &#186;C under shaking conditions for aeration, then dilute 1:31 in fresh LB and continue growth for 3.5 hr. At this time-point, the cultures reach the late log phase and bacteria are highly invasive. A &#8216;roller drum&#8217; is convenient to incubate test tube cultures with aeration.</li>
</ol>
5. Infection of HeLa cells by Salmonella and Gold-BSA-Rhodamine NPs Pulse Chase-labeling (see Figure 1 for scheme)
<ol>
	<li>Measure OD600 of the sub-cultured bacteria and dilute to OD600 = 0.2 in 1 ml PBS (~3 &#215; 108 cfu/ml), add appropriate amounts of bacteria to HeLa cells in 8-well chamber slides to achieve a multiplicity of infection (MOI) of 100.</li>
	<li>Incubate for 30 min in the cell incubator, wash 3 times with PBS to remove non-internalized bacteria (this time point was set as 0 hr post-infection, or 0 hr p.i.). Add 300 &#181;l of fresh culture medium containing 100 &#181;g/ml gentamicin and maintain for 1 hr. Then replace the medium with fresh medium containing 10 &#181;g/ml gentamicin for the rest of the incubation time.</li>
	<li>After incubation with culture medium containing 100 &#181;g/ml gentamicin for 1 hr, the medium is replaced by imaging medium (Eagles MEM without FCS, L-glutamine, phenol red and sodium bicarbonate, with 30 mM HEPES, pH 7.4) containing 10 &#181;g/ml gentamicin. Gold-BSA-rhodamine NPs are added to HeLa cells to obtain a final concentration of OD520 = 0.1. 
	NOTE: Gold-BSA-rhodamine NPs may also be added to cells before infection, or at various time-points p.i.</li>
	<li>After 1 hr incubation, remove the medium, wash 3 times with PBS, and add 300 &#181;l of fresh imaging medium containing 10% FCS and 10 &#181;g/ml gentamicin for the rest of the incubation time. 
	NOTE: Duration of incubation may vary depending on concentration of NPs and cell lines used. For RAW264.7 macrophages, we observed that 30 min incubation with NPs at a concentration of OD520 = 0.05 allowed sufficient internalization.</li>
</ol>
6. Imaging
<ol>
	<li>Use a confocal imaging system such as a confocal laser-scanning (CLSM) or spinning disc (SD) microscope with a humidified environment chamber for high resolution imaging at different time-points.</li>
	<li>Switch on the temperature control system and wait until it is stable. Optimize the imaging settings such as magnification, scanning speed, resolution, Z-stack etc. Use appropriate excitation/emission settings for GFP and gold-BSA-rhodamine NPs. For this protocol, use a 400 Hz scanning speed, resolution of 512 x 512 pixels and Z-step size of 0.25 &#181;m. Excite GFP and gold-BSA-rhodamine using an Ar laser (488 nm) and a HeNe laser (543 nm), respectively. Include a bright-field (BF) channel for observing the shape of the cells. For other microscopy systems and infection conditions, the setting have to be adjusted accordingly. 
	NOTE: Use the same Ar laser as the light source of BF channel and signal was detected with a photo multiplier (PMT) detector.</li>
	<li>At indicated time-points p.i., mount the 8-well chamber slides containing infected cells on the microscope stage and record images.</li>
</ol>
7. Analysis of Images
<ol>
	<li>Use microscopy image analysis software (see Materials and Equipment&#8217;s Table) to analyze the remodeling of the endo-lysosomal system by intracellular Salmonella. Open the data by clicking &#8216;Open&#8217; and choosing the file. 
	NOTE: Alternatively, open source software packages such as ImageJ or FIJI may be used to quantitative image analyses.</li>
	<li>In the objects toolbar of the Surpass view click on the icon<img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> to add a new surface item. Click on <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>To analyze a Region of Interest (ROI), select segment a ROI. In a viewing area, a rectangle-bordered section overlaid on image is representing the ROI. Enter the values in the corresponding x-, y-, and z- fields, or directly click on the arrows in the preview rectangle to modify size and position of the ROI. Then click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>As a source channel, select Channel 3 (signal for Gold-BSA-Rhodamine NPs). Check the smooth option to set up the smoothness of the resulting area. Define a value manually or accept the automatically generated value. For Threshold, select the Absolute Intensity option.</li>
	<li>For the threshold adjustment, select the manual option and set a value. In the viewing area, a surface threshold preview is displayed in gray.</li>
	<li>On the tab Classify Surfaces the resulting surface can be filtered by various criteria. A default filter is &#8216;number of voxels &#62;10&#8217;, and other filters can also be included by clicking &#8216;add&#8217;. If a filtering is not necessary, then delete all filters by clicking on the delete button. Click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next).</li>
	<li>To complete Surface creation, click on <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon3.jpg" /> (Finish). In the Object list, now un-check the box for the item Volume and new created surface is displayed in viewing area.</li>
	<li>Export the statistics. Now, in the Surpass view the color of the subjects vary from purple to red according to their area. And the &#8216;Plot Numbers Area&#8217; table shows the statistics information (e.g., ID number and area of objects). Export all statistics of surface 1 to an Excel file by click <img alt="Icon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Save).</li>
</ol>

