Abstract
Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are incubated with listeria for 30 min but in invasion assay the cell growth is arrested at several time points after infection to monitor the growth and survival rate of listeria in cells.
Keywords: Adhesion assay, Invasion assay, Listeria, Caco-2 cells
Background
Listeria monocytogenes being a facultative intracellular bacterium can enter, survive and multiply in both phagocytic and non-phagocytic cells and this property of the bacterium has been extensively studied and understood. Being a foodborne pathogen it enters the blood stream in humans via intestines by crossing intestinal barriers. Thus human intestinal cells are used as an in vitro medium to study adhesion and intracellular survival of Listeria monocytogenes. Human colon adenocarcinoma cells, Caco-2 cells have been used extensively as a model for intestinal barrier (Angelis and Turco, 2011).The protocol described in this article explains the procedure to grow and maintain Caco-2 cells and then infect them to study adhesion and invasion properties of listerial species. This protocol can also be used with minor changes for cells like HT-29 (human colorectal adenocarcinoma cells) and Tc7 cells (a subclone of the Caco-2 cell line have) which have also been used to study Listeria. These assays are generally used to compare the pathogenicity of listerial mutants with wide type(WT) strains (Reddy and Lawrence, 2014). Adhesion assay is straight forward wherein the bacteria are incubated with Caco-2 cells for 30 min and the bacterial counts for mutants and WT strains are compared to observe any alteration in adhesion properties as a result of mutation. Bacterial counts obtained at different time points during invasion assay give more information about the survival of listeria in the human cells and the comparison of these counts between mutants and WT strains gives information about the changes in the adaptation of listeria after mutation in human cells. This protocol has been successfully used previously to study the adhesion and invasion properties of listerial strains (Jaradat and Bhunia, 2003; Lecuit, 2005; Sambuy et al., 2005; Reddy et al., 2016).
Materials and Reagents
Equipment
Procedure
Data analysis
Note: Data can be analyzed by performing Student’s t-test using Microsoft Excel.
Notes
Recipes
Acknowledgments
This protocol was adapted from the previously published studies (Reddy et al., 2016). This project was funded by USDA ARS Agreement #58-6402-2729, which is operated under USDA CRIS project MIS501170, ‘Mississippi Center for Food Safety and Post-Harvest Technology’.
References
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