Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.

, while, Gulten and Sacchettini reported that monomeric CarD binds RNAP β in 1:1 stoichiometry (Fig. 1a) 18,19 . Tth CarD has been crystallized as a monomer (Fig. 1b) 20 and in-solution data for the oligomeric state is unavailable. In case of Mxa CdnL, NMR and analytical ultracentrifugation studies have indicated that monomers and dimers co-exist in solution (Fig. 1c) 21 . Furthermore, bacterial two hybrid and size-exclusion studies have suggested that N-terminal region of CdnL interacts with itself and forms homodimers in solution 21,24 . The solution structure of Mxa CdnL reveals high flexibility in the first six N-terminal residues 21 . Superposing the N-terminal domains of the Mtb CarD and its homologs, reveals differences in the conformational states of CarD, thus, highlighting the flexible nature of linker in the protein (Fig. 1d). Srivastava et al. reported that Tth CarD is rigidly maintained 20 whereas our comparative analysis of all the full-length structures of CarD_CdnL family of proteins suggest that CarD may adopt functionally relevant multiple conformational states in solution.

CarD exists as a homodimer in solution.
To further resolve the oligomeric state of CarD in solution, we performed synchrotron based size exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS) experiments. Since the crystal structure of the C-terminal his-tagged CarD (CarD His ) was reported previously 19 , we used CarD His for performing SEC-SAXS experiments and for performing all the other assays, we used both native and CarD His . CarD eluted as a single peak in the gel filtration chromatography having consistent values of radius of gyration (R g ) across the peak (Fig. 2a inset, Supplementary Fig. 1a). The SAXS scattering intensity profile [I(Q)] as a function of Q, Guinier plot, Kratky Plot and normalized pair-distribution function [P(r)], are all shown in the Fig. 2(a-d). Analysis of the Guinier plots over a low q (between 0.004 and 0.18 Å −1 ), reveals that the purified protein was free of aggregation and inter-particle interference effects (Fig. 2b). Analysis of the Kratky plot and Porod-Debye plots 25 suggests that CarD is well-folded globular protein (Fig. 2c). The Porod volume estimated from the SEC-SAXS data is 66,457 Å 3 . The estimated molecular weight (41 kDa) is in close agreement with the expected molecular weight of homodimeric CarD (38 kDa). The native-PAGE experiments were consistent with the SEC-SAXS results, suggesting the presence of dimeric CarD in solution ( Supplementary  Fig. 1b). In addition, we did not observe any monomeric species of CarD at three different concentrations (8,30 and 80 μM) tested in the analytical size exclusion experiments ( Supplementary Fig. 1c). The R g obtained from Guinier analysis (R g = 22.9 ± 0.064 Å) matches closely with the real space R g derived from P(r) analysis (R g = 21.6 ± 0.084 Å). The R g and D max (maximum dimension) calculated for the monomeric CarD (PDB ID 4KBM, coordinates extracted for CarD alone) were 17.6 ± 0.049 and 60 Å, respectively whereas the R g and D max calculated for dimeric CarD (PDB ID 4ILU) were 22.9 and 72 Å, respectively (Fig. 2d). The R g and D max values obtained from the SAXS analysis match well with the R g and D max values calculated for the dimeric CarD using FoXS server 26 and CRYSOL 27 , respectively. DAMMIF 28 was used to generate 20 ab initio models which were superimposed, averaged and filtered by DAMAVER 29 to generate the final model for envelope generation. We then superposed the available crystal structures of Mtb CarD (PDB ID 4ILU and 4KBM) into the SAXS derived envelope using SUPCOMB 30 (Fig. 3). The domain-swapped dimeric crystal structure (Fig. 3a) superposed well whereas large unoccupied volumes were present when the monomeric CarD structure was superposed in the SAXS envelope (Fig. 3b). The NSD (normalized spatial discrepancy) values calculated using SUPCOMB for dimeric and monomeric CarD were 0.96 and 3.2, respectively, suggesting better fit for the dimeric CarD. Furthermore, the FoXS web server was used to compute the theoretical scattering profile from the crystal structures and compare it with the experimentally obtained scattering profile. The SAXS data acquired for CarD matches well with the theoretical scattering profile computed from the X-ray crystal structure of dimeric CarD (χ = 1.23) as compared to the monomeric CarD (χ = 1.48). Hence, our SAXS data further demonstrates the existence of homodimeric CarD in solution. Taken together these results show that CarD is monodisperse, homogeneous and exists as a homodimer in solution at the concentrations tested in our study.
