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Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis .

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ABSTRACT

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

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Effects of the introduction of, or replacement with, oligohistidines on PorZ function.(a) Location of insertions (i) and substitutions (>) of consecutive residues by polyhistidines at the junction (residues G680-G691, over yellow background) between CTD (green font) and the preceding domain of PorZ (F677i8H, Q678i6H, S683 > 6 H, A686 > 6 H, L689 > 6 H and D690i6H) or at the C-terminus (I770 > 6 H, I770i6H, and R776i8H). β-strands are indicated above the alignment. (b–e) Wild-type and mutant strains were grown to OD600 = 1.0 and whole cultures were subjected to Western blot analysis with anti-PorZ (b), anti-polyhistidine (c), anti-Rgp (d) and anti-Kgp (e) antibodies. (f) The same strains were used for gingipain activity assays. (g) The level of surface exposure of PorZ in various mutants was analyzed by flow cytometry using anti-PorZ antibodies (red) and negative isotype control (blue). Representative histograms are shown from three independent experiments.
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f8: Effects of the introduction of, or replacement with, oligohistidines on PorZ function.(a) Location of insertions (i) and substitutions (>) of consecutive residues by polyhistidines at the junction (residues G680-G691, over yellow background) between CTD (green font) and the preceding domain of PorZ (F677i8H, Q678i6H, S683 > 6 H, A686 > 6 H, L689 > 6 H and D690i6H) or at the C-terminus (I770 > 6 H, I770i6H, and R776i8H). β-strands are indicated above the alignment. (b–e) Wild-type and mutant strains were grown to OD600 = 1.0 and whole cultures were subjected to Western blot analysis with anti-PorZ (b), anti-polyhistidine (c), anti-Rgp (d) and anti-Kgp (e) antibodies. (f) The same strains were used for gingipain activity assays. (g) The level of surface exposure of PorZ in various mutants was analyzed by flow cytometry using anti-PorZ antibodies (red) and negative isotype control (blue). Representative histograms are shown from three independent experiments.

Mentions: As to which variant of PorZ is found on the cell surface, the relative molecular mass of a PorZ-immunoreactive band in SDS-PAGE was ~80 kDa, which suggests that the protein is full length, without the signal peptide (theoretic molecular mass: 81 kDa). To verify this contention, we constructed P. gingivalis mutant strain R776i8H, which expresses PorZ with an octahistidine at the C-terminus (see also the next section). This mutant possesses a secretory phenotype that is indistinguishable from the wild type, as determined by colony pigmentation (data not shown), cellular distribution (Fig. 7e) and gingipain activity (Fig. 8f). Western blot analysis with anti-His-tag antibodies revealed reactivity to a band of ~80 kDa, which confirmed the presence of intact CTD in the mature PorZ protein (Fig. 7g). This observation is consistent with proteomics data reporting that PorZ appears to retain its CTD and does not undergo A-LPS modification as seen in other T9SS cargos27.


Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis .
Effects of the introduction of, or replacement with, oligohistidines on PorZ function.(a) Location of insertions (i) and substitutions (>) of consecutive residues by polyhistidines at the junction (residues G680-G691, over yellow background) between CTD (green font) and the preceding domain of PorZ (F677i8H, Q678i6H, S683 > 6 H, A686 > 6 H, L689 > 6 H and D690i6H) or at the C-terminus (I770 > 6 H, I770i6H, and R776i8H). β-strands are indicated above the alignment. (b–e) Wild-type and mutant strains were grown to OD600 = 1.0 and whole cultures were subjected to Western blot analysis with anti-PorZ (b), anti-polyhistidine (c), anti-Rgp (d) and anti-Kgp (e) antibodies. (f) The same strains were used for gingipain activity assays. (g) The level of surface exposure of PorZ in various mutants was analyzed by flow cytometry using anti-PorZ antibodies (red) and negative isotype control (blue). Representative histograms are shown from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121618&req=5

f8: Effects of the introduction of, or replacement with, oligohistidines on PorZ function.(a) Location of insertions (i) and substitutions (>) of consecutive residues by polyhistidines at the junction (residues G680-G691, over yellow background) between CTD (green font) and the preceding domain of PorZ (F677i8H, Q678i6H, S683 > 6 H, A686 > 6 H, L689 > 6 H and D690i6H) or at the C-terminus (I770 > 6 H, I770i6H, and R776i8H). β-strands are indicated above the alignment. (b–e) Wild-type and mutant strains were grown to OD600 = 1.0 and whole cultures were subjected to Western blot analysis with anti-PorZ (b), anti-polyhistidine (c), anti-Rgp (d) and anti-Kgp (e) antibodies. (f) The same strains were used for gingipain activity assays. (g) The level of surface exposure of PorZ in various mutants was analyzed by flow cytometry using anti-PorZ antibodies (red) and negative isotype control (blue). Representative histograms are shown from three independent experiments.
Mentions: As to which variant of PorZ is found on the cell surface, the relative molecular mass of a PorZ-immunoreactive band in SDS-PAGE was ~80 kDa, which suggests that the protein is full length, without the signal peptide (theoretic molecular mass: 81 kDa). To verify this contention, we constructed P. gingivalis mutant strain R776i8H, which expresses PorZ with an octahistidine at the C-terminus (see also the next section). This mutant possesses a secretory phenotype that is indistinguishable from the wild type, as determined by colony pigmentation (data not shown), cellular distribution (Fig. 7e) and gingipain activity (Fig. 8f). Western blot analysis with anti-His-tag antibodies revealed reactivity to a band of ~80 kDa, which confirmed the presence of intact CTD in the mature PorZ protein (Fig. 7g). This observation is consistent with proteomics data reporting that PorZ appears to retain its CTD and does not undergo A-LPS modification as seen in other T9SS cargos27.

View Article: PubMed Central - PubMed

ABSTRACT

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

No MeSH data available.


Related in: MedlinePlus