Gonococcal porin IB activates NF-kappaB in human urethral epithelium and increases the expression of host antiapoptotic factors

Abstract

Infection of human urethral epithelial cells (UECs) with Neisseria gonorrhoeae increases the transcription of several host antiapoptotic genes, including bfl-1, cox-2, and c-IAP-2. In order to identify the bacterial factor(s) responsible for eliciting these changes, the transcriptional status of apoptotic machinery was monitored in UECs challenged with certain gonococcal membrane components. Initially, we observed that infection of UECs with gentamicin-killed gonococci increased the expression of the antiapoptotic Bcl-2 family member, bfl-1. This observation indicated that viable, replicating bacteria are not required for induction of antiapoptotic gene expression. Confirming this observation, treatment of UECs with purified gonococcal membrane increased the expression of bfl-1, cox-2, and c-IAP-2. This finding suggested that a factor or multiple factors present in the outer membrane (OM) are responsible for altering UEC antiapoptotic gene expression. Interestingly, treatment of UECs with gonococcal porin IB (PorB IB), a major constituent of the OM, significantly increased the transcription of bfl-1, cox-2, and c-IAP-2. The upregulation of these genes by PorB IB was determined to be dependent on NF-kappaB activation, as inhibiting NF-kappaB blocked induced expression of these genes. This work demonstrates the altered expression of host apoptotic factors in response to gonococcal PorB IB and supports a model whereby UEC cell death may be modulated as a potential mechanism of bacterial survival and proliferation.

FIG. 1.

Increased expression of bfl-1 in urethral epithelium infected with live or gentamicin-killed N. gonorrhoeae. UECs received medium alone (uninfected) or were challenged for 4 h with live (1291) or gentamicin-killed (1291+Gent) gonococci. After infection, total RNA was harvested, and cDNA was synthesized. PCR analysis then demonstrated that expression of the antiapoptotic Bcl-2 family member bfl-1 is increased in UECs infected with either live or gentamicin-killed gonococci. Panel 1, 18S rRNA internal control; panel 2, bfl-1; panel 3, reverse transcriptase-negative controls.

FIG. 2.

Analysis of antiapoptotic gene expression in UECs treated with purified gonococcal membrane. (A) RT-PCR analysis of antiapoptotic gene expression in UECs either left untreated or treated for 4 h with 10 μg of purified gonococcal outer membrane (1291 OM) per ml. Panel 1, 18S rRNA gene; panel 2, bfl-1; panel 3, c-IAP-2; panel 4, cox-2. (B) Real-time PCR analysis of bfl-1 expression showing an approximately fourfold increase in expression (P < 0.001) in 1291 OM-treated UECs (gray bars), while no change in expression occurred with the 18S rRNA control (white bars). (C) Real-time PCR analysis of cox-2 expression (hatched bars) demonstrating an approximately eightfold upregulation (P = 0.025) in 1291 OM-treated UECs. The 18S rRNA control (white bars) showed no change in expression in the different conditions. Real-time PCR data are shown as the mean from three independent reactions per condition.

FIG. 3.

Analysis of the effects of various gonococcal OM components on the expression of bfl-1. (A) UECs received medium alone (Uninfected) or were infected for 4 h with either wild-type N. gonorrhoeae strain FA1090 (WT), a FA1090 mutant lacking expression of the opacity-associated proteins (Δopa), or a FA1090 mutant lacking pili (Δpil). RT-PCR revealed the increased expression of bfl-1 (panel 2) in UECs infected with wild-type, Δopa, or Δpil gonococci, indicating that Opa and pili are not required to elicit the increase in host antiapoptotic gene expression. No change in expression was observed in the 18S rRNA control (panel 1). (B) UECs received medium alone (Untreated) or were treated for 4 h with either untreated gonococcal LOS diluted directly into culture medium [1291 LOS (in solution)] or LOS treated with 50 mM NaOH (NaOH-LOS). RT-PCR analysis of bfl-1 (panel 2) and 18S rRNA gene (panel 1) was performed. C) UECs were left untreated or treated for 4 h with magnetic beads coated with 1, 10, or 100 μg of purified gonococcal LOS [1291 LOS (+beads)] per ml. RT-PCR demonstrated that 18S rRNA gene (panel 1) and bfl-1 (panel 2) expression is not altered in UECs treated with gonococcal LOS.

FIG. 4.

