Imaging Flow Cytometry Analysis of CEACAM Binding to Opa-Expressing Neisseria gonorrhoeae.

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.

Quantifying Carcinoembryonic Antigen-like Cell Adhesion Molecule-Targeted Liposome Delivery Using Imaging Flow Cytometry.

Carcinoembryonic antigen-like cell adhesion molecules (CEACAMs) are human cell-surface proteins that can exhibit increased expression on tumor cells and are thus a potential target for novel tumor-seeking therapeutic delivery methods. We hypothesize that engineered nanoparticles containing a known interaction partner of CEACAM, Neisseria gonorrhoeae outer membrane protein Opa, can be used to deliver cargo to specific cellular targets. In this study, the cell association and uptake of protein-free liposomes and Opa proteoliposomes into CEACAM-expressing cells were measured using imaging flow cytometry. A size-dependent internalization of liposomes into HeLa cells was observed through endocytic pathways. Opa-dependent, CEACAM1-mediated uptake of liposomes into HeLa cells was observed, with limited colocalization with endosomal and lysosomal trafficking compartments. Given the overexpression of CEACAM1 on several distinct cancers and interest in using CEACAM1 as a component in treatment strategies, these results support further pursuit of investigating Opa-dependent specificity and the internalization mechanism for therapeutic delivery.

Gonococcal Colony Typing.

Gonococcal colony typing is part a science and part an art that has been central to studies which have identified crucial virulence antigens and also demonstrated the ability of the bacteria to undergo rapid phase and antigenic variation. Without this fundamental work, modern molecular biological studies of gonococcal pathogenesis would not have been possible. Indeed colony typing is still essential when performing biological experiments with clinical and laboratory isolates and for monitoring their outcome. In this chapter, the methods for performing colony typing and techniques to optimize the process are described.