Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins

Abstract

Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells.

FIG. 1.

N. gonorrhoeae infection prevents STS-induced mitochondrial depolarization and caspase-3 activation in End/E6E7 cells. (A) Mitochondrial membrane depolarization was determined by staining with rhodamine 123 and fluorescence-activated cell sorter analysis. The shaded gray histogram represents uninfected cells, the gray line represents cells infected with N. gonorrhoeae at an MOI of 100 for 48 h, the black line represents uninfected cells with the addition of 1 μM STS at 24 h, and the dashed line represents cells infected with N. gonorrhoeae at an MOI of 100 for a total of 48 h, with the addition of STS at 24 h. Results are representative of three experiments performed in duplicate. (B) Caspase-3 activation was determined in End/E6E7 cells following infection with N. gonorrhoeae FA1090B at various MOIs (100, 10, and 1), as indicated, for 48 h. STS (1 μM) was added to the cultures at 24 h postinfection, where indicated. Cell lysates were tested for caspase-3 activity by incubation with a fluorogenic substrate, and fluorescence units were determined by excitation at 365 nm and emission detection at 465 nm (indicated on the y axis). The data represent combined means from three individual experiments performed in duplicate; error bars indicate the standard errors of the means.

FIG. 2.

N. gonorrhoeae infection protects End/E6E7 cells from STS-induced DNA degradation. End/E6E7 cells were left uninfected or infected with N. gonorrhoeae FA1090B at an MOI of 100 for 48 h (A), and some infections were followed by the addition of 1 μM STS at 24 h (B). Hypodiploid DNA content, indicative of DNA degradation, was examined by PI staining and fluorescence-activated cell sorter analysis. The percentage of cells with hypodiploid DNA is indicated in each histogram. Data are representative of three duplicate experiments.

FIG. 3.

The antiapoptotic response of N. gonorrhoeae-stimulated End/E6E7 cells is specific to cells in association with bacteria. (A) Confocal microscopy was performed on End/E6E7 cell cultures infected with N. gonorrhoeae F62-GFP at an MOI of 100 for 24 h and stained with a red fluorescent plasma membrane marker, DiI. The image represents a 1-μm z-stack section (6 of 12). Bar = 10 μm. (B) EndE6/E7 cells were infected with N. gonorrhoeae F62-GFP at an MOI of 100 for 24 h, followed by the addition of 1 μM STS for an additional 22 h, as indicated. Uninfected cells were used as a control, and PI staining was used to determine DNA content. Flow cytometry was performed by gating on GFP⁺ cells from the infected cell cultures. The GFP⁻ population was also assessed for PI fluorescence by analysis of a “dead gate” following flow cytometry. Apoptosis is expressed on the y axis as the percentage of total cells containing hypodiploid DNA. Samples not treated with STS had hypodiploid DNA levels of <3% (data not shown). Results represent the means for triplicate samples from two individual experiments. Error bars represent standard deviations.

FIG. 4.

The antiapoptotic response of N. gonorrhoeae-stimulated End/E6E7 cells is dependent on live bacteria. N. gonorrhoeae FA1090B was incubated with End/E6E7 cells at an MOI of 100 for 24 h prior to the addition of 1 μM STS for an additional 22 h. Mitochondrial membrane potential was measured by staining with rhodamine (R123) followed by analysis of fluorescence intensity by flow cytometry. (A) Infection with live N. gonorrhoeae (GC). (B) Infection with heat-killed N. gonorrhoeae. (C) Prolonged gentamicin treatment during infection. Shaded gray histograms represent uninfected cells (plus gentamicin in panel C), thin black lines represent uninfected cells treated with STS (plus gentamicin in panel C), thick black lines represent cells incubated with N. gonorrhoeae (live, heat killed, or gentamicin treated), and dashed lines represent cells incubated with N. gonorrhoeae and treated with STS. Results are representative of three experiments performed in triplicate.

FIG. 5.

The canonical pathway of NF-κB is activated and sustained upon stimulation of End/E6E7 cells with N. gonorrhoeae FA1090B. End/E6E7 cells were left untreated and uninfected, infected with gonococcal (GC) strain FA1090B at an MOI of 100, or treated with 25 ng/ml TNF-α for 1 h. A Trans-AM ELISA kit was used to detect NF-κB nuclear translocation, and the colorimetric reaction was read as the optical density (OD) at 450 nm. Nuclear proteins were incubated alone (gray bars), with an oligonucleotide containing a mutated NF-κB consensus sequence (black bars), or with an oligonucleotide containing a wild-type NF-κB consensus sequence for competitive binding (white bars). The p65 (A) and p50 (B) subunits were detected in nuclear extracts from 1-h samples following treatment with gonococci or TNF. Untreated, uninfected cells showed a basal nuclear level of each subunit. Error bars represent standard deviations for triplicate experimental samples.

FIG. 6.

Expression of host genes and proteins following infection of End/E6E7 cells with N. gonorrhoeae. Gene expression was measured for representative genes associated with the inhibition of apoptosis (A), the induction of apoptosis (B), and inflammation (C). Transcript levels are represented as changes compared to uninfected control RNA. Genes were normalized to the β-2 microglobulin (b-2-M) housekeeping control. Results represent the means for four experimental samples. *, P < 0.05; **, P < 0.005 (Student's t test). Black bars, infection with live bacteria for 8 h; white bars, treatment with heat-killed bacteria for 8 h. (D) End/E6E7 cells were infected with N. gonorrhoeae FA1090B at an MOI of 100, and at 8 h and 24 h postinfection, whole-cell lysates were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 20 μg of total protein, followed by Western blot analysis using anti-Bfl-1, -c-FLIP, -cIAP-2, and -β-2-microglobulin antibodies. GC, N. gonorrhoeae.