Figure 1.
P. gingivalis strain 381 utilizes endogenous lipid A 1- and 4′- phosphatase activities to modify lipid A species and evade TLR4 activation.
Lipid A isolated from P. gingivalis wild-type strain 381 (A) or lipid A mutant strains PG1587381 (B) and PG1773381 (C) were examined by MALDI-TOF MS. Arrows indicate the predominant lipid A species that are expressed for each strain (A–C). The major lipid A structures examined in this study have been identified in P. gingivalis as previously described (D–G) [19].
Figure 2.
Lipid A phosphatase activity contributes to the ability of P. gingivalis to evade host innate immune defenses.
HEK cells overexpressing mouse TLR4-MD2 (A–B) or TLR2 (D–E) were stimulated with corresponding LPS concentration purified from each strain (A,D) or whole bacterium (B,E) overnight. Relative NFkB activity indicates inducible firefly luciferase activity over the media control. Wild-type 381 and the lipid A mutants PG1587381 and PG1773381 were grown in BHI broth cultures overnight in the presence or absence of polymyxin B (PMB 1, 5, 10, 25, 50 µg/mL). Growth was measured by spectrophotometry (OD660) (C). Wild-type P. gingivalis (solid), PG1587381 (dotted), and PG1773381 (dashed). Graphs show the mean ± SEM of triplicate wells and are representative of two independent experiments. (See also Figure S1).
Figure 3.
Expression of antagonistic or inert lipid A attenuates the production of NFkB-dependent proinflammatory mediators.
BMDMs from C57BL/6 were stimulated with P. gingivalis wild-type strain 381 (381) or the lipid A mutant strains PG1587381 (1587) and PG1773381 (1773) at an MOI of 1 (white) 10 (gray) and 100 (black). Levels of TNFα (A), KC (B), and IL-6 (C) were assayed by ELISA at 24 h. BMDMs from wild-type C57BL/6 mice (white), TLR2-deficient (gray), or TLR4-deficient (black) were stimulated with P. gingivalis wild-type strain 381 or the lipid A mutant strains at an MOI of 100 (D) or MOI of 10 (E–F) and levels of TNFα (D) KC (E) and IL-6 (F) were assessed by ELISA at 24 h (See also Figure S2). Cells treated with E. coli LPS (100 ng/mL) or Pam3CysSk4 (1 µg/mL) for 5 h served as a positive control. Graphs show the mean ± SEM of triplicate wells and are representative of three independent experiments. *p<.05 **p≤.001 ***p<.0001; ANOVA with Bonferroni's posttest.
Figure 4.
P. gingivalis evades activation of the inflammasome through expression of modified lipid A species.
C57BL/6 WT BMDM were stimulated with P. gingivalis wild-type strain 381 or the lipid A mutant strains PG1587381 and PG1773381 at an MOI of 1 (white), 10 (gray) or 100 (black). Levels of IL-1β (A) and IL-1α (B) were assayed by ELISA at 24 h. LDH release (Promega) was assayed in BMDM stimulated with P. gingivalis wild-type strain 381 or the lipid A mutants at an MOI 100 at 2 h (white), 6 h (gray) and 24 h (black) (C). BMDM from wild-type (white bars) and caspase-11-deficient (black bars) mice were stimulated with P. gingivalis wild-type strain 381 or the lipid A mutants at an MOI of 100 and levels of IL-1β (D) and IL-1α (E) were assessed by ELISA at 24 h. Viable counts (CFU) of internalized P. gingivalis were determined by plating serial dilutions of macrophage lysates on blood agar plates at 2 h (white) and 6 h (gray) (F). Western blot analysis was performed on BMDM cell lysates to assess levels of the proform of IL-1β, pro Caspase-1 and β-actin at 24 h (G). Levels of mature IL-1β and active caspase-1 (p10) were detected in cell supernatants. BMDMs treated with media alone (−) or with E. coli LPS (100 ng/mL) for 5 h and then ATP (5 mM) for 20 m (+) served as the negative and positive control respectively. Graphs depict the mean ± SEM of triplicate wells and are representative of at least three independent experiments. * p<.05 **p≤.001 ***p<.0001; two-tailed unpaired t-tests. (See also Figure S2).
Figure 5.
Expression of immunologically silent or antagonistic lipid A structures exacerbates atherosclerotic plaque progression in the innominate artery.
Innominate arteries of ApoE−/− mice were imaged by MRA at baseline (wk 0) and at 8 and 16 wks after first oral infection. The temporal change in luminal area (mm2) was calculated for individual mice normalized to baseline luminal area (n = 10–12/group) (A). Sham-infected ApoE−/− (orange); 381-infected ApoE−/− (black); PG1587381-infected ApoE−/− (blue); PG1773381- infected ApoE−/− (red). Inset - the temporal change in luminal area calculated for individual mice normalized to 8 wk luminal area (n = 10–12/group). *p≤.01 **p<.001 ***p<.0001; two-tailed unpaired t-tests compared to sham-infected and PG1587381-infected mice. (B) Representative images of the innominate artery with F4/80 staining (macrophages stain brown) and hematoxylin counterstaining for each group at 10× and 40× (n = 3/group) (See also Figure S3).
Figure 6.
Expression of immunologically silent or antagonistic lipid A structures exacerbates atherosclerotic plaque progression in the aorta.
Plaque area was determined from Oil Red O staining for lipids in en face aortic lesions 16 wk after first infection from (A) sham-infected, (B) wild-type 381, (C) PG1587381, and (D) PG1773381. (E) Quantification of lipid content within total aorta was calculated using ImageJ software (NIH) (n = 8/group). * p<.05; two-tailed unpaired t-tests. (See also Figure S4).
Figure 7.
Expression of immunologically silent, agonistic or antagonistic lipid A does not alter the ability of the pathogen to induce oral bone loss.
Maxillae were dissected (n = 8/group) 16 wk post initial infection and scanned on a micro CT 40 apparatus. Using AMIRA software, three-dimensional images were generated from micro CT scans. Representative images from (A) sham-infected mouse indicating no bone loss, (B) P. gingivalis wild-type strain 381-induced bone loss, (C) PG1587381, and (D) PG1773381. (E) Quantification of bone loss. ***p<.001; two-tailed unpaired t-tests.
Figure 8.
P. gingivalis dysregulates host cell immune activation facilitating systemic inflammation.
The host predominantly senses P. gingivalis infection through engagement of TLR2 while the involvement of TLR4-dependent recognition is significantly impaired. Expression of antagonistic or immunologically inert lipid A by P. gingivalis attenuates production of proinflammatory mediators and prevents activation of the inflammasome (potentially through evasion of an unknown sensor) that facilitates intracellular survival. These events allow the pathogen to disseminate and to exacerbate systemic inflammation. In contrast, increased immunostimulatory potential at TLR4, through expression of an agonistic lipid A moiety, results in increased production of proinflammatory mediators, inflammasome activation, and reduced survival of the bacterium in macrophages leading to attenuated systemic inflammation.