Effects of Various Flavonoids on the α-Synuclein Fibrillation Process

α-Synuclein aggregation and fibrillation are closely associated with the formation of Lewy bodies in neurons and are implicated in the causative pathogenesis of Parkinson's disease and other synucleinopathies. Currently, there is no approved therapeutic agent directed toward preventing the protein aggregation, which has been recently shown to have a key role in the cytotoxic nature of amyloidogenic proteins. Flavonoids, known as plant pigments, belong to a broad family of polyphenolic compounds. Over 4,000 flavonoids have been identified from various plants and foodstuffs derived from plants and have been demonstrated as potential neuroprotective agents. In this study 48 flavonoids belonging to several classes with structures differing in the position of double bonds and ring substituents were tested for their ability to inhibit the fibrillation of α-synuclein in vitro. A variety of flavonoids inhibited α-synuclein fibrillation, and most of the strong inhibitory flavonoids were also found to disaggregate preformed fibrils.

Introduction
Parkinson's disease (PD) is a chronic progressive disease, characterized clinically by resting tremor, bradykinesia (slowness in initiating movements), and rigidity. As only a small number of cases are diagnosed before the age of 50, PD is typically considered an aging disease, with a prevalence of approximately 2% after the age of 65 [1]. The cause of this neurodegenerative disease is still mysterious, although considerable evidence suggests a multifactorial etiology involving genetic susceptibility and environmental factors.
In PD, neuronal death is localized to dopaminergic neurons in the substantia nigra region of the brain stem and precedes appearance of symptoms. It is believed that ca. 70% of neurons may have died by the time symptoms become apparent [2]. Diagnosis can only be definitively confirmed post mortem, by histopathological examination for the pigmented neuron loss and the presence of Lewy bodies (LBs) and Lewy neuritis (LNs) [3]. LBs are spherical protein inclusions found in the cytoplasm of surviving nigral neurons consisting of a dense core surrounded by a halo of radiating fibrils of α-synuclein, the major component of LBs [4]. A variety of other proteins have also been identified in LBs [5,6]. However, both the mechanism underlying the formation of LBs and their pathogenic relevance are still unclear [7,8].
Substantial evidence suggests that the aggregation of αsynuclein is a critical step in the etiology of PD [9][10][11]. The following observations are among the most compelling for the involvement of α-synuclein and its aggregation in PD: (i) fibrils of α-synuclein are observed in LBs and LNs, the characteristic hallmarks of the PD pathology [4,12,13]; (ii) three missense mutations, A53T, A30P, and E46K in αsynuclein, lead to familial early onset Parkinson's disease [14][15][16]; A53T and A30P were known to increase the aggregation propensity of α-synuclein [17,18]; (iii) genetic studies have shown that several cases of familial early onset PD are caused by overexpression of α-synuclein due to duplication or triplication of the α-synuclein gene locus (SNCA) [19,20].

