This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Initiative 796 (SFB796) to A.L. and C.B., and through the ERA-NET PathoGenoMics 3rd call to A.L.

1. Generation of Stable Cell Lines Expressing the Protein of Interest
<ol>
	<li>Prepare media by adding heat-inactivated FCS and 1% Penicillin/Streptomycin to commercially available Dulbecco&#180;s Modified Eagle Medium (DMEM) supplemented with GlutaMAX-I, Pyruvate, and 4.5&#160;g/L D-Glucose.</li>
	<li>Cultivate HEK293 cells in media at 37&#160;&#176;C in 5%&#160;CO2. Sub-cultivate cells every third day. Remove the media and resuspend the cells in 15 ml fresh media. Pipette 1 ml into a new 75&#160;cm2 cell culture flask and add 14 ml media.</li>
	<li>Analyze the cell number by using a hemocytometer.</li>
	<li>Seed HEK293 cells in a 6-well plate at a density of 2 x 105 cells per well and incubate for 24 hr.</li>
	<li>For transfection use the protocol provided by the manufacturer of the transfection reagent. Transfect cells with pEGFP-C2 or pEGFP-C2-CaeB using the transfection reagent. Prior to use, warm up the transfection reagent and the media required to RT. Use a 3:1 ratio of transfection reagent to DNA.
	<ol>
		<li>In detail, pipette 1.5 &#181;l of transfection reagent directly into 50 &#181;l of serum-free DMEM medium. For complex formation, pipette 0.5&#160;&#181;g of DNA encoding GFP or GFP-CaeB to the transfection reagent-containing mixture and incubate for 15&#160;min at RT.</li>
		<li>Transfect cells by adding the reaction mixture in a drop-wise manner and incubate at 37&#160;&#176;C in 5%&#160;CO2 for 24 hr.</li>
	</ol>
	</li>
	<li>Add 1&#160;mg/ml geneticin for selection of GFP-positive clones at 37&#160;&#176;C in 5%&#160;CO2.</li>
	<li>After 6&#160;days, isolate single cells by flow cytometry sorting in a 96-well plate harboring medium and HEK293 supernatant in a 1:1 ratio. Generate HEK293 supernatant from cultured confluent HEK293 cells that were centrifuged for 15&#160;min at 4,966 x g.</li>
	<li>On the following day, replace culture medium with medium containing 1.5&#160;mg/ml geneticin. Change media once a week. If the cells form a confluent monolayer, transfer them to a 24-well plate. Sub-cultivate cells twice a week in a ratio of 1:5 in media containing 1.5&#160;mg/ml geneticin. Finally, analyze the percentage of GFP-positive cells by immunoblot analysis and flow cytometry.</li>
</ol>
2. Analysis of Stable Cell Lines by Immunoblot Analysis
<ol>
	<li>Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 &#181;l of 2x Laemmli sample buffer, heat for 5&#160;min at 95&#160;&#176;C and store at -20&#160;&#176;C.</li>
	<li>Load approximately 4 x 104 cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 &#181;l&#160;of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180&#160;V for 45-60&#160;min with 1x&#160;Laemmli running buffer.</li>
	<li>Blot proteins onto PVDF membranes (pore size of 0.45&#160;&#181;m) at a constant voltage of 16&#160;V for 60&#160;min using a Trans-Blot SD Semi-Dry Transfer Cell.</li>
	<li>Block membranes in 10 ml of 5%&#160;nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.</li>
	<li>Dilute 4 &#181;l of anti-GFP antibody in 4 ml of MPT and incubate membranes O/N at 4&#160;&#176;C.</li>
	<li>Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.</li>
	<li>Incubate membranes with 0.8 &#181;l of horseradish peroxidase-conjugated secondary antibodies in 4 ml of MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 2.6) and detect horseradish peroxidase activity with a commercial kit according to the manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands.</li>
</ol>
3. Analyze the Cells Using a Flow Cytometer.
<ol>
	<li>Resuspend cells in PBS. Use HEK293 cells as negative control to adjust forward scatter (FSC) and side scatter (SSC) so that the cells are on scale. Draw a gate on living cells by excluding cell doublets, aggregates and cell debris.</li>