CarD undergoes reversible thermal folding in solution. Taken together, the crystal structure as well as the solution studies, both confirms the presence of domain-swapping and dimerization in CarD. We assume that such a domain-swapped dimer could form by partial unfolding of two monomers, followed by refolding and assembly into an intertwined form 31,32 . To test this hypothesis, we performed thermal denaturation experiments. Consistently, circular dichroism (CD) spectra revealed that CarD undergoes reversible thermal melting/folding with the presence of isosbestic point at 204 nm (Fig. 4a). The deconvolution of the CD spectra obtained as a function of temperature using the BeStSel program 33 , revealed a ~4-fold decrease in the α-helical content (both regular and distorted) when temperature was increased from 20 to 95 °C (Fig. 4b). On the other hand, there was a ~ 2.5-fold increase in the parallel and anti-parallel β-sheet content (primarily, right-handed twist angle increased by ~3-fold (Supplementary Fig. 2) with the increase in temperature. Interestingly, with the decrease in temperature (95 °C to 20 °C), the changes in the secondary structural contents showed the reverse trend (Fig. 4b). The presence of highly flexible six N-terminal residues as evident in the solution structure of Mxa CdnL 21 and the ability to undergo reversible thermal denaturation strongly suggest that CarD may undergo domain-swapping by partial unfolding followed by re-assembly with its counterpart.
CarD has a propensity to form higher order oligomers in solution. Domain-swapping in proteins has been implicated in the formation for higher order oligomers and even closed spherical shells [34][35][36][37] . We aimed to investigate whether domain-swapped CarD oligomers can further self-associate to form higher order oligomers in solution. We used mass spectrometry to determine the mass of native CarD. The MALDI-TOF linear mass spectrum of the intact CarD shows peaks corresponding to monomeric, dimeric, and trimeric species of CarD (Fig. 5a). In order to re-confirm our results, we also performed cross-linking experiments using BS 3 [bis(sulfosuccinimidyl)suberate chemical cross linker. BS 3 contains an amine-reactive N-hydroxysulfosuccinimide (NHS) ester at each end of an 8-carbon spacer arm. We incubated CarD with 30-fold and 50-fold molar excess of BS 3 for 1 h at 4 °C and later quenched the reactions by addition of 50 mM Tris-HCl pH 8.0. Bands corresponding to the dimeric and higher oligomeric state species were observed on the SDS-PAGE (Fig. 5b). Taken together, these data suggest that CarD has a propensity not just to dimerize but also to form higher order oligomeric species in solution.

CarD forms SDS-resistant higher order oligomers upon thermal denaturation.
There is a strong correlation between the ability of a protein to form domain-swapped dimers and amyloids 38,39 . In several instances, domain-swapped oligomers serve as precursors for the formation of the amyloid fibrils, including a prion protein 40,41 , cystatinC 42 and GB1 43 . To further investigate whether higher order oligomeric species of CarD form amyloid-like fibrils, we performed thermal denaturation experiments. With the increase in temperature, dimeric CarD further self-associates to form higher order oligomers as suggested by the native-PAGE analysis (Fig. 5c). CarD (50 μM) was incubated at 50 °C for 10 min and samples were analyzed on SDS-PAGE. Though the predominant population was monomeric, SDS-resistant dimers and higher order oligomers were formed at low efficiency as observed on the SDS-PAGE, a characteristic feature of the amyloids or pili proteins 44 (Fig. 5d).