Parkinson's Disease
Several possible mechanisms for the observed cytotoxicity of amyloidogenic assemblies have been suggested. One mechanism suggests a direct effect of amyloidogenic assemblies on cell membrane structure and integrity, either through the pore formation [21][22][23][24][25][26], or via the destabilization of amyloidogenic bundles [27,28].
Currently, no preventive therapy is available for PD. It is believed that compounds that slow and/or prevent aggregation and fibrillation of α-synuclein could represent potential drug leads for PD prevention. Therefore, many of the latest therapeutic strategies are aimed at inhibiting fibril formation and at promoting fibril clearance. They include the use of antibodies, synthetic peptides, molecular chaperones, and chemical compounds. In the past few years small organic molecules, especially certain polyphenolic compounds, have been extensively tested for their ability to inhibit fibril formation in vitro, particularly in relation to Aβ deposition [29], the formation of protease-resistant forms of the prion protein [30], the aggregation of huntingtin [31], and the heparin-induced formation of tau filaments [32,33]. An analysis of these reports shows that certain polyphenols dramatically inhibit cell death in the amyloidogenic cytotoxicity assays.
Less is known about effects of polyphenols and other small organic molecules on α-synuclein fibril formation. Only the antituberculosis drug rifampicin and Chinese herbal drug baicalein from Scutellaria baicalensis have been analyzed and shown to inhibit α-synuclein fibrillation [36][37][38].
Flavonoids have been known as plant pigments for over a century and belong to a broad class of polyphenolic compounds. Over 4,000 flavonoids have been identified from various plants [39] and foodstuffs derived from plants, forming substantial constituents of the human diet. The intake of flavonoids is in the range of 50-800 mg/day, depending on the consumption of vegetables and fruits and of specific beverages, such as red wine, tea, and unfiltered beer [40]. Most of the flavonoids are very potent antioxidants, which has often been associated with their health-related effect. Due to their low redox potentials (0.23 < E7 < 0.75 V) [41], flavonoids (Fl-OH) are thermodynamically able to reduce highly oxidizing free radicals with redox potentials in the range 2.13-1.0 V [42], by hydrogen atom donation: where R • represents superoxide anion, peroxyl, alkoxyl, and hydroxyl radicals [43][44][45]. The aroxyl radical (Fl-O • ) may react with a second radical, acquiring a stable quinone structure (Figure 1(b)). Scavenging of free radicals plays a considerable role in the antioxidant activity of flavonoid compounds [46]. In recent years, flavonoids, being potent free radical scavengers, have attracted a tremendous interest as possible therapeutics against free radical-mediated diseases associated with the oxidative stress [47]. Oxidative stress is an imbalanced state where excessive quantities of reactive oxygen species (ROS), such as superoxide radical anion, hydrogen peroxide, and hydroxyl radical, are present at levels higher than required for normal cell function and overwhelm endogenous antioxidant capacity and repair [48,49]. Oxidative stress has been linked to cancer, aging, atherosclerosis, ischemic injury, inflammation, and neurodegenerative diseases such as Parkinson's and Alzheimer's diseases [50][51][52][53].
As recently reviewed by Pietta [34], several attempts have been made by numerous authors to establish the relationship between flavonoid structure and their radical-scavenging activity [54][55][56][57]. In general, the radical-scavenging activities of flavonoids depend on the molecular structure and the substitution pattern of hydroxyl groups, that is, on the availability of phenolic hydrogens and on the possibility of stabilization of the resulting phenoxyl radicals via hydrogen bonding or by expanded electron delocalization [58][59][60]. Besides the radical scavenging activity, the ability of flavonoids to chelate (bind) metal ions also contributes to their antioxidant activity in vitro. The proposed binding sites for trace metals to flavonoids are basically consistent with the structural requirements for effective radical scavenging, suggesting that metal chelation and antioxidant activity are intimately linked.
Oxidative stress, transition metal accumulation, and inflammation appear to play a role in the pathology of several neurodegenerative diseases, including Parkinson's disease. Accumulated evidence has suggested that dietary flavonoids could be potential neuroprotective agents. For example, the consumption of flavonoid-rich blueberries or strawberries can reverse cognitive and motor behavior deficits in rats [62], and intake of antioxidant flavonoids is associated with a lower incidence of dementia [63]. EGCG, a flavonoid from green tea, has been shown to be neuroprotective in an MPTP-induced animal model of dopaminergic degeneration by Levites et al. [64]. The data from Griffioen et al. also demonstrated that several flavonoids counteract the cytotoxic properties of α-synuclein aggregates directly in a cellular context [65].
The properties of flavonoids such as their abundance in plants and human diet, variety in composition and structure, and antioxidative and neuroprotective biological activities make these compounds very attractive targets while screening small organic molecules that can interact with αsynuclein protein and inhibit its aggregation. These natural chemical compounds can be potentially used as therapeutic drugs in preventing of neurodegeneration in Parkinson's disease. Therefore, we analyzed a flavonoid library containing 48 compounds from several classes for their ability to inhibit α-synuclein fibril formation in vitro. Many flavonoids were shown to inhibit α-synuclein fibrillation and aggregation. Importantly, the majority of the strong inhibitors also disaggregate the preformed fibrils.

Materials.
All flavonoids were obtained from IND-OFINE chemical Co., Inc. Thioflavin T (ThT) was obtained from Sigma-Aldrich. All other chemicals were analytical grade and obtained from Fisher chemicals or VWR scientific.

Expression and Purification of α-Synuclein.
Wild type (WT) human recombinant α-synuclein-containing plasmid pRK172 (obtained from R. Jakes and M. Goedert) was transfected into Escherichia coli BL21 (DE3) cells. Protein was induced and purified as described previously [66]. Protein concentration was determined spectrophotometrically.

