Name: bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem
Uploaded: 2015-10-01T13:00:00
Description: Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

The following text describes a stepwise protocol to perform optimized Psm ES4326 syringe infiltration assay in Arabidopsis. Major procedures of this assay are represented in a simplified flowchart (Figure 1).
1. Plant Growth Conditions
<ol>
	<li>Sow seeds
	<ol>
		<li>Prepare 2 pots (4 in&#160;diameter, 3.75 in&#160;tall) loosely filled with soil and water pots by soaking them from the bottom O/N before draining the excess water.</li>
		<li>Sow 50-100 Arabidopsis see...

With decreasing available farmland and increasing population, researchers around the world are challenged with pressing needs for crop improvement. The yield can be greatly influenced by various biotic and abiotic stresses. Among them, pathogen infection is one of the leading causes of crop yield reduction, responsible for approximately 12% losses in the U.S. alone45. To resolve this issue, massive research has been conducted in the model Arabidopsis - P. syringae pathosystem to comprehensively charac...

Due to their sessile nature, plants are constantly threatened by a plethora of pathogens exhibiting various lifestyles and nutritional strategies1. To a first approximation, biotrophic pathogens maintain their host alive to retrieve nutrients, while necrotrophic pathogens actively secret toxins and enzymes to kill host tissue and feed on the dead cells1. Another group of pathogens, termed hemibiotrophs, begins their infection course with the biotrophic stage and shifts to the necrotrophic stage upon reaching a certain threshold of pathogen accumulation2. In order to effectively defend themselves against these microorganisms, plants have evolved a complicated innate immune system equipped with multiple surveillance mechanisms to detect the pathogen attack and trigger localized programmed cell death3 as well as Systemic Acquired Resistance (SAR)4. Current research is focused on characterizing the essential signaling components and cross-talks within the plant immune system5.

As proposed in the &#8220;Zig-Zag&#8221; model5, the first layer of the plant innate immune response requires the presence of plasma membrane-localized Pattern Recognition Receptors (PRRs) to detect the invasion of a microbe. PRRs are able to recognize Microbe-Associated Molecular Patterns (MAMPs) and establish MAMP-Triggered Immunity (MTI)6. Besides inducing a transcriptional upregulation of genes encoding antimicrobial PR proteins7, MTI leads to a variety of events that arrest pathogen growth, including the production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), deposition of callose to the cell wall as well as the activation of multiple kinase signaling pathways8.
Until now, several MAMPs have been identified to trigger MTI in Arabidopsis, including bacterial flg229 (a 22 amino acid fragment derived from flagellin), elf1810 (18 amino acids from the bacterial translation elongation factor Tu) and a structural cell wall component peptidoglycans11. To establish a successful infection, some specialized pathogens have evolved the ability to secret virulence effector proteins into the intracellular or intercellular spaces, and consequently repress MTI and trigger Effector-Triggered Susceptibility (ETS)12,13. For instance, virulence effectors can inactivate Mitogen-Activated Protein Kinase (MAPK) phosphorylation cascades of MTI to induce the disease development within the infected tissue14-16. During the dynamic co-evolution between hosts and pathogens, plants also developed the counterattack strategy to recognize the effector proteins and attenuate the pathogen virulence molecules17. This direct or indirect effector recognition is mediated by disease resistance (R) proteins18. Most of them are members of NB-LRR (Nucleotide Binding and Leucine-Rich Repeats) family19. The perception of an avirulent effector by an R protein elicits a stronger and broader immune response characterized as Effector-Triggered Immunity (ETI)20. Besides inducing the expression of defense genes21 and the production of defense metabolites22, ETI often leads to a rapid localized programmed cell death known as Hypersensitive Response (HR) to restrict the pathogen from spreading into the adjacent tissue3.
In addition to the localized programmed cell death23, plants are capable of initiating a long-term and system-wide immune response termed Systemic Acquired Resistance (SAR)4. Upon challenge with a biotrophic pathogen, plant cells trigger the biosynthesis and accumulation of an endogenous phytohormone salicylic acid (SA) and PR proteins in both local and systemic tissues24. Through this process, a heightened state of preparedness is achieved in the uninfected leaves that allows for mounting faster defense responses during a subsequent infection by a broad spectrum of pathogens24. SA and its synthetic analogs such as benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA) are capable of chemically inducing Salicylic Acid (SA)-Triggered Immunity (STI) upon external application24. Nonexpressor of Pathogenesis-Related genes 1 (NPR1) is proposed to be one of the SA receptors and functions as a major transcriptional regulator during SA-mediated defense response in both local and systemic tissues21,25,26. It has been conclusively demonstrated that NPR1 is required for SAR establishment and the loss of NPR1 leads to dramatic susceptibility towards Pseudomonas syringae25.
To extensively characterize the molecular contribution of plant components in the plant-pathogen interactions, multiple bioassays have been developed to measure specific defense events, including ROS burst27, callose deposition28, defense genes expression and accumulation of their protein products21. While these individual assays can provide insights into a specific form of the plant immune response, none of them, however, are able to represent the complete defense response on the whole plant level. Conversely, quantification of pathogen growth after infection provides an overall estimation of immune response at the organismal level. Therefore, the development and optimization of a precise and highly standardized pathogen inoculation assay is critical to fuel up the research and discoveries on the Arabidopsis immune responses.
Pseudomonas syringae, a Gram-negative bacterium, was identified as a phytopathogen capable of causing disease in a range of plant hosts including Arabidopsis29. As the model plant-pathogen system, the Arabidopsis - P. syringae interaction has been widely applied to understand the molecular mechanisms underlying plant defense responses29. Until now, over 50 P. syringae pathovars have been identified based on their ability to infect different plant species30 . P. syringae pv. tomato DC3000(Pst DC3000)31and P. syringae pv. maculicola ES4326 (Psm ES4326)32 are the two most widely used and extensively characterized virulent strains. Aside from being recognized by the plant and triggering the MTI response, Pst DC3000 and Psm ES4326 are capable of secreting virulent effector proteins to suppress MTI and trigger ETS to favor the pathogen growth31,33.
To functionally dissect the interaction between Arabidopsis and P. syringae, multiple&#160;pathogen infection methods have been developed based on the pathogen delivery approach. For soil-grown plants, pathogen can be delivered by syringe infiltration, vacuum infiltration, dip inoculation and spray inoculation29,34. Recently, seedling flood-inoculation assay was developed to perform large-scale screens on tissue culture plates-grown young Arabidopsis plants35. Syringe infiltration, as one of the most commonly used approach, manually delivers the pathogen into the apoplast through the natural leaf openings termed stomata29. Through this approach, equal amounts of P. syringae can be infiltrated into the infected leaf and the strength of plant immune response is inversely correlated to the pathogen growth levels. Therefore, quantification of pathogen growth serves as an optimal approach to evaluate the immune function at the whole plant level. In addition, syringe infiltration can distinguish the local and systemic tissue, which can be applicable in characterizing the molecular mechanisms underlying SAR36.
In the following protocol, we describe an optimized syringe infiltration assay with Psm ES4326 to screen Arabidopsis mutants for enhanced disease susceptibility (EDS). This protocol will employ two Arabidopsis genotypes: a wild-type ecotype Columbia-0 (Col-0) plants (control) and npr1-1 loss-of-function&#160;mutants (hypersusceptible) that will be infected with virulent bacterial strain Psm ES432637. The npr1-1 mutant carries a point mutation within the ankyrin-repeat consensus sequence of the NPR1 molecule, which alters the highly conserved histidine to tyrosine and renders the protein non-functional25. In addition, a number of modifications of the syringe infiltration assay are described that allow quantification of defects in the specific layers of immune response, including MTI and STI.

