Bacterial Model Membranes Reshape Fibrillation of a FunctionalAmyloid ProteinRavit Malishev,†,§ Razan Abbasi,‡,§ Raz Jelinek,*,† and Liraz Chai*,‡

†Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel‡Institute of Chemistry, The Hebrew University of Jerusalem and The Center for Nanoscience and Nanotechnology, Edmond J. SafraCampus, Jerusalem 91904, Israel

*S Supporting Information

ABSTRACT: Biofilms are aggregates of cells that form surface-associated communities.The cells in biofilms are interconnected with an extracellular matrix, a network that ismade mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellularmatrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiatetheir constructive function from disease-related amyloid fibers. Recent functional amyloidassembly studies have neglected their interaction with membranes, despite their nativeformation in a cellular environment. Here, we use TasA, a major matrix protein in biofilmsof the soil bacterium Bacillus subtilis, as a model functional amyloid protein and askwhether the bacterial functional amyloid interacts with membranes. Using biochemical,spectroscopic, and microscopic tools, we show that TasA interacts distinctively withbacterial model membranes and that this interaction mutually influences the morphologyand structure of the protein and the membranes. At the protein level, fibers of similarstructure 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. Atthe membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform moreconsiderably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial morethan eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formationpathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilmdrugs to the protein−membrane interface.

Biofilms are aggregates of cells that form surface-associatedcommunities that grow on natural and synthetic surfaces.1

Biofilms can be harmful, for example, when they develop oncatheters, artificial implants, and water pipes, but they can bebeneficial as well, for example, when they promote plant growthas they develop on plant roots2 and when (together with mold)they make rich-flavored cheese rind.3 Once formed, they arevery hard to eradicate as they are 10−50 times more resistant toantibiotics relative to planktonic cells, partly for reasons relatedto their extracellular matrix (ECM).4,5 The ECM is a networkof biopolymers that comprises more than 90% of the dry weightof biofilms.4 The exact composition of the bacterial ECM variesbetween species, but a feature common to all matrices is thatthey are made of polysaccharides, proteins, and sometimesnucleic acids.4,6 The proteins often form fibrillar appendagesthat are amyloid-like, for example, the proteins curli (Escherichiacoli),7 FapC (Pseudomonas),8 PSMs (Staphylococcus aureus),9,10

and TasA that is secreted by the soil bacterium, Gram-positiveand spore-forming, Bacillus subtilis.11,12

In recent years, many studies of functional amyloids,including those of TasA, have focused on their structural andaggregation properties in solution.12−16 However, despite thefact that TasA fibers have been observed close to and/orattached to the surface of the bacterial cells,17 the actual roles of

the bacterial cells’ surfaces in the assembly and structuralproperties of TasA have not been addressed. This scantknowledge base stands in sharp contrast to the huge body ofwork aimed at deciphering amyloid protein−membraneinteractions in prominent neurodegenerative diseases, such asAlzheimer’s and Parkinson’s disease.18−20 In these degenerativediseases, soluble intermediate oligomers aggregate into fibers,and it is now widely accepted that lipid bilayers may act as aneffective catalyst of fibrillogenesis, providing a generic environ-ment where protein molecules adopt conformation andorientation to promote their assembly into protofibrillar andfibrillar structures.18,21 Furthermore, the intermediate oligomershave been related by several bilayer disruption events, such asdetergent and carpeting effects or pore formation.22−24

The thrust of this study is to decipher the interplay betweenbacterial membranes and TasA in aggregation conditions. Wehave previously shown that TasA can be purified from B. subtilisin the form of natively structured, soluble oligomers.12 Toinduce aggregation of a folded protein in vitro, it is oftennecessary to work under denaturing conditions (such as

Received: January 2, 2018Revised: February 18, 2018Published: March 22, 2018

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© XXXX American Chemical Society A DOI: 10.1021/acs.biochem.8b00002Biochemistry XXXX, XXX, XXX−XXX

elevated temperatures, high denaturant concentration, or lowpH) and allow the protein to refold.25,26 Following previousprotocols for amyloid and functional amyloid proteinaggregation,27−30 we chose to work in an acidic solution ofpH ⩽3. We have therefore tested here the TasA fibrillation inthe absence and presence of vesicle bilayers after the addition ofacid. While employing model membranes mimicking bacterialmembranes31 and eukaryotic cell membranes32 (Figures 1A and1B), we demonstrate that TasA (sequence presented in Figure1C) interacts distinctively with bacterial-mimicking bilayers andthat this interaction influences the TasA aggregation pathwaysas well as the membrane morphology and fluidity. Thisimportant in vitro finding could open new avenues for acomprehensive understanding of bacterial biofilm assembly.

