O-Polysaccharide Plays a Major Role on the Virulence and Immunostimulatory Potential of Aggregatibacter actinomycetemcomitans During Periodontal Infection

Abstract

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium with high immunostimulatory and pathogenic potential involved in the onset and progression of periodontitis, a chronic disease characterized by aberrant immune responses followed by tooth-supporting bone resorption, which eventually leads to tooth loss. While several studies have provided evidence related to the virulence factors of A. actinomycetemcomitans involved in the host cell death and immune evasion, such as its most studied primate-specific virulence factor, leukotoxin, the role of specific lipopolysaccharide (LPS) domains remain poorly understood. Here, we analyzed the role of the immunodominant domain of the LPS of A. actinomycetemcomitans termed O-polysaccharide (O-PS), which differentiates the distinct bacterial serotypes based on its antigenicity. To determine the role of the O-PS in the immunogenicity and virulence of A. actinomycetemcomitans during periodontitis, we analyzed the in vivo and in vitro effect of an O-PS-defective transposon mutant serotype b strain, characterized by the deletion of the rmlC gene encoding the α-L-rhamnose sugar biosynthetic enzyme. Induction of experimental periodontitis using the O-PS-defective rmlC mutant strain resulted in lower tooth-supporting bone resorption, infiltration of Th1, Th17, and Th22 lymphocytes, and expression of Ahr, Il1b, Il17, Il23, Tlr4, and RANKL (Tnfsf11) in the periodontal lesions as compared with the wild-type A. actinomycetemcomitans strain. In addition, the O-PS-defective rmlC mutant strain led to impaired activation of antigen-presenting cells, with less expression of the co-stimulatory molecules CD40 and CD80 in B lymphocytes and dendritic cells, and downregulated expression of Tnfa and Il1b in splenocytes. In conclusion, these data demonstrate that the O-PS from the serotype b of A. actinomycetemcomitans plays a key role in the capacity of the bacterium to prime oral innate and adaptive immune responses, by triggering the Th1 and Th17-driven tooth-supporting bone resorption during periodontitis.

Keywords: Aggregatibacter actinomycetemcomitans; LPS; O-PS; O-polysaccharide; T-lymphocytes; bone resorption; lipopolysaccharide; periodontitis.

Figure 1

Lipopolysaccharide (LPS) analysis and alveolar bone loss triggered by the different strains of A. actinomycetemcomitans. (A) Diagram illustrating the protocol for the bacteria-induced periodontitis model. (B) LPS profiles obtained by SDS-PAGE analysis of the VT1169 wild-type strain and the rmlC and waaL mutant strains, as well as the rmlC/rmlC⁺ and waaL/waaL⁺ complemented strains. (C) Schematic view of the A. actinomycetemcomitans serotype b O-polysaccharide (O-PS) biosynthetic pathway, highlighting the modified enzymes and the O-PS profile obtained by Tang et al. (26) and used in the present study. (D) The 2% agarose gel of the amplification products obtained with specific primers for the strain VT1169, from PCR analysis of the V3-V6 variable region of the 16S rDNA. (E) Sagittal slices of the 2D-reconstruction by μCT showing the alveolar bone loss determined as the CEJ-ABC linear distance in the interdental area between the first and second molar (white-red lines) in mice infected with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains and the sham-infected and untreated controls. (F) Vestibular view of the 3D-reconstruction by μCT showing the alveolar bone loss, determined as the CEJ-ABC linear distance in the distal surface of the distal root of the first molar in the same experimental conditions described in (E). Yellow double-arrow lines represent the CEJ-ABC linear distance (CEJ marked as a dashed white line and ABC marked as a dashed red line). (G) Quantification of the alveolar bone loss determined as the CEJ-ABC linear distance in the distal surface of the distal root of the first molar in mice infected with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains and the sham-infected and untreated controls (UT). (H) Quantification of the alveolar bone loss determined as the percentage of remnant alveolar bone in mice infected with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains and the sham-infected and UT controls, considering the average of remnant alveolar bone in UT mice as 100%. (I) Sagittal slices of the 2D-reconstruction by μCT showing the alveolar bone loss determined as the CEJ-ABC linear distance in the interdental area between the first and second molar (white-red lines) in mice infected with the A. actinomycetemcomitans VT1169 and ATCC 23718 strains. (J) Vestibular view of the 3D-reconstruction by μCT showing the alveolar bone loss, determined as the CEJ-ABC linear distance in the distal surface of the distal root of the first molar in the same experimental conditions described in (I). Yellow double-arrow lines represent the CEJ-ABC linear distance (CEJ marked as a dashed white line and ABC marked as a dashed red line). (K) Quantification of the alveolar bone loss determined as the CEJ-ABC linear distance in the distal surface of the distal root of the first molar in mice infected with the A. actinomycetemcomitans VT1169 and ATCC 23718 strains and UT controls. (L) Quantification of the alveolar bone loss determined as the percentage of remnant alveolar bone in mice infected with the A. actinomycetemcomitans VT1169 and ATCC 23718 strains and UT controls, considering the average of remnant alveolar bone in UT mice as 100%. Mean ± SD, one-way ANOVA and Tukey post-hoc test, **p < 0.01, ****p < 0.0001. Error bars represent SEM in all panels. ABC, alveolar bone crest; CEJ, cement-enamel junction; CFU, colony-forming units; cLNs, cervical lymph nodes; FC, flow cytometry; ns, no significant; LPS, lipopolysaccharide; O-PS, O-polysaccharide; OS, oligosaccharide; PPTs, palatal periodontal tissues.

