Tau misfolding and aggregation follows a seeding nucleation mechanism that can be modeled in vitro using purified recombinant tau protein incubated in the presence of heparin at physiological pH and temperature37,38. For our experiments, we used full-length human tau protein containing 4 microtubule binding repeats (4 R) and 2 N-terminal inserts (2 N). The 2N4R tau is the largest form of tau and the most active in promoting microtubule assembly12,13. In most tauopathies the 4R tau is the predominant form of the protein in the aggregates12,13. Incubation of tau at a concentration of 50 µM in 10 mM HEPES, pH 7.4 containing 100 mM NaCl and 12.5 µM heparin at 37 °C with constant agitation led to the formation of amyloid aggregates, detectable by the fluorescence emission of thioflavin T (ThT) (Fig. 1A). ThT is an amyloid-binding molecule, which emits fluorescence when bound to the aggregates and is widely used to characterize the kinetic of amyloid formation39. Under these conditions, tau form ThT-positive aggregates (Fig. 1A,B, Supplementary Fig. S1) with a lag phase of around 15 h (Fig. 1A). To measure the amount of aggregated tau, we centrifuged the samples and evaluated the tau signal in pellet and supernatant by western blot. As shown in Fig. 1C, most of the protein was recovered in the pellet and appears as a smear of high molecular weight bands. This result suggests that, under the conditions used the majority of tau was forming part of large aggregates. To use this aggregated material as seeds, we sonicated the preparation in order to generate seeding-competent short fibrils, as described in recent publications to produce tau pre-formed fibrils (tau-PFF)40,41. Analysis by transmission electron microscopy (TEM), revealed that this preparation contains short unbranched amyloid-like fibrils of different sizes and the expected width of ~10 nm (Fig. 1D). One of the typical biological activities of tau aggregates is their ability to seed aggregation of monomeric tau. To test this property, we incubated tau at a lower concentration (22 µM), with less heparin (4.4 µM) and lower temperature (20 °C) in order to slow down spontaneous aggregation and observe a clear seeding activity (Fig. 1E). Addition of different quantities of PFF tau aggregates (sonicated fibrils) accelerated tau aggregation in a concentration-dependent manner (Fig. 1E). Indeed, the lag phase was directly proportional to the logarithmic amount of tau aggregates added as seeds (Fig. 1F). The lag phase was measured as the time in which aggregation begins, which is defined as the moment when the ThT fluorescence reaches a threshold of 40 fluorescence units.

Figure 1 Tau seeding aggregation assay. (A) Full-length Tau seeds were prepared by incubating tau monomer (50 µM) with 12.5 µM heparin in 10 mM HEPES pH 7.4, 100 mM NaCl for 5 days at 37 °C with shaking. Aggregation was monitored by ThT fluorescence. (B) Tau aggregates exhibit the typical ThT fluorescence spectrum with a maximum around 495 nm when excited at 435 nm. (C) The aggregation state was further confirmed by sedimentation followed by western blot, showing that the majority of tau appeared in the pellet in the form of large molecular weight bands. (D) the morphological characteristics of the tau aggregates were studied by transmission electron microscopy after negative staining with uranyl acetate. (E) The Tau aggregation assay was performed on 96 well plates using 22 µM Tau monomer, 4.4 µM heparin, 10 µM Thioflavin T, using cyclic agitation (1 min shaking at 500 rpm followed by 29 min without shaking). Aggregation was followed over time by ThT fluorescence using a plate spectrofluorometer (excitation: 435; emmision: 485). Graph show the mean and SD of three replicates. (F) Relationship between the quantity of tau oligomers and the Tau-PMCA signal (time to reach 50% aggregation). Full size image

Using this tau aggregation assay, we examined whether tau fibril formation could be accelerated in the presence of extracellular DNA from various species including bacteria, yeast and human. For the experiments, monomeric tau was incubated with preparations containing 100 ng of DNA extracted from different bacterial species including Pseudomonas aeruginosa (PA), Tetzosporium hominis (TH), Tetzerella alzheimeri (TA), Escherichia coli ATCC 25922 (EC25), Escherichia coli ATCC 472217 (EC47), Porphyromonas gingivalis (PG), Borrelia burgdorferi (BB). We also incubated tau with the same amount of DNA extracted from Candida albicans (CA) and human samples. The results showed that DNA from various (but not all) bacterial species significantly promoted tau aggregation (Fig. 2A). Conversely, addition of eukaryotic DNA, such as from yeast or human cells, had a much lower effect in promoting tau aggregation. To compare the magnitude of the effect of these different DNA extracts on the kinetic of aggregation, we measured the lag phase, defined as the time at which aggregation begins, which is experimentally determined as the time when ThT fluorescence reaches a value >40 fluorescent units (equivalent to ~2-folds the background levels after subtraction of the blank)42. Comparisons of the lag phases indicate that the largest promoting effect (shorter lag phase) was obtained in the presence of Tetzerella alzheimeri, Escherichia coli ATCC 25922, and Escherichia coli 472217 (Fig. 2B). Interestingly, Tetzerella alzheimeri (VT-16-1752 gen.nov, sp.nov) is a new species which was isolated from the oral cavity of a patient with AD. Moderate promoting effect was observed with Porphyromonas gingivalis and Borrelia burgdorferi, and no significant effect was detectable for Pseudomonas aeruginosa and Tetzosporium hominis43 (Fig. 2B).