■ MATERIALS AND METHODS

Materials. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DOPG), cardiolipin (heart, bovine), 1,2-dioleoyl-sn-glycero-3-phosphocholine (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)(N-NBD-PE) were purchased from Avanti Polar Lipids.Thioflavin T (ThT), sodium hydrosulfite, and sodiumphosphate monobasic were purchased from Sigma-Aldrich(Rehovot, Israel). 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenylhexatriene(DPH) were obtained from Molecular Probes, Inc. (Eugene,Oregon). A11 antioligomer antibody was obtained fromRhenium (Modi’in, Israel). Horseradish peroxidase conjugated

anti-rabbit IgG (HRP) was purchased from ZOTAL (Tel Aviv,Israel).

Protein Purification. TasA was purified from the B. subtilisdouble mutant strain sinR eps, as previously reported.11,12

Briefly, cells were grown overnight in Msgg broth at 37 °C.After centrifugation the pellet was collected, and the proteinwas extracted from the pellet using sonication in a salineextraction buffer. The supernatant was separated from the pelletusing an additional centrifugation, and the supernatant wasfiltered through a 0.45 μm poly(ether sulfone) bottle-top filter.The crude extract was concentrated using a GE HealthcareVivaspin sample concentrator (10 kDa MWCO) and furthercleaned using gel filtration.

Vesicle Preparation. Vesicles consisting of DOPE/DOPG/CL and DOPC/cholesterol/sphingomyelin (DOPC/Chol/Sph) were prepared by dissolving the lipid componentsin chloroform/ethanol (1:1 v/v) and drying them together invacuum. Small unilamellar vesicles (SUVs; DOPE/DOPG/CLand DOPC/cholesterol/sphingomyelin, 0.45:0.45:0.1 and0.67:0.25:0.08 molar ratios, respectively) were prepared in 10mM sodium phosphate (pH 7.4) by probe-sonication (power,130 W; frequency, 20 kHz; at 20% amplitude) of the aqueouslipid mixtures at room temperature for 10 min. Vesiclesuspensions were kept for 1 h at room temperature to stabilizethem prior to usage.

Thioflavin T (ThT) Fluorescence Assay. ThT fluores-cence measurements were conducted at 25 °C using black,clear-bottom, 96-well plates (Costar) on a Synergy H1 platereader (Biotek). The aggregation reaction mixture contained1−1.5 μM TasA, 0.3 mM DOPE/DOPG/CL or DOPC/cholesterol/sphingomyelin lipid vesicles, and 25 μM ThT. Data

Figure 1. Composition of bacterial and eukaryotic model membranes and the sequence of the extracellular protein TasA. (A,B) Composition ofbacterial (PE/PE/CL) and eukaryotic (DOPC/Chol/Sph) model vesicles. The bacterial-mimicking membranes are composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), and cardiolipin (PE/PG/CL, 0.45:0.45:0.1molar ratio), and the eukaryotic-mimicking membranes are composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, andsphingomyelin (DOPC/Chol/Sph, 0.67:0.25:0.08 molar ratio). (C) The sequence of TasA (234 residues), an extracellular fiber-forming functionalamyloid protein secreted by B. subtilis.

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were collected with or without vesicles after adjusting the pH to2.5 using formic acid; control experiments were conducted atpH 7.4. The device was programmed to record fluorescenceintensity every 2 min for 3−4 h under orbital shakingconditions between measurements. Excitation and emissionwavelengths were 430 and 480 nm, respectively. Thefluorescence curves of representative measurements weresmoothed by using a five-point adjacent averaging.Cryogenic Transmission Electron Microscopy (cryo-

