This work was supported by Massachusetts General Hospital Department of Medicine Internal Funds (J.M.T., M.K.M, M.L.C., J.M.V.), National Institute of Biomedical Imaging and Bioengineering grant T32EB006348 (C.E.C.), Massachusetts General Hospital's Center for Computational and Integrative Biology development fund and AI062773 (R.J.H.), grants AI062773, DK83756, and DK 043351 (R.J.X.), NSF 0643745 (M.J.L.), NIH R21CA133576 (M.J.L.), and National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) AI057999 (J.M.V.). We thank Nicholas C. Yoder for helpful discussions, and Charles Felts (RPI, Inc.) for technical assistance.

1. Culture conditions of pathogens for optical trapping
<ol>
	<li>Grow A. fumigatus (B-5233/RGD12-8) on a semi-solid agar media containing SBD (Sabouraud dextrose) at 30&deg;C for 3 days. </li>
	<li>Grow C. albicans (SC5314) in YPD (Yeast-Peptone Dextrose) liquid culture containing 100 &mu;g/mL ampicillin overnight in a shaker incubator at 30&deg;C.</li>
</ol>
2. Preparation of pathogens for fluorescent labeling
<ol>
	<li>Harvest desired amount of pathogens and transfer to a 1.5 mL reaction tube.</li>
	<li>Add 300 &mu;L of phosphate buffered saline (PBS) to reaction tube.</li>
	<li>Sonicate mixture for 30 seconds.</li>
	<li>Centrifuge at 4000 rpm for 1 minute.</li>
	<li>Aspirate supernatant, leaving pellet undisturbed.</li>
	<li>Repeat (2.4) and (2.5) two more times.</li>
	<li>Resuspend in 500&mu;L of PBS.</li>
</ol>
3. Labeling of pathogens with fluorescent dye
<ol>
	<li>Dissolve 1 mg of dye of interest (e.g. Alexa Fluor 488, Alexa Fluor 647) in 100 &mu;L dimethylformamide (DMF) (concentration of 10 mg/mL).</li>
	<li>Add 3 &mu;L of dye mixture to reaction tubes containing washed pathogens.</li>
	<li>Rotate or shake sample at 37&deg;C for 1 hour.</li>
	<li>Wash sample with PBS 3X by centrifuging at 4000 rpm for 1 minute.</li>
	<li>Resuspend in 300 &mu;L of PBS.</li>
</ol>
4. Harvesting T-cells from whole blood 
<ol>
	<li>Obtain whole blood (fresh).</li>
	<li>Warm blood, PBS + 2% fetal bovine serum (FBS), and histopaque to room temperature.</li>
	<li>Add RosetteSep Human CD4+ T Cell Enrichment Cocktail at 50 &mu;L/mL of whole blood.</li>
	<li>Rotate sample and incubate for 20 minutes at room temperature.</li>
	<li>Dilute sample with an equal volume of PBS + 2% FBS and mix gently.</li>
	<li>Layer diluted sample on top of histopaque, minimizing mixing</li>
	<li>Centrifuge for 20 minutes at 1200 x g at room temperature with the brake off.</li>
	<li>Remove the enriched cells.</li>
	<li>Wash the enriched cells 2x with PBS + 2% FBS solution.</li>
	<li>	Lyse red blood cells for 2 minutes with red blood cell lysing buffer</li>
	<li>	Add 10 mL of PBS + 2% FBS and centrifuge lysed red blood cells at 1500 rpm for 5 minutes</li>
	<li>	Aspirate supernatant, careful not to disturb the pellet</li>
	<li>	Resuspend in IMDM media containing 10% fetal bovine serum</li>
</ol>
5. Preparation of RAW 264.7 macrophages into chamber slides
<ol>
	<li>Prepare DMEM (Dulbecco's modified Eagle's medium) to contain 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.</li>
	<li>Warm media, trypsin, and PBS in warm bath to 37&deg;C.</li>
	<li>Wash plate 2x with PBS</li>
	<li>Aspirate PBS between each wash.</li>
	<li>Add 5 mL trypsin to plate to cover surface (for a 10 cm tissue culture plate). </li>
	<li>Incubate for 5 min at 37&deg;C.</li>
	<li>Gently knock side of plate to detach cells from plate surface. Be careful not to splash trypsin outside of the plate.</li>
	<li>Add 5 mL or equivalent amount of media to trypsin.</li>
	<li>Aspirate mixture into a reaction tube.</li>
	<li>	Centrifuge at 1000 x g for 3 minutes.</li>
	<li>	Aspirate media, careful not to disturb the pellet.</li>
	<li>	Resuspend in 10 mL of media.</li>
	<li>	Add 400 &mu;L of media to each chamber of the chamber slide.</li>
