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Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease.

Besbes A, Le Goff S, Antunes A, Terrade A, Hong E, Giorgini D, Taha MK, Deghmane AE - PLoS Pathog. (2015)

Bottom Line: However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis.We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells.In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Invasive Bacterial Infections Unit, Paris, France.

ABSTRACT
Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Non-ST-11 isolates release α-peptide-lacking IgA protease.(A) Subcellular localisation of IgA protease in infected cells. Hec-1B cells were infected with GFP-expressing LNP19995 or LNP21019 (green) or left uninfected. After 12 h of infection, cells were fixed with 4% PFA, permeabilised and stained with anti-IgaP polyclonal serum and Texas Red (TR)-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI. Fluorescence was analyzed using immunofluorescence microscopy. Data are representative of three independent experiments. (B) Analysis of ST-11 and non-ST-11 isolates by PCR using primers specific to α-peptide sub-domain. PCR products were amplified from genomic DNA of strains indicated above the gel, using the couple of primers alphaFwNhe / alphaRevSma. PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. PCR amplification generated amplicons of ~ 1500 bp in all ST-11 isolates, while amplicon sizes ranged between 687 and 770 bp in non-ST-11 isolates. Molecular sizes (kb) are indicated in the left side. (C) Upper panel. Non scaled schematic representation of the passenger subdomains of meningococcal IgA protease from isolates LNP19995 (ST-11) and LNP21019 (non-ST-11). The positions of autocatalytic processing sites and their sequences (PPSP) are indicated. Arrows indicate positions of NLSs in α-peptide of the ST-11 isolate. Lower panel. Five hundred nanograms of the C-terminal His6-tagged passenger domain of the strains LNP19995 (IgaPα19995) or LNP21019 (IgaPα21019) were mixed with 5 or 10 μg of MSPs. After 3 h, the reaction mixtures were analyzed with immunoblot using anti-His tag mAb. The different cleavage products are indicated by arrows. Mw indicates the molecular weight. (D) IgA protease of ST-11 isolates restores the capacity of non-ST-11 isolates in cleaving nuclear p65. Each of the 19995Δiga and 21019Δiga were complemented with the heterologous iga allele of the WT strain LNP21019 and LNP19995, respectively. Hec-1-B cells were infected for 12 h with the parental WT strains, the isogenic iga knock-out mutant strains or the heterologous complemented strains. Nuclear fractions prepared from infected cells, were resolved by SDS-PAGE and were probed with anti-p65 mAb (N-terminal specific) or polyclonal serum anti-IgaP. LPS-treated cells were used as positive control for nuclear translocation of NF-κB. Immunoblot with anti histone H3 was used as loading control.
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ppat.1005078.g006: Non-ST-11 isolates release α-peptide-lacking IgA protease.(A) Subcellular localisation of IgA protease in infected cells. Hec-1B cells were infected with GFP-expressing LNP19995 or LNP21019 (green) or left uninfected. After 12 h of infection, cells were fixed with 4% PFA, permeabilised and stained with anti-IgaP polyclonal serum and Texas Red (TR)-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI. Fluorescence was analyzed using immunofluorescence microscopy. Data are representative of three independent experiments. (B) Analysis of ST-11 and non-ST-11 isolates by PCR using primers specific to α-peptide sub-domain. PCR products were amplified from genomic DNA of strains indicated above the gel, using the couple of primers alphaFwNhe / alphaRevSma. PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. PCR amplification generated amplicons of ~ 1500 bp in all ST-11 isolates, while amplicon sizes ranged between 687 and 770 bp in non-ST-11 isolates. Molecular sizes (kb) are indicated in the left side. (C) Upper panel. Non scaled schematic representation of the passenger subdomains of meningococcal IgA protease from isolates LNP19995 (ST-11) and LNP21019 (non-ST-11). The positions of autocatalytic processing sites and their sequences (PPSP) are indicated. Arrows indicate positions of NLSs in α-peptide of the ST-11 isolate. Lower panel. Five hundred nanograms of the C-terminal His6-tagged passenger domain of the strains LNP19995 (IgaPα19995) or LNP21019 (IgaPα21019) were mixed with 5 or 10 μg of MSPs. After 3 h, the reaction mixtures were analyzed with immunoblot using anti-His tag mAb. The different cleavage products are indicated by arrows. Mw indicates the molecular weight. (D) IgA protease of ST-11 isolates restores the capacity of non-ST-11 isolates in cleaving nuclear p65. Each of the 19995Δiga and 21019Δiga were complemented with the heterologous iga allele of the WT strain LNP21019 and LNP19995, respectively. Hec-1-B cells were infected for 12 h with the parental WT strains, the isogenic iga knock-out mutant strains or the heterologous complemented strains. Nuclear fractions prepared from infected cells, were resolved by SDS-PAGE and were probed with anti-p65 mAb (N-terminal specific) or polyclonal serum anti-IgaP. LPS-treated cells were used as positive control for nuclear translocation of NF-κB. Immunoblot with anti histone H3 was used as loading control.