TEM). Aliquots were taken from the ThT reaction mixturesafter incubation for 3 h. A 3 μL droplet of the reaction mixturewas deposited on a glow-discharged TEM grid (300 mesh CuLacey substrate grid; Ted Pella). The excess liquid was blottedoff with a filter paper, and the specimen was rapidly plungedinto liquid ethane pre-cooled with liquid nitrogen in acontrolled environment using a Vitrobot Mark IV (FEI). Thevitrified samples were transferred to a cryo-specimen holder(Gatan 626) and examined at −177 °C using a FEI Tecnai 12G2 TWIN TEM instrument operated at 120 kV in low-dosemode. Grids were imaged at a few micrometers under focus toincrease phase contrast. The images were recorded with a 4K ×4K FEI Eagle CCD camera.Circular Dichroism (CD) Spectroscopy. CD spectra were

recorded in the range of 190−260 nm at room temperature ona Jasco J-715 spectropolarimeter, using 10 mm quartz cuvettes.Solutions (400 μL) were composed of 1−1.5 μM TasA in a 10mM potassium phosphate buffer, pH 7.4, in the absence orpresence of DOPE/DOPG/CL or DOPC/cholesterol/sphin-gomyelin (final concentration 0.3 mM). CD was measured atpH 7.4 or at pH 2.5, with the latter obtained by addingconcentrated formic acid. Spectra were recorded every 30 minfor 2 h. CD signals resulting from vesicles and buffer weresubtracted from the corresponding spectra.Fourier Transform Infrared (FTIR) Spectroscopy. FTIR

spectra were measured with a Thermo Scientific is50 FTIRspectromter equipped with a DTGS detector. Samplescontaining 0.09 mM TasA in the absence or presence of PE/PG/CL or DOPC/cholesterol/sphingomyelin lipid vesicles(final concentration 1 mM) were incubated for 3 h at 4 °C. A10 μL sample was then placed in a Biocell (Biotools, Inc.)33

with CaF2 windows and a 6 μm path length, and 256 scans inabsorbance mode were collected. The data were collected at pH7.4 or after adjusting the pH to 2.5 using HCl or formic acid. A256-scan measurement in ambient air served as a background.The final spectra were obtained after subtracting thecorresponding solution (lacking the protein) spectra, as wellas subtracting a water vapor spectrum, and three to fourmeasurements were then averaged. Similar FTIR spectra wererecorded for samples taken directly from solution after mixingby pipetting or for samples taken from a pellet aftercentrifugation (9600g for 5 min).Fluorescence Anisotropy. The fluorescence probes TMA-

DPH and DPH were incorporated into the SUVs (DOPE/DOPG/CL or DOPC/choles tero l/sphingomyel in ,0.45:0.45:0.1 and 0.67:0.25:0.08 molar ratios, respectively) byadding the dye dissolved in THF (1 mM) to the vesicles (finalconcentration 0.3 mM) to a final concentration of 1.25 μM.After 30 min of incubation at 30 °C, TMA-DPH or DPHfluorescence anisotropy was measured at λex = 360 nm and λem= 430 nm using a FL920 spectrofluorimeter (Edinburgh Co.,Edinburgh, U.K.). Data were collected before and after additionof TasA (final concentration 1 μM) at pH 7.4 or at pH 2.5, withthe latter obtained by adding concentrated formic acid.

Anisotropy values were automatically calculated by thespectrofluorimeter software using the equation r = (IVV −GIVV)/(IVV + 2GIVH). G = IVH/IHH, where IVV is with theexcitation and emission polarizers mounted vertically and IHH iswith the excitation and emission polarizers mounted horizon-tally. IHV corresponds to the excitation polarizer mountedhorizontally and the emission polarizer mounted vertically. IVHcorresponds to the excitation polarizer mounted vertically andthe emission polarizer mounted horizontally. Each experimentwas repeated at least three times. Results are presented as themean ± SEM (*P < 0.05).

Giant Unilamellar Vesicles Labeled with N-NBD-PE.Giant unilamellar vesicles (GUVs) were prepared through therapid evaporation method.34 Briefly, GUVs comprising DOPE/DOPG/CL or DOPC/cho les te ro l/sph ingomye l in(0.45:0.45:0.1 and 0.67:0.25:0.08 molar ratios, respectively)were prepared via the dissolution of the lipid constituents inchloroform/ethanol (1:1 v/v), and subsequent addition of themixture to a round-bottom flask (250 mL) containing NBD-PE(1:500) and chloroform (1 mL). The aqueous phase (5 mL of0.1 M sucrose, 0.1 mM KCl, 50 mM Tris solution, pH 7.4) wasthen carefully added along the flask walls. The organic solventwas 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 wasobtained with a volume of approximately 5 mL.