The formation of SDS-resistant higher-order oligomers led us to hypothesize that CarD may also form amyloids. We performed bioinformatics analysis using PASTA 2.0 web server to predict "Presence of Amyloid STructural Aggregation" in CarD 45 . The number of amyloid fibril regions predicted by PASTA is 2 and the best aggregation pairing energy is −5.406. According to Trovato et al., 46   suggesting that the region involved in domain-swapping may also be crucial for the formation of amyloid fibrils. We created a CarDtr construct (CarD  ) and performed thermal denaturation experiments. Upon thermal denaturation, no visible precipitates were observed in CarDtr and no SDS-resistant higher order oligomers were detected on the SDS-PAGE, suggesting that N-terminal residues are crucial for the formation of SDS-resistant higher order oligomers ( Supplementary Fig. 3).

CarD forms amyloid-like fibrils in solution.
Our results indicated that N-terminal region of CarD encompassing β1 is involved in both domain-swapping 19 as well as in the formation of SDS-resistant higher order oligomers, so, we hypothesized that it may be crucial for the amyloid-fibril formation as well. So, we further investigated the ability of CarD to form amyloid-like fibrils using combination of ThioflavinT (ThT) fluorescence assay and transmission electron microscopy experiments.
The presence of amyloid fibrils was detected by ThT dye based assay, a fluorescent probe commonly used to monitor the formation of amyloid fibrils in vitro 47 . Upon binding amyloid fibrils, ThT gives a strong fluorescence signal at approximately 482 nm when excited at 440 nm. We incubated varying concentrations of CarD (5, 10 and 100 μM) with ThT at 37 °C for 15 min and observed a significant increase in fluorescence intensity (Fig. 6a). On the other hand, when we incubated CarDtr (100 μM) with ThT, no significant increase in fluorescence intensity was observed (Fig. 6a), thus, indicating that the N-terminal region encompassing β1 is essential for amyloid formation. Further, the kinetics of formation of amyloid fibrils by CarD (5 µM) was monitored by ThT fluorescence at 37 °C for a period of 24 h (Fig. 6b). The fibrillation pattern resembles a characteristic sigmoidal curve consisting of a long lag (nucleation) phase, an elongation phase followed by a stationary phase. While the higher concentrations of CarD (100 μM) reached saturation within about 200 mins of incubation at 37 °C, the lower concentrations of CarD showed a comparatively longer lag phase. To further test amyloidogenic property and to reduce the lag phase, we performed the seeding experiments by adding preformed nuclei or seeds (5% or 10%) to the sample. As expected, addition of seeds reduced the lag phase significantly (Fig. 6b) as compared to the unseeded CarD.
The formation of CarD amyloid-like fibrils was further confirmed by transmission electron microscopy (TEM). Electron micrographs reveal the presence of several branched fibrils (protofibrils), ~40-50 Å in diameter when imaged after 10 h of incubation (Fig. 6c). The aggregated amyloid fibrils, known as "spikes" were also visible in electron micrographs (Fig. 6d). However, we did not observe any fibrils for CarDtr sample in EM studies under identical conditions. ThioflavinS (ThS), being cell permeable, is generally used to detect the presence of intracellular amyloid-like structures in live bacterial cells 48,49 . CarD is essential in Mycobacteria but absent in E. coli, hence, we used E. coli as an expression host for investigating in vivo amyloidogenic property of CarD. The bacterial (E. coli) cells carrying empty expression vector, over-expressing CarD or CarDtr were stained with ThS and monitored using confocal microscopy. E. coli cells expressing CarD exhibit strong green fluorescence along with the presence of several strong foci in majority of the cells thus, suggesting that CarD adopts amyloid-like conformations within the bacterial cells (Fig. 7b). In contrast, the E. coli cells carrying empty vector exhibit only residual background fluorescence (Fig. 7a). Furthermore, we could observe significant cell elongation in the bacterial cells expressing CarD. This observation correlates well with earlier studies which demonstrate that aggregation of the amyloidogenic proteins inside bacteria promotes severe cell division defects 50 . However, we did not observe cell elongation in cells over-expressing CarDtr compared to control cells. The in vivo confocal imaging of the E. coli cells over expressing CarDtr, stained with ThS, did not show strong fluorescent foci suggesting that CarDtr has a reduced ability to form intracellular amyloid inclusions (Fig. 7c).