Figure 2 Effect of DNA extracted from diverse sources on tau aggregation. To study the effect of DNA on tau aggregation, monomeric tau (22 µM) under the conditions described in Fig. 1E, was incubated with preparations containing 100 ng of DNA extracted from different bacterial species including Pseudomonas aeruginosa (PA), Tetzosporium hominis (TH), Tetzerella alzheimeri (TA), Escherichia coli ATCC 25922 (EC25), Escherichia coli ATCC 472217 (EC47), Porphyromonas gingivalis (PG), Borrelia burgdorferi (BB). We also incubated tau with same amount of DNA extracted from Candida albicans (CA) and human samples. In all experiments the signal at time zero, corresponding to buffer + DNA + heparin + ThT + monomeric tau was substracted from the values. (A) tau aggregation was monitored over time by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate (except for control without seeds that was performed in quintuplicate). (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.01; **P < 0.001. Full size image

We then aimed to confirm whether the promoting activity of bacterial DNA is dose dependent. We found that DNA of E. coli ATCC 25922 and P. gingivalis at concentrations of 1000 to 10 ng significantly accelerated Tau aggregation relative to controls. The promoting activity of E. coli ATCC 25922 (Fig. 3) and especially P. gingivalis (Fig. 4) was lower than that of tau seeds. A dose dependent effect was more clearly observed only for addition of P. gingivalis DNA, perhaps because of the higher efficiency of E. coli ATCC 25922, which may require lower concentrations to observe a dose-dependency.

Figure 3 Influence of different concentration of E. coli ATCC 25922 DNA on tau aggregation. To study whether the promoting effect of E. coli DNA can be observed at different concentrations of DNA, we incubated monomeric tau under the conditions described above (Figs. 1E and 2) with 1000, 100 and 10 ng of DNA extracted from E. coli ATCC 25922. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.01; **P < 0.001. Full size image

Figure 4 Dose-dependent effect of DNA from Porphyromonas gingivalis on tau aggregation. Monomeric tau was incubated under the conditions described above (Figs. 1E and 2) with 1000, 100 and 10 ng of DNA extracted from P. gingivalis. Сontrol probes of 1000 ng of P. gingivalis DNA were treated with DNase I to remove DNA from the sample. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.05; **P < 0.001. Full size image

Notably treatment of P. gingivalis DNA samples with DNAse I (Fig. 4A, green line) led to a complete prevention of Tau aggregation, pointing out the specificity of the DNA effect on protein misfolding. This result strongly indicate that the promoting effect is due to DNA and not other small contaminant that might be present in the preparation.

To confirm the effect of DNA on promoting tau misfolding and aggregation, we employed two additional methodologies to examine the presence and quantity of tau aggregates after incubation with DNA. For these studies we incubated monomeric tau with 1000 ng of P. gingivalis DNA and measured formation of amyloid-like aggregates by sedimentation assay and TEM, as well as by the ThT assay. The result of the ThT assay (Fig. 5A) was very similar as that described in Fig. 4A, indicating the reproducibility of the result. Aliquots were taken after 300 h of incubation and analyzed by TEM after negative staining. The study showed that in the presence of P. gingivalis DNA tau formed an abundant amount of ~10 nm amyloid-like fibrils as observed by TEM, whereas in the absence of DNA, no fibrils were observed (Fig. 5B). Tau aggregation was also monitored by a sedimentation assay employing centrifugation to separate soluble from aggregated tau and measuring the amount of protein in pellet and supernatant by a dot blot assay. The result indicate that after 300 hours of incubation in the presence of P. gingivalis a large amount of tau was found as aggregates in the pellet fraction, whereas in the absence of DNA, a small amount of the protein was aggregated (Fig. 5C, left panel). To measure the extent of aggregation in a more quantitative manner, we compared the dot blot signal with that obtained in the same membrane with different concentrations of recombinant monomeric tau (Fig. 5C, right panel). Considering the amount loaded in the blots and the dilution used, we estimated that >80% of tau protein was detectable in the pellet fraction (aggregated) for the experiment done in the presence of DNA, whereas only ~10% of tau incubated alone was aggregated. Finally, to analyze whether DNA was interacting with tau aggregates, we measured the UV absorbance of the pellet fraction. The data showed that in the tubes incubated with DNA, a peak at ~260 nm was observed, in addition to the protein peak at 280 nm, suggesting the presence of DNA in this fraction (Fig. 5D). Overall, these results fully support our experiments using ThT fluorescence, and strongly indicate that bacterial DNA promotes tau misfolding and aggregation.

Figure 5 Effect of DNA from Porphyromonas gingivalis on tau aggregation measured by TEM and sedimentation assay. Monomeric tau was incubated under the conditions described above (Figs. 1E, 2 and 4) with 1000 ng of DNA extracted from P. gingivalis. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) Aliquots taken after 300 h of incubation were taken and loaded into TEM grids and stained with uranyl acetate, as indicated in methods. Scale bar corresponds to 50 nm. (C) The aggregation state was further confirmed by sedimentation followed by dot blot. The left panel shows the signal obtained in the pellet fraction after 300 h of incubation for individual wells or the pool of the samples in the presence and absence of DNA. For this experiment, the pellet was resuspended in 200 µl of the same buffer used for aggregation (10 mM Hepes pH 7.4, 100 mM NaCl) and 2 µl of a 8-fold dilution of this sample was loaded in the membrane. The right panel shows the dot blot signal of distinct concentrations of recombinant monomeric tau. (D) The UV spectra of solubilized pellet was measured between 240 and 300 nm for the pool of replicates incubated alone or in the presence of 1000 ng of PG DNA. Full size image

Analyses of the DNA sizes used in the study via electrophoresis showed no visible differences between DNA from different microorganisms consisting of bands with sizes ranging from 20 to 30 kb. The only difference was noticed in the human DNA, which, in addition to 20–30 kb bands, had larger fragments at the start (Fig. 6).