Confocal Fluorescence Microscopy. GUVs (final con-centration 0.3 mM) were measured in the absence or presenceof 2 μM TasA at pH 7.4 or at pH 3 using a PerkinElmerUltraVIEW system (PerkinElmer Life Sciences Inc., MA, USA)equipped with an Axiovert-200 M (Zeiss, Germany) micro-scope and a Plan-Neofluar 63 1.4× oil objective. The excitationwavelength of 488 nm was generated by an Ar/Kr laser. Weperformed this experiment three times and visualized similarimages in different locations in the sample. For technicalreasons, due to the constant motion of the GUVs, it wasdifficult to take snapshots of short time scale (approximatelyseveral minutes) events, and therefore, we show representativeimages.

Dot Blot Assay. Oligomers of TasA at pH 2.5 wereincubated in the absence or presence of DOPE/DOPG/CL orDOPC/cholesterol/sphingomyelin lipid vesicles, and oligomersof Aβ42 were prepared via previously described methods.35

Both types of oligomers were probed by the oligomer-specificpolyclonal antibody (pAb) A11. The resulting solution wasmaintained at room temperature for up to 10 min. Periodically,2 μL aliquots (the concentrations of TasA and Aβ42 were 1and 45 μM, respectively) were applied to nitrocellulosemembranes. The membranes were blocked for 1 h with 5%nonfat milk in 10 mM Tris-buffered saline (TBS), followed byincubation with A11 at a 1:1000 dilution in TBS containing 5%nonfat milk, followed by appropriate horseradish peroxidase-linked secondary polyclonal antibodies, and developed using anenhanced chemiluminescence (ECL) reagent kit (GE Health-care).

Optical Density Measurements at 280 nm (OD280).We used a Nanodrop (ND-1000) to measure the TasAabsorbance at 280 nm (OD280). Formic acid was added tosamples containing TasA (in 10 mM potassium phosphatebuffer, pH 7.4) in the absence and presence of 0.3 mM vesicles(eukaryotic or bacterial model membranes, as described above,final concentration in 10 mM potassium phosphate buffer) toadjust the solution pH to 2.5. OD280 measurements of

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supernatant aliquots were taken before the addition of acid and3 h after the addition of acid (without pipetting).

■ RESULTSTasA Assembly and Fibrillation Are Affected by

Bacterial-Mimicking Membrane Vesicles. We initiallyevaluated whether membrane vesicles affected the aggregationof TasA oligomers. Utilizing dot blot assay with the A11polyclonal antibody (pAb), which recognizes prefibrillaroligomers en route to fibrillation36 (Figure 2A), we observed

the TasA oligomers’ reaction with A11 after 15 min in theacidic solutions. Purified TasA oligomers reacted positively withA11, indicating that there are still abundant A11-bindingoligomeric species in the solution (for a positive control weused Aβ42, Figure 2A). A similar reaction of TasA with A11 hasbeen observed upon the addition of eukaryotic-mimickingDOPC/Chol/Sph vesicles, but in the presence of the bacterial-

mimicking vesicle bilayers comprising PE/PG/CL (see Figure1A for composition and lipid structure), A11 staining wasnegligible, indicating a low abundance of A11-binding TasAoligomers. We note that we have performed the dot blotexperiments at pH 7.4 (Figure S1 in the SupportingInformation), and under these conditions there was a similarinteraction of TasA with the A11 antibody in the absence andpresence of the membranes.Witnessing a more enhanced reaction with A11 during TasA