Figure 2

Th1/Th17/Th22-related cytokines, RANKL, and TLR expression within the periodontal tissues induced by the different strains of A. actinomycetemcomitans. (A) Heatmap of mRNA expression levels for Th1, Th17, Th22, and T regulatory-related cytokines and RANKL quantified by qRT-PCR. Bar plots showing relative expression of (B) Th1-related Ifng and Il1b, (C) Th17-related Il6, Il17, and Il23, (D) Th22-related Il22 and Ahr, (E) T regulatory-related Il10, (F) RANKL (Tnfsf11), and (G) Tlr2 and Tlr4 mRNAs analyses in periodontal lesions infected with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains (n=4). The data were pooled from three independent experiments. For relative expression, the mRNA expression in untreated (UT) mice was considered as 0, as a reference for fold-change in expression. Mean ± SD, one-way ANOVA and Tukey post-hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001, ns, non-significant. Error bars represent SEM in all panels. Ahr, aryl hydrocarbon receptor; Il, interleukin; Ifng, interferon-gamma; Tlr, toll-like receptor; RANKL (Tnfsf11), receptor activator of nuclear-factor κB ligand.

Figure 3

T-bet⁺, RORγt⁺, and AhR⁺ T lymphocytes detection within the periodontal tissues induced by the different strains of A. actinomycetemcomitans. Cells obtained from periodontal tissues were analyzed by flow cytometry. (A) Gating strategy used for lineage determination of single/live/CD45⁺CD3⁺CD4⁺ T lymphocytes. Flow cytometric quantification of (B, C) CD45⁺CD3⁺CD4⁺T-bet⁺ Th1 lymphocytes, (D, E) CD45⁺CD3⁺CD4⁺RORγt⁺ Th17 lymphocytes, and (F, G) CD45⁺CD3⁺CD4⁺AhR⁺ Th22 lymphocytes in periodontal lesions upon infection with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains, or the untreated (UT) controls. Plots are representative of three independent experiments (n=3). Mean ± SD, one-way ANOVA and Holm-Sidak post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ns, non-significant. Error bars represent SEM in all panels.

Figure 4

T-bet⁺, RORγt⁺, and AhR⁺ T lymphocytes detection within the cervical lymph nodes induced by the different strains of A. actinomycetemcomitans. Cells obtained from cervical lymph nodes were analyzed by flow cytometry. (A) Comparison of the number of single/live/CD45⁺CD3⁺CD4⁺ T lymphocytes between periodontal tissues and cervical lymph nodes. Flow cytometric quantification of (B, C) CD45⁺CD3⁺CD4⁺T-bet⁺ Th1 lymphocytes, (D, E) CD45⁺CD3⁺CD4⁺RORγt⁺ Th17 lymphocytes, and (F, G) CD45⁺CD3⁺CD4⁺AhR⁺ Th22 lymphocytes in periodontal lesions upon infection with the A. actinomycetemcomitans VT1169, rmlC, or rmlC/rmlC⁺ strains, or the untreated (UT) controls. Data were pooled from three independent experiments (n=3). Mean ± SD, one-way ANOVA and Holm-Sidak post-hoc test, *p < 0.05. Error bars represent SEM in all panels.

Figure 5

B-lymphocyte, dendritic cell, and splenocyte responses against the different strains of A. actinomycetemcomitans. (A) tSNE map of 11-parameter flow cytometry data from untreated (UT) mice, color-coded as follows: B-cells in red, T-cells in cyan, innate lymphoid cells (ILCs) in orange, macrophages in light green, dendritic cells in dark green, monocytes in brown, and neutrophils in purple. (B) Percentage of each cell population described in (A). Data pooled from two independent experiments (n=4). The MFI levels of the co-stimulatory molecules CD40 and CD80 are shown in (C) for B lymphocytes, (D) for dendritic cells, and (E) macrophages stimulated 20 h with the A. actinomycetemcomitans VT1169, rmlC, rmlC/rmlC⁺, waaL, or waaL/waaL⁺ strains, and the E. coli-derived LPS-stimulated and UT cells, used as controls. The histograms representing the MFI levels of the co-stimulatory molecules CD40 and CD80 are shown in (F) for B lymphocytes and (G) for dendritic cells, under the same conditions experimental described in (C-E). Data pooled from three different experiments (n=7). (H) Heatmap and (I) bar plots of qRT-PCR analysis of Tnfa, Il1b, Il17, and Il23 transcripts in total splenocytes stimulated 8 h with the A. actinomycetemcomitans VT1169, rmlC, rmlC/rmlC⁺, waaL, or waaL/waaL⁺ strains, and the E. coli-derived LPS-stimulated and UT cells, used as controls (n=3). Data pooled from two independent experiments. Mean ± SD, one-way ANOVA and Tukey post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM in all panels. Il, interleukin; LPS, E. coli-derived lipopolysaccharide; Tlr, toll-like receptor; Tnfa, tumor necrosis factor-alpha.