Bacterial model membranes reshape fibrillation of a functional amyloid

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Bacterial model membranes reshape fibrillation of a functional amyloid protein Ravit Malishev, Razan Abbasi, Raz Jelinek, and Liraz Chai Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00002 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Biochemistry

Bacterial model membranes reshape fibrillation of a functional amyloid protein Ravit Malishev1‡, Razan Abbasi2‡, Raz Jelinek1* and Liraz Chai2* 1

Department of Chemistry, the Ben Gurion University of the Negev, Beer Sheva 84105, Israel

2

Institute of Chemistry, the Hebrew University of Jerusalem and &The center for Nanoscience

and Nanotechnology, Edmond J. Safra Campus, Jerusalem 91904, Israel ‡These authors contributed equally * To whom correspondence should be addressed: Dr. Liraz Chai, Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra campus, Jerusalem 91904, Israel. Telephone: +972-2-6585303, Fax. +972-2-5660425. Email. [email protected]; Prof. Raz Jelinek, Department of Chemistry, the Ben Gurion University of the Negev, Beer Sheva 84105, Israel. Telephone: +972-52-6839384, Fax. +972-8-647-2943, Email: [email protected]

KEYWORDS: functional amyloid; Bacillus subtilis; biofilm; membrane biophysics; protein aggregation

ACS Paragon Plus Environment

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ABSTRACT

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers.

Recent studies of functional amyloid assembly have

neglected their interaction with membranes, despite their native assembly in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein, and ask whether a bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the protein and the membranes’ morphology and structure. At the protein's level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane's level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of anti-biofilm drugs to the protein-membrane interface.


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Biochemistry

INTRODUCTION Biofilms are aggregates of cells that form surface-associated communities that grow on natural and synthetic surfaces1. Biofilm can be harmful, for example when they develop on catheters, artificial implants and water pipes, but they can be beneficial for example when they promote plant growth as they develop on plant roots2, and when (together with mold) they make the richflavored cheese rind3. Once formed, they are very hard to eradicate as they are 10 to 50 times more resistant to antibiotics relative to planktonic cells, partly for reasons related with their extracellular matrix (ECM)4, 5. The ECM is a network of biopolymers that comprises more than 90% of the dry weight of biofilms4. The exact composition of the bacterial ECM varies between species but a common feature to all matrices is that they are made of polysaccharides, proteins and sometimes nucleic acids4, 6. The proteins often form fibrillar appendages that are amyloidlike, for example the protein curli (E.coli)7, FapC (Pseudomonas)8, PSMs (Staphylococcus aureus)9, 10, and TasA that is secreted by the soil bacterium, Gram positive and spore forming, Bacillus subtilis 11, 12. In recent years, many studies of functional amyloids, including those of TasA, have focused on their structural and aggregation properties in solution

12-16

. However, despite the fact that TasA

fibers have been observed close to and/or attached onto the surface of the bacterial cells

17

, the

actual roles of the bacterial cell’s surface, in the assembly and structural properties TasA have not been addressed. This scant knowledgebase stands in sharp contrast to the huge body of work aimed at deciphering amyloid protein – membrane interactions in prominent neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease

18-20

. In these degenerative diseases,

soluble intermediate oligomers aggregate into fibers and it is now widely accepted that lipid bilayers may act as an effective catalyst of fibrillogenesis, providing a generic environment


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where protein molecules adopt conformation and orientation promoting their assembly into protofibrillar and fibrillar structures

18, 21

. Furthermore, the intermediate oligomers have been

related with several bilayer disruption events such as detergent and carpeting effects or pore formation 22-24. The thrust of this study is to decipher the interplay between bacterial membranes and TasA in aggregation conditions. We have previously shown that TasA can be purified from Bacillus subtilis in the form of natively structured, soluble oligomers12. To induce aggregation of a folded protein in vitro, it is often necessary to work under denaturing conditions (such as elevated temperatures, high denaturant concentration, or low pH) and allow the protein to refold25,

26

.

Figure 1. Composition of bacterial and eukaryotic model membranes and the sequence of the extracellular protein TasA. (a,b) Composition of bacterial (PE/PE/CL) and eukaryotic (DOPC/Chol/Sph) model vesicles. The bacterial mimic membranes are composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-snglycero-3-phospho-(1'-rac-glycerol) (DOPG), and Cardiolipin (PE/PG/CL, 0.45:0.45:0.1 mole ratio) and the eukaryotic mimic membranes are composed of 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC), sphingomyelin and cholesterol (DOPC/Chol/Sph 4 ACSThe Paragon Plus Environment 0.67:0.25:0.08 molar ratio). (c) sequence of TasA (234 residues), an extracellular fiber-forming functional amyloid protein secreted by Bacillus subtilis.

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Biochemistry

Following previous protocols for amyloid and functional amyloid protein aggregation27-30, we chose to work in an acidic solution of pH < 3. We have therefore tested here the TasA fibrillation in the absence and presence of vesicle bilayers after the addition of acid. Employing model membranes mimicking bacterial membranes

31

and eukaryotic cell membranes

32

, respectively

(figures 1A, 1B), we demonstrate that TasA (sequence presented in figure 1C) interacts distinctively with bacterial-mimicking bilayers and that this interaction influences the TasA aggregation pathways as well as the membrane morphology and fluidity. This important in vitro finding could open new avenues for a comprehensive understanding of bacterial biofilm assembly. MATERIALS AND METHODS Materials. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero3-phospho-(1'-rac-glycerol) (DOPG), Cardiolipin (Heart, Bovine), 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), sphingomyelin (brain, porcine), cholesterol (ovine wool, >98%) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7- nitro-2-1,3-benzoxadiazol-4-yl) (NNBD-PE) were purchased from Avanti Polar Lipids. Thioflavin T (ThT), sodium hydrosulfite and sodium phosphate monobasic were purchased from Sigma-Aldrich (Rehovot, Israel). 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenylhexatriene (DPH) were obtained from Molecular Probes, Inc. (Eugene, Oregon).