Apoptotic regulator expression in UECs treated with purified gonococcal PorB IB. (A) UECs (maintained in the absence of serum) received medium alone (Untreated) or were treated for 4 h with 5 μg of gonococcal porin IB (PorB IB) per ml. RT-PCR analysis demonstrated the increased expression of antiapoptotic genes in PorB IB-treated UECs. Panel 1, 18S rRNA gene; panel 2, bfl-1; panel 3, c-IAP-2; panel 4, cox-2. (B) Western panel analysis of Bfl-1 in untreated urethral epithelium and cells treated for 2 or 4 h with 5 μg of PorB IB per ml. Molecular size markers (in kilodaltons) are indicated on the right. (C) RT-PCR analysis of proapoptotic gene expression in untreated and PorB IB-treated UECs. Panel 1, 18S rRNA control; panel 2, bax; panel 3, bak. (D) Real-time PCR analysis of 18S rRNA gene (white bars) and bfl-1 (gray bars; P < 0.01) in untreated and PorB IB-treated UECs. (E) Real-time PCR analysis of the 18S rRNA gene (white bars) and cox-2 (hatched bars; P < 0.01) expression in untreated and PorB IB-treated UECs.

FIG. 5.

Activation of NF-κB in gonococcus-infected and PorB IB-treated UECs. (A) UECs were left untreated, infected with N. gonorrhoeae (1291), or treated for 4 h with purified gonococcal porin IB (PorB IB). Dual-luciferase assays were then performed to measure the activity of NF-κB in UECs. The relative NF-κB activity, plotted as the ratio of the firefly luciferase to Renilla luciferase reporter expression, is graphed. The values are the means ± standard errors of the means (error bars) for three experiments. (B) UECs received medium alone (untreated) or were infected for 4 h with N. gonorrhoeae (1291) or were pretreated for 1 h with 10 μM MG-132 to inhibit NF-κB activation prior to the 4-h infection with the gonococcus (MG132 - 1291). RT-PCR analysis was then performed to analyze the expression of the 18S rRNA control (panel 1), bfl-1 (panel 2), or cox-2 (panel 3). (C) Real-time PCR analysis of 18S rRNA (white bars) and bfl-1 (gray bars) gene expression in (bar 1) untreated UECs, (bar 2) gonococcus-infected cells (1291), (bar 3) cells treated with 1 μM MG-132 prior to infection with 1291, (bar 4) cells treated with 10 μM MG-132 prior to infection with 1291, or (bar 5) cells treated with 10 μM MG-132 alone. (D) Real-time PCR analysis of 18S rRNA (white bars) and bfl-1 (gray bars) gene expression in (bar 1) untreated UECs, (bar 2) cells treated with 5 μg of PorB IB per ml for 4 h, (bar 3) cells treated with 10 μM MG-132 prior to treatment with 5 μg of PorB IB per ml, (bar 4) cells treated with 50 ng of PorB IB per ml, (bar 5) cells treated with 0.1 μM MG-132 prior to PorB IB treatment, (bar 6) cells treated with 1 μM MG-132 prior to PorB IB treatment, (bar 7) cells treated with 10 μM MG-132 prior to PorB IB treatment, or (bar 8) cells treated with 10 μM MG-132 alone.

FIG. 6.

Model of the regulation of urethral epithelial programmed cell death after infection with N. gonorrhoeae. (A) Infection of UECs with N. gonorrhoeae or treatment with purified gonococcal PorB IB activates NF-κB and increases the expression of antiapoptotic factors that can function to regulate apoptosis initiated by either the extrinsic or intrinsic death pathway. Infection with the gonococcus does not alter the expression status of key proapoptotic members of the Bcl-2 family. IAP, inhibition of apoptosis; AIF, apoptosis-inducing factor. (B) The increased expression of antiapoptotic factors (e.g., Bfl-1) by gonococcal porin (pIB) may prevent the opening of a Bax-mediated VDAC pore, sequestering cyt c in the intermitochondrial membrane space (IMMS). (C) In the absence of key antiapoptotic regulators, proapoptotic Bcl-2 family members (i.e., Bax) may interact with and promote the open conformation of gonococcal pIB, allowing for the release of cyt c. Increased levels of antiapoptotic factors (i.e., Bfl-1) may maintain pIB in a closed conformation either by interacting directly with pIB or by preventing its association with Bax or other proapoptotic factors. OMM and IMM, outer and inner mitochondrial membranes, respectively. (D) Gonococcal pIB may translocate to the outer mitochondrial membrane (OMM) of UECs and interact directly with VDAC, preventing the release of cyt c through an otherwise open VDAC pore. It is also possible, but unlikely, that Bfl-1 may interact directly with VDAC to influence the size of the VDAC channel.