Discussion
In the current study, we attempted to resolve the oligomeric state of CarD in solution and therefore, determined its solution structure. Consistent with our previous findings 19 , we report that CarD exists predominantly as a dimer in solution at μM range of concentrations and has a tendency to form higher order oligomers. Concentration-dependent homodimerization has been previously reported in Mxa CarD 51 . However, Mtb CarD is the only example where the domain-swapped dimeric structure has been reported for the CarD_CdnL_TRCF family of transcriptional regulators 19 . The mechanisms or the factors which drive domain-swapping in CarD are currently unknown. One of the proposed mechanisms involved in domain-swapping is the ability of the proteins to partially unfold and re-fold in an inter-twined form along with their counter-part 32 . Our CD spectroscopy data suggests possible role of partial unfolding/refolding in mediating domain-swapping in CarD. We also report and demonstrate an interesting yet peculiar, amyloidogenic property of Mtb CarD, both in vivo as well as physiologically relevant conditions in in vitro (5 μM). We observed that deleting the N-terminal fourteen residues involved in domain-swapping in CarD results in loss of self-association to form SDS-resistant higher order oligomers and hampers its ability to form amyloids in solution. Though in our solution studies we did not observe monomeric form of CarD under tested concentrations, Asmat et al., 52 have reported a small population of monomeric CarD in gel filtration experiments. The crystal structure of 1:1 stoichiometric complex of Mtb RNAP β1β2/CarD complex suggests that CarD exists as monomer as well in solution 18 . The protein sample in this study was prepared using co-expression and co-purification strategy suggesting probably in the presence of the binding partner, CarD exist as a monomer. CarD and RNAP population is similar in cells under normal growth conditions 11 . However, the levels of CarD transcripts increase from 5-to 20-fold under varying stress conditions 10 . So, under stress conditions CarD levels may probably reach in higher μM concentrations (>5 μM) which is in excess of its binding partner RNAP and hence, may self-associate to form homodimer. Further, self-association leads to the Based on our data, we propose a possible model for the formation of CarD amyloids where two N-terminal domains, having unpaired β strands probably self-associate by β-augmentation to form amyloid fibrils (Fig. 8). Interestingly, in the crystal structure of domain-swapped homodimeric Mtb CarD, this unpaired β3 was observed to interact with the flexible C-terminal His-tag and the latter also adopted a β strand conformation 19 . However, CarD His and tagless CarD share similar functional properties suggesting there is no significant effect of histidine tag on the CarD function ( Supplementary Fig. 4-6).
Usually amyloids are associated with the human diseases 53 but there are several instances of functional bacterial amyloids formed by extracellular proteins such as Curli proteins from E. coli 54 , Type IV Pili from Mtb 44 , TasA from Bacillus subtilis 55 and Microcin E492 from Klebsiella pneumoniae 56 . The presence of amyloidogenic properties in bacterial cytosolic proteins especially soluble transcription factors has been discovered recently 23,57,58 . Cbo transcriptional terminator Rho 23 , has been shown to form amyloids intracellularly in yeast and E. coli models. So, this field is still at its infancy and offers a wide opportunity to explore physiological relevance of amyloidogenesis in bacterial cytosolic proteins. Interestingly, while our manuscript was under communication, Takacs et al., analyzed the structures of all non-amyloid proteins present in PDB and predicted >500 soluble proteins with amyloid-like substructures, one of them being Tth CarD (PDBID: 4L5G) (https://arxiv.org/abs/1805.09758). Based on our results, we report that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils. As Mtb has a tendency to stay in a dormant/latent state for decades, hence there is a possibility of the formation of intracellular CarD amyloids over such a long period of incubation. So, it would be interesting to explore the physiological role, if any, of the amyloid-like fibrils formed by Mtb CarD. Future structural and functional studies are required to gain mechanistic insights into the phenomenon of domain-swapping mediated oligomerization and amyloidogenesis in CarD and similar behaving proteins.