	<li>	Add 5 &mu;L of cell suspension to each chamber.</li>
	<li>	Grow overnight in incubator at 37&deg;C with 5% CO2.</li>	
</ol>
6. Addition of pathogens to sample
<ol>
	<li>Pipet 5-10 &mu;L of labeled pathogens of interest (from steps 1-3) into chamber.</li>
	<li>Mix thoroughly by pipeting up and down, careful not to touch the bottom of the chamber to disturb the adhered macrophages.</li>
</ol>
7. Loading sample onto spinning disk confocal microscope (in video, scan through components)
<ol>
	<li>Turn on all components to spinning disk confocal microscope.</li>
	<li>Align microscope for DIC imaging.</li>
	<li>Remove chamber slide from incubator.</li>
	<li>Insert chamber slide into specialized stage.</li>
	<li>Remove top of chamber slide (necessary for DIC imaging).</li>
</ol>
8. Preparation for optical trapping (in video, scan through components and how it's integrated into microscope)
<ol>
	<li>Turn on shutter for optical trap.</li>
	<li>Turn on IR laser.</li>
	<li>Open shutter (on the laser) to optical trap.</li>
	<li>Confirm shutter in front of IR laser is closed by checking with IR card.</li>
</ol>
9. Selection and manipulation of pathogen with optical trap
<ol>
	<li>Focus on macrophages on adhered slide.</li>
	<li>Find pathogens freely fluctuating in solution adjacent to macrophages. </li>
	<li>Move stage such that the pathogen is in the vicinity of the trap.</li>
	<li>Open shutter and engage the trap.</li>
	<li>Move sample to bring macrophages into contact with the stationary trapped pathogen.</li>
	<li>Image with spinning disk confocal microscope, either in DIC, fluorescence, or combination of both. Typically, trapping is done in DIC, and real-time imaging is done with fluorescence.</li>
</ol>
10. Representative Results: 
To exert full spatial and temporal control of pathogens and beads in an in vivo and in vitro environment, we designed a custom-built trapping apparatus integrated onto a spinning disk confocal microscope (schematic shown in Fig. 1). At full strength, the laser provided 350 mW of power and after coupling the light with the various optical components in the optical trap apparatus, &tilde;80 mW of power at the TIRF objective, as measured with a power meter, was used to form the trap in the chamber. 
In order to position an object in the chamber relative to the trapped object, the stage was moved while holding the trapped object stationary with the trapping laser. The stage was moved at slow enough velocities, so that the drag force on the trapped particle did not exceed the maximum trapping force. 
Separate populations of C. albicans (typical size - &tilde;5 &mu;m) were labeled with each of three colors (AF488, AF568, and AF647, corresponding to green, blue, and red in the figure, respectively) to illustrate imaging with multiple fluorescence channels while simultaneously optically trapping the pathogen. A single C. albicans was trapped and moved in a square pattern through a cluster of other yeast, as indicated by gray arrows, demonstrating the ability to capture and manipulate the specific location of a single pathogen chosen by the operator even in a crowded environment (Fig. 2A). 
To illustrate further the flexibility of this system to trap the different shape morphologies exhibited by pathogenic organisms, the optical tweezer was also able to hold and situate a C. albicans particle with a pseudohyphae. The C. albicans labeled with AF647 (red) was moved along a trajectory as outlined by the white arrow and placed next to fluorescent GFP-LC3-RAW cells (Fig. 2B). The yeast portion of the C. albicans was trapped as the pseudohyphae trailed along.
