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

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.

Neisseria gonorrhoeaescavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation

“…Given the importance of CEACAM3 to phagocytic killing of Opa+ Gc by neutrophils, we tested the hypothesis that Gc sialylation disrupted OpaD-CEACAM3 engagement. First, sialylated or nonsialylated OpaD was incubated with a Glutathione S-transferase (GST)-tagged N-terminus of human CEACAM3 (N-CEACAM3), and CEACAM3 binding was measured by flow cytometry using anti-GST fluorescence (66). Lst-dependent sialylation did not significantly affect OpaD binding of N-CEACAM3 ( Fig 4A ).…”

“…Gc in this study are in the FA1090 background, constitutively expressing the pilin variant 1-81-S2 and with in-frame deletions of all opa genes (Opaless) (48). Opaless Gc with non-phase variable, constitutively expressed opaD has been described previously (OpaD) (48); Opa60 and OpaI were constructed similarly (49, 66).…”

“…Samples were examined using ImageStream X Mark II imaging flow cytometer and analyzed using INSPIRE and IDEAS v. 6.2 software packages (Amnis Luminex Corporation). Focused, singlet TIV+ Gc were gated as in (66). AF647+ gate was set in reference to TIV+ Gc without 6B4.…”

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils

“…Preincubation of OpaD+ Gc with serum suppressed the resulting neutrophil ROS response in a concentration-dependent manner, with serum concentrations of 10% and higher abrogating ROS production (Fig 1C). The suppressive effect of serum on neutrophil ROS production was replicated with a different isogenic derivative of Opaless Gc that constitutively expresses the Opa60 protein, which also binds to CEACAMs 1 and 3 [19,30], as well as Gc of strain 1291 [27] expressing undefined Opa proteins (S2 Fig) . Given that complement is a major opsonic activity in serum, we tested whether the effects observed with serum on Opa+ Gc were complement-dependent. Heat-inactivated serum behaved identically to untreated serum in OpaD+ Gc-mediated suppression of neutrophil ROS (Fig 1D).…”

“…N-CEACAM binding to Gc. Binding of recombinant N-termini of CEACAM1 or CEA-CAM3 to Gc, with and without incubation with C4BP, was measured by imaging flow cytometry, based on fluorescence of mouse anti-GST antibody p1A12 (Biolegend) as in [30].…”