A11 anti-oligomer

antibody was obtained from Rhenium (Modi'in, Israel). Horseradish peroxidase conjugated antirabbit IgG (HRP) was purchased from ZOTAL (Tel-Aviv, Israel). Protein purification. TasA was purified from the B. subtilis double mutant strain sinR eps, as previously reported

11, 12

. Briefly, cells were grown overnight in Msgg broth at 37°C. After


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centrifugation the pellet has been collected and the protein was extracted from the pellet using sonication in saline extraction buffer. The supernatant was separated from the pellet using an additional centrifugation and the supernatant was filtered through a 0.45um polyethersulfone bottle-top filter. The crude extract was concentrated using a GE Healthcare Vivaspin sample concentrator (10KDa MWCO) and further cleaned using gel filtration. Vesicle

Preparation.

sphingomyelin/cholesterol

Vesicles were

consisting

prepared

by

of

DOPE/DOPG/CL

dissolving

the

lipid

and

DOPC/

components

in

chloroform/ethanol (1:1, v/v) and drying together in vacuum. Small unilamellar vesicles (SUVs; DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin 0.45:0.45:0.1, 0.67:0.25:0.08 mole ratio, respectively) were prepared in 10 mM sodium phosphate (pH 7.4) by probe-sonication (power: 130 w, frequency: 20 kHz, at 20 % amplitude) of the aqueous lipid mixtures at room temperature for 10 min. Vesicle suspensions were kept for 1h at room temperature to stabilize prior to usage. Thioflavin T (ThT) Fluorescence Assay. ThT fluorescence measurements were conducted at 25 °C using black, clear bottom, 96-well plates (Costar) on a Synergy H1 plate reader (Biotek). The aggregation reaction contained 1-1.5 µM TasA, 0.3 mM DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin lipid vesicles, 25µM ThT. Data were collected with or without vesicles after adjusting the pH to 2.5 using formic acid; control experiments were conducted at pH 7.4. The device was programmed to record fluorescence intensity every two minutes for 3 – 4 hours under orbital shaking conditions between measurements. Excitation and emission wavelengths were 430 and 480 nm, respectively. The fluorescence curves of representative measurements were smoothed by using a five-point adjacent averaging.


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Biochemistry

Cryogenic transmission electron microscopy (cryo-TEM). Aliquots were taken from the ThT reaction mixtures after 3 h incubation. A 3µL droplet of the reaction mixture was deposited on a glow-discharged TEM grid (300 mesh Cu Lacey substrate grid; Ted Pella). The excess liquid was blotted off with a filter paper, and the specimen was rapidly plunged into liquid ethane pre-cooled with liquid nitrogen in a controlled environment using Vitrobot Mark IV (FEI). The vitrified samples were transferred to a cryo-specimen holder (Gatan model 626) and examined at -177 ºC using a FEI Tecnai 12 G2 TWIN TEM operated at 120 kV in low-dose mode. Grids were imaged a few micrometers under focus to increase phase contrast. The images were recorded with a 4k x 4k FEI Eagle CCD camera. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded in the range of 190–260 nm at room temperature on a Jasco J-715 spectropolarimeter, using 10-mm quartz cuvettes. Solutions composed of 400 µL contained 1-1.5 µM TasA in 10 mM potassium phosphate buffer, pH 7.4, in the absence or presence DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin (final concentration 0.3 mM). CD was measured at pH 7.4 or at pH 2.5, with the latter obtained by adding concentrated formic acid. Spectra were recorded every 30 min for 2 h. CD signals resulting from vesicles and buffer were subtracted from the corresponding spectra. Fourier Transform InfraRed (FTIR) spectroscopy. FTIR spectra were measured with a Thermo Scientific is50 FT-IR spectromter equipped with a DTGS detector. Samples containing 0.09mM TasA in the absence or presence of PE/PG/CL and DOPC/Chol/ sph lipid vesicles (final concentration 1 mM) were incubated for three hours at 4°C. A 10 µL sample was then placed in a Biocell (Biotools, Inc.)33 with CaF2 windows and 6 µm path length and 256 scans in absorbance mode were collected. The data were collected at pH 7.4 or after adjusting the pH to


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2.5 using HCl or formic acid. A 256 scan measurement in ambient air served as a background. The final spectra were obtained after subtracting the corresponding solution (lacking the protein) spectra as well as subtracting a water vapor spectrum and three to four measurements were then averaged. Similar FTIR spectra were recorded for samples taken directly from solution after mixing by pipetting or for samples taken from a pellet after centrifugation (9,600 xg for 5 minutes). Fluorescence Anisotropy. The fluorescence probes TMA-DPH or DPH were incorporated into the SUVs (DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin 0.45:0.45:0.1, 0.67:0.25:0.08 mole ratio, respectively) by adding the dye dissolved in THF (1 mM) to vesicles (final concentration 0.3 mM) up to a final concentration of 1.25 µM. After 30 min of incubation at 30°C TMA-DPH or DPH fluorescence anisotropy were measured at λex=360 nm λem=430 using a FL920 spectrofluorimeter (Edinburgh Co., Edinburgh, UK). Data were collected before and after addition of TasA [final concentration 1 µm] at pH 7.4 or at pH 2.5, with the latter obtain by adding concentrated formic acid. Anisotropy values were automatically calculated by the spectrofluorimeter software using the equation: r = (IVV – GIVV) / (IVV + 2GIVH), G = IVH / IHH in which IVV is with excitation and emission polarizers mounted vertically; IHH corresponds to the excitation and emission polarizers mounted horizontally; IHV is the excitation polarizer horizontal and the emission polarizer vertical; IVH requires the excitation polarizer vertical and emission polarizer horizontal. Each experiment was repeated at least three times. Results are presented as means ± SEM; (*P < 0.05). Giant Unilamellar Vesicles Labelled With N-NBD-PE. Giant vesicles (GUVs) were prepared through the rapid evaporation method 34. Briefly, GUVs comprising DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin (0.45:0.45:0.1, 0.67:0.25:0.08 mole ratio, respectively)