	<li>Adjust the photomultiplier tube (PMT) gain so that the unstained cells are on the far left of the histogram for the FL1 channel (488 nm argon laser).</li>
	<li>To analyze the fluorescence intensity of HEK293-GFP and HEK293-GFP-CaeB cells open the histogram of the FL1 channel and acquire 10,000 events on the gated population.</li>
	<li>Analyze the data using commercial FACS analysis software.</li>
</ol>
4. Transfection of the Stable Cell Line with the Inducible Expression Vector System
<ol>
	<li>Seed HEK293 cells stably expressing GFP or GFP-CaeB in a 12-well plate at a density of 1 x 105 cells/well.</li>
	<li>19 hr post-seeding, co-transfect the cells with regulator plasmid and response plasmid either encoding Bax or activated caspase&#160;3.
	<ol>
		<li>Co-transfect the stable HEK293 cells with 100&#160;ng of pWHE125-P regulator plasmid (Figure 2) and 100&#160;ng of the response plasmids pWHE655-hBax or pWHE655-revCasp3 (Figure 3) and 0.4 &#181;l of polyethylenimine.</li>
		<li>Prepare the DNA and polyethylenimine transfection reagent each in 75 &#181;l of Opti-MEM medium and incubate for exactly 5&#160;min at RT. For complex formation, incubate both mixtures together for 15&#160;min at RT.</li>
	</ol>
	</li>
	<li>Add the polyethylenimine/DNA solution drop-wise to the cells and incubate at 37&#160;&#176;C in 5%&#160;CO2.</li>
</ol>
5. Induction of Apoptosis
<ol>
	<li>5 hr post-transfection, induce expression of the pro-apoptotic proteins by addition of 1&#160;&#181;g/ml doxycycline to the culture medium. Incubate cells for 18 hr at 37&#160;&#176;C in 5%&#160;CO2.</li>
</ol>
6. Analysis of Host Cell Apoptosis by Immunoblot Analysis
<ol>
	<li>Inspect cells prior to harvesting under the light microscope to check for cell death induction. Apoptotic cells are detectible by the presence of apoptotic bodies surrounding the dying cell.</li>
	<li>Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 &#181;l of 2x Laemmli sample buffer, heat for 5&#160;min at 95&#160;&#176;C and store at -20&#160;&#176;C.</li>
	<li>Load approximately 4 x 104 cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 &#181;l of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180&#160;V for 45-60&#160;min with 1x&#160;Laemmli running buffer.</li>
	<li>Blot proteins onto PVDF membranes (pore size of 0.45&#160;&#181;m) at a constant voltage of 16&#160;V for 60&#160;min using a Trans-Blot SD Semi-Dry Transfer Cell.</li>
	<li>Block membranes in 10 ml of 5%&#160;nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.</li>
	<li>Dilute 4 &#181;l of anti-cleaved poly ADP-ribose polymerase (PARP) antibody in 4 ml of MPT and incubate O/N at 4&#160;&#176;C.</li>
	<li>Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.</li>
	<li>Incubate membranes with 0.8 &#181;l horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7) and detect horseradish peroxidase activity with a commercial kit according to manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands.</li>
	<li>Remove bound antibodies by 30&#160;min incubation at RT with 7 ml Western Blot Stripping Buffer.</li>
	<li>After three washes with PBS/0.05% Tween 20 (as described in 6.7), probe the membranes with 4 &#181;l of anti-actin antibody in 4 ml of MPT O/N at 4&#160;&#176;C.</li>
	<li>After three washing steps with MPT (as described in 6.7), incubate the membranes with 0.8 &#181;l of horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml of MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7 and detect horseradish peroxidase activity with a commercial kit according to the manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands. 
	Note: All incubation steps were performed on a plate shaker for the indicated time and temperature.</li>
</ol>

