Engulfment of Neisseria 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.

FIG. 1.

Kinetics of neisserial uptake mediated by CEACAM family receptors. Stably transfected HeLa cell lines expressing the indicated CEACAM receptors were infected by centrifuging Opa₅₇-expressing N. gonorrhoeae N313 onto HeLa cell monolayers, washed to remove nonadherent bacteria, incubated at 37°C for the indicated times, and then fixed to arrest the infection process. Bacterial association was quantified by fluorescence microscopy following differential staining of intracellular and extracellular bacteria. (A) Mean intracellular (black bars) and total associated (intracellular plus extracellular; white bars) numbers of bacteria per cell, calculated based on a total of 100 HeLa cells analyzed per sample. Standard deviations indicate variations among 12 to 22 microscopic fields counted per sample, depending on the density of the infected monolayer. Note different y-axis scale for CEACAM5. (B) Proportions of associated bacteria that were intracellular (mean and standard deviation per field).

FIG. 2.

Effects of kinase inhibitors on CEACAM receptor-mediated uptake of N. gonorrhoeae. HeLa cell lines stably expressing the indicated CEACAM receptors were infected in the presence of the indicated concentrations of the broad-spectrum serine kinase inhibitor staurosporine (Stauro) (A), the broad-spectrum tyrosine kinase inhibitor genistein (Genist) (B), or the Src family kinase-specific inhibitor PP2 (C). In each experiment, the effects of several inhibitor concentrations were tested by gentamicin assays, and the highest concentrations of inhibitors used were also assessed by differential staining of intracellular and extracellular bacteria for fluorescence microscopy. For staurosporine and genistein, results obtained by microscopy (data not shown) reflected those obtained with the gentamicin assays (A and B). For PP2, the inhibition of neisserial uptake mediated by CEACAM3 was apparent by microscopy (C) but was observed only inconsistently in gentamicin assays (data not shown). For the gentamicin assays (A and B), values indicate 10⁴ (for associated) and 10² (for intracellular) means and standard deviations of triplicate samples and are representative of at least three independent experiments. For microscopy experiments (C), values indicate the mean and standard deviation per cell calculated from three groups of 15 cells and are representative of at least two independent experiments. In each panel, asterisks indicate samples that were significantly different (P < 0.01) from parallel untreated samples.

FIG. 3.

Tyrosine phosphorylation of CEACAM receptors following neisserial infection. HeLa cell lines stably expressing the indicated CEACAM receptors were either infected with N. gonorrhoeae expressing the HSPG-specific Opa₅₀ or CEACAM-specific Opa₅₂ proteins or left uninfected (uninf) as outlined in Materials and Methods. Phosphotyrosine-containing proteins (P-Tyr) and CEACAM receptors were immunoprecipitated from cellular lysates, and recovered CEACAM receptors were detected by immunoblot analysis. Schematic illustrations of individual CEACAM receptors reflect the relative sizes and presence of transmembrane and cytoplasmic domains (CEACAM1 and CEACAM3) or GPI anchors (CEACAM5 and CEACAM6); potential tyrosine phosphorylation sites are indicated by stars. The molecular mass estimates listed are based on the electrophoretic mobilities of proteins isolated from stably transfected HeLa cell lines used throughout this study and are consistent with the sizes of proteins displayed in the lower panels.

FIG. 4.

Role of the cytoskeleton in neisserial uptake mediated by CEACAM family receptors. Stably transfected HeLa cell lines expressing various CEACAM receptors were infected with N. gonorrhoeae expressing the CEACAM-specific Opa₅₇ protein in the presence of the indicated concentrations of the actin microfilament-disrupting agent cytochalasin D (CytoD) or the tubulin-depolymerizing agent nocodazole. Internalized bacteria were detected by either gentamicin assays (A and B) or microscopy of samples following differential staining of intracellular (Intra) and extracellular bacteria (C). Cytochalasin D had similar effects on neisserial uptake regardless of whether gentamicin assays (A) or microscopy (data not shown) was used. In contrast, nocodazole increased the recovery of bacteria following gentamicin treatment (B) but tended either to have little effect on or to reduce the number of intracellular bacteria apparent by fluorescence microscopy (C). For gentamicin assays, values indicate 10⁴ (for associated) and 10² (for intracellular) means and standard deviations of triplicate samples and are representative of at least three independent experiments. For microscopy experiments, values indicate the mean and standard deviation calculated from four independent experiments; the proportion of bacteria that were internalized in the absence (−) or presence (+) of nocodazole is listed below. In each panel, asterisks indicate samples that were significantly different (P < 0.01) from parallel untreated samples.