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Biochemistry

were prepared through dissolving the lipid constituents in chloroform/ ethanol (1:1, v/v), subsequently adding to round-bottom flask (250 ml) containing NBD-PE (1:500) and chloroform (1 ml). The aqueous phase (5 ml of 0.1 M sucrose, 0.1 mM KCl, 50 mMTris solution, pH 7.4) was then carefully added along the flask walls. The organic solvent was removed in a rotary evaporator under reduced pressure (final pressure 40 mbar) at room temperature and 40 rpm. After evaporation for 4–5 min, an opalescent fluid was obtained with a volume of approximately 5 ml. Confocal Fluorescence Microscopy. GUVs (final concentration 0.3 mM) were measured in the absence or presence of 2 µm TasA at pH 7.4 or at pH 3 using PerkinElmer UltraVIEW system (PerkinElmer Life Sciences Inc., MA, USA) equipped with Axiovert-200 M (Zeiss, Germany) microscope and a Plan-Neofluar 63 1.4/×oil objective. The excitation wavelength of 488 nm was generated by an Ar/Kr laser. We performed this experiment three times and visualized similar images in different locations in the sample. For technical reasons, due to the constant motion of the GUVs, it was difficult to take snap shots of short time scale (~ minutes) events and therefore we show representative images. Dot Blot Assay. Oligomers of TasA at pH 2.5 were incubated in the absence or presence of DOPE/DOPG/CL and DOPC/cholesterol/ sphingomyelin lipid vesicles and oligomers of Aβ42 were prepared as previously described methods

35

. Both, probed by the oligomer-specific

polyclonal antibody (pAb) A11. The resulting solution was maintained at room temperature up to 10 min. Periodically, 2-µL aliquots (the concentration of TasA and Aβ42, were 1 µM, 45 µM, respectively) were applied to nitrocellulose membranes. The membranes were blocked for 1h with 5% nonfat milk in 10 mM Tris-buffered saline (TBS) followed by incubation with A11 at


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1:1000 dilution in TBS containing 5% nonfat milk followed by appropriate horseradish peroxidase-linked secondary polyclonal antibodies and developed using an Enhanced Chemiluminescence (ECL) reagent kit (GE Healthcare). Optical Density measurements at 280 nm (OD280). We used a Nanodrop (ND-1000) to measure the TasA absorbance at 280 nm (OD280). Formic acid was added to samples containing TasA (in 10 mM potassium phosphate buffer, pH 7.4) in the absence and presence of 0.3 mM vesicles (eukaryotic and bacterial model membranes, as described above, final concentration in 10 mM potassium phosphate buffer) to adjust the solution pH to 2.5. OD280 measurements of supernatant aliquots were taken without pipetting before the addition of acid and after 3 hours from the addition of acid (without pipetting).

RESULTS TasA assembly and fibrillation are affected by bacterial-mimic membrane vesicles We initially evaluated whether membrane vesicles affected the aggregation of TasA oligomers. Utilizing dot blot assay with the A11 polyclonal antibody (pAb), that recognizes prefibrillar oligomers en route to fibrillation 36 (Figure 2A), we observed the TasA oligomers’ reaction with A11 after 15 minutes in the acidic solutions. Purified TasA oligomers reacted positively with A11, indicating that there are still abundant A11-binding oligomeric species in the solution (for a positive control we used Aβ42, figure 2A). A similar reaction of TasA with A11 has been observed upon the addition of eukaryotic - mimic vesicles DOPC/Chol/Sp, but in the presence of the bacterial - mimic vesicle bilayers comprising PE/PG/CL (see figure 1A for composition and


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Biochemistry

lipid structure) A11 staining was negligible, indicating a low abundance of A11 – binding TasA oligomers. We note that we have performed the dot blot experiments at pH 7.4 (Figure S1 in the Supplementary Information), and under these conditions there was a similar interaction of TasA with the A11 antibody in the absence and presence of the membranes. Witnessing the more enhanced reaction with A11 during TasA aggregation in the presence of biomimetic bacterial in comparison with eukaryotic membranes, we aimed to probe the effect of lipid bilayers on protein aggregation along time. We carried out fluorescence thioflavin-T (ThT) assays in an acidic solution in the absence and in the presence of vesicle bilayers comprising different lipid compositions (Figure 2B). ThT fluorescence has been widely employed for measuring the assembly process of β-sheet-containing amyloid fibrils

37

. Figure 2B reveals

major differences of TasA fibrillation depending on solution composition. Gradual increase in ThT fluorescence signal is observed in an acidic solution (no vesicles present) reflecting fibril

Figure 2. The effect of membranes of the assembly and fibrillation of TasA. (a) Dot blot assay using the oligomer-sequence-specific A11 polyclonal antibody 15 minutes after a pH adjustment to 2.5. Darker color indicates larger oligomer abundance in solution. From left to right: a TasA spot that was incubated with A11 in the absence (‘Acid’) and presence of PE/PG/CL and DOPC/Chol/Sph vesicles. Right: Aβ42 (positive control) that was incubated with A11 under oligomerization conditions (see experimental procedures for details). (b) Average ThT fluorescence of 1 µM TasA that was recorded after adjusting the pH to 2.5 in the absence or presence of PE/PG/CL and DOPC/Chol/Sph vesicles. Dashed line, TasA in buffer; solid line, TasA in PE/PG/CL; dotted line, TasA in DOPC/Chol/Sph. The gray shadow shows the scatter in the measurements. 11 ACS Paragon Plus Environment

