eoe sub articles

EoE Sub Articles

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols may be used for a number of anaerobic pathogens. Although HUVECs are used, this protocol may be used for other eukaryotic cells both immune and non-immune.
1. Anaerobic Chamber Use and Maintenance
Note: P. gingivalis is an anaerobe sensitive to normal levels ...

a bacterial infection assay for studying anaerobic bacteria-host cell interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

<img alt="Figure 1" src="/files/ftp_upload/21795/21795_Figure1.jpg" /> 
Figure 1. Anaerobic vinyl chamber and its components. (A) A vinyl anaerobic chamber sealed completely from atmospheric oxygen provides workspace for two individuals at a time (32 in x 78 in). It contains an incubator set at 37 &#176;C (back middle). (B) An airlock is used for the transfer of items from the lab environment to the anaerobic chamber. Pictured is an ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

Begin with a culture of endothelial cells.
Replace the media with anaerobic media. Incubate in an anaerobic chamber for temperature equilibration.&#160;
Add Porphyromonas gingivalis, a pathogenic anaerobic bacterium, and incubate briefly.
The bacteria possess proteinaceous projections, known as fimbriae, which bind to specific adhesion molecules on the endothelial cells, facilitating bacterial adherence.
This binding triggers intracellular signaling cascades, resulting in actin cytoskeleton rearrangement and bacterial internalization into phagosomes.
Post-incubation, remove media. Wash with an anaerobic buffer to remove non-adhered extracellular bacteria.
Add a mild detergent to permeabilize the host cell membranes and induce cell lysis, releasing internalized and surface-attached bacteria without causing damage.
Dilute the lysate-containing bacteria with media and prepare serial dilutions.
Plate these dilutions on blood agar plates. Incubate under anaerobic conditions to allow Porphyromonas gingivalis to proliferate and form colonies.
Post-incubation, count the colony-forming units (CFUs) on each plate to quantify surface-attached and internalized bacteria, indicating bacteria-host cell interactions.