We also positioned Aspergillus fumigatus next to a RAW mouse macrophage cell in order to analyze the absolute time frame of phagocytosis with this particular cell line and pathogen (Fig. 3A). Once the pathogen makes contact with the cell, the trap is turned off, and time-lapse imaging is employed to observe the subsequent cellular events (Fig. 3B). The optical trap was also used to capture primary T-cells isolated from blood and directed adjacent to beads coated with anti-CD3 antibody in order for the T-cell to form an immunological synapse with the bead (Fig. 4), show the additional versatility to directly trap and manipulate immune cells. 
<img src="/files/ftp_upload/3123/3123fig1.jpg" alt="Figure 1"/> Figure 1. Overview and schematic of combined optical trap and spinning disk confocal microscope setup. Instrument layout showing the trapping beam (purple), illumination path for brightfield imaging (orange), fluorescence excitation beam (red), fluorescence emission (green), charge-coupled device (CCD) camera, electron-multiplying charge coupled device (EM-CCD) camera, dichroic mirrors (D1 and D2), mirrors (M1 and M2), and lenses (L1, L2, L3, L4). All other components of the trap-confocal microscope system are labeled in the figure.
<img src="/files/ftp_upload/3123/3123fig2.jpg" alt="Figure 2"/> Figure 2. (A) Fluorescence images of trapped and manipulated C. albicans. A trapped fluorescently-labeled (Alexa Fluor 488 (AF488), blue) organism in a field of other fluorescently-labeled C. albicans (AF568, green and AF647, red). The stage is moved around the trapped, blue CA particle as indicated by the gray arrows. (B) Trapped pseudo-hyphal form of C. albicans next to GFP-LC3 RAW cell. Fluorescently labeled psuedohyphal form of CA (red, AF647) optically trapped and moved adjacent to GFP-LC3 expressed RAW macrophages. The CA organism is moved along the trajectory as indicated by the white arrow.
<img src="/files/ftp_upload/3123/3123fig3.jpg" alt="Figure 3"/> Figure 3. Trapping and positioning of A. fumigatus next to a phagocytic RAW cell. (A) Brightfield images of a trapped A. fumigatus, as indicated by the white arrow, moved and positioned along the path as indicated by the red arrow. The trapped pathogen is slightly out of focus due to the trap pushing the organism slightly above the focal plane. A. fumigatus is moved until it is placed adjacent to the desired RAW cell. (B) Fluorescence imaging of phagocytosis of A. fumigatus by RAW cell. After the trapped pathogen is placed next to a RAW cell, the phagocytosis process is activated. At 30 s, the membrane of the RAW cell starts to change and form a cup around the particle. At 60 s, the cup is fully formed. From 90s to 150s, the A. fumigatus is engulfed, and by 180 s, the particle is fully internalized
<img src="/files/ftp_upload/3123/3123fig4.jpg" alt="Figure 4"/> Figure 4. Trapping primary T-cell to form synapse with anti-CD3 coated beads. DIC images of a primary T-cell moved by the optical trap to a position next to a bead coated with anti-CD3 antibody. The cell then forms an immunological synapse, which cannot be easily discerned in the images.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev Sci Instrum. 75, 2787-2809 (2004).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+neutral+particles+using+lasers.">Optical trapping and manipulation of neutral particles using lasers.</a> Proc Natl Acad Sci USA. 94, 4853-4860 (1997).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+and+trapping+of+particles+by+radiation+pressure.">Acceleration and trapping of particles by radiation pressure.</a> Phys Rev Lett. 24, 156-159 (1970).</li><li>Ashkin, A., Dziedzic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+viruses+and+bacteria+Science.">Optical trapping and manipulation of viruses and bacteria Science.</a> Nature. 235, 1517-1520 (1987).</li><li>Khalil, A. 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No conflicts of interest declared.