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formation from TasA oligomers12 (Figure 2B, dashed line). The ThT curve lacks a lag phase, commonly observed with disease-related

38

as well as functional amyloid proteins39,

40

. A

possible explanation for the lack of the lag phase is our use of TasA oligomers (rather than monomers) that either makes the lag phase too short to be probed or causes its elimination altogether. A lower ThT signal (Figure 2B, dotted line) is observed when TasA interacts with mimic eukaryotic membranes

41

. The lower ThT fluorescence might be ascribed to lower

fibrillation, but also to interference of the vesicles with ThT binding onto the fibrils of TasA, and/or less access of ThT to the fibrils associated with the vesicle bilayers, as compared to fibrils in solution. However, significantly enhanced ThT fluorescence is apparent upon incubating TasA in the presence of PE/PG/CL vesicle mimickingbacterial membrane composition

31

(Figure 2B,

solid line). The distinct effect of bacterial - mimic lipid bilayers on TasA fibrillation pathways observed from the ThT data in Figure 2B is indicative of a more pronounced structural transition into β sheet under these conditions 37.

TasA – membranes interaction affects fiber morphology Figure 2 shows the impact of vesicle bilayers on TasA aggregation. We further investigated the effect of the protein - membrane interaction on the morphology of both protein and membranes. Cryogenic transmission electron microscopy (cryo-TEM) was applied to gain visual insight into the morphology of TasA aggregates. Consistent with the increase in ThT signal (Figure 2B), abundant tangled fibrils were apparent after 4-hour incubation of TasA in the acidic solution (Figure 3A). Similar fibril networks were apparent in the cryo-TEM image of TasA incubated with DOPC/Chol/Sph vesicles (Figure 3C), however notable abundant spherical vesicles appeared in proximity and/or associated with the fibrils.


Significantly different fibril

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Biochemistry

morphology is observed following TasA incubation with PE/PG/CL vesicles (Figure 3B), revealing disordered aggregates.

Figure 3. TasA – membranes interaction affects fiber morphology. Cryo-TEM images of TasA samples after 3 h incubation in the absence and presence of PE/PG/CL and DOPC/Chol/Sph vesicles. (a). TasA fibrils formed at pH 2.5; (b). TasA with PE/PG/CL; (c). TasA with DOPC/Chol/Sph. Scale bars correspond to 200 nm.

TasA – membranes interaction affects membrane morphology and fluidity To examine the effect of TasA on the lipid membranes, we used confocal microscopy to visualize the morphology of giant vesicles (GUVs), harboring the fluorescent dye ,2-dimyristoylsn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)

(NPD-PE),

in

the

presence of TasA. The fluorescence microscopy images in Figures 4A-4D revealed timedependent structural effects following addition of TasA to NBD-PE/PE/PG/CL GUVs (mole ratio of 0.45:0.45:0.1:0.002). Prior to addition of TasA, the vesicles exhibited spherical morphology and uniform surface organization (Figure 4A). However, abundant dark domains formed on the vesicle surface immediately after adding TasA to the vesicle solution (Figure 4B). The dark spots disappeared after ~ 1 min, while distortions of the spherical shapes became noticeable a few minutes later (Figure 4C-4D). The microscopy results in Figures 4A – 4D


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indicate the occurrence of a pronounced instantaneous interaction of TasA with the bacterialmimic vesicle bilayers that is followed by a more gradual vesicle remodeling. Importantly, fluorescence microscopy experiments examining GUVs comprising DOPC/Chol/Sph indicated no discernible structural effects following addition of TasA to the vesicles (Figures 4E-4H), underscoring the distinct interactions between TasA and bacterial-mimicking bilayers. Observing the effect of membranes on TasA aggregation (figures 2,3) and the effect of TasA on membrane morphology (figure 4), we asked whether and to what extent the protein penetrates into the membranes. To answer this question, we used fluorescence anisotropy measurements utilizing PE/PG/CL and DOPC/Chol/Sph lipid vesicles which also contained the fluorescent dyes trimethylammonium-diphenylhexatriene (TMA-DPH) and 1,6-diphenylhexatriene (DPH). DPH has been employed as a membrane fluidity probe through monitoring its fluorescence anisotropy; larger anisotropy indicates larger membrane rigidity

42

. The bilayer location of the dye in each

vesicle system enables probing the effects of TasA in different membrane regions. Specifically, the TMA substituent in TMA-DPH acts as a surface anchor that localizes the probe close to the

Figure 4. TasA – membranes interaction affects membrane morphology. Confocal fluorescence microscopy images of PE/PG/CL/NPD-PE GUVs (a-d) and of DOPC/Chol /Sph/NPD-PE GUVs (e-h) before (a, e), and following addition of 2 µM TasA, immediately after the protein addition (t ~ 1 minute, b, f), one (c, g) and 10 minutes (d, h) after adjusting the pH to 2.5. Scale bars in all images correspond to 10 µm (a- d) and to 5 µm (e-h). ACS Paragon Plus Environment

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Biochemistry

membrane interface43, while the highly hydrophobic DPH resides primarily deeper within the bilayer interior, sensing the dynamic properties of the acyl chain environments43 (schematic, figure 5A). The bar diagram in Figure 5B shows that prior to the addition of TasA the bacterial – mimic membranes were more fluid than the eukaryotic – mimic membranes, as expected from membranes of highly unsaturated phospholipids. The eukaryotic – mimic membranes contain cholesterol and sphingomyelin that oppositely affect membrane fluidity, leading to more rigid membranes relative to the bacterial – mimic membranes. The addition of TasA resulted with an increased TMA-DPH fluorescence anisotropy in the presence of both PE/PG/CL and DOPC/Chol/Sph lipid vesicles, suggesting that both membrane surfaces interact with TasA upon aggregation44. This interpretation is consistent with the microscopy data in Figure 3 above. Significantly, however, Figure 5B shows that the anisotropy of DPH alone, undergone a more pronounced increase upon addition of TasA in case of the PE/PG/CL vesicles compared to DOPC/Chol/Sph vesicles. The larger DPH anisotropy is indicative of a mobility reduction of the lipid acyl chains closer to the DPH probe, which is inserted deeper into the bilayer. This result

Figure 5. TasA – membranes interaction affects membrane fluidity. (a) Schematic representation of the DPH location across the membranes. TMA-DPH, DPH sense membrane fluidity at the surface, interior of the membranes, respectively. (b) Fluorescence anisotropy of TMA-DPH and DPH embedded in PE/PG/CL or DOPC/Chol/Sph vesicles in the presence and absence of TasA, as indicated in the figure, following a pH adjustment and incubation for 5 minutes. Results are presented as means ± SEM; (*P < 0.05).