The authors have nothing to disclose.

Many pathogenic bacteria harbor secretion systems to secrete or translocate bacterial effector proteins into the host cell. These effector proteins have the capacity to modulate processes and pathways in the host cell, allowing the bacteria to survive and replicate within their respective intracellular niche. Understanding the biochemical activities and the molecular mechanisms of the effector proteins will help towards a better understanding of pathogenicity and may help to develop new therapeutic tools to combat diseas...

The virulence of many Gram-negative bacterial pathogens depends on specialized secretion systems to hijack eukaryotic host cells. Bacteria use these secretion systems to inject bacterial virulence proteins (effectors) into the host cell to modulate a variety of cellular and biochemical activities. The study of effector proteins has not only provided remarkable insight into fundamental aspects of host/pathogen interactions but also into the basic biology of eukaryotic cells1. Modulation of host cell apoptosis has been shown to be an important virulence mechanism for many intracellular pathogens, and a number of effector proteins modulating apoptosis have been identified2-9. However, their precise molecular mechanisms of activity remain elusive in many cases.
Apoptosis, a form of programmed cell death, plays an important role in immune responses to infection10. Two main pathways leading to apoptosis have been identified: targeting the mitochondria (intrinsic apoptosis) or direct transduction of the signal via cell death receptors at the plasma membrane (extrinsic apoptosis). The intrinsic or mitochondria-mediated cell death pathway is triggered by intracellular signals and involves the activation of Bax and Bak, two pro-apoptotic members of the Bcl-2 family. This family is composed of pro- and anti-apoptotic regulator proteins that control cell death11-14. Activation of apoptosis leads to oligomerization of Bax and Bak followed by subsequent permeabilization of the mitochondrial outer membrane, resulting in cytochrome C release into the cytoplasm. Cytochrome C release initiates activation of the effector caspases 3 and 7 through activation of caspase 9 in the apoptosome15. This leads to proteolysis of selected substrates that, among others, results in the exposure of phosphatidylserine on the cell surface16 and frees a dedicated DNase that fragments chromatin17,18.
In order to determine where within the apoptotic cascade an individual effector protein interferes, an inducible expression system was employed19. Regulatory systems for conditional expression of transgenes have been an invaluable tool in analyzing a protein&#8217;s function within the cell or its importance for tissue, organ and organism development, as well as during initiation, progression and maintenance of disease20-23. Typically, inducible control systems, such as the Tet system24 employed here, form an artificial transcription unit (see Figure 1). One component is an artificially engineered transcription factor called tTA (tetracycline-dependent transcription activator), formed by fusion of the bacterial transcription repressor TetR25 to a mammalian protein domain that mediates transcriptional activation or silencing 24,26. The second component is a hybrid promoter, termed TRE (tetracycline-responsive element), consisting of a eukaryotic minimal promoter, containing at least a TATA-box and a transcription initiation site, joined to multiple repeats of the cognate DNA-binding site for TetR, tetO24,25. The third component is the natural ligand of TetR, tetracycline or one of its derivatives, such as anhydrotetracycline or doxycycline25. Upon ligand addition to the culture medium, TetR loses its affinity for tetO and dissociates from the TRE. As a result, transcription of the target gene is abolished. Transgene expression can, thus, be tightly controlled in a time- and dose-dependent manner in both cell culture and in animals20,23,24. With tTA, transgene expression occurs constitutively, except in the presence of a tetracycline. This can be a disadvantage in the study of cytotoxic or oncogenic proteins because tetracycline first has to be removed from the system, before transgene expression occurs and the target protein&#8216;s effects on the cell can be monitored. This can be time-consuming and is not always complete, especially in transgenic animals27. To address this limitation, a TetR mutant with an inverse response to the presence of doxycycline was used to generate a new transcription factor, rtTA (reverse tTA)28. It only binds to the TRE and, concomitantly, activates transcription in the presence of doxycycline. Residual leakiness of the system, i.e., transgene expression in the absence of TRE-bound transcription factor, originating either (i) from position effects at a genomic integration site, (ii) from the TRE itself29, or (iii) from non-specific binding of tTA/rtTA28, was addressed by introducing an additional transcriptional silencer, termed tTS (tetracycline-dependent transcriptional silencer)30 to the system. It forms a dual regulator network together with rtTA (see Figure 1). In the absence of doxycycline, tTS binds to TRE and actively shuts down any remaining transcription. In the presence of doxycycline, tTS dissociates from TRE and rtTA binds simultaneously inducing expression of the target gene. This additional layer of stringency is often necessary to express highly active cytotoxic proteins31-34.
Using this tightly controlled dual-regulator system, the apoptotic cascade can be initiated at a defined step allowing analysis of whether the given effector protein can interfere with apoptosis induction. This method can not only be used to study the anti-apoptotic activity of bacterial effector proteins but also for the inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>life technologies</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>life technologies</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365752001</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Roth</td>
			<td>CP11.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polyscienes</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>165-8000EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP&#160;</td>
			<td>life technologies</td>
			<td>A6455</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cleaved PARP</td>
			<td>BD Bioscience</td>
			<td>611038</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin</td>
			<td>Sigma Aldrich</td>
			<td>A2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-045</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Plus Western Blot Stripping Buffer</td>
			<td>Thermo Scientific</td>
			<td>46428</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

First, HEK293 cell lines stably expressing the protein of interest (CaeB) as a GFP-fusion protein were established. As a control, HEK293 cell lines stably expressing GFP were also generated. Expression of GFP and GFP-CaeB was verified by immunoblot analysis. The representative immunoblot (Figure 4A) demonstrates stable and clearly detectable expression of GFP and GFP-CaeB. However, this assay cannot determine whether all cells express GFP or GFP-CaeB. Therefore, the stably transfected HEK293 cell lines w...

Watch this Scientific Journal Video about Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling at JoVE.com

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

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 ...

applying-an-inducible-expression-system-to-study-interference

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JoVE Journal

Immunology and Infection

9.1K Views.  Friedrich-Alexander-Universität. 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.

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

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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.

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...