Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

Stephen S Dominy, Casey Lynch, Florian Ermini, Malgorzata Benedyk, Agata Marczyk, Andrei Konradi, Mai Nguyen, Ursula Haditsch, Debasish Raha, Christina Griffin, Leslie J Holsinger, Shirin Arastu-Kapur, Samer Kaba, Alexander Lee, Mark I Ryder, Barbara Potempa, Piotr Mydel, Annelie Hellvard, Karina Adamowicz, Hatice Hasturk, Glenn D Walker, Eric C Reynolds, Richard L M Faull, Maurice A Curtis, Mike Dragunow, Jan Potempa, Stephen S Dominy, Casey Lynch, Florian Ermini, Malgorzata Benedyk, Agata Marczyk, Andrei Konradi, Mai Nguyen, Ursula Haditsch, Debasish Raha, Christina Griffin, Leslie J Holsinger, Shirin Arastu-Kapur, Samer Kaba, Alexander Lee, Mark I Ryder, Barbara Potempa, Piotr Mydel, Annelie Hellvard, Karina Adamowicz, Hatice Hasturk, Glenn D Walker, Eric C Reynolds, Richard L M Faull, Maurice A Curtis, Mike Dragunow, Jan Potempa

Abstract

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Figures

Fig. 1. Gingipain IR in brain correlates…
Fig. 1. Gingipain IR in brain correlates with AD diagnosis and pathology.
(A and B) Representative TMA NVD005 containing brain tissue cores from the MTG of AD patients and controls probed for RgpB (A) and Kgp (B) with antibodies CAB101 and CAB102, respectively. Higher magnification of representative tissue cores reveals higher neuronal RgpB-IR and Kgp-IR in AD tissue cores than in control cores. (C) RgpB-IR and (D) Kgp-IR data from TMAs NVD005 and NVD003 show significantly higher load in AD brain compared to controls. Mann-Whitney test, ***P < 0.0001; presented as geometric mean ± 95% confidence interval, n = 99 (C) and n = 104 (D). (E and F) Tau load correlates to RgpB load (Spearman r = 0.674, P < 0.0001, n = 84) (E) and Kgp load (Spearman r = 0.563, P < 0.0001, n = 89) (F). Blue, control; red, AD. (G and H) Ubiquitin load, a marker of AD pathology, correlates to RgpB load (blue, control; red, AD; Spearman r = 0.786, P < 0.0001, n = 99) (G) and Kgp load (Spearman r = 0.572, P < 0.0001, n = 104) (H). (I) RgpB load correlates with Kgp load (Spearman r = 0.610, P < 0.0001, n = 99).
Fig. 2. RgpB colocalizes with neurons and…
Fig. 2. RgpB colocalizes with neurons and pathology in AD hippocampus.
(A) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). (B) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. (C) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. (D) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. (E) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
Fig. 3. Identification of P. gingivalis –specific…
Fig. 3. Identification of P. gingivalis–specific protein and DNA in cortex from control and AD patients.
(A) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. (B) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgpcat), along with higher– and lower–molecular weight Kgp species seen in (A). (C) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
Fig. 4. Detection of P. gingivalis in…
Fig. 4. Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects.
(A) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. (B) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. (C) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. (D) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
Fig. 5. P. gingivalis and gingipains fragment…
Fig. 5. P. gingivalis and gingipains fragment tau.
(A) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 (P.g.) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A). Uninfected SH-SY5Y cells (No P.g.) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. (B) Densitometry analysis of the total tau WB images. (C) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. (D) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
Fig. 6. Small-molecule gingipain inhibitors protect neuronal…
Fig. 6. Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis– and gingipain-induced toxicity in vitro and in vivo.
(A) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. (B) AlamarBlue viability assay shows that P. gingivalis (P.g.) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis. (C) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. (D) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (*P < 0.05, n = 14). All graphs show the mean with SEM error bars.
Fig. 7. P. gingivalis invasion of the…
Fig. 7. P. gingivalis invasion of the brain induces an Aβ1–42 response that is blocked by gingipain inhibition in mice.
(A) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane (P.g.), P. gingivalis W83 was used as a positive control. (B) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ1–42 levels compared to mock-infected mice (***P < 0.001, n = 40). (C) RgpB-IR (red) colocalized with Aβ1–42-IR (green) on the surface of P. gingivalis (D) Aβ1–42, but not Aβ1–40 or Aβ1–42 scrambled, decreased viability of P. gingivalis (***P < 0.001, n = 12). (E) Study design to quantitate the effect of gingipain inhibitors on brain P. gingivalis load. (F) qPCR results showed a substantial P. gingivalis copy number in the brain at 5 weeks, increasing 10-fold at 10 weeks (Inf. 10 week). All treatment groups showed a significant decrease in P. gingivalis load compared to vehicle-treated Inf. 10 week mice (***P < 0.0001, n = 63). Treatment with the Kgp inhibitor COR271 resulted in a 90% reduction of P. gingivalis copy number. Comparing treatment groups to baseline infection at the beginning of treatment (Inf. 5 week) showed a significant reduction with COR271 and COR286 (##P < 0.01, #P < 0.05) but not with moxifloxacin. (G) The number of Gad67+ interneurons in the dentate gyrus of the hippocampus was significantly decreased in the Inf. 10 week group (*P < 0.05, n = 120). This decrease was reduced in all treatment groups, with COR271 and COR286 trending to better protection than moxifloxacin. (F) Geometric mean with 95% confidence interval. (B), (D), and (G) show the mean with SEM error bars.
Fig. 8. COR388 target engagement and dose-dependent…
Fig. 8. COR388 target engagement and dose-dependent effects on brain P. gingivalis, Aβ1–42, and TNFα in mice.
(A) COR553 fluorescent activity probe for Kgp. (B) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). (C) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. (D) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. (E) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. (F) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. (G) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. (H) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. (I) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. (J to L) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. ***P < 0.001, **P < 0.01, *P < 0.05, t test with Dunn’s multiple comparison correction; n = 39.

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