Materials and Methods
Cloning, over-expression, and protein purification. The tagless construct of CarD was PCR amplified from Mtb genomic DNA and cloned into pYUB28b vector between NcoI and HindIII sites. The sequence of the clones was further confirmed by DNA sequencing. The positive clone of tagless CarD was transformed and expressed in E. coli strain BL21(DE3). The CarD overexpressing cells were grown in 1 L Luria Bertani (LB) media supplemented with Hygromycin (150 μg/mL) at 37 °C. When the cells reached OD 600 = 0.5, the expression was induced by adding 0.3 mM IPTG and cells were further incubated for 16 h at 16 °C. The cells were harvested by centrifugation at 4 °C and the cell pellet was resuspended in 20 mM MES pH 6.0 and subjected to sonication. This was followed by centrifugation at 16,000 × g for 30 min at 4 °C. The supernatant was loaded immediately on DEAE-ion exchange chromatographic column pre-equilibrated with 20 mM MES pH 6.0. CarD eluted in the same buffer (20 mM MES pH 6.0) with the linear gradient from 0 to 1 M NaCl. The fractions corresponding to each peak were loaded on SDS-PAGE and the purified CarD fractions were pooled, concentrated and dialyzed in 20 mM HEPES pH 7.5 and 150 mM NaCl for further studies. The N-terminal deletion construct of CarD (CarD  , referred as CarDtr), was cloned in pNIC28-Bsa4 vector between NdeI and NotI sites. The His-tagged constructs of CarD (CarD His and CarDtr) were purified as per the protocol in Kaur et al. 19 , except in this study, we used 20 mM HEPES pH 7.5 and 150 mM NaCl in all the purification steps. The identity of the protein samples were confirmed by MALDI-TOF mass spectrometry. The purity of all the protein samples were >95% as judged by the SDS-PAGE analysis. All the protein samples were further purified using gel filtration chromatography. The purified protein samples (500 μL each) were injected in Superdex S200 10/300 GL column pre-equilibrated with 20 mM HEPES pH 7.5 and 150 mM NaCl using AKTA purifier FPLC system (GE Healthcare) with a flow rate of 0.5 mL/min.

Size-exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS).
SEC-SAXS was performed at the Diamond Light Source, Harwell, UK at B21 beamline. Inline SEC-SAXS data was collected using an Agilent HPLC equipped with Shodex column. Data were recorded on a Pilatus 2 M detector and purified samples of CarD (277 μM) were loaded onto the Shodex column pre-equilibrated in the running buffer of 20 mM HEPES pH 7.5 and 150 mM NaCl, at a flow rate of 0.018 mL/min at 4 °C. The primary reduction of the SAXS data and data processing were performed using ScÅtter (http://www.bioisis.net/ScÅtter) to obtain the radius of gyration (R g ), the maximum particle dimension (D max ), the excluded particle volume (Vp) and the pair distribution function [P(r)]. The molecular mass of the scattering particles was calculated using a method described by Rambo et al. 59 . Low resolution 3-D ab initio models were generated using DAMMIF software 28 using the slow mode. Averaging the results of 20 independent runs using DAMAVER 29 generated the dummy atom models which were superposed with the monomeric and domain-swapped dimeric crystal structures of Mtb CarD using SUPCOMB 30 . Structural visualization, analysis and figure preparation were performed using PyMoL (Schrödinger, LLC). FoXS web server 26 was used to fit the experimentally derived scattering profile to the theoretically calculated scattering profile from the crystal structure. FoXS web server 26 and CRYSOL 27 were used for calculating R g and D max from the crystal structure and the experimental obtained SAXS data.
CD spectroscopy and thermal melt assays. CarD or CarD His (10 μM each) were dialyzed overnight into 10 mM sodium phosphate buffer (pH 7.5). CarD or CarD His in a 1 mm path-length quartz cuvette were used to collect CD spectra in the 195-250 nm and 190-250 nm wavelength range, respectively, using JASCO J-810 spectrometer. Thermal melt experiments were performed from 20 to 95 °C and 95 to 20 °C temperature ranges with ramp rate of 1 °C/min. The spectra were recorded at every 5 °C with a scanning speed of 100 nm/min. The secondary structural content was deconvoluted using BeStSel Server 33 .