Mentions: Cells exhibited sustained activation of NF-κB when infected by the non-ST-11 strain LNP21019 with a transient activation of JNK and apoptotic level close to uninfected cells (LNP21019 in Figs 5A, 5B and S4C). To understand why non-ST-11 isolates did not alter the nuclear activity of p65/RelA in vivo, we examined the expression and subcellular localisation of IgA protease of these isolates. Unlike ST-11 isolates, only the 100 kDa-IgaP was detected in the MSP fractions prepared from non-ST-11 isolates that could be also found in the cytosolic fractions of infected cells (S3A Fig, right panel), suggesting that non-ST-11 isolates were unable to release IgaP linked to α-peptide, although a priori express NalP (S3A Fig). These findings were also confirmed by immunofluorescence microscopy examination. Indeed, after 12 h of infection, the fluorescent signal specific to IgA protease (red) appeared in the cytosolic compartment but a substantial signal colocalized also with the nuclear compartment (blue) of cells infected with the ST-11 isolate LNP19995 (appears as magenta in merged panel). In contrast, IgA protease of the non-ST-11 isolate LNP21019 appeared as a punctuate pattern exclusively localized in the cytosolic space (Fig 6A). These results suggest that IgA protease released from non-ST-11 isolates did not present a defect in cell internalisation but rather in nuclear translocation. It is worth noting that purified IgaP sub-domain of the non-ST-11 isolate LNP21019 (IgaP21019), as MSP prepared from different non-ST-11 isolates, exhibited an in vitro cleavage activity of p65/RelA comparable to ST-11 isolates (Fig 4C, lower panel), ruling out a defect in the protease activity. We then compared α-peptide encoding regions of non-ST-11 isolates to those from ST-11 isolates. From all ST-11 isolates examined, PCR amplification yielded a single PCR band of ~ 1500 bp (Fig 6B). Amino acid-derived sequence analysis revealed the presence of two bipartite NLS sequences flanked by two cleavage sites CS1 and CS2 (Figs 6C and S6). Of the 20 non-ST-11 isolates examined, all yielded a single α-peptide PCR product with a smaller length than those resulted from ST-11 isolates (Fig 6B). This difference resulted from the deletion of ∼813 bp (271 amino-acids) encompassing the NLS sequences (S6 Fig). Indeed, DsRed fused to α-peptide of the carriage isolate remained in the cytosolic compartment (Fig 4B, lower panel) suggesting impaired nuclear transport. Importantly, all isolates shared the cleavage site CS1 separating IgaP and α-peptide (Igα). However, the alternative cleavage site CS2 downstream α-peptide was missing in all non-ST-11 isolates (S6 Fig). These observations were confirmed experimentally by incubating the purified C-terminal His6-tagged passenger domains IgaPα of LNP19995 (1500 residues extending from A28 to T1527) and LNP21019 (1235 residues extending from A28 to T1262) with the MSPs of LNP19995 and LNP21019, respectively (Fig 6C). Cleavage of LNP19995 passenger domain generated two His6-tagged fragments of ~ 60 kDa (that resulted from cleavage at the site CS1) and ~14 kDa (that resulted from the cleavage at site CS2) in presence of increasing amounts of MSPs. These fragments correspond to Igaα (~44–45 kDa) associated to the linker domain (~14 kDa); and the linker domain alone, respectively (Fig 6C, left panel). However, only one His6-tagged fragment of ~ 31 kDa (corresponding to Igaα associated to the linker) was generated from the cleavage at CS1 of LNP21019 passenger domain (Fig 6C, right panel). These results suggest that proteolytic cleavage of IgA protease may therefore occur at two cleavage sites in ST-11 isolates LNP19995: CS1 autocleavage site (separating the protease domain from the α peptide) and CS2 autocleavage site (separating the α peptide from the translocator domain). However, the proteolytic cleavage may occur at the unique CS1 site, leading to release of α-peptide-lacking IgA protease in non-ST-11 isolates.


Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease.