aggregation in the presence of biomimetic bacterial membranesin comparison with eukaryotic membranes, we aimed to probethe effect of lipid bilayers on protein aggregation over time. Wecarried out fluorescence thioflavin T (ThT) assays in an acidicsolution in the absence and in the presence of vesicle bilayerscomprising different lipid compositions (Figure 2B). ThTfluorescence has been widely employed for measuring theassembly process of β sheet-containing amyloid fibrils.37 Figure2B reveals major differences of TasA fibrillation depending onsolution composition. A gradual increase in the ThTfluorescence signal is observed in an acidic solution (novesicles present), reflecting fibril formation from TasAoligomers12 (Figure 2B, dashed line). The ThT curve lacks alag phase, commonly observed with disease-related,38 as well asfunctional, amyloid proteins.39,40 A possible explanation for thelack of the lag phase is our use of TasA oligomers (rather thanmonomers) that either make the lag phase too short to beprobed or cause its elimination altogether. A lower ThT signal(Figure 2B, dotted line) is observed when TasA interacts witheukaryotic-mimicking membranes.41 The lower ThT fluores-cence might be ascribed to lower fibrillation or to interferenceof the vesicles with ThT binding to the fibrils of TasA and/orless access of ThT to the fibrils associated with the vesiclebilayers, as compared to fibrils in solution. However,significantly enhanced ThT fluorescence is apparent uponincubating TasA in the presence of PE/PG/CL vesiclesmimicking bacterial membrane composition31 (Figure 2B,solid line). The distinct effect of bacterial-mimicking lipidbilayers on TasA fibrillation pathways observed from the ThTdata in Figure 2B is indicative of a more pronounced structuraltransition into β sheets under these conditions.37

TasA−Membrane Interaction Affects Fiber Morphol-ogy. Figure 2 shows the impact of vesicle bilayers on TasAaggregation. We further investigated the effect of the protein−membrane interaction on the morphology of both protein andmembranes. Cryogenic transmission electron microscopy(cryo-TEM) was applied to gain visual insight into themorphology of TasA aggregates. Consistent with the increase

Figure 2. The effect of membranes on the assembly and fibrillation ofTasA. (A) Dot blot assay using the oligomer sequence-specific A11polyclonal antibody 10 min after a pH adjustment to 2.5. Darker colorindicates a larger oligomer abundance in solution. From left to right: aTasA spot that was incubated with A11 in the absence (“acid”) andpresence of PE/PG/CL and DOPC/Chol/Sph vesicles. Right: Aβ42(positive control) that was incubated with A11 under oligomerizationconditions (see Materials and Methods for details). (B) Average ThTfluorescence of 1 μM TasA that was recorded after adjusting the pH to2.5 in the absence or presence of PE/PG/CL and DOPC/Chol/Sphvesicles. Dashed line, TasA in buffer; solid line, TasA in PE/PG/CL;dotted line, TasA in DOPC/Chol/Sph. The gray shadow shows thescatter in the measurements.

Figure 3. TasA−membrane interaction affects fiber morphology. Cryo-TEM images of TasA samples after incubation for 3 h in the absence andpresence 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.

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in ThT signal (Figure 2B), abundant tangled fibrils wereapparent after incubation of TasA in the acidic solution for 3 h(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 vesiclesappeared in proximity to and/or associated with the fibrils.Significantly different fibril morphology is observed followingincubation of TasA with PE/PG/CL vesicles (Figure 3B),revealing disordered aggregates.TasA: Membrane Interaction Affects Membrane

Morphology and Fluidity. To examine the effect of TasAon the lipid membranes, we used confocal microscopy tovisualize the morphology of giant unilamellar vesicles (GUVs)harboring the fluorescent dye 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(NPD-PE) in the presence of TasA. The fluorescencemicroscopy images in Figures 4A−4D revealed time-dependentstructural effects following addition of TasA to NBD-PE/PE/PG/CL GUVs (molar ratio of 0.45:0.45:0.1:0.002). Prior toaddition of TasA, the vesicles exhibited spherical morphologyand uniform surface organization (Figure 4A). However,abundant dark domains formed on the vesicle surfaceimmediately after adding TasA to the vesicle solution (Figure4B). The dark spots disappeared after ∼1 min, while distortionsof the spherical shapes became noticeable a few minutes later(Figures 4C and 4D). The microscopy results in Figures4A−4D indicate the occurrence of a pronounced instantaneousinteraction of TasA with the bacterial-mimicking vesicle bilayersthat is followed by a more gradual vesicle remodeling.Importantly, fluorescence microscopy experiments examining

GUVs comprising DOPC/Chol/Sph indicated no discerniblestructural effects following addition of TasA to the vesicles(Figures 4E−4H), underscoring the distinct interactionsbetween TasA and bacterial-mimicking bilayers.Observing the effect of membranes on TasA aggregation

