Engulfment ofNeisseria gonorrhoeae: Revealing Distinct Processes of Bacterial Entry by Individual Carcinoembryonic Antigen-Related Cellular Adhesion Molecule Family Receptors

Abstract

Individual Neisseria gonorrhoeae colony opacity-associated (Opa) protein variants can bind up to four different carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) receptors. Most human cells encountered by gonococci express a combination of CEACAM receptors, thereby complicating the elucidation of intracellular signaling pathways triggered by individual receptors. Here, we compare the process of bacterial engulfment by a panel of stably transfected HeLa epithelial cell lines expressing each CEACAM receptor in isolation. CEACAM1 and CEACAM3 each contain proteinaceous transmembrane and cytoplasmic domains; however, the processes of neisserial uptake mediated by these receptors differ with respect to their susceptibilities to both tyrosine kinase inhibitors and the actin microfilament-disrupting agent cytochalasin D. Neisserial uptake mediated by glycosylphosphatidylinositol (GPI)-anchored CEACAM5 and CEACAM6 was not significantly affected by any of a broad spectrum of inhibitors tested. However, cleavage of the GPI anchor by phosphatidylinositol-specific phospholipase C reduced bacterial uptake by HeLa cells expressing CEACAM5, consistent with a single zipper-like mechanism of uptake mediated by this receptor. Regardless of the CEACAM receptor expressed, internalized gonococci were effectively killed by a microtubule-dependent process that required acidification of the bacterium-containing phagosome. Given the phase-variable nature of neisserial Opa proteins, these results indicate that the mechanism of bacterial engulfment and the cellular response to gonococcal infection depend on both the receptor specificities of the neisserial Opa protein variants expressed and the spectrum of CEACAM receptors present on target cells, each of which determines the combination of receptors ultimately engaged.Despite the existence of effective antibiotic therapy to clear the bacterium, recent estimates indicate that ϳ62 million new Neisseria gonorrhoeae infections occur each year (46). Infection likely begins with attachment of the gonococcal type IV pilus to the apical surface of mucosal epithelial cells (26,43). Pilus retraction (27) then allows the colony opacity-associated (Opa) adhesins to confer a tight association between the bacteria and apically oriented host cellular receptors. These interactions facilitate bacterial entry into and transcellular transcytosis through epithelial cells and entry into the subepithelial compartment (11,21,26,45), presumably allowing the establishment of localized and/or disseminated infection (11,19).Neisserial Opa proteins are integral outer membrane proteins that are predicted to span the lipid bilayer eight times with four surface-exposed loops (24). Individual gonococcal strains possess ϳ11 different opa alleles, each of which may encode functionally and/or antigenically distinct variants (reviewed in reference 13). The expression of each allele is phase variable due to frequent RecA-independent DNA rearrangements occurring within a pentanucleotide repeat sequence pres...

Quantification of bacterial invasion into adherent cells by flow cytometry

“…3B). In contrast, gentamicin assaybased viable counts of gonococci internalized via CEA-CAM3 decline rapidly, consistent with previous reports (McCaw et al, 2004). These results further stress the limitations of antibiotic protection assays, where the read-out (recovered viable bacteria) is influenced by both the efficiency of uptake as well as rate of intracellular killing or multiplication of the bacteria.…”

“…3A). These results demonstrated that bacteria taken up via CEACAM3 are rapidly killed inside human epithelial cells and similar observations have been reported by others (McCaw et al, 2004;Schmitter et al, 2004). Importantly, when the flow cytometric analysis was performed on samples infected and gentamicin treated identical to the ones used in the antibiotic protection assay, the fluorescence signal obtained 30, 60 and 120 min after gentamicin addition did not decrease (Fig.…”

“…This is particularly evident with receptors that are specialized in phagocytosis and efficiently deliver their cargo to lysosomal compartments (Booth et al, 2003). CEACAM3 represents a phagocytic receptor that not only efficiently internalizes bound particles, but also initiates the elimination of the internalized bacteria (McCaw et al, 2004;Schmitter et al, 2004). Importantly, determination of bacterial uptake by the flow cytometric approach should, theoretically, not be influenced by intracellular killing, as within a reasonable time the CFSE-label should stay intact.…”

Mast cells, which interact with Escherichia coli, up-regulate genes associated with innate immunity and become less responsive to FcεRI-mediated activation

“…6). CEACAMs mediate binding to and engulfment of Neisseria gonorrhoeae [12] and E. coli [35] by epithelial cells. We show that huMC express CEACAM1 and may be involved in internalization and engulfment of E. coli.…”

“…These molecules included the ␤1 integrins and the newly characterized family of CEACAM molecules. CEACAM1, a receptor known to bind bacteria in other cell types [12], was up-regulated threefold at Fig. 4.…”