Besbes A, Le Goff S, Antunes A, Terrade A, Hong E, Giorgini D, Taha MK, Deghmane AE - PLoS Pathog. (2015)

Non-ST-11 isolates release α-peptide-lacking IgA protease.(A) Subcellular localisation of IgA protease in infected cells. Hec-1B cells were infected with GFP-expressing LNP19995 or LNP21019 (green) or left uninfected. After 12 h of infection, cells were fixed with 4% PFA, permeabilised and stained with anti-IgaP polyclonal serum and Texas Red (TR)-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI. Fluorescence was analyzed using immunofluorescence microscopy. Data are representative of three independent experiments. (B) Analysis of ST-11 and non-ST-11 isolates by PCR using primers specific to α-peptide sub-domain. PCR products were amplified from genomic DNA of strains indicated above the gel, using the couple of primers alphaFwNhe / alphaRevSma. PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. PCR amplification generated amplicons of ~ 1500 bp in all ST-11 isolates, while amplicon sizes ranged between 687 and 770 bp in non-ST-11 isolates. Molecular sizes (kb) are indicated in the left side. (C) Upper panel. Non scaled schematic representation of the passenger subdomains of meningococcal IgA protease from isolates LNP19995 (ST-11) and LNP21019 (non-ST-11). The positions of autocatalytic processing sites and their sequences (PPSP) are indicated. Arrows indicate positions of NLSs in α-peptide of the ST-11 isolate. Lower panel. Five hundred nanograms of the C-terminal His6-tagged passenger domain of the strains LNP19995 (IgaPα19995) or LNP21019 (IgaPα21019) were mixed with 5 or 10 μg of MSPs. After 3 h, the reaction mixtures were analyzed with immunoblot using anti-His tag mAb. The different cleavage products are indicated by arrows. Mw indicates the molecular weight. (D) IgA protease of ST-11 isolates restores the capacity of non-ST-11 isolates in cleaving nuclear p65. Each of the 19995Δiga and 21019Δiga were complemented with the heterologous iga allele of the WT strain LNP21019 and LNP19995, respectively. Hec-1-B cells were infected for 12 h with the parental WT strains, the isogenic iga knock-out mutant strains or the heterologous complemented strains. Nuclear fractions prepared from infected cells, were resolved by SDS-PAGE and were probed with anti-p65 mAb (N-terminal specific) or polyclonal serum anti-IgaP. LPS-treated cells were used as positive control for nuclear translocation of NF-κB. Immunoblot with anti histone H3 was used as loading control.
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ppat.1005078.g006: Non-ST-11 isolates release α-peptide-lacking IgA protease.(A) Subcellular localisation of IgA protease in infected cells. Hec-1B cells were infected with GFP-expressing LNP19995 or LNP21019 (green) or left uninfected. After 12 h of infection, cells were fixed with 4% PFA, permeabilised and stained with anti-IgaP polyclonal serum and Texas Red (TR)-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI. Fluorescence was analyzed using immunofluorescence microscopy. Data are representative of three independent experiments. (B) Analysis of ST-11 and non-ST-11 isolates by PCR using primers specific to α-peptide sub-domain. PCR products were amplified from genomic DNA of strains indicated above the gel, using the couple of primers alphaFwNhe / alphaRevSma. PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. PCR amplification generated amplicons of ~ 1500 bp in all ST-11 isolates, while amplicon sizes ranged between 687 and 770 bp in non-ST-11 isolates. Molecular sizes (kb) are indicated in the left side. (C) Upper panel. Non scaled schematic representation of the passenger subdomains of meningococcal IgA protease from isolates LNP19995 (ST-11) and LNP21019 (non-ST-11). The positions of autocatalytic processing sites and their sequences (PPSP) are indicated. Arrows indicate positions of NLSs in α-peptide of the ST-11 isolate. Lower panel. Five hundred nanograms of the C-terminal His6-tagged passenger domain of the strains LNP19995 (IgaPα19995) or LNP21019 (IgaPα21019) were mixed with 5 or 10 μg of MSPs. After 3 h, the reaction mixtures were analyzed with immunoblot using anti-His tag mAb. The different cleavage products are indicated by arrows. Mw indicates the molecular weight. (D) IgA protease of ST-11 isolates restores the capacity of non-ST-11 isolates in cleaving nuclear p65. Each of the 19995Δiga and 21019Δiga were complemented with the heterologous iga allele of the WT strain LNP21019 and LNP19995, respectively. Hec-1-B cells were infected for 12 h with the parental WT strains, the isogenic iga knock-out mutant strains or the heterologous complemented strains. Nuclear fractions prepared from infected cells, were resolved by SDS-PAGE and were probed with anti-p65 mAb (N-terminal specific) or polyclonal serum anti-IgaP. LPS-treated cells were used as positive control for nuclear translocation of NF-κB. Immunoblot with anti histone H3 was used as loading control.
