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Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

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The effect of Ngo infection  on the activation of transcription factors.  (A) HeLa cells were analyzed in a gel retardation assay using a 32P-labeled H-2K  gene NF-κB–binding site oligonucleotide. Nuclear extracts were prepared at  different time points after infection with  the Ngo P+ strain and the DNA-binding  activity was analyzed (lanes 1–5). The  identity of the NF-κB transcription factor was investigated by competition  with increasing amounts of the unlabeled  oligonucleotide (lanes 6–9). The composition of the Ngo-induced NF-κB complex was investigated by antibody supershifting and inhibition using different amounts (0.5 μl and 2 μl) of anti-p50 (lanes 10 and 11), anti-p65  (lanes 12 and 13), or anti–c-Rel antisera (lanes 14 and 15). (B) The NF-κB DNA-binding activity was assayed in response to Ngo strains, different  MOIs, and different time points (lanes 1–5). As controls, HeLa cells were  stimulated with TNF-α (10 ng/ml) or treated with LPS (10 μg/ml). (C)  AP-1 DNA-binding activity was investigated in response to Ngo infection  at different time points (lanes 1–5) using a 32P-labeled AP-1 DNA-binding site oligonucleotide as a probe. As controls cells were stimulated with  PMA (40 nM) or treated with LPS (10 μg/ml). (D) DNA-binding activity at the CRE, C/EBP, and octamer binding sites were studied in extracts from Ngo P+ strain–infected HeLa cells at different time points  (lanes 1–5). Only sections of the autoradiograms containing the protein– DNA complexes are shown. The position of protein–DNA complexes  are indicated with arrows.
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Figure 2: The effect of Ngo infection on the activation of transcription factors. (A) HeLa cells were analyzed in a gel retardation assay using a 32P-labeled H-2K gene NF-κB–binding site oligonucleotide. Nuclear extracts were prepared at different time points after infection with the Ngo P+ strain and the DNA-binding activity was analyzed (lanes 1–5). The identity of the NF-κB transcription factor was investigated by competition with increasing amounts of the unlabeled oligonucleotide (lanes 6–9). The composition of the Ngo-induced NF-κB complex was investigated by antibody supershifting and inhibition using different amounts (0.5 μl and 2 μl) of anti-p50 (lanes 10 and 11), anti-p65 (lanes 12 and 13), or anti–c-Rel antisera (lanes 14 and 15). (B) The NF-κB DNA-binding activity was assayed in response to Ngo strains, different MOIs, and different time points (lanes 1–5). As controls, HeLa cells were stimulated with TNF-α (10 ng/ml) or treated with LPS (10 μg/ml). (C) AP-1 DNA-binding activity was investigated in response to Ngo infection at different time points (lanes 1–5) using a 32P-labeled AP-1 DNA-binding site oligonucleotide as a probe. As controls cells were stimulated with PMA (40 nM) or treated with LPS (10 μg/ml). (D) DNA-binding activity at the CRE, C/EBP, and octamer binding sites were studied in extracts from Ngo P+ strain–infected HeLa cells at different time points (lanes 1–5). Only sections of the autoradiograms containing the protein– DNA complexes are shown. The position of protein–DNA complexes are indicated with arrows.

Mentions: Subconfluent monolayers of HeLa cells were infected with Ngo. At different time points after challenge, nuclear protein extracts were prepared and analyzed for the levels of cellular transcription factor DNA-binding activity by using a panel of radiolabeled oligonucleotides corresponding to the DNA-binding sites of five transcription factor families (NF-κB, AP-1, CRE, C/EBP, and octamer factors). As Fig. 2 A shows, when the NF-κB–binding site (H-2K) was used for the electrophoretic mobility shift assay (EMSA), an enhanced binding of nuclear proteins was observed in HeLa cells within 10 min after infection with the P+ strain (Fig. 2 A, lanes 1 and 2). The DNA-binding activity increased within 90 min, and was already reduced after 180 min (Fig. 2 A, lanes 3–5). To examine the specificity of the DNA-binding capability induced by adherence of the P+ strain, nonlabeled double-stranded oligonucleotide was added for competition. A decrease in the amount of bound complex was observed as the concentrations of unlabeled NF-κB consensus sequence increased (Fig. 2 A, lanes 6–9). The most prominent form of transcription factor NF-κB has been described as a heterodimer consisting of two proteins, p50 and p65 (23). Furthermore, members of the NF-κB/rel family of proteins can form homodimers and heterodimers (24). The nature of the proteins that bind to the κB sequence were characterized using supershift assays. Experiments were performed in which the nuclear extracts were preincubated with either an anti-p50, anti-p65, or anti-c-Rel antiserum before addition of the 32P-labeled oligonucleotide. The results shown in Fig. 2 A (lanes 10–15) indicate that the anti-p50 antibody (0.5 μl and 2 μl antiserum, lanes 10 and 11) and the anti-p65 antibody (0.5 μl and 2 μl antiserum, lanes 12 and 13) led to a significant reduction and to supershifts of the NF-κB complex. The anti-c-Rel antibody affected only slightly the NF-κB–DNA complex (0.5 μl and 2 μl antiserum, lanes 14 and 15). Therefore, both p50 and p65 represent the predominant protein species in the κB DNA-binding activity present in P+ strain–infected HeLa cells.


Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

The effect of Ngo infection  on the activation of transcription factors.  (A) HeLa cells were analyzed in a gel retardation assay using a 32P-labeled H-2K  gene NF-κB–binding site oligonucleotide. Nuclear extracts were prepared at  different time points after infection with  the Ngo P+ strain and the DNA-binding  activity was analyzed (lanes 1–5). The  identity of the NF-κB transcription factor was investigated by competition  with increasing amounts of the unlabeled  oligonucleotide (lanes 6–9). The composition of the Ngo-induced NF-κB complex was investigated by antibody supershifting and inhibition using different amounts (0.5 μl and 2 μl) of anti-p50 (lanes 10 and 11), anti-p65  (lanes 12 and 13), or anti–c-Rel antisera (lanes 14 and 15). (B) The NF-κB DNA-binding activity was assayed in response to Ngo strains, different  MOIs, and different time points (lanes 1–5). As controls, HeLa cells were  stimulated with TNF-α (10 ng/ml) or treated with LPS (10 μg/ml). (C)  AP-1 DNA-binding activity was investigated in response to Ngo infection  at different time points (lanes 1–5) using a 32P-labeled AP-1 DNA-binding site oligonucleotide as a probe. As controls cells were stimulated with  PMA (40 nM) or treated with LPS (10 μg/ml). (D) DNA-binding activity at the CRE, C/EBP, and octamer binding sites were studied in extracts from Ngo P+ strain–infected HeLa cells at different time points  (lanes 1–5). Only sections of the autoradiograms containing the protein– DNA complexes are shown. The position of protein–DNA complexes  are indicated with arrows.
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Related In: Results  -  Collection

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Figure 2: The effect of Ngo infection on the activation of transcription factors. (A) HeLa cells were analyzed in a gel retardation assay using a 32P-labeled H-2K gene NF-κB–binding site oligonucleotide. Nuclear extracts were prepared at different time points after infection with the Ngo P+ strain and the DNA-binding activity was analyzed (lanes 1–5). The identity of the NF-κB transcription factor was investigated by competition with increasing amounts of the unlabeled oligonucleotide (lanes 6–9). The composition of the Ngo-induced NF-κB complex was investigated by antibody supershifting and inhibition using different amounts (0.5 μl and 2 μl) of anti-p50 (lanes 10 and 11), anti-p65 (lanes 12 and 13), or anti–c-Rel antisera (lanes 14 and 15). (B) The NF-κB DNA-binding activity was assayed in response to Ngo strains, different MOIs, and different time points (lanes 1–5). As controls, HeLa cells were stimulated with TNF-α (10 ng/ml) or treated with LPS (10 μg/ml). (C) AP-1 DNA-binding activity was investigated in response to Ngo infection at different time points (lanes 1–5) using a 32P-labeled AP-1 DNA-binding site oligonucleotide as a probe. As controls cells were stimulated with PMA (40 nM) or treated with LPS (10 μg/ml). (D) DNA-binding activity at the CRE, C/EBP, and octamer binding sites were studied in extracts from Ngo P+ strain–infected HeLa cells at different time points (lanes 1–5). Only sections of the autoradiograms containing the protein– DNA complexes are shown. The position of protein–DNA complexes are indicated with arrows.
Mentions: Subconfluent monolayers of HeLa cells were infected with Ngo. At different time points after challenge, nuclear protein extracts were prepared and analyzed for the levels of cellular transcription factor DNA-binding activity by using a panel of radiolabeled oligonucleotides corresponding to the DNA-binding sites of five transcription factor families (NF-κB, AP-1, CRE, C/EBP, and octamer factors). As Fig. 2 A shows, when the NF-κB–binding site (H-2K) was used for the electrophoretic mobility shift assay (EMSA), an enhanced binding of nuclear proteins was observed in HeLa cells within 10 min after infection with the P+ strain (Fig. 2 A, lanes 1 and 2). The DNA-binding activity increased within 90 min, and was already reduced after 180 min (Fig. 2 A, lanes 3–5). To examine the specificity of the DNA-binding capability induced by adherence of the P+ strain, nonlabeled double-stranded oligonucleotide was added for competition. A decrease in the amount of bound complex was observed as the concentrations of unlabeled NF-κB consensus sequence increased (Fig. 2 A, lanes 6–9). The most prominent form of transcription factor NF-κB has been described as a heterodimer consisting of two proteins, p50 and p65 (23). Furthermore, members of the NF-κB/rel family of proteins can form homodimers and heterodimers (24). The nature of the proteins that bind to the κB sequence were characterized using supershift assays. Experiments were performed in which the nuclear extracts were preincubated with either an anti-p50, anti-p65, or anti-c-Rel antiserum before addition of the 32P-labeled oligonucleotide. The results shown in Fig. 2 A (lanes 10–15) indicate that the anti-p50 antibody (0.5 μl and 2 μl antiserum, lanes 10 and 11) and the anti-p65 antibody (0.5 μl and 2 μl antiserum, lanes 12 and 13) led to a significant reduction and to supershifts of the NF-κB complex. The anti-c-Rel antibody affected only slightly the NF-κB–DNA complex (0.5 μl and 2 μl antiserum, lanes 14 and 15). Therefore, both p50 and p65 represent the predominant protein species in the κB DNA-binding activity present in P+ strain–infected HeLa cells.

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Show MeSH
Related in: MedlinePlus