(Figures 2 and 3) and the effect of TasA on membranemorphology (Figure 4), we asked whether and to what extentthe protein penetrates into the membranes. To answer thisquestion, we used fluorescence anisotropy measurementsutilizing PE/PG/CL and DOPC/Chol/Sph lipid vesicles thatalso contained the fluorescent dyes trimethylammonium-diphenylhexatriene (TMA-DPH) and 1,6-diphenylhexatriene(DPH). DPH has been employed as a membrane fluidity probethrough monitoring its fluorescence anisotropy; larger aniso-tropy indicates larger membrane rigidity.42 The bilayer locationof the dye in each vesicle system enables probing of the effectsof TasA in different membrane regions. Specifically, the TMAsubstituent in TMA-DPH acts as a surface anchor that localizesthe probe close to the membrane interface,43 while the highlyhydrophobic DPH resides primarily deeper within the bilayerinterior, to allow for sensing the dynamic properties of the acylchain environments43 (schematic, Figure 5A). The bar diagramin Figure 5B shows that prior to the addition of TasA thebacterial-mimicking membranes were more fluid than theeukaryotic-mimicking membranes, as expected from mem-branes of highly unsaturated phospholipids. The eukaryotic-mimicking membranes contain cholesterol and sphingomyelinthat oppositely affect membrane fluidity, leading to more rigidmembranes relative to the bacterial-mimicking membranes. Theaddition of TasA resulted in an increased TMA-DPH

Figure 4. TasA−membrane 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 proteinaddition (t < 1 min, B, F), 1 min (C, G) and 10 min (D, H) after adjusting the pH to 2.5. Scale bars for use in all images correspond to 10 μm (A−D) and 5 μm (E−H).

Figure 5. TasA−membrane interaction affects membrane fluidity. (A) Schematic representation of the DPH location across the membranes. TMA-DPH and DPH are used to sense membrane fluidity at the surface and in the interior of the membranes, respectively. (B) Fluorescence anisotropy ofTMA-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 min. Results are presented as means ± SEM (*P < 0.05).

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fluorescence anisotropy in the presence of both PE/PG/CLand DOPC/Chol/Sph lipid vesicles, suggesting that bothmembrane surfaces interact with TasA upon aggregation.44

This interpretation is consistent with the microscopy data inFigure 3. Significantly, however, Figure 5B shows that theanisotropy of DPH alone undergoes a more pronouncedincrease upon addition of TasA in the case of the PE/PG/CLvesicles compared to that with the DOPC/Chol/Sph vesicles.The larger DPH anisotropy is indicative of a mobility reductionof the lipid acyl chains closer to the DPH probe, which isinserted deeper into the bilayer. This result suggests that TasApenetrated deeper into the bacterial-mimicking bilayers.45

TasA Structural Development Is Affected by Bacterial-Mimicking Membrane Vesicles. In light of the TasA−membrane interaction presented in Figures 2−5, we furthercharacterized the secondary structure of TasA using circulardichroism (CD) and FTIR spectroscopy (Figure 6). Figures6A−6C depict the CD spectra of TasA before and after theaddition of acid in the absence (A) and in the presence of PE/PG/CL vesicles mimicking bacterial membranes (B) orDOPC/Chol/Sph vesicles eukaryotic-mimicking cell mem-branes (C). CD spectroscopy provides information aboutsolution conformations of the proteins, specifically thedevelopment of prominent structural elements such as αhelices and β sheets.46 Similarly to the ThT and A11 pAbresults in Figure 2, Figures 6A−6C demonstrate that TasAunderwent distinctive structural changes in the presence of PE/PG/CL vesicles. Specifically, the 208 and 222 nm peaks in theCD spectrum of TasA suggest that it is predominantly α helicalupon dissolution in a buffer of pH 7.4.12 In acidic solutionswithout membrane vesicles (Figure 6A) or upon incubationwith DOPC/Chol/Sph vesicles (Figure 6C), these peaksdiminish in intensity. Loss of the CD signal could originateeither from scattering of the TasA aggregates or from theirsettling. Measurement of the absorbance at 280 nm of TasA inthe sample before and after aggregation in acid shows (TableS1) that indeed most of the protein settled in the absence of

vesicles (90% settles) and in the presence of DOPC/Chol/Sphvesicles (80% settles). However, in the presence of PE/PG/CLvesicles the TasA aggregates did not settle, probably becausethey are smaller than those formed in the absence of vesicles orin the presence of the mimicking eukaryotic ones (as shown inFigure 3). The CD measurement of these aggregates revealed apronounced negative peak at 218 nm as well as an increase insignal intensity at 195 nm (despite some possible scattering, seeTable S1), underscoring significant β sheet formation (Figure6B).We then used FTIR spectroscopy to measure the structure of