FIG. 5.

Effect of PI-PLC on CEACAM receptor-mediated uptake of N. gonorrhoeae. Opa₅₇-expressing gonococci were centrifuged onto CEACAM-expressing HeLa cell lines to promote binding; the samples were immediately washed to remove nonadherent bacteria. The samples then were treated with PI-PLC, which cleaves the GPI anchor of surface-expressed CEACAM5 without significant effects on other CEACAMs, prior to being incubated at 37°C. Values indicate 10⁴ (for associated) and 10² (for intracellular) means and standard deviations of triplicate samples and are representative of at least three independent experiments. The asterisk indicates a sample that was significantly different (P < 0.06) from parallel untreated samples.

FIG. 6.

Intracellular survival of N. gonorrhoeae following CEACAM receptor-mediated uptake. HeLa cell lines expressing the indicated receptors were infected for 3 h, the samples were treated with gentamicin for 2 h to kill extracellular gonococci, the antibiotic was removed by washing, and then the antibiotic was reapplied for the final 2 h before eukaryotic cell lysis with saponin to recover and quantify viable intracellular bacteria by dilution plating. Similar results were obtained when the antibiotic was applied after 3 h of infection and maintained until saponin treatment (data not shown).

FIG. 7.

Influence of phagosomal acidification on the intracellular survival of N. gonorrhoeae. (A) Stably transfected HeLa cell lines expressing the indicated CEACAM receptors were infected in the presence or in the absence of concanamycin A (conA), which prevents phagosomal acidification by inhibiting vacuolar (H⁺) ATPases. After 3 h of infection, viable associated and intracellular bacteria were recovered by gentamicin assays. Values indicate the means and standard deviations of triplicate samples; the values obtained for each cell line in the absence of concanamycin A are shown as 100% recovery of total associated or intracellular bacteria. Results are representative of at least three independent experiments. Concanamycin A treatment had no obvious effect on the rate of neisserial uptake, as observed by differential staining of intracellular and extracellular bacteria for fluorescence microscopy (data not shown); these results indicate that the increased recovery of intracellular bacteria in gentamicin assays reflects the increased survival of internalized gonococci. Asterisks indicate samples that were significantly different (P < 0.01) from parallel untreated samples. (B) Growth of N. gonorrhoeae in culture media adjusted to the indicated pHs. At the indicated times, samples were removed and bacterial culture densities were assessed by dilution plating. The results presented are those obtained with Opa₅₇-expressing N. gonorrhoeae N313; however, no significant difference was apparent when Opa⁻ N. gonorrhoeae N302 or Opa₅₂-expressing N. gonorrhoeae N309 was used in parallel (data not shown). Similar results were obtained when neisserial culture density was monitored by absorbance rather than dilution plating (data not shown).

FIG. 8.

Schematic model summarizing CEACAM receptor-mediated uptake by transfected HeLa cell lines. Events apparent during the engulfment of N. gonorrhoeae expressing CEACAM-specific Opa proteins by HeLa-CEACAM1, HeLa-CEACAM3, HeLa-CEACAM5, and HeLa-CEACAM6 cells are described in Discussion. Broken arrows indicate that a Syk tyrosine kinase is involved in CEACAM3 receptor-mediated uptake of N. gonorrhoeae by CEACAM3-expressing DT40 B cells (8) and, presumably, neutrophils, but is not expressed by HeLa cells (5). Note that CEACAM1 and CEACAM3 possess transmembrane and cytoplasmic domains containing tyrosine residues that are phosphorylated (indicated by “-P” adjacent to the receptor), either constitutively (CEACAM1) or in response to neisserial binding (CEACAM3), while CEACAM5 and CEACAM6 are anchored via GPI moieties. Once engulfed, maturation of the gonococcus-containing phagosome appears to be similar in the HeLa cell lines. PI, phosphatidylinositol; PLCγ, phospholipase Cγ.