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suggests that TasA penetrated deeper into the bacterial-mimic bilayers 45.

TasA structural development is affected by bacterial-mimic membrane vesicles In light of the TasA – membrane interaction presented in Figures 2 -5 we further characterized the secondary structure of TasA using circular dichroism (CD) and FTIR spectroscopy (Figure 6). Figures 6A-6C depict the CD spectra of TasA before and after the addition of acid in the absence (A) and in the presence of PE/PG/CL vesicles mimicking bacterial membranes (B) or DOPC/Chol/Sph vesicles mimicking eukaryotic cell membranes (C). CD spectroscopy provides

Figure 6. TasA secondary structure is affected by the TasA- membrane interaction. Secondary structure change of TasA monitored by CD spectroscopy following the addition of acid in the absence (a) or presence (b–c) of vesicles. a) TasA in acid. b) TasA with PE/PG/CL. c) TasA with DOPC/Chol/Sph. (d) FTIR spectra of TasA following a pH adjustment to 2.5 (black) and in the presence of DOPC/Chol/Sph (red) and PE/PG/CL vesicles (blue). The arrows point at the ~ 1622 cm-1, 1742 cm-1 shoulders exhibited upon the interaction of TasA with the PE/PG/CL vesicles, and with both model membranes, respectively. (e) Second derivatives of the spectra in (d). The arrow points at the 1622 cm-1 peak, characteristic of intermolecular β sheet structure. ACS Paragon Plus Environment

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information on solution conformations of proteins, specifically the development of prominent structural elements such as α helices and β sheets 46. Similarly to the ThT and A11 pAb results in Figure 2, Figures 6A-6C demonstrate that TasA underwent distinctive structural change in the presence of PE/PG/CL vesicles. Specifically, the 208, 222 nm peaks in the CD spectrum of TasA suggest that it is predominantly α helical upon dissolution in buffer of pH 7.412. In acidic solutions without membrane vesicles (Figure 6A) or upon incubation with DOPC/Chol/Sph vesicles (Figure 6C), these peaks diminish in intensity. Loss of the CD signal could originate either from scattering of the TasA aggregates or from their settling. Measuring the absorbance at 280 mn of TasA in the sample before and after aggregation in acid (Table S1 in Supporting information), show that indeed most of the protein settled in the absence of vesicles (90% settles) and in the presence of DOPC/Chol/Sph vesicles (80% settles). However, in the presence of PE/PG/CL vesicles the TasA aggregates did not settle, probably because they are smaller than those formed in the absence of vesicles or in the presence of the mimic eukaryotic ones (as shown in Figure 3). The CD measurement of these aggregates revealed a pronounced negative peak at 218 nm as well as increase in signal intensity at 195 nm (despite some possible scattering, see Table S1), underscoring significant β-sheet formation (Figure 6B). We then used FTIR spectroscopy in order to measure the structure of the settled TasA aggregates, that could not be probed with CD measurements, in addition to the spectra of the soluble TasA aggregates. Figure 6D shows the FTIR spectra of TasA and their corresponding second derivatives (figure 6E) after the addition of acid in the absence (black) and in the presence of DOPC/Chol/Sph (red) and PE/PG/CL (blue) vesicles. The spectra of the protein in the absence of vesicles and in the presence of eukaryotic – like vesicles are similar. Both spectra peak in the amide I region at ~1648 cm-1 that is typical of carbonyl in α helices47, originating


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either from the residual oligomers in solution (see table S1) and/or from a residual α helical structure in the aggregates. The second derivatives of these spectra show additional peaks at ~ 1636 cm-1, 1690 cm-1, suggestive of some β sheet contribution to the settling aggregates, in correspondence with the ThT data in figure 2B. In the presence of the bacterial – model vesicles, TasA also shows a residual α helical structure, according to the amide I peak at ~ 1652 cm-1. Based on the dotblot results (figure 2A), this residual α helicity can be attributed to the aggregates as there are very little A11 – binding oligomers left in solution in the presence of the mimic bacterial membranes. Similarly to the aggregates that were formed without vesicles and with the mimic eukaryotic vesicles, here as well the second derivative of the FTIR spectrum shows two additional peaks at ~ 1684, 1695 cm-1, both are indicative of β sheet secondary structure. However, this spectrum also shows a more apparent shoulder at ~ 1622 cm-1, which is characteristic of intermolecular β sheets in aggregated protein47. Interestingly, the interaction of TasA with both membrane types yields a ~ 1742 cm-1 shoulder, which are typical for hydrogen bonded side chains of the amino acids Asp and Glu48. This additional carbonyl stretch possibly suggests that when TasA – membrane interaction causes these two amino acids to change position from the protein's (or fiber's) core to the protein's surface where they are free to hydrogen bond with water. DISCUSSION The central question addressed in this study concerns the contribution of membranes in shaping the fibrillation pathway of TasA, a functional amyloid protein constituting a prominent component of the biofilm matrix of Bacillus subtilis.