Mentions: Cells exhibited sustained activation of NF-κB when infected by the non-ST-11 strain LNP21019 with a transient activation of JNK and apoptotic level close to uninfected cells (LNP21019 in Figs 5A, 5B and S4C). To understand why non-ST-11 isolates did not alter the nuclear activity of p65/RelA in vivo, we examined the expression and subcellular localisation of IgA protease of these isolates. Unlike ST-11 isolates, only the 100 kDa-IgaP was detected in the MSP fractions prepared from non-ST-11 isolates that could be also found in the cytosolic fractions of infected cells (S3A Fig, right panel), suggesting that non-ST-11 isolates were unable to release IgaP linked to α-peptide, although a priori express NalP (S3A Fig). These findings were also confirmed by immunofluorescence microscopy examination. Indeed, after 12 h of infection, the fluorescent signal specific to IgA protease (red) appeared in the cytosolic compartment but a substantial signal colocalized also with the nuclear compartment (blue) of cells infected with the ST-11 isolate LNP19995 (appears as magenta in merged panel). In contrast, IgA protease of the non-ST-11 isolate LNP21019 appeared as a punctuate pattern exclusively localized in the cytosolic space (Fig 6A). These results suggest that IgA protease released from non-ST-11 isolates did not present a defect in cell internalisation but rather in nuclear translocation. It is worth noting that purified IgaP sub-domain of the non-ST-11 isolate LNP21019 (IgaP21019), as MSP prepared from different non-ST-11 isolates, exhibited an in vitro cleavage activity of p65/RelA comparable to ST-11 isolates (Fig 4C, lower panel), ruling out a defect in the protease activity. We then compared α-peptide encoding regions of non-ST-11 isolates to those from ST-11 isolates. From all ST-11 isolates examined, PCR amplification yielded a single PCR band of ~ 1500 bp (Fig 6B). Amino acid-derived sequence analysis revealed the presence of two bipartite NLS sequences flanked by two cleavage sites CS1 and CS2 (Figs 6C and S6). Of the 20 non-ST-11 isolates examined, all yielded a single α-peptide PCR product with a smaller length than those resulted from ST-11 isolates (Fig 6B). This difference resulted from the deletion of ∼813 bp (271 amino-acids) encompassing the NLS sequences (S6 Fig). Indeed, DsRed fused to α-peptide of the carriage isolate remained in the cytosolic compartment (Fig 4B, lower panel) suggesting impaired nuclear transport. Importantly, all isolates shared the cleavage site CS1 separating IgaP and α-peptide (Igα). However, the alternative cleavage site CS2 downstream α-peptide was missing in all non-ST-11 isolates (S6 Fig). These observations were confirmed experimentally by incubating the purified C-terminal His6-tagged passenger domains IgaPα of LNP19995 (1500 residues extending from A28 to T1527) and LNP21019 (1235 residues extending from A28 to T1262) with the MSPs of LNP19995 and LNP21019, respectively (Fig 6C). Cleavage of LNP19995 passenger domain generated two His6-tagged fragments of ~ 60 kDa (that resulted from cleavage at the site CS1) and ~14 kDa (that resulted from the cleavage at site CS2) in presence of increasing amounts of MSPs. These fragments correspond to Igaα (~44–45 kDa) associated to the linker domain (~14 kDa); and the linker domain alone, respectively (Fig 6C, left panel). However, only one His6-tagged fragment of ~ 31 kDa (corresponding to Igaα associated to the linker) was generated from the cleavage at CS1 of LNP21019 passenger domain (Fig 6C, right panel). These results suggest that proteolytic cleavage of IgA protease may therefore occur at two cleavage sites in ST-11 isolates LNP19995: CS1 autocleavage site (separating the protease domain from the α peptide) and CS2 autocleavage site (separating the α peptide from the translocator domain). However, the proteolytic cleavage may occur at the unique CS1 site, leading to release of α-peptide-lacking IgA protease in non-ST-11 isolates.

Bottom Line: However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis.We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells.In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Invasive Bacterial Infections Unit, Paris, France.

ABSTRACT
Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.

No MeSH data available.


Related in: MedlinePlus