detection of bacterial adherence to host cells using fluorescence imaging

Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

For bacterial adherence assay, wash the previously cultured A549 cell monolayers three times by adding 100 microliters of warm PBS to each well, and gently pipette up and down. Then, discard PBS. Alternatively, after adding PBS, wait for ten seconds and then, remove PBS using vacuum.
To determine the kinetics of bacterial association, overlay the cells with 100 microliters of desired concentrations of bacterial suspension with different multiplicity of infection. Spin down the bacteria and incubate the infected A549 cells at 37 degrees Celsius and 5% carbon dioxide for an additional one hour. Remove the unbound bacteria by washing the monolayers five times with warm PBS as demonstrated.
Next, fix the cells by adding 100 microliters of 4% formaldehyde in all the wells of a 96-well plate on the ice. After 15 minutes, wash the plate three times with PBS to remove the fixation solution, staining the nuclei with 50 nanograms per milliliter DAPI stain for 10 minutes at room temperature.
After washing the cells with PBS, cover the infected A549 cells with 100 microliters of PBS to avoid drying, and then, store the plate at 4 degrees Celsius for up to two days in the dark or proceed for imaging.

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Host Defense Mechanism

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1. Bacterial adherence and host cell staining
<ol>
	<li>First, wash the seeded A549 cell monolayers three times with warm 1x PBS. For each wash, add 100 &#956;L of 1x PBS to each well, gently pipette up and down three times, dispose of 1x PBS or wait for 10 s after addition, and then vacuum to remove 1x PBS. To determine the kinetics of bacterial association, overlay the cells with 100 &#181;L of desired concentrations of bacterial suspension with different MOIs (0, 1, 10, and 100). Spin down the bacteria at 200 x g for 10 min and incubate the infected A549 cells at 37 &#176;C, 5% CO2 for an additional 1 h. 
	NOTE: In this experiment, each condition had a technical triplicate.</li>
	<li>Remove the unbound bacteria by washing the monolayers five times with warm 1x PBS as described above. Add 100 &#181;L of 4% formaldehyde (in 1x PBS) into each well of the 96-well plates to fix the cells. Let the plates sit on ice for 15 min and then wash off the fixation solution using 1x PBS three times.</li>
	<li>To stain the nuclei, add 50 ng/mL of 4&#8242;,6-diamidino-2-phenylindole (DAPI) and incubate it for 10 min at RT. After incubation, wash the wells three times using 1x PBS. Cover the infected A549 cells with 100 &#181;L of 1x PBS to avoid drying. Process for the next step or store the plate at 4 &#176;C for up to 2 days in the dark.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS</td>
			<td>VWR</td>
			<td>45001-130</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher</td>
			<td>62248</td>
			<td>Host cell staining dye</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3882</td>
			<td>Half area well, flat clear bottom</td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL 185</td>
			<td>Mammalian cell line</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>OD600 DiluPhotometer</td>
			<td>IMPLEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Typical soy broth</td>
			<td>Growcells</td>
			<td>MBPE-4040</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12k medium</td>
			<td>ATCC&#160;</td>
			<td>302004</td>
			<td>A549 cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Yang, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells.</a> J. Vis. Exp. (2021)
This video demonstrates the bacterial adherence assay to evaluate GFP-tagged bacterial adherence to epithelial cell culture to study the dynamics of host-pathogen interactions using fluorescence microscopy-based imaging.

1. Bacterial adherence and host cell staining

	First, wash the seeded A549 cell monolayers three times with warm 1x PBS. For each wash, add 100 &#956;L ...

Take a multi-well plate containing adherent epithelial cell monolayer susceptible to bacterial adherence.
Add varying numbers of fluorescent protein-tagged Pseudomonas aeruginosa, a pathogenic bacterium, to achieve the desired bacteria-to-epithelial cell ratio.
Centrifuge to bring the bacteria closer to the epithelial cells, facilitating interactions.
Type IV pili, proteinaceous fibers on the bacteria, play a pivotal role by binding to specific glycolipids on the epithelial cells, mediating bacterial adherence to the host cells.
Post-incubation, wash with a buffer to remove non-adherent bacteria. Fix the cells to preserve the cell structure.
Add DAPI, a fluorescent stain that binds strongly to A-T-rich DNA regions, staining the nucleus. Cover the cells with buffer to prevent cell drying.
Acquire images of the GFP-labeled bacteria attached to epithelial cells with DAPI-stained nucleus. Enumerate the adherent bacteria on a single epithelial cell.
Increased adherent bacteria per host cell in a dose-dependent manner, enables quantification of host-pathogen interactions.

33 ビュー. 蛍光イメージングによる宿主細胞への細菌付着の検出

ビデオ: 蛍光イメージングによる宿主細胞への細菌付着の検出 - 実験

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

に関与する宿主因子のハイスループットスクリーニングのための顕微鏡ベースのアッセイ<em&gt;ブルセラ</em&gt; HeLa細胞の感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

JoVE Journal

免疫・感染学

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

アメーバを用いた急速なイメージに基づく細菌の病原性試験

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

生物工学

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

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Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging