In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Summary

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Abstract

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells¹. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'². Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories^3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.

Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787⁵, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before⁶.

Protocol

1. Preliminary: Bacterial strains and epithelial cells.

Manipulations of cells and bacteria are performed aseptically, under a laminar flow hood.

1. Freshly isolate the E. coli strains 2787, 2787ΔaidA, and C600 from glycerol stocks on Lysogeny Broth (LB) agar plates (1% tryptone, 0.5% sodium chloride, 0.5% yeast extract, 1.5% agar) and grow at 37°C. To minimize variability in the assay, it is advised to always use freshly plated strains and to keep the strains at 4°C on sealed Petri plates only for a maximum of a couple of weeks.
2. Culture HEp-2 cells (ATCC CCL-23) in high glucose Dulbecco’s Modified Eag....

Discussion

This protocol describes a standard bacterial adherence assay that can be modified to study invasion (e.g. using gentamicin protection assay³). The counting of colony forming units allows quantification, by comparison with approaches relying on standard visualization techniques under a microscope, such as Giemsa staining. The latter only gives a qualitative view of adhesion but is often a useful complement since it can differentiate various patterns of adhesion and give mechanistical insights⁹. For i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High glucose DMEM	GIBCO, by Life Technologies	12430-054
DDPBS	GIBCO, by Life Technologies	14190-144
Bovine Growth Serum	Hyclone	SH3054
Penicillin/Streptomycin	GIBCO, by Life Technologies	15140-148
24 well plates	Corning	3337
Triton X-100	Fisher Scientific	BP151-100