the settled TasA aggregates, which could not be probed withCD measurements, in addition to the spectra of the solubleTasA aggregates. Figure 6D shows the FTIR spectra of TasAand their corresponding second derivatives (Figure 6E) afterthe addition of acid in the absence (black) and in the presenceof DOPC/Chol/Sph (red) and PE/PG/CL (blue) vesicles.The spectra of the protein in the absence of vesicles and in thepresence of eukaryotic-mimicking vesicles are similar. Bothspectra peak in the amide I region at ∼1648 cm−1, which istypical of carbonyl in α helices,47 originating from the residualoligomers in solution (see Table S1) and/or from a residual αhelical structure in the aggregates. The second derivatives ofthese spectra show additional peaks at ∼1636 and 1690 cm−1,suggestive of some β sheet contribution to the settlingaggregates, which is in correspondence with the ThT data inFigure 2B. In the presence of the bacterial-model vesicles, TasAalso shows a residual α helical structure, according to the amideI peak at ∼1652 cm−1. On the basis of the dot blot results(Figure 2A), this residual α helicity can be attributed to theaggregates as there are very few A11-binding oligomers left insolution in the presence of the bacterial-mimicking membranes.Similar to the aggregates that were formed without vesicles andwith the eukaryotic-mimicking vesicles, here, the secondderivative values of the FTIR spectrum show two additionalpeaks at ∼1684 and 1695 cm−1, both indicative of a β sheetsecondary structure. However, this spectrum also shows a more

Figure 6. TasA secondary structure is affected by the TasA−membrane interaction. Secondary structure change of TasA monitored by CDspectroscopy following the addition of acid in the absence (A) or presence (B and 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 (blue) vesicles. The arrows point at the ∼1622 and 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 an intermolecular β sheet structure.

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apparent shoulder at ∼1622 cm−1, which is characteristic ofintermolecular β sheets in aggregated protein.47 Interestingly,the interaction of TasA with both membrane types yields a∼1742 cm−1 shoulder, which is typical for hydrogen bondedside chains of the amino acids Asp and Glu.48 This additionalcarbonyl stretch possibly suggests that the TasA−membraneinteraction causes these two amino acids to change positionfrom the protein’s (or fiber’s) core to the protein’s surfacewhere they are free to hydrogen bond with water.

■ DISCUSSION

The central question addressed in this study concerns thecontribution of membranes in shaping the fibrillation pathwayof TasA, a functional amyloid protein constituting a prominentcomponent of the biofilm matrix of B. subtilis. Thespectroscopic and microscopic experiments in Figures 2−6 allreveal a distinct difference between the interaction of TasA withvesicles comprised of PE/PG/CL which mimics bacterialmembranes and with vesicles comprised of cholesterol andphospholipids 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 presence of eukaryotic-mimicking membranes(bottom schematic). These fibers are similar in structure(Figures 6D and 6E), and they both settle when formed (TableS1). The cryo-TEM images (Figure 3) and fluorescenceanisotropy (Figure 5B) clearly indicate that TasA interactswith the eukaryotic-mimicking vesicles and that this interactionis limited to the membranes’ surface and does not involve asignificant change in either the protein’s morphology (Figure3C) or the membranes’ fluidity (Figure 5B). In contrast, theTasA−PE/PG/CL bacterial-mimicking membrane vesicles’interaction results in the formation of disordered aggregatesrather than fibers (Figure 3B) as well as in the deformation ofthe membranes’ morphology (middle schematic).Previous studies of the interactions between membranes and

amyloid proteins such as Aβ49,50 or islet amyloid polypeptide

(IAPP)51 have shown that the initial protein−membraneaffinity is electrostatic (either charge−charge or charge−dipole)and that this interaction depends on the protein’s isoelectricpoint and the membrane’s pKa. Further protein−membraneinteractions may either promote fibrillation or lead to theformation of amorphous aggregates.49 Fibrillation occurs whenfiber-promoting segments in the protein are exposed to thesolution, whereas amorphous aggregation often results frominsertion of hydrophobic protein segments into the membrane.Protein insertion into the membrane would disrupt themembrane, as well as delay or inhibit fiber formation.49,50