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			<td>MetroMix 360&#160;</td>
			<td>Grosouth</td>
			<td>SNGMM360</td>
			<td></td>
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			<td>Large pots</td>
			<td>Grosouth</td>
			<td>TEKUVCC10TC</td>
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			<td>12x6 Inserts</td>
			<td>Grosouth</td>
			<td>LM1206</td>
			<td></td>
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			<td>11x21 Flats with no holes</td>
			<td>Grosouth</td>
			<td>LM1020</td>
			<td></td>
		</tr>
		<tr>
			<td>11x21 Flats with holes</td>
			<td>Grosouth</td>
			<td>LM1020H</td>
			<td></td>
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			<td>Vinyl propagation domes</td>
			<td>Grosouth</td>
			<td>CW-221</td>
			<td></td>
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		<tr>
			<td>Proteose Peptone</td>
			<td>Fisher Scientific</td>
			<td>DF0122-17-4</td>
			<td></td>
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			<td>Potassium Phosphate Dibasic Trihydrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>151946</td>
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			<td>Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>A360-500</td>
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			<td>Streptomycin sulfate</td>
			<td>Bio Basic Inc</td>
			<td>SB0494</td>
			<td></td>
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			<td>100x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
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			<td>150x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80150</td>
			<td></td>
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			<td>Rectangular plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-450&#160;</td>
			<td></td>
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			<td>MgCl2 Hexahydrate</td>
			<td>Bio Basic Inc</td>
			<td>MB0328</td>
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			<td>Glycerol</td>
			<td>Bio Basic Inc</td>
			<td>GB0232</td>
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			<td>MgSO4&#160;</td>
			<td>Bio Basic Inc</td>
			<td>MN1988</td>
			<td></td>
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			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>NC9992493&#160;</td>
			<td></td>
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			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td></td>
		</tr>
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			<td>Grinding tubes&#160;</td>
			<td>Denville Scientific</td>
			<td>B1257</td>
			<td></td>
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			<td>Caps for grinding tubes</td>
			<td>Denville Scientific</td>
			<td>B1254</td>
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			<td>Stainless steel grinding ball</td>
			<td>Fisher Scientific</td>
			<td>2150</td>
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			<td>96-well plate&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-556-008</td>
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			<td>Sodium Salicylate</td>
			<td>Sigma Aldrich</td>
			<td>s3007-1kg</td>
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		<tr>
			<td>flg22 (QRLSTGSRINSAKDDAAGLQIA)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
		</tr>
		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
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		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td>146308</td>
			<td></td>
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			<td>Biophotometer plus</td>
			<td>Eppendorf</td>
			<td>952000006</td>
			<td></td>
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		<tr>
			<td>PowerGen High-Throughput Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>02-215-503</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu spin micro centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (10-100 &#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.043</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (30-300&#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.060</td>
			<td></td>
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		<tr>
			<td>Pipette (20&#181;l)</td>
			<td>Eppendorf</td>
			<td>3120 000.038</td>
			<td></td>
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			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>3552-HR</td>
			<td></td>
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			<td>Sharpie permanent marker</td>
			<td>Staples</td>
			<td>507130</td>
			<td></td>
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			<td>1.5 mL tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
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		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>08-890</td>
			<td></td>
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bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

The protocol we describe here represents an optimized P. syringae syringe infiltration assay to quantitatively evaluate the immune response in Arabidopsis plants. As illustrated in Figure 1, the syringe infiltration of Psm ES4326 is followed by pathogen extraction and quantification via serial dilutions and colonies enumeration.
As described in Step 3 within the protocol text, Enhanced Disease Susceptibility (EDS) against Psm ES4326 can be a...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

bacterial-leaf-infiltration-assay-for-fine-characterization-plant

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Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...