Intact Mass Spectrometry analysis. Purified CarD (50 μM) or CarD His or CarDtr were mixed with sinapinic acid matrix solution (50% Acetonitrile/50% water with 0.1% Trifluoroacetic acid) in 1:1 ratio and the mix was spotted on the MALDI plate. The spots were air dried and data were acquired in linear and positive ion mode keeping the laser intensity between 3000-3400 V (AB Sciex TOF/TOF 5800). The data were analyzed using Data Explorer software version 4.9 of Applied Biosystems. Native-PAGE analysis. Samples of purified CarD (50 μM) and CarD His (50 to 716 μM) were incubated from 4 to 60 °C for 30 min. The heated samples were mixed with 2× non-denaturing gel loading dye (200 mM Tris-HCl, pH 6.8, 0.2% bromophenol blue and 20% glycerol). The continuous native-PAGE (10%) was cast and pre-run at 10 V/cm for 30 min at 4 °C. Electrophoresis was performed in 1× native-PAGE running buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine) according to the standard method at 4 °C for 2 h. The gels were stained using Coomassie brilliant blue followed by destaining using standard protocol. Thermal denaturation. To perform thermal denaturation experiments, CarD (50 μM), CarD His (100 µM) or CarDtr (100 µM) were incubated at 50 °C for 10 min. The samples were then immediately mixed with 2× SDS loading dye and resolved on the 15% SDS-PAGE. The protein bands were visualized using standard staining and destaining protocols.
Thioflavin T (ThT) fluorescence and fibrillation assays. Native tagless CarD (5, 10 and 100 μM), CarD His (100 μM) and CarDtr (100 μM) were mixed with 10-fold molar excess of ThT and incubated for 15 min at 37 °C. Seeding experiments were performed where 5% and 10% seeds were transferred from the 100 μM CarD sample incubated at 37 °C for 5 h to 5 μM CarD. The ThT fluorescence intensity was measured using excitation wavelength of 440 nm and emission wavelength of 482 nm. The fluorescence measurements were recorded using Synergy H1 Hybrid multi-mode microplate reader. The kinetics for the formation of amyloid fibrils by CarD and CarD His were recorded for a period of 24 h. The microplate reader was set at constant temperature of 37 °C and readings were acquired every 2 min with continuous orbital shaking between reads over a period of 24 h. The top of the each well was sealed using adhesive tape to minimize the rate of evaporation.

Transmission Electron Microscopy (TEM).
Aliquots of 20 μL from samples containing CarD or CarDtr stained with ThT fluorescent dye were pipetted on a carbon-coated 300-mesh copper grid (Polysciences). Samples were diluted five-fold using 20 mM HEPES pH 7.5 and 150 mM NaCl buffer before loading on the grids. The excess sample was blotted and the grids were incubated with uranyl acetate solution (2%) for 2 min at room temperature followed by air drying. The grids were imaged using a JEM 2100 transmission electron microscope, operated at 200 keV (JEOL). Thioflavin S (ThS) staining of live bacterial cells. E. coli BL21(DE3) cells harboring CarD or CarDtr expression vector and empty expression vector control were grown at 37 °C in LB medium until optical density (OD 600 ) reached 0.5. The protein expression was induced by addition of 0.3 mM IPTG and the cultures were further grown at 37 °C for 5 h after induction. Thereafter, 0.5 mL of each bacterial culture was centrifuged at 6000 × g, 25 °C for 2 min. The bacterial cultures were washed thrice with 1 × PBS, followed by their fixation using 4% paraformaldehyde at 37 °C for 30 min. The fixed bacterial cultures were washed thrice with 1 × PBS and further incubated with 125 μM ThioflavinS (ThS, Catalog number T1892, Sigma) at 37 °C for 30 min in the dark. The bacterial cultures were again resuspended and washed thrice with 1 × PBS. 10 μL of the sample was placed on top of the glass slide, covered with a coverslip and air-dried. The images were acquired using confocal fluorescence microscope (Nikon A1R), using a 100 × oil-immersion objective and 1 Airy unit aperture. The ThS stained samples were excited using an excitation wavelength of 405 nm and an emission wavelength of 482 nm.