Name: double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions
Uploaded: 2021-07-10T14:00:00
Description: Watch this Scientific Journal Video about Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions at JoVE.com

This work was supported by Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP 2010/19547-1; 2018/03677-5) to MMAB, by Funda&#231;&#227;o de Apoio ao Ensino, Pesquisa e Assist&#234;ncia- FAEPA to MMAB and by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior- Brasil (CAPES) - finance code 001. CG-C received a master and doctoral fellowship from CAPES and LAMT-S received doctoral fellowship from CNPq. We thank Elizabete R. Milani for confocal microscopy assistance and Dr. Dario Zamboni for providing LLC-MK2 cells (Ribeirao Preto Medical School, USP).

1. Cell and parasite cultures
<ol>
	<li>Grow LLC-MK2 (Rhesus Monkey Kidney Epithelial) cells from the American Type Culture Collection (CCL-7) in a 25 cm2&#160;cell culture flask containing in RPMI medium supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) and antibiotics (100 U/mL Penicillin and 100 &#181;g/mL Streptomycin) at 37 &#176;C in 5% CO2 17.</li>
	<li>Infect LLC-MK2 cells with Trypanosoma cruzi (Y strain) according to a previous protocol <sup...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+affecting+cellular+invasion+and+escape+from+the+parasitophorous+vacuole+by+different+infective+forms+of+Trypanosoma+cruzi.">Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 98 (7), 953-958 (2003).</li><li>de Souza, W., de Carvalho, T. M., Barrias, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+Trypanosoma+cruzi:+Host+Cell+Interaction.">Review on Trypanosoma cruzi: Host Cell Interaction.</a> International Journal of Cell Biology. 2010, 295394 (2010).</li><li>Burleigh, B. A., Woolsey, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signalling+and+Trypanosoma+cruzi+invasion.">Cell signalling and Trypanosoma cruzi invasion.</a> Cellular Microbiology. 4 (11), 701-711 (2002).</li><li>Baqui, M. M. A., Takata, C. S., Milder, R. V., Pudles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+giant+protein+associated+with+the+anterior+pole+of+a+trypanosomatid+cell+body+skeleton.">A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton.</a> European Journal of Cell Biology. 70, 243-249 (1996).</li><li>Baqui, M. M., Milder, R., Mortara, R. 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Here, we present a protocol to perform double immunolabeling in Trypanosoma cruzi infected cells using two different antibodies from the same host species. To study, with more detail, the implications of the infection, structures in the host cell such as the nucleus or cytosolic organelles can be labeled using this protocol. Also, it can be used in the post-embedding thin section immunogold labeling method. This approach helps to overcome the obstacle of having few antibodies available to study trypanosomatids a...

