Characterization of <i>Treponema denticola</i> Mutants Defective in the Major Antigenic Proteins, Msp and TmpC

<div><p><i>Treponema denticola</i>, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteinswere Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of <i>T</i>. <i>denticola</i>, especially cell surface properties.</p></div

Cell surface hydrophobicity.

<p>(A) Cell surface hydrophobicity was quantified by measuring the partition coefficient of <i>T</i>. <i>denticola</i> cells between the aqueous and <i>n</i>-hexadecane phases. The data presented show a representative result from two independent experiments and are expressed as the means ± SD (n = 3). A Dunnett’s test was performed to compare the wild type and mutants (**, <i>P</i><0.01). (B) Colony appearance of <i>T</i>. <i>denticola</i> strains cultivated on a medium containing 2.5% agar. The wild type and Δ<i>tmpC</i> were smooth in appearance, whereas Δ<i>msp</i> were rough and clearly different, indicating that Δ<i>msp</i> has a hydrophobic surface.</p

Novel fimbrilin PGN_1808 in <i>Porphyromonas gingivalis</i>

<div><p><i>Porphyromonas gingivalis</i>, a periodontopathic gram-negative anaerobic bacterium, generally expresses two types of fimbriae, FimA and Mfa1. However, a novel potential fimbrilin, PGN_1808, in <i>P</i>. <i>gingivalis</i> strain ATCC 33277 was recently identified by an <i>in silico</i> structural homology search. In this study, we experimentally examined whether the protein formed a fimbrial structure. Anion-exchange chromatography showed that the elution peak of the protein was not identical to those of the major fimbrilins of FimA and Mfa1, indicating that PGN_1808 is not a component of these fimbriae. Electrophoretic analyses showed that PGN_1808 formed a polymer, although it was detergent and heat labile compared to FimA and Mfa1. Transmission electron microscopy showed filamentous structures (2‒3 nm × 200‒400 nm) on the cell surfaces of a PGN_1808-overexpressing <i>P</i>. <i>gingivalis</i> mutant (deficient in both FimA and Mfa1 fimbriae) and in the PGN_1808 fraction. PGN_1808 was detected in 81 of 84 wild-type strains of <i>P</i>. <i>gingivalis</i> by western blotting, suggesting that the protein is generally present in <i>P</i>. <i>gingivalis</i>.</p></div

Subcellular localization of TDE2508.

<p>The whole cell lysate (WCL), soluble fraction (Sol) and envelope fraction (Env) of <i>T</i>. <i>denticola</i> ATCC 35405 were separated by SDS-PAGE with CBB-staining (A) and analyzed by Western blotting with anti-TDE2508 antiserum (B). The envelope fraction was further fractionated by differential solubilization in 0, 0.25, 0.5, 1, 2, 4, and 8% Triton X-100, and the soluble fractions (lanes 1–7, respectively) and insoluble fractions (lanes 8–14, respectively) were subjected to Western blot analysis with anti-<i>T</i>. <i>denticola</i> whole cell antiserum (C) and anti-TDE2508 antiserum (D). The grey, white and black arrowheads denote Msp, TmpC and TDE2508, respectively. M denotes a standard marker.</p

Biofilm formation. <i>T</i>. <i>denticola</i> ATCC 35405 (Wild) and <i>tde2508</i>-deletion mutant (Δ<i>2508</i>) were incubated on a polystyrene plate for 2 days.

<p>After washing, the adherent cells were collected and quantified using the optical density (OD) at 600 nm (A). Data represent means and standard deviations of two experiments performed in triplicate. Additionally, the adherent cells were observed by scanning electron microscopy (B).</p

Protein patterns of <i>T</i>. <i>denticola</i> strains.

<p>The whole-cell lysates of the wild type (WT), Δ<i>tmpC,</i> and Δ<i>msp</i> were applied to SDS-PAGE (5–20% gradient gel) followed by CBB-staining (A) and western blotting using anti-<i>T</i>. <i>denticola</i> antiserum (B). Panel (A) shows that TDE1072 (a) and Msp (c) were absent in Δ<i>msp</i>. Western blot analysis confirmed deficiencies of TmpC (d) and Msp (c) in Δ<i>tmpC</i> and Δ<i>msp</i>, respectively. The arrowhead b in panel A was identified as OppA. Molecular-weight standards (kDa) are shown at the left.</p

Deduced amino acid sequence of PGN_1808.

<p>The LipoP program predicted that the N-terminal 20 amino acids were a signal peptide (underlined). N-terminal sequencing of the 50-kDa PGN_1808 monomer band in the SDS-PAGE analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173541#pone.0173541.g002" target="_blank">Fig 2A</a>) showed that the N-terminal amino acid was Gly-56, indicating that the N-terminal 55 amino acids were removed for processing (highlighted in gray).</p

Reactivity of sera of mice injected with <i>T</i>. <i>denticola</i> strains to cellular components of <i>T</i>. <i>denticola</i>.

<p>The whole-cell lysate of wild-type <i>T</i>. <i>denticola</i> was applied to SDS-PAGE (11% gel) and blotted on a PVDF membrane. The PVDF membrane was cut into strips and reacted with each serum sample collected at 3 weeks after injection. As a negative control, sera of mice injected with PBS were pooled and subjected to immunoreaction. Numbers denote individual mice. Major bands were indicated by a, b, c, and d and were predicted to react to TDE1072, OppA, Msp, and TmpC, respectively. A molecular-weight standard (kDa) is shown at the left.</p

Structure and polymerization models.

<p>PGN_1808 without the signal sequence (the N-terminal 20 amino acids) was submitted to structure homology-modeling (A). The color (orange to blue) indicates the QMEAN score, which indicates the reliability of the model (low to high quality, respectively) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173541#pone.0173541.ref023" target="_blank">23</a>]. The N-terminal sequence (from the 21st to 55th amino acid), surrounded by the black dotted line, was removed for processing (see text). PGN_1808 is predicted to polymerize by donor-strand complementation and cleft-mediated anchorage (B). White letter N denotes the N-terminal region.</p

Fractionation of bacterial cell lysates.

<p><i>P</i>. <i>gingivalis</i> TDC60 lysates were fractionated by anion-exchange chromatography after ammonium sulfate precipitation (A). Fractions 30 to 50 (10 μl each) were subjected to SDS-PAGE, followed by CBB staining (B) and western blotting using anti-FimA, anti-Mfa1, and anti-PGN_1808 antisera (C). We demonstrated reproducibility by repeating the fractionation experiment three times (data not shown). Numbers in left indicate molecular weights (kDa).</p