On the basis of these previous studies, we speculate that thepreliminary TasA−membrane interaction is electrostatic. TasAis positively charged while both membranes are zwitterionic atpH ∼2.5 (the isoelectric point of TasA is ∼5; see Table S2 forrelevant pKa values), and therefore, the TasA−membranesurface interaction is dipole−charge. Following the initialattachment, TasA penetrates into the bacterial membranesdeeper than into the eukaryotic membranes (based on theresults presented in Figures 3−5) due to their larger relativefluidity, as shown in Figure 5B. In addition to changes inmembrane fluidity, we have also observed changes in the TasAfibers’ structure. When TasA interacts with the bacterialmembranes it exhibits some transition into a β sheet, and thistransition is similar in the absence of membranes and in thepresence of eukaryotic-mimicking membranes (Figures 2B and6). However, in the presence of bacterial-mimicking mem-branes, TasA gains different β sheet features, as shown by theFTIR measurements (Figure 6), as well as by a larger ThTfluorescence signal (Figure 2B). Buried inside a membrane in astable β sheet conformation, TasA may be less prone toaggregation, and this may lead to the deformed fibers observedin the presence of bacterial membranes (Figure 3B).In real biofilms, TasA is exposed to a complex environment

made of other proteins, polysaccharides, and cells, withconstantly changing external physical conditions, such astemperature, pH, and salinity. Attempting to understand the

Figure 7. Schematic illustration of a suggested mechanism of TasA−membrane interaction. TasA oligomers aggregate upon a reduction of pH insolution (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 themembrane disruption upon the interaction with TasA, our results also indicate that the bacterial-like membranes stabilize the protein in a β sheetform.

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aggregation of TasA in situ is, therefore, rather complicatedsince control of a large number of parameters is difficult.Previous studies have shown that the aggregation of TasA isaffected by the auxiliary protein TapA,17 as well as by externalconditions such as pH in solution.12 This study reduces thecomplex biofilm into a well-defined system composed of TasAand membranes, and our results indicate that in addition to thepresence of the protein TapA and the external pH, TasAaggregation is also affected by membranes of both eukaryoticand bacterial compositions and that the effect is different andmore pronounced in case of the bacterial membranes. Taking aleap back to real biofilms, our analysis suggests that in additionto the protein TapA17 and external pH,12 bacterial membranesmay participate in the aggregation process of TasA in situ andthat this process is pH dependent. Interestingly, there isevidence from another bacterium, S. aureus, that adsorption ofproteins to the cell surface in biofilms is pH dependent.52

Whether or not this is the case within B. subtilis biofilms has yetto be determined.In conclusion, our results provide important evidence for

morphological and structural remodeling of TasA aggregates bybilayers mimicking bacterial membranes. TasA aggregation iscritical in the formation and stability of B. subtilis biofilms, andtherefore, the principals that were demonstrated in our in vitrofindings could serve as the basis for new antibiofilm solutionsthat target the TasA−membrane interactions.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bio-chem.8b00002.

Dot blot assay of TasA and membranes at pH 7.4 and 2.5(Figure S1); absorbance at 280 nm (OD280) of TasA inthe supernatant and pellet after acidifying the solution inthe absence and presence of membranes (Table S1); andpKa values of the functional groups in the phospholipidsused in this study (Table S2) (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: Liraz.chai@mail.huji.ac.il. Telephone: +972-2-6585305. Fax: +972-2-5660425.*E-mail: razj@bgu.ac.il. Telephone: +972-52-6839384. Fax:+972-8-647-2943.ORCIDRaz Jelinek: 0000-0002-0336-1384Liraz Chai: 0000-0001-9644-4455Author Contributions§These authors contributed equally. The manuscript waswritten through contributions of all authors. All authors havegiven approval to the final version of the manuscript.FundingThis work was funded by an ISF grant to L.C. (1150/14).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Dr. Y. Kalisman and Dr. A. Upcher for their helpwith TEM. We are grateful to M. Yellen for help with confocalmicroscopy.

■ ABBREVIATIONSECM, extracellular matrix; cryo-TEM, cryogenic transmissionelectron microscopy; CD, circular dichroism; FTIR, Fouriertransform infrared; TMA-DPH, trimethylammonium-diphenyl-hexatriene; DPH, 1,6-diphenylhexatriene; SUVs, small uni-lamellar vesicles; GUVs, giant unilamellar vesicles; Asp, asparticacid; Glu, glutamic acid

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