The spectroscopic and microscopic

experiments in Figures 2-6 - all reveal distinct difference between the interaction of TasA with


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Biochemistry

vesicles comprising PE/PG/CL which mimic bacterial membranes, and its interaction with vesicles comprising cholesterol and phospholipids designed to mimic eukaryotic cell membranes. These results are summarized in the schematic in figure 7. Tangled TasA fibers are formed in the absence (top schematic) and in the presence of eukaryotic – mimic membranes (Bottom schematic). These fibers are similar in structure (figures 6D, 6E) and they both settle when formed (table S1 in supporting Information). The cryo-TEM images (Figure 3) and fluorescence anisotropy (Figure 5B) clearly indicate that TasA interacts with the eukaryotic – mimic vesicles and that this interaction is limited to the membranes’ surface and it does not involve a significant change neither in the protein’s morphology (figure

Figure 7. Schematic illustration of a suggested mechanism of TasA – membrane interaction. TasA oligomers aggregate upon a reduction of pH in solution (top, ‘acid’). When vesicles are added to the solution, TasA attaches to their surface (middle, ‘PE/PG/CL’ and bottom ‘DOPC/Chol/sph’) but only in vesicles that mimic the bacterial composition (PE/PG/CL) does the protein significantly penetrate into the membrane. In addition to the membrane disruption upon the interaction with TasA, our results also indicate that the bacterial – like membranes stabilize the protein in a β sheet form.


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3C) nor in the membranes’ fluidity (figure 5B). In contrast, the TasA - PE/PG/CL bacterial membrane mimicking vesicles’ interaction results in the formation of disordered aggregates rather than fibers (figure 3B) as well as in the deformation of the membranes’ morphology (middle schematic). Previous studies of the interactions between membranes and amyloid proteins such as Aβ (49, 50

) or Islet Amyloid polypeptide (IAPP) 51 have shown that the initial protein - membrane affinity

is electrostatic (either charge – charge or charge – dipole), and that this interaction depends on the protein's isoelectric point and membrane's pKa. Further protein – membrane interactions may either promote fibrillation, or lead to the formation of amorphous aggregates 49. Fibrillation occurs when fiber-promoting segments in the protein are exposed towards the solution, whereas amorphous aggregation often results from insertion of hydrophobic protein segments into the membrane. Protein insertion into the membrane would disrupt the membrane as well as delay or inhibit fiber formation 49, 50. Based on these previous studies, we speculate that the preliminary TasA –membrane interaction is electrostatic. TasA is positively charged while both membranes are zwitterionic at pH ~ 2.5 (the isoelectric point of TasA is ~ 5 and see table S2 of the relevant pKa values in the Supporting information) and therefore the TasA – membrane surface interaction is dipole – charge. Following the initial attachment, TasA penetrates deeper into the bacterial membranes than into the eukaryotic membranes (based on the results presented in figures 3-5) due to their larger relative fluidity, as shown in figure 5B. In addition to changes in membrane fluidity, we have also observed changes in the TasA fibers' structure. When TasA interacts with the bacterial membranes it exhibits some transition into a β sheet and this transition is similar in the absence


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of membranes and in the presence of eukaryotic-mimic membranes (figures 2B, figure 6). However, in the presence of bacterial – mimic membranes, TasA gains different β sheet features, as shown by the FTIR measurements (figure 6) as well as by a larger ThT fluorescence signal (figure 2B). Buried inside a membrane in a stable β sheet conformation, TasA may be less prone to aggregation and this may lead to the deformed fibers observed in the presence of bacterial membranes (figure 3B). In real biofilms, TasA is exposed to a complex environment made of other proteins, polysaccharides, and cells, with constantly-changing external physical conditions such as temperature, pH, and salinity. Attempting to understand the aggregation of TasA in situ is therefore rather complicated since control of a large number of parameters is difficult. Previous studies have shown that the aggregation of TasA is affected by the auxiliary protein TapA well as by external conditions such and pH in solution

12

17

as

. This study reduces the complex

biofilm into a well-defined system composed of TasA and membranes, and our results indicate that in addition to the presence of the protein TapA and the external pH, TasA aggregation is also affected by membranes of both eukaryotic and bacterial compositions and that the effect is different and more pronounced in case of the bacterial membranes. Taking a leap back to real biofilms, our analysis suggests that in addition to the protein TapA

17

and external pH

12

,

bacterial membranes may participate in the aggregation process of TasA in situ and that this process is pH - dependent. Interestingly, there is evidence from another bacterium, Staphylococcus aureus, that adsorption of proteins to the cell-surface in biofilms is pH dependent

52

. Whether or not this is the case within Bacillus subtilis biofilms is yet to be

determined.


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In conclusion, our results provide important evidence for morphological and structural remodeling of TasA aggregates by bilayers mimicking bacterial membranes. TasA aggregation is critical in the formation and stability of Bacillus subtilis biofilms and therefore the principals that were demonstrated in our in vitro findings could serve the basis for new antibiofilm solutions that target the TasA – membrane interactions.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was funded by ISF grant to L.C (1150/14). Acknowledgement We thank Dr.Y Kalisman and Dr. A Upcher for their help with TEM. We are grateful to M Yellen for help with confocal microscopy. Supporting information This manuscript is accompanied with a supporting information file that includes supplementary figures and tables: dotblot assay of TasA and membranes in pH 7.4 and pH 2.5; a supplementary table showing the Absorbance at 280 nm (OD280) of TasA in the supernatant and pellet after acidifying the solution in the absence and presence of membranes; a supplementary table S1 that summarizes the pKa values of the functional groups in the phospholipids used in this study.


