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1.
Figure 2

Figure 2. Characterizations of the TDE0471 mutant (Tde471mut) and its complemented strain (Tde471com). From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

(A) Immunoblotting analysis of Td35405, Tde471mut, and Tde471com strains. Equivalent amounts of Td35405, Tde471mut, and Tde471com whole cell lysates were analyzed by SDS-PAGE and then probed with αTdneu, a specific antibody against Tdneu. The flagellin protein, FlaA, was used as a sample loading control and detected by αFlaA, a specific antibody against FlaA. (B) Filter paper spot test of Td35405, Tde471mut, and Tde471com strains. The assay was carried out as described in using the whole cell lysates of the three strains.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
2.
Figure 5

Figure 5. Growth curves of Td35405, Tde471mut, and Tde471com strains in TYGVS medium (A) and the serum growth medium (B). From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

The serum growth medium contains 50% heat-inactivated rabbit serum supplemented with 6.0 μg/ml TPP. Cell counting was repeated in triplicate with at least three independent samples. The results are expressed as the mean ± SEM. The data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison at p < 0.01.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
3.
Figure 8

Figure 8. Evaluating the virulence of Td35405, Tde471mut, and Tde471com strains using a mouse-skin-infection model. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

The infectious studies were carried out as previously documented (). 109 bacterial cells were subcutaneously injected into each mouse. Three different mouse strains were included: (A) BALB/C mice; (B) The complement-deficient mice (B6.129S4-C3tm1Crr/J) and its parental wild-type C57BL/6 mice. Ten days post-infection, the sizes of the abscesses were measured. The average sizes of the observed abscesses were calculated. The statistic differences between groups were analyzed by t-test, two-tailed at p < 0.01

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
4.
Figure 4

Figure 4. Lectin blot analysis of rTdneu-treated human serum. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

(A) Normal human serum (NHS) was treated with various amounts of rTdneu at 37°C for 1 h. The resultant samples were separated on SDS-PAGE gels followed by Coomassie blue staining; (B-D) The NHS was treated with 0.2 μg rTdneu or the same amount of rTdMneu at 37°C for 3 hours. The resultant samples were separated on SDS-PAGE gels, transferred to PVDF membranes, and probed with biotin-labeled SNA (B, 0.2 μg/ml), MAA (C, 2 μg/ml), or ConA (D, 0.5 μg/ml). The final concentrations of NHS in the reactions were 0.15% for SNA and ConA, and 1.5% for MAA.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
5.
Figure 6

Figure 6. Survival rates of Td35405, Tde471mut, and Tde471com strains in serum. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

The strains were co-incubated with either 25% normal human serum (NHS) or heat-inactivated human serum (HIS) for 1 h at 37°C. The number of surviving bacteria was counted using a Petroff Hausser counting chamber. The survival rates were calculated as follows: the total number of living cells in NHS divided by the total number of living cells in HIS. Cell counting was repeated in triplicate with at least three independent samples, and the results are expressed as the mean ± SEM. The data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison at p < 0.01.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
6.
Figure 3

Figure 3. Localization of Tdneu. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

(A) Immunoblotting analysis of proteinase K-treated Td35405 whole-cell lysates. The spirochetes were either incubated with proteinase K (240 μg/ml) or PBS at 37°C for 1 h. The resultant samples were separated on SDS-PAGE and then probed with αTdneu and αFlaA. (B) IFA analysis. Td35405 and Tde471mut cells were fixed with methanol, stained with αTdneu, and counterstained with a goat-anti-rat Texas red antibody as previously described. The micrographs were taken under DIC light microcopy or fluorescence microscopy with a tetramethylrhodamine isothiocyanate (TRITC) emission filter, and the resultant images were merged. (C) Detection of Tdneu in the supernatants prepared from the cultures of Td35405, Tde471mut, and Tde471com strains by immunoblotting analysis. FlaA was used as a sample preparation control. (D) Detecting neuraminidase activity in the supernatants by the filter paper spot test as described in .

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
7.
Figure 7

Figure 7. Deposition of membrane attack complex (MAC) on Td35405, Tde471mut, and Tde471com strains. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

106 cells of Td35405, Tde471mut, or Tde471com were co-incubated with 25% NHS for 20 min at 37°C. As a control, 10 mM EDTA was added to the reactions to block complement activation. The resultant serum-treated cells (approximately 5 × 104) were subjected to SDS-PAGE, followed by coomassie blue staining (A), or probed with four different antibodies as labeled: (B) αTd, a rat polyclonal antibody against Td35405; (C) αC3, a polyclonal antibody against C3; (D) αC9, a monoclonal antibody against C9; and (E) αC5-9, a monoclonal antibody against C5-9 complex. Notably, complement components were detected only in the serum-treated Tde471mut, but not in the serum-treated Td35405 and Tde471com strains. The complement factors detected by the antibodies and their molecular weights are indicated.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.
8.
Figure 1

Figure 1. TDE0471 (Tdneu) enzymatic assay. From: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola.

(A) Filter paper spot sialidase test of rTdneu. The substrate 4-MUNANA was treated with: rTdneu (spot 1, from the left), truncated rTdneu (rTdMneu, spot 2), and Neu5Ac2en (spots 3 and 4). The image was processed using the ChemiDoc XRS system (Bio-Rad) with an excitation wavelength of 302 nm and an emission wavelength of 548 nm. (B) rTdneu is active in a wide pH range. For this assay, the spot test was carried out from pH 3.0 to pH 9.0. The release of 4-MU (the product 4-MUNANA) was quantified based on a standard curve (fluorescence intensity vs. defined amounts of 4-MU). The data shown are the means from at least three samples. Error bars represent ± the standard error of the mean (SEM). The amounts of 4-MU (the product of 4-MUNANA) were determined using a standard curve. (C) Kinetic analysis of rTdneu. This assay was carried out according to a standard method for enzyme kinetics studies. The saturation curves were fitted to Michaelis-Menten kinetics using GraphPad Prism (GraphPad Software), and Km and Vmax were calculated. (D) Lectin blot analysis of rTdneu. The assay was performed as previously described (;). Biotin-labeled SNA, MAA, and DSA lectins were used to detect terminal α2,6- and α2,3-linked sialic acid and galactose linked to GlcNAc of human α-1 acid glycoprotein (AGP), respectively. The C. perfringens neuraminidase NanH was used as a positive control, and rTdMneu was used as a negative control. Arrows point to the products detected by the three lectins.

Kurni Kurniyati, et al. Mol Microbiol. ;89(5):10.1111/mmi.12311.

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