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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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Characterization of the ΔPorZ secretory phenotype.(a) Pigmentation on blood agar of P. gingivalis wild type (W83), ΔPorZ, and in trans porZ-complemented ΔPorZ (PorZ+) strains. Enzymatic activity of (b) Rgps, (c) Kgp, and (d) dipeptidyl peptidase IV (DPPIV) and prolyl tripeptidyl peptidase (PtpA) in whole cultures, fractionated washed cells or growth medium as determined with specific synthetic substrates. Cultures were adjusted to OD600 = 1.0 prior to testing and processing, and results shown correspond to triplicate experiments. Significant differences between the wild type and mutants are indicated by *P < 0.05 and ****P < 0.0001.
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f1: Characterization of the ΔPorZ secretory phenotype.(a) Pigmentation on blood agar of P. gingivalis wild type (W83), ΔPorZ, and in trans porZ-complemented ΔPorZ (PorZ+) strains. Enzymatic activity of (b) Rgps, (c) Kgp, and (d) dipeptidyl peptidase IV (DPPIV) and prolyl tripeptidyl peptidase (PtpA) in whole cultures, fractionated washed cells or growth medium as determined with specific synthetic substrates. Cultures were adjusted to OD600 = 1.0 prior to testing and processing, and results shown correspond to triplicate experiments. Significant differences between the wild type and mutants are indicated by *P < 0.05 and ****P < 0.0001.

Mentions: An isogenic deletion mutant of the porZ gene, ΔPorZ, was created by homologous recombination to assess its effect on T9SS cargo secretion and posttranslational processing. Deletion had a negligible effect on the P. gingivalis growth rate in complex media (Supplementary Fig. S1). However, on blood agar, the mutant could not accumulate heme on the cell surface and therefore yielded non-pigmented colonies (Fig. 1a). This is attributable to gingipains, which are secreted T9SS cargos that are essential for hemoglobin degradation and heme recruitment293031. Therefore, lack of pigmentation in ΔPorZ suggested failure of functional gingipain secretion and activation. Indeed, we found that ΔPorZ was deficient in extracellular Kgp and Rgp gingipain activities when compared to the wild-type P. gingivalis strain W83 (hereafter, “wild type”; Fig. 1b,c). In contrast, dipeptidyl peptidase IV and prolyl tripeptidyl peptidase A, which are surface enzymes but not secreted by T9SS, were produced and transported to the bacterial surface in significantly higher amounts than in the wild type (Fig. 1d). A similar response had been previously observed in an inactivation mutant of an essential T9SS component, PorT32. This presumably reflects general upregulation of peptidolytic enzymes as a response to the absence of functional gingipains, which account for 85% of the extracellular proteolytic activity of P. gingivalis33. Reconstitution in trans of the porZ gene in the ΔPorZ mutant—yielding PorZ+—restored both pigmentation and proteolytic activity to wild-type levels (Fig. 1a–d).


Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis .
Characterization of the ΔPorZ secretory phenotype.(a) Pigmentation on blood agar of P. gingivalis wild type (W83), ΔPorZ, and in trans porZ-complemented ΔPorZ (PorZ+) strains. Enzymatic activity of (b) Rgps, (c) Kgp, and (d) dipeptidyl peptidase IV (DPPIV) and prolyl tripeptidyl peptidase (PtpA) in whole cultures, fractionated washed cells or growth medium as determined with specific synthetic substrates. Cultures were adjusted to OD600 = 1.0 prior to testing and processing, and results shown correspond to triplicate experiments. Significant differences between the wild type and mutants are indicated by *P < 0.05 and ****P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Characterization of the ΔPorZ secretory phenotype.(a) Pigmentation on blood agar of P. gingivalis wild type (W83), ΔPorZ, and in trans porZ-complemented ΔPorZ (PorZ+) strains. Enzymatic activity of (b) Rgps, (c) Kgp, and (d) dipeptidyl peptidase IV (DPPIV) and prolyl tripeptidyl peptidase (PtpA) in whole cultures, fractionated washed cells or growth medium as determined with specific synthetic substrates. Cultures were adjusted to OD600 = 1.0 prior to testing and processing, and results shown correspond to triplicate experiments. Significant differences between the wild type and mutants are indicated by *P < 0.05 and ****P < 0.0001.
Mentions: An isogenic deletion mutant of the porZ gene, ΔPorZ, was created by homologous recombination to assess its effect on T9SS cargo secretion and posttranslational processing. Deletion had a negligible effect on the P. gingivalis growth rate in complex media (Supplementary Fig. S1). However, on blood agar, the mutant could not accumulate heme on the cell surface and therefore yielded non-pigmented colonies (Fig. 1a). This is attributable to gingipains, which are secreted T9SS cargos that are essential for hemoglobin degradation and heme recruitment293031. Therefore, lack of pigmentation in ΔPorZ suggested failure of functional gingipain secretion and activation. Indeed, we found that ΔPorZ was deficient in extracellular Kgp and Rgp gingipain activities when compared to the wild-type P. gingivalis strain W83 (hereafter, “wild type”; Fig. 1b,c). In contrast, dipeptidyl peptidase IV and prolyl tripeptidyl peptidase A, which are surface enzymes but not secreted by T9SS, were produced and transported to the bacterial surface in significantly higher amounts than in the wild type (Fig. 1d). A similar response had been previously observed in an inactivation mutant of an essential T9SS component, PorT32. This presumably reflects general upregulation of peptidolytic enzymes as a response to the absence of functional gingipains, which account for 85% of the extracellular proteolytic activity of P. gingivalis33. Reconstitution in trans of the porZ gene in the ΔPorZ mutant—yielding PorZ+—restored both pigmentation and proteolytic activity to wild-type levels (Fig. 1a–d).

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 &beta;-propeller domains and a C-terminal &beta;-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