Use of a modified plaque assay for live observation of Shigella flexneri infection in HeLa cells.

Session

Poster Session 1

Authors

Karla Valenzuela (1), Brendan Leung (1), Zhenyu Cheng (1)

Affiliations

1. Dalhousie University

Keywords

Shigella flexneri

Plaque assay

Live Microscopy

Abstract text

Shigella flexneri is an intracellular bacterium that infects colonic epithelial cells causing inflammatory colitis in humans. Shigella multiplies in the host cell cytoplasm and disseminate into adjacent cells by induction of an actin tail. Shigella pathogenesis has been extensively studied in cell lines as there are no animal models available. Plaque assays are commonly used to determine the titer of animal viruses that can induce lysis of mammalian cells. But they can also be implemented for the study of Shigella pathogenesis. Plaques or areas of infection are formed as Shigella spreads in the monolayer. To date, plaque assays are performed in the presence of a dense agarose overlay that restricts extracellular diffusion of Shigella but also impedes real time observation of infection. We describe a modified plaque assay that allows live observation of Shigella cell-to-cell spreading and cell death induction. Fluorescent HeLa cells were infected with fluorescent bacteria and the cells were covered with Methocel, a translucent yet dense substance, supplemented with DAPI to analyze cell death. Control cells were covered with media without Methocel. Cell-to-cell spreading of Shigella was recorded in real time and also plaque number and area were quantified. We found fewer secondary areas of infection in the cells covered with Methocell compared to cells without it. Also, the area of the plaques was smaller and correlated better to the area of cell death. These indicates that the Methocel overlay can improve detection of primary infection points, genuine cell-to-cell spreading and cell death induction. Additionally, we successfully recorded Shigella spreading in real time. Stable expression of F-tractin-GFP in the HeLa cells allowed observation of actin tail induction by Shigella. The modified plaque assay described here can also be used to monitor other aspects of Shigella virulence and host cell responses to virulence factors. Furthermore, this assay could be implemented to study other intracellular pathogens, such as, Listeria, Rickettsia and Chlamydia. 

Shigella flexneri, a Gram-negative intracellular bacterium, is the causative agent of Shigellosis. This pathogen infects colonic epithelial cells causing inflammatory colitis in humans. Despite many efforts there are not licenced vaccines available to prevent Shigellosis and some species have developed antibiotic resistance. Further studies are needed to better understand Shigella pathogenesis in order to develop effective antimicrobial therapies. Since Shigellosis cannot be fully recapitulated in any small animal model, cultured cells have served as model system to study the different aspects of Shigella virulence. Plaque-based assays are commonly used to determine the titer of animal viruses that can induce lysis of mammalian cells. As the virus spreads, cells are lysed forming plaques on the monolayer of cells that can be counted and measured. Plaque assays have been adapted to study Shigella cell-to-cell spreading. These assays are performed in the presence of a dense agarose overlay that restricts extracellular diffusion of Shigella, followed by fixation and staining of the monolayers. In these conditions bacterial cell-to-cell spreading cannot be observed in real time. Here we describe a modified plaque assay that allows live observation of Shigella cell-to-cell spreading and cell death induction. 

F-tractin-GFP expressing HeLa cells were used in this study. Cells were seeded at a density of 5 x 10⁵ cells/mL on 96-well plates or 4 well glass-bottom dishes. Next day, a fluorescent ds-RED Shigella culture that had reached an OD₆₀₀ of 0.5 was diluted 1 in 1000 times in DMEM. Cells were infected with the diluted culture for 30 min. After this time, cells were treated with gentamycin 100 ug/ml diluted in DMEM for 15 min to eliminate extracellular bacteria. The monolayers were then washed three times with PBS and covered with an overlay made with 0.4% Methocel diluted in Fluorobrite, supplemented with DAPI (0.1 ng/mL) to identify dead cells. Cells incubated with Fluorobrite supplemented with DAPI only were used as controls. The monolayers were imaged using a spinning disk or a widefield microscope every 15 minutes or at a final time point respectively. Image analysis were performed using ImageJ 2.1.0.

Shigella induced the formation of plaques in HeLa cells regardless of the presence of Methocel after 10 to 12 hours of infection. However, the monolayers that were not covered with Methocel showed an increase in the number of secondary areas of infection compared to Methocel treated monolayers (64% and 11% respectively). Also, the area of the plaques was smaller in the cells overlayed with Methocel. Induction of cell death was observed in both conditions and the size of the area of dead cells correlated with the area of Shigella spreading. These results show that Methocel can reduce Shigella spreading by blocking diffusion in the media. Therefore, Methocel overlay can improve detection of primary infection points, genuine cell-to-cell spreading and cell death induction. Additionally, by using this methodology we successfully recorded Shigella spreading in real time. Shigella infects a new cell every 30 to 45 min during the first 3 hours of infection. After this time Shigella spreading decreases showing a new infection every 2 to 2.5 hours. Stable expression of F-tractin-GFP in the HeLa cells allowed observation of the various modifications that Shigella induces in the cell actin cytoskeleton. As previously described, we observed that actin tail induction propels the bacterium from one cell to the next one propagating infection.    

The modified plaque assay described here allowed observation of Shigella spreading in real time as well as reliable quantification of number and size of plaques. This method also allowed easy quantification of cell death caused by Shigella. In contrast to the agarose regularly used in plaque assays, Methocel has the advantage of being translucent, thus permitting live observation of the infection process. Also, the methodology we describe does not require a fixation step, thus making the assay simpler and more accurate to determine the areas of Shigella dissemination. Similarly, this protocol could be used to examine other aspects of Shigella virulence and host cell responses to virulence factors. Furthermore, the modified plaque assay could be implemented to study other intracellular pathogens, such as, Listeria, Rickettsia and Chlamydia.