In this work we use an optical trap to capture pathogens with dimensions between 3 &mu;m - 5 &mu;m. Although pathogens of interest to our lab typically have these dimensions, the optical tweezer system described here is flexible to trap a large range of sizes. Indeed optical traps have been used to capture particles ranging from single atoms to cells approximately 10 &mu;m in diameter. Additionally, this optical trapping system was able to capture particles of various shapes: spherical, elliptical, and extremely elon...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>A. fumigatus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Albino strain, B-5233/RGD12-8, gift from K.J. Kwon-Chung, NIH</td>
		</tr>
		<tr>
			<td>C. albicans</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>SSY50-B mutant, gift from Eleftherios Mylonakis, MGH; SC5314 strain, gift from Gerald Fink, Whitehead Institute</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A20000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>Sigma</td>
			<td>D4551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fresh blood</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Gift from R.J.W. Heath, MGH, HMS</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>Model Ti-E </td>
		</tr>
		<tr>
			<td>Trapping laser, ChromaLase</td>
			<td>Blue Sky Research</td>
			<td>CLAS-106-STF02-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence excitation laser</td>
			<td>Coherent</td>
			<td>&nbsp;</td>
			<td>Model Innova 70C </td>
		</tr>
		<tr>
			<td>Breadboards for trapping components</td>
			<td>Thorlabs</td>
			<td>MB1224, MB1218</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical air table</td>
			<td>Technical Manufacturing Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electronic shutter with pedal control</td>
			<td>Uniblitz</td>
			<td>&nbsp;</td>
			<td>Purchased from Vincent Associates, Rochester, NY</td>
		</tr>
		<tr>
			<td>Singlemode optical fiber</td>
			<td>Oz Optics</td>
			<td>PMJ-3S3S-1064-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber positioner</td>
			<td>Thorlabs</td>
			<td>PAF-X-5-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber collimator</td>
			<td>Oz Optics</td>
			<td>HPUCO-23-1064-P-25AC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lenses for telescope</td>
			<td>Thorlabs</td>
			<td>AC254-150-B</td>
			<td>Focal length of 150 mm</td>
		</tr>
		<tr>
			<td>Translation stages (x, y, z)</td>
			<td>Newport</td>
			<td>M-461-XYZ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR dichroic mirror</td>
			<td>Chroma</td>
			<td>ET750-sp-2p8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Objective lens (100X)</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>NA = 1.49, oil immersion, TIRF objective</td>
		</tr>
		<tr>
			<td>Confocal head</td>
			<td>Yokogawa</td>
			<td>CSU-XI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polarizer</td>
			<td>Nikon</td>
			<td>MEN51941</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Wollaston prism</td>
			<td>Nikon</td>
			<td>MBH76190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EM-CCD camera</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CCD camera (ORCA ER)</td>
			<td>Hamamatsu</td>
			<td>C4742-80-12AG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Ludl</td>
			<td>99A353</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Sutter</td>
			<td>LB10-NWE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chambered coverglass</td>
			<td>Lab-Tek/Nunc</td>
			<td>155409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynabeads</td>
			<td>Invitrogen</td>
			<td>111-51D</td>
			<td>Coated with anti-CD3</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's medium (DMEM)</td>
			<td>Invitrogen/Gibco</td>
			<td>10313</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen/Gibco</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
			<tr>
			<td>L-glutamine</td>
			<td>Invitrogen/Gibco</td>
			<td>25030-081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (HyClone)</td>
			<td>ThermoScientific</td>
			<td>SH30071.03</td>
			<td>&nbsp;</td>
		</tr>		
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

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

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 ...

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JoVE Journal

Immunology and Infection

12.0K vistas.  Massachusetts General Hospital, Harvard Medical School. Se describe un método para seleccionar de forma individual, manipular, y los agentes patógenos de imágenes en vivo con una trampa óptica acoplada a un microscopio de disco giratorio. La trampa óptica proporciona un control espacial y temporal de los organismos y los coloca junto a las células huésped. Microscopía de fluorescencia captura dinámica de interacciones intercelulares con perturbación mínima para las células.

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

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 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

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 ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...