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ABBREVIATIONS Extracellular matrix, ECM Cryo-TEM, cryogenic transmission electron microscopy CD, circular dichroism FTIR, fourier transform infrared TMA-DPH, trimethylammonium-diphenylhexatriene DPH, 1,6-diphenylhexatriene Small Unilamellar Vesicles, SUVs Giant Unilamellar Vesicles, GUVs Asp, Aspartic acid Glu, Glutamic acid Table of Contents image

Bacterial membrane

Eukaryotic membrane


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REFERENCES [1] Romeo, T. (2008) Bacterial biofilms. Preface, Vol. 322, 1 ed., Springer-Verlag Berlin Heidelberg. [2] Raaijmakers, J. M., and Mazzola, M. (2012) Diversity and Natural Functions of Antibiotics Produced by Beneficial and Plant Pathogenic Bacteria, Annual Review of Phytopathology 50, 403-424. [3] Wolfe, B. E., Button, J. E., Santarelli, M., and Dutton, R. J. (2014) Cheese rind communities provide tractable systems for in situ and in vitro studies of microbial diversity, Cell 158, 422-433. [4] Flemming, H., and Wingender, J. (2010) The biofilm matrix. Nat Rev Microbiol 8: 623–633. [5] Mulcahy, H., Charron-Mazenod, L., and Lewenza, S. (2008) Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms, PLoS Pathog 4, e1000213. [6] Branda, S. S., Vik, Å., Friedman, L., and Kolter, R. (2005) Biofilms: the matrix revisited, Trends in microbiology 13, 20-26. [7] Chapman, M. R., Robinson, L. S., Pinkner, J. S., Roth, R., Heuser, J., Hammar, M., Normark, S., and Hultgren, S. J. (2002) Role of Escherichia coli curli operons in directing amyloid fiber formation, Science (New York, N.Y.) 295, 851-855. [8] Zeng, G., Vad, B. S., Dueholm, M. S., Christiansen, G., Nilsson, M., Tolker-Nielsen, T., Nielsen, P. H., Meyer, R. L., and Otzen, D. E. (2014) Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness, Frontiers in microbiology 6, 1099-1099. [9] Tayeb-Fligelman, E., Tabachnikov, O., Moshe, A., Goldshmidt-Tran, O., Sawaya, M. R., Coquelle, N., Colletier, J.-P., and Landau, M. (2017) The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril, Science 355, 831-833. [10] Schwartz, K., Syed, A. K., Stephenson, R. E., Rickard, A. H., and Boles, B. R. (2012) Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms, PLoS pathogens 8, e1002744. [11] Romero, D., Aguilar, C., Losick, R., and Kolter, R. (2010) Amyloid fibers provide structural integrity to Bacillus subtilis biofilms, Proceedings of the National Academy of Sciences 107, 2230-2234. [12] Chai, L., Romero, D., Kayatekin, C., Akabayov, B., Vlamakis, H., Losick, R., and Kolter, R. (2013) Isolation, characterization, and aggregation of a structured bacterial matrix precursor, Journal of Biological Chemistry 288, 17559-17568. [13] Bednarska, N. G., Schymkowitz, J., Rousseau, F., and Van Eldere, J. (2013) Protein aggregation in bacteria: the thin boundary between functionality and toxicity, Microbiology 159, 1795-1806. [14] Blanco, L. P., Evans, M. L., Smith, D. R., Badtke, M. P., and Chapman, M. R. (2012) Diversity, biogenesis and function of microbial amyloids, Trends in microbiology 20, 6673. [15] Goyal, P., Krasteva, P. V., Van Gerven, N., Gubellini, F., Van den Broeck, I., TroupiotisTsaïlaki, A., Jonckheere, W., Péhau-Arnaudet, G., Pinkner, J. S., and Chapman, M. R. (2014) Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG, Nature 516, 250-253.


24

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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[16] Shu, Q., Krezel, A. M., Cusumano, Z. T., Pinkner, J. S., Klein, R., Hultgren, S. J., and Frieden, C. (2016) Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation, Proc Natl Acad Sci U S A 113, 7130-7135. [17] Romero, D., Vlamakis, H., Losick, R., and Kolter, R. (2011) An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms, Molecular microbiology 80, 1155-1168. [18] Kotler, S. A., Walsh, P., Brender, J. R., and Ramamoorthy, A. (2014) Differences between amyloid-β aggregation in solution and on the membrane: insights into elucidation of the mechanistic details of Alzheimer's disease, Chemical Society Reviews 43, 6692-6700. [19] Malishev, R., Nandi, S., Kolusheva, S., Shaham-Niv, S., Gazit, E., and Jelinek, R. (2016) Bacoside-A, an anti-amyloid natural substance, inhibits membrane disruption by the amyloidogenic determinant of prion protein through accelerating fibril formation, Biochimica et Biophysica Acta (BBA)-Biomembranes 1858, 2208-2214. [20] Auluck, P. K., Caraveo, G., and Lindquist, S. (2010) α-Synuclein: membrane interactions and toxicity in Parkinson's disease, Annual review of cell and developmental biology 26, 211-233. [21] Walsh, P., Vanderlee, G., Yau, J., Campeau, J., Sim, V. L., Yip, C. M., and Sharpe, S. (2014) The mechanism of membrane disruption by cytotoxic amyloid oligomers formed by prion protein (106–126) is dependent on bilayer composition, Journal of Biological Chemistry 289, 10419-10430. [22] Malishev, R., Nandi, S., Kolusheva, S., Levi-Kalisman, Y., Klärner, F.-G., Schrader, T., Bitan, G., and Jelinek, R. (2015) Toxicity Inhibitors Protect Lipid Membranes from Disruption by Aβ42, ACS chemical neuroscience 6, 1860-1869. [23] Relini, A., Marano, N., and Gliozzi, A. (2014) Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers, Advances in colloid and interface science 207, 81-92. [24] Andreasen, M., Lorenzen, N., and Otzen, D. (2015) Interactions between misfolded protein oligomers and membranes: A central topic in neurodegenerative diseases?, Biochimica et Biophysica Acta (BBA)-Biomembranes 1848, 1897-1907. [25] Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G., and Dobson, C. M. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils, Proceedings of the National Academy of Sciences of the United States of America 96, 3590-3594. [26] Guijarro, J. I., Sunde, M., Jones, J. A., Campbell, I. D., and Dobson, C. M. (1998) Amyloid fibril formation by an SH3 domain, Proc Natl Acad Sci U S A 95, 4224-4228. [27] Dueholm, M. S., Nielsen, S. B., Hein, K. L., Nissen, P., Chapman, M., Christiansen, G., Nielsen, P. H., and Otzen, D. E. (2011) Fibrillation of the major curli subunit CsgA under a wide range of conditions implies a robust design of aggregation, Biochemistry 50, 8281-8290. [28] Bader, R., Bamford, R., Zurdo, J., Luisi, B. F., and Dobson, C. M. (2006) Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation, J Mol Biol 356, 189-208. [29] Manno, M., Craparo, E. F., Martorana, V., Bulone, D., and San Biagio, P. L. (2006) Kinetics of Insulin Aggregation: Disentanglement of Amyloid Fibrillation from LargeSize Cluster Formation, Biophysical Journal 90, 4585-4591.


