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Published on: October 24, 2010
Imaging Flow Cytometry Analysis of CEACAM Binding to Opa-Expressing Neisseria gonorrhoeae
Lacie M Werner1, Allison Palmer1, Asya Smirnov1
1Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, 22903, USA.
Abstract:
Human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are a family of receptors that mediate intercellular interactions. Pathogenic bacteria have ligands that bind CEACAMs on human cells. Neisseria gonorrhoeae (Gc) encodes numerous unique outer membrane opacity-associated (Opa) proteins that are ligands for one or more CEACAMs. CEACAMs that are expressed on epithelial cells facilitate Gc colonization, while those expressed on neutrophils affect phagocytosis and consequent intracellular survival of Gc. Since Opa protein expression is phase-variable, variations in receptor tropism affect how individual bacteria within a population interact with host cells. Here we report the development of a rapid, quantitative method for collecting and analyzing fluorescence intensity data from thousands of cells in a population using imaging flow cytometry to detect N-CEACAM bound to the surface of Opa-expressing Gc. We use this method to confirm previous findings regarding Opa-CEACAM interactions and to examine the receptor-ligand interactions of Gc expressing other Opa proteins, as well as for other N-CEACAM proteins. © 2020 International Society for Advancement of Cytometry.
Insights
This study introduces a new imaging flow cytometry method to analyze bacterial interactions with human CEACAM receptors. The technique quantifies how Neisseria gonorrhoeae Opa proteins bind to CEACAMs on host cells, advancing our understanding of bacterial colonization and host defense.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) mediate cell interactions.
- Pathogenic bacteria, like Neisseria gonorrhoeae (Gc), utilize ligands such as Opa proteins to bind CEACAMs on host cells.
- CEACAMs on epithelial cells aid Gc colonization, while those on neutrophils influence phagocytosis and bacterial survival.
Purpose of the Study:
- To develop a rapid, quantitative method for analyzing CEACAM-host cell interactions with bacteria.
- To detect and quantify N-CEACAM bound to the surface of Opa-expressing Gc.
- To validate previous findings and explore new Opa-CEACAM and Gc-CEACAM interactions.
Main Methods:
- Development of a quantitative imaging flow cytometry method.
- Analysis of fluorescence intensity data from thousands of cells.
- Detection of N-CEACAM bound to Opa-expressing Gc surface.
Main Results:
- Successful development of a rapid and quantitative assay for bacterial-host receptor interactions.
- Confirmation of previously established Opa-CEACAM binding specificities.
- Characterization of interactions involving diverse Opa proteins and N-CEACAM variants.
Conclusions:
- The new method provides a powerful tool for studying bacterial adhesin-receptor interactions.
- This approach enhances understanding of how bacterial phase-variable proteins influence host cell tropism.
- The findings contribute to insights into Gc pathogenesis and host immune responses.
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