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1.
Fig. 8

Fig. 8. Glycosylation deficiency impairs bacterial survival. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

Bacterial loads in gingival tissues from mice infected mice with T. forsythia wild-type or ED1 strains on day 10 postinfection as determined by quantitative real-time PCR. The bars and error bars indicate the means and standard deviations for four mice in each group; *, p< 0.05.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
2.
Fig. 6

Fig. 6. T. forsythia wild-type strain induces increased osteoclastic activity in alveolar bone. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

(A) Representative histological sections showing TRAP+ cells from sham- and bacteria-infected mice. (B) Average number of TRAP+ cells in 10 high power magnification fields/slide (4 mice/group). Statistically significant differences between groups is indicated by; **, p<0.01.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
3.
Fig. 9

Fig. 9. A model depicting the role of T. forsythia surface glycosylation in pathogenesis. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

The S-layer glycoproteins modulate DC effector functions to suppress Th17 responses, promoting bacterial persistence in the oral cavity. Bacteria then exploit TLR2 signaling to favor dominance of Th2 responses. The TLR2-Th2 inflammatory axis causes tissue destruction and drives osteoclastogenesis. Concurrently, inflammation promotes bacterial growth by making available peptides, heme and other factors as nutrients.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
4.
Fig. 1

Fig. 1. Alteration of the glycosylation pattern of surface proteins of T. forsythia by deletion of the wecC gene results in an altered but intact S-layer and no changes in LPS. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

(A) TEM of negatively stained ultrathin sections of wild-type and ED1 mutant strains. Arrowheads indicate the S-layer structure covering the outer membrane.
(B) Outer membrane preparations of wild-type and ED1 strains were separated on SDS-PAGE (4-8%) gels and stained with silver (left), ProQ Emerald glycostain (middle) and probed with an anti-S-layer antibody after western blotting (right)
(C) LPS was extracted and separated on a 10% Urea SDS-PAGE gel and silver stained

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
5.
Fig. 2

Fig. 2. T. forsythia surface glycosylation differentially regulates cytokine expression in macrophages and dendritic cells. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

Inflammatory cytokine levels (A. IL-1 β, IL-6 and IL-10; B. IL12p40, IL12p70-, IL-23) were examined by ELISA in supernatants of mouse DC’s and peritoneal macrophages following challenge with either the wild-type (Tf WT) or the glycosylation deficient mutant (ED1) of T. forsythia. The data show the means ± standard deviations of triplicate determinations in one of 3 independent sets of experiments that yielded similar results; statistically significant differences between the groups are indicated by asterisks (***, p<0.001; **, p<0.01; *, p< 0.05).

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
6.
Fig. 7

Fig. 7. T. forsythia glycosylation deficient ED1 mutant induces enhanced lymphocytes/neutrophil infiltration following infection. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

(A) Immunohistochemical staining for total inflammatory cells (CD45 positive) and neutrophils in gingival tissue 3 weeks following infection. All images are representative of 400x magnification. (B) Slide images were viewed with Aperio Image Scope viewing software and the inter-dental areas from the first to third molar were used to quantify inflammatory cells. Bar graphs showing number of CD45 positive and neutrophil antibody positive cells. Statistically significant differences between the respective T. forsythia-infected and sham-infected groups is indicated by; ***, p<0.001; **, p<0.01; *, p< 0.05, n.s, not significant.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
7.
Fig. 5

Fig. 5. T. forsythia glycosylation deficient ED1 mutant induces Th17 cells following infection. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

Production of IFN-γ, IL-5 and IL-17 in cervical lymph nodes of mice. Mice (n=4) were infected 3 times at 48 h intervals. 72 h after the final infection, cLN cells were stimulated with anti-CD3 and anti-CD28 Abs for 48 h. Cells were then stimulated with ionomycin and PMA for 4-6 h prior to intracellular staining for IL-5, IFN-γ or IL-17 (A) Representative flow cytometry dot plots of CD4+ T cells from sham and bacteria infected mice intracellularly stained for IL-5, IFN-γ or IL-17. (B) Bar graphs showing percentages (mean and standard deviations) of specific cytokine positive T cells for each group. Statistically significant differences between the groups is indicated by; *, p< 0.05, n.s, not significant.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
8.
Fig. 3

Fig. 3. Abrogation of surface glycosylation in T. forsythia causes enhanced dendritic cell association. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

Association of FITC-labeled wild-type (Tf WT) and ED1 strains was assessed by flow cytometry. (A) Representative traces of bacteria bound/engulfed by BMDCs are demarcated by the thick grey (wild-type strain) and dark (ED1 strain) lines. Autofluorescence signal is demarcated by the thin line (untreated BMDCs) and corresponds to BMDCs without bound/ingested bacteria. (B) Bar graphs show percentages of BMDCs in gates M1 (representing the background autofluorescence and unbound bacteria) and M2 (representing brightly fluorescent cell population with bound and/or internalized FITC-labeled bacteria. (C) Bar graphs represent the mean fluorescent intensities (±S.D.). (D) Bacterial killing by DCs. Dcs were infected with bacteria (wild-type or ED1) at an MOI of 10. The number of viable bacteria were determined (CFU) at indicated time points post infection. Data represent mean CFU (±S.D.) per 105 DC cells of triplicate readings from two independent experiments. ***P<0.001.

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.
9.
Fig. 4

Fig. 4. T. forsythia wild-type strain is more virulent than the glycosylation deficient ED1 mutant strain in a mouse model of periodontitis. From: A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

(A) Specific and cross-reactive antibodies are elevated in mice following oral infection. Sera from mice after 6 weeks of first infection (sham, Tf WT, or ED1) were analyzed for T. forsythia specific IgG by ELISA. Antibody levels are presented as log2 titers. Data represents means and standard deviations for each group (n=8), and statistical differences between the group means was determined (*, p<0.05 vs sham-infected group; #, p<0.05 vs Tf infected group). (B & C) ED1 mutant causes significantly less bone loss compared to the wild-type strain. Mice (n = 8) were infected by oral gavage with 6 doses (109 cells/dose) of either T. forsythia wild-type strain (109 cells/dose; Tf WT) or T. forsythia ED1 mutant or sham-infected. (B) Alveolar bone destruction was assessed after 6 weeks by measuring the distance from the ABC to the CEJ at 14 maxillary buccal sites per mouse (R1-R7 = right jaw; L1-L7 = left jaw). (C) Net bone loss induced by wild-type or ED1 bacteria was calculated as mean total ABC-CEJ distance of bacterially infected group minus mean total ABC-CEJ distance of the sham-infected group. The data show that the net bone loss caused by the wild-type strain is significantly higher than that by the ED1 mutant. Bars indicate means and standard deviations. Data were analyzed by a Mann-Whitney unpaired t test, and statistically significant differences are indicated with an asterisk (***, p<0.001; **, p<0.01; n. s. not significant).

Rajendra P. Settem, et al. Mucosal Immunol. ;6(2):415-426.

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