To study the interaction of the pathogen with the host cell at the cellular level provides essential information on the underlying causes of the disease since different groups,&#160;such as viruses, bacteria, and protozoa,&#160;can infect most host cell types1,2,3,4. It can also help develop and identify potential therapeutic targets that can slow or inhibit the growth of the pathogen. In live conditions, the produced antibodies are responsible for recognizing self-components, antigens from viruses, bacterial components or products, fungi, parasites, and others5.
For this purpose, antibodies are widely used tools, mainly for understanding the location and function of cellular structures and proteins. Several studies using multiple antibody labeling demonstrate that additional blocking steps contribute to the specificity of the immunolocalization. In addition, most described protocols use specific commercial monoclonal antibodies, including antibodies from the same host species6,7,8,9,10,11,12,13,14.
Usually, double labeling immunofluorescence uses two antibodies raised in different species to stain the cell structures of interest or the pathogens and the host cells to see the interaction between them. However, this can be a problem when no commercial monoclonal or polyclonal antibodies specific for some pathogens are available to perform the double labeling. Also, there are commercially available antibody conjugation kits, and it is possible to conjugate the primary antibodies directly to the fluorophore by a succinimidyl ester reaction15. The problem is that these kits are often expensive, and it is necessary to have enough antibodies to label them. Knowing this, we successfully developed a double immunofluorescence method using two different antibodies raised in the same species to study protein localization in Trypanosoma brucei16. However, for intracellular parasites, including Trypanosoma cruzi, this approach has not been demonstrated. Here, we show how to perform double labeling immunofluorescence to study intracellular T. cruzi parasites and the host cell using primary antibodies raised in the same species without cross-reactions. Besides this method, a triple immunofluorescence labeling has been established with the addition of the third antibody from a different species. These approaches help when the source of antibodies is limited and can be used in any cell type.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 - IgG2b antibody</td>
			<td>Life technologies, USA</td>
			<td>A21141</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>AffiniPure Rabbit anti-mouse IgG (H+L)</td>
			<td>Jackson Immunoresearch, USA</td>
			<td>315-005-003</td>
			<td>Anti-mouse antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 - IgG F (ab&#39;)2 (H+L) antibody</td>
			<td>Life technologies, USA</td>
			<td>A11017</td>
			<td>Goat anti mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 IgG1 antibody</td>
			<td>Life technologies, USA</td>
			<td>A21125</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 - IgG F (ab&#39;)2 (H+L) antibody</td>
			<td>Life technologies, USA</td>
			<td>A21237</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>Anti-hnRNPA1 antibody IgG2b</td>
			<td>Sigma-Aldrich, USA</td>
			<td>R4528</td>
			<td>Mouse antibody</td>
		</tr>
		<tr>
			<td>anti-TcFAZ (T. cruzi FAZ protein) antibody</td>
			<td>Our lab</td>
			<td>In-house</td>
			<td>Mouse antibody</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A2153-10G</td>
			<td>Albumin protein</td>
		</tr>
		<tr>
			<td>Detergent Igepal CA-630</td>
			<td>Sigma-Aldrich, USA</td>
			<td>I3021</td>
			<td>Nonionic Detergent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo fisher scientific, USA</td>
			<td>12657-029</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco, Thermo fisher scientific, USA</td>
			<td>15140-122</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Phalloidin Alexa Fluor 594</td>
			<td>Life technologies, USA</td>
			<td>A12381</td>
			<td>Actin marker</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade with DAPI</td>
			<td>Life technologies, USA</td>
			<td>P36935</td>
			<td>Mounting media reagent</td>
		</tr>
		<tr>
			<td>RPMI 1640 1X with L-glutamine</td>
			<td>Corning, USA</td>
			<td>10-040-CV</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T4049-100ML</td>
			<td>Bioreagent</td>
		</tr>
	</tbody>
</table>

double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, we show how to study host-parasite interactions by immunofluorescence when the source of antibodies is limited due to the unavailability of commercial antibodies that recognize specific structures and proteins in trypanosomatids.
Among trypanosomatids, T. cruzi has one of the most complex life cycles involving various development stages between vertebrate and invertebrate hosts 19. During the T. cruzi life cycle, at an early stage of mammalian in...

Watch this Scientific Journal Video about Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions at JoVE.com