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No conflicts of interest declared.

The endo-lysosomal system of mammalian cells controls important physiologic processes, including nutrient absorption, hormone-mediated signal transduction, immune surveillance, and antigen presentation17. Up to now, a variety of markers have been used for labeling of the endocytic pathway and tracking studies. For example, LysoTracker probes are fluorescent acidotropic probes developed by Molecular Probes (Life Technologies, USA) for lysosome labeling, which can selectively accumulate in cellular compartments ...

Fluorescent nanoparticles (NPs), including metal NPs, quantum dots, polymer NPs, silica NPs, carbon dots, etc., have attracted considerable attention during the past decades1,2. Compared to traditional organic dyes, fluorescent NPs show desirable chemical, optical and mechanical properties, such as strong signal strength, resistance to photobleaching and high biocompatibility3,4. These advantages make them the method of choice for intracellular sensing and live cell imaging. Furthermore, a variety of electron-dense NPs are visible by electron microscopy (EM), facilitating their use for correlated microscopic analysis, which allows combination of live cell tracking with light microscopy (LM) and higher resolution at ultrastructural level with EM5. For example, gold NPs have been long time efficiently used as biosensors in living cells for sensitive diagnosis as well as in the field of immuno-labeling6. Recent studies indicate that gold NPs with different size and shape can be readily uptake by a large variety of cell lines and routinely transport through the endosomal pathway, therefore have great potential being applied for intracellular vesicle transportation tracking and the endo-lysosomal system labeling7,8.
Microbial pathogens, such as Salmonella enterica, Shigella flexneri and Listeria monocytogenes, have developed different mechanisms to invade non-phagocytic host cells9. After being internalized, the pathogens, either localized in the cytosol or sequestered in membrane-bound compartments, interact extensively with their host environments and modulate these to favor their own survival10. For instance, Salmonella enterica resides and replicates within an intracellular phagosomal compartment termed the Salmonella-containing vacuole (SCV) upon infection11. The maturing SCV traffics towards the Golgi apparatus, undergoing continuous interactions with the endocytic pathway, and induces formation of extensive tubular structures, such as Salmonella-induced filaments (SIF), sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules, etc.12-14. Studying how these bacterial pathogens manipulate host-cell pathways is essential to understanding infectious disease.
Here, gold-BSA-rhodamine NPs were used as fluid tracers to label the host cellular endo-lysosomal system, and the human gastrointestinal pathogen Salmonella enterica serovar Typhimurium (Salmonella) was used as a model bacterium to study the interactions of the pathogen with the host endocytic pathway. Intracellular gold-BSA-rhodamine NPs in non-infected cells and cells infected with WT Salmonella or mutant strains were imaged by a confocal laser-scanning microscope (CLSM). Then Imaris software was used to quantify the distribution of NPs, indicating that Salmonella infection induced extreme rearrangement of the endosomes/lysosomes. Following the description of this method, analogous experiments can be designed to track long-term fate of the internalized NPs and to investigate the influence of various exogenous substances or endogenous factors on the endocytic pathway of eukaryotic cells.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

Gold NPs were generated through a well-established method via reduction of chloroauric acid by citrate and tannic acid. As shown in Figure 2A, the synthesized gold NPs were quasi-spherical in shape with a size of approximately 10 nm. BSA-coating and rhodamine-labeling did not influence their morphology or size (Figure 2B).
It has been reported that gold NPs can be readily taken up by various mammalian cells and ended up in the endocytic systems7. In...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

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Immunology and Infection

9.9K Views.  Universität Osnabrück. This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Video: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

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

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

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

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...