25

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

[30] Hsieh, M. C., Liang, C., Mehta, A. K., Lynn, D. G., and Grover, M. A. (2017) Multistep Conformation Selection in Amyloid Assembly, J Am Chem Soc 139, 17007-17010. [31] Sohlenkamp, C., and Geiger, O. (2015) Bacterial membrane lipids: diversity in structures and pathways, FEMS microbiology reviews, fuv008. [32] van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008) Membrane lipids: where they are and how they behave, Nature reviews. Molecular cell biology 9, 112-124. [33] Shanmugasundaram, M., Kurouski, D., Wan, W., Stubbs, G., Dukor, R. K., Nafie, L. A., and Lednev, I. K. (2015) Rapid Filament Supramolecular Chirality Reversal of HET-s (218–289) Prion Fibrils Driven by pH Elevation, The Journal of Physical Chemistry B 119, 8521-8525. [34] Moscho, A., Orwar, O., Chiu, D. T., Modi, B. P., and Zare, R. N. (1996) Rapid preparation of giant unilamellar vesicles, Proceedings of the National Academy of Sciences 93, 11443-11447. [35] Necula, M., Kayed, R., Milton, S., and Glabe, C. G. (2007) Small molecule inhibitors of aggregation indicate that amyloid β oligomerization and fibrillization pathways are independent and distinct, Journal of Biological Chemistry 282, 10311-10324. [36] Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science 300, 486-489. [37] Biancalana, M., and Koide, S. (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils, Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1804, 1405-1412. [38] Galvagnion, C., Buell, A. K., Meisl, G., Michaels, T. C., Vendruscolo, M., Knowles, T. P., and Dobson, C. M. (2015) Lipid vesicles trigger alpha-synuclein aggregation by stimulating primary nucleation, Nat Chem Biol 11, 229-234. [39] Shu, Q., Crick, S. L., Pinkner, J. S., Ford, B., Hultgren, S. J., and Frieden, C. (2012) The E. coli CsgB nucleator of curli assembles to beta-sheet oligomers that alter the CsgA fibrillization mechanism, Proc Natl Acad Sci U S A 109, 6502-6507. [40] Zhou, Y., Smith, D. R., Hufnagel, D. A., and Chapman, M. R. (2013) Experimental manipulation of the microbial functional amyloid called curli, Methods Mol Biol 966, 5375. [41] Simons, K., and Sampaio, J. L. (2011) Membrane organization and lipid rafts, Cold Spring Harbor perspectives in biology 3, a004697. [42] Lentz, B. R. (1993) Use of fluorescent probes to monitor molecular order and motions within liposome bilayers, Chemistry and physics of lipids 64, 99-116. [43] do Canto, A. M., Robalo, J. R., Santos, P. D., Carvalho, A. J. P., Ramalho, J. P., and Loura, L. M. (2016) Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study, Biochimica et Biophysica Acta (BBA)Biomembranes 1858, 2647-2661. [44] Nandi, S., Malishev, R., Bhunia, S. K., Kolusheva, S., Jopp, J., and Jelinek, R. (2016) LipidBilayer Dynamics Probed by a Carbon Dot-Phospholipid Conjugate, Biophysical journal 110, 2016-2025. [45] Pereira, F. B., Goñi, F. M., Muga, A., and Nieva, J. L. (1997) Permeabilization and fusion of uncharged lipid vesicles induced by the HIV-1 fusion peptide adopting an extended conformation: dose and sequence effects, Biophysical Journal 73, 1977-1986.


26

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Biochemistry

[46] Greenfield, N. J. (2006) Using circular dichroism spectra to estimate protein secondary structure, Nature protocols 1, 2876-2890. [47] Barth, A. (2007) Infrared spectroscopy of proteins, Biochimica et Biophysica Acta (BBA) Bioenergetics 1767, 1073-1101. [48] Barth, A. (2000) The infrared absorption of amino acid side chains, Progress in Biophysics and Molecular Biology 74, 141-173. [49] Terakawa, M. S., Yagi, H., Adachi, M., Lee, Y.-H., and Goto, Y. (2015) Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40), The Journal of Biological Chemistry 290, 815-826. [50] Sabaté, R., Espargaró, A., Barbosa-Barros, L., Ventura, S., and Estelrich, J. (2012) Effect of the surface charge of artificial model membranes on the aggregation of amyloid βpeptide, Biochimie 94, 1730-1738. [51] Caillon, L., Lequin, O., and Khemtemourian, L. (2013) Evaluation of membrane models and their composition for islet amyloid polypeptide-membrane aggregation, Biochim Biophys Acta 1828, 2091-2098. [52] Foulston, L., Elsholz, A. K., DeFrancesco, A. S., and Losick, R. (2014) The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH, MBio 5, e01667-01614.


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Bacterial model membranes reshape fibrillation of a functional amyloid

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