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

In this protocol, two antibodies from the same host can be used together in the immunofluorescence assay to study host cell and pathogen interactions. Since there are only few commercial antibodies available to recognize specific cell structures and proteins in parasites, this protocol can be very useful. This is an easy-to-perform double-labeling immunofluorescence protocol that uses polyclonal and monoclonal antibodies raised in the same species.Many researchers may be unaware that this is possible. This approach helps when the source of antibodies is limited. It can be used to detect the pathogens in the host cell proteins in infected host cells and can also be applied to free living organisms.This is a simple protocol, require one blocking step between the first and second pair of antibodies. The procedure will be demonstrated by Dr.Camila Gachet-Castro and PhD student Lays Trajano-Silva from my laboratory. Three days after infecting LLC-MK2 cells with Trypanosoma cruzi, collect the supernatant from the infected cells in a 15 milliliter cell culture conical tube.Centrifuge the sample to lower cell debris, then place the tube at room temperature to allow the trypomastigotes to swim to the supernatant. After 10 minutes, collect the supernatant in a new conical tube, then centrifuge the sample and discard the supernatant before resuspending the pellet containing the parasites in complete RPMI medium. Add LLC-MK2 cells in 24-well plates containing UV sterilized rounded coverslips and allow the&m to settle for 16 hours.Then to infect the cells, add supernatant containing T.Cruzi to each well and incubate for six hours. Next, wash the coverslips containing the infected and non-infected cells five times with PBS, then fix the cells with 2%paraformaldehyde in PBS. After 10 minutes, wash the coverslips three times for five minutes each with PBS and then permeabilize the coverslips with non-ionic detergent for 10 minutes.After three five-minute PBS washes, incubate the coverslips in blocking solution for 30 minutes, followed by another 30-minute incubation with either a mouse monoclonal or polyclonal antibody. After PBS washes, incubate the coverslips for 30 minutes with secondary antibody in blocking solution and phalloidin to stain actin filaments in the host cell. Then wash the coverslips with PBS three times again before applying a small amount of anti-fade mounting reagent with DAPI medium to the surface of the slide.Using forceps, gently tilt the coverslip in the medium to prevent bubble formation. For double labeling, after incubating the coverslips in blocking solution as demonstrated previously, incubate them in the mouse polyclonal antibody for 30 minutes. Next, wash the coverslips three times for five minutes each with PBS before incubating them with the secondary antibody for 30 minutes.Then following three PBS washes, perform a second blocking step with AffiniPure rabbit anti-mouse IgG diluted in blocking solution. After 30 minutes, wash the coverslips with PBS again before incubating them with the mouse monoclonal antibody for another 30 minutes. Next, after three PBS washes, incubate the coverslips with goat anti-mouse IgG 2B antibody and phalloidin.Then following three PBS washes, apply anti-fade mounting reagent as demonstrated previously. For triple labeling, after blocking the coverslips and incubating with the mouse polyclonal antibody, followed by goat anti-mouse antibody, start a new incubation with rabbit polyclonal antibody in blocking solution for 30 minutes. Then after three PBS washes, incubate the coverslips with goat anti-rabbit antibody for 30 minutes.Next, following three PBS washes, perform a blocking step with AffiniPure rabbit anti-mouse antibody diluted in blocking solution for 30 minutes. Then wash the coverslips again before incubating with any mouse monoclonal IgG subclass antibody for 30 minutes. Following three PBS washes, incubate the coverslips for 30 minutes with a specific pair of goat anti-mouse IgG subclass antibodies.After incubation, wash the coverslips three times for five minutes each with PBS. Analyze the immunofluorescent samples using a confocal microscope with a 63X oil immersion objective and detect the fluorescence with a photomultiplier tube and hybrid detector. Then acquire all confocal images with separated channels and perform image processing using Adobe Photoshop.These confocal microscopy images show results with the control experiments of infected and non-infected cells highlighting the specificity of the antibodies in the host cell and the internalized parasite. The mouse polyclonal antibody anti-Trypanosoma cruzi FAZ recognized the T.cruzi giant protein in the flagellum attachment zone at the parasite flagellum, but not in the host cell. Distribution of the heterogeneous nuclear ribonucleoprotein A1 in the nuclei was observed using a commercial mouse monoclonal antibody that recognizes only the host mammalian cells, but not the parasite.Also, the host and parasite nuclei and the parasite kinetoplasts were stained with DAPI and host F-actin was stained with phalloidin conjugated to Alexa 594, confirming the specificity of the antibodies. Double-labeling immunofluorescence shows the protein distributions in the host and the parasite analyzed by confocal microscopy. The labeling suggested no cross-reaction between antibodies using this methodology.In summary, the protocol described here to study host-pathogen interactions presents a basic and elaborate technique that can be adapted to any combination of antibodies. This approach makes immunofluorescence cost effective and can help study the localization of two or more proteins of interest when few antibodies are available.

double-labeling-immunofluorescence-using-antibodies-from-same-species

Research

JoVE Journal

Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...