Aggregation/Fibrillation Studies and ThT Assays.
Lyophylized α-synuclein was prepared for use by dissolution at high pH (approximately 10.5) in dilute NaOH for 5 to 10 minutes and adjusted to pH 7.4 prior to centrifugation at 95,000 rpm with a Beckman Airfuge ultracentrifuge to remove any aggregated material. A 5-fold dilution with buffer (see text for specific conditions used) was followed with adjustment to the desired pH. Flavonoids were dissolved in DMSO to make a stock solution at a concentration of 5, 10, or 20 mm and to make a final solution of protein and flavonoid in 1% DMSO unless indicated otherwise. All of the protein final solutions consisted of PBS with 0.02% NaN 3 added to avoid bacteria growth.
For plate assays, the protein solution (0.5 mg/mL or 35 μM) was mixed with flavonoids to give a final concentration of 50 μM. Three replicates of each sample were prepared in a 96-well plate with a volume of 120 μL per well and a 3 mm Teflon bead to provide agitation (120 rpm, 20 mm diameter). The assays were performed in a fluorescent plate reader (Labsystems Fluoroskan Ascent CF). Measurements were taken at 30-minute intervals with excitation at 444 nm and emission monitored at 485 nm.
For manual assays, the protein solution (1 mg/mL or 70 μM) was mixed with flavonoids to give a final concentration of 100 μM. Protein samples were stirred at 37 • C with a mini-Teflon stir bar. Aliquots of 5 or 10 μL were removed from the incubated solution and added to 1 mL of 10 μM ThT solutions in 20 mm phosphate buffer (pH 7.4) as a function of time to monitor the fibrillation kinetics. ThT fluorescence was recorded at 482 nm with excitation at 450 nm and slits of 5 nm for both excitation and emission using a FluoroMax-3 spectrofluorometer (Jobin Yvon Horiba).

Electron Microscopy Measurements.
Transmission electron microscopy (TEM) was used to estimate the size and structural morphology of α-synuclein. Aliquots of 5 μL sample were deposited on Formvar-coated 300 mesh copper grids (Ted Pella) and incubated for 5 to 10 minutes. Salts were washed out with distilled water, and samples were dried, negatively stained with 1% (w/v) uranyl acetate, and visualized on a JEOL JEM-100B transmission electron microscopy operated at 80 kV. Typical magnifications ranged from ×75,000 to 300,000. The grids were thoroughly examined to obtain an overall evaluation of the samples. Images were produced with Gattan Digital Micrograph software.

Chromatography: Size Exclusion (SEC) HPLC Measure-
ments. An aliquot of each sample was removed from the α-synuclein incubation at various times, and the insoluble material was removed by centrifugation for 20 minutes at 14,000 rpm. Sample volumes of 40 μL of supernatant were eluted from a TSK-GEL G4000SWXL size exclusion column (7.8 mm inner diameter × 30 cm) in 20 mm phosphate buffer, pH 7.0, and 100 mm Na 2 SO 4 using a Waters 2695 separations module with a Waters 996 photodiode array detector. The HPLC system was controlled, and data were collected and analyzed by Millennium software. The column was eluted at a flow rate of 0.5 mL/min, and the absorbance of the mobile phase was monitored over the wavelength range from 220 to 450 nm with a bandwidth of 1.2 nm. The retention times were calibrated with the following protein molecular mass standards: ribonuclease A (13.6 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (bovine serum) (67 kDa), and aldolase (158 kDa). The void volume was determined with blue dextran 2000 (∼2000 kDa).

Electrospray Ionization Mass Spectrometry (ESI-MS).
Samples for mass spectrometry analysis were desalted with C8 reverse phase column and eluted out with 80% acetonitrile adjusted with TFA to a final pH at 2.0. Injection was carried out via a Harvard Apparatus (Holliston, MA) syringe pump at a flow rate of 20 μL/min. Mass spectra were obtained using Micromass ZMD electrospray mass spectrometer operating in positive ionization mode. The source temperature was set to 80 • C, and the capillary voltage was 3.25 kV. Protein molecular weight was determined from m/z by Masslynx software.

Fibrillation Kinetics of WT α-Synuclein in the Presence of Various Flavonoids.
In this study, 48 flavonoids belonging to several classes with structures differing in ring substituents and in the nature/extent of alkylation have been tested for their ability to inhibit the fibrillation of α-synuclein in vitro. These flavonoids include 18 flavones, 12 flavonols, 4 flavanones, 6 isoflavones, 2 dihydroflavonols, 3 catechins, and 3 anthraquinones. Flavonols and flavones accounted for the majority of the study because of their wide distribution in foods. Table S1 (see Table S1 in Supplementary Materials available online at doi:10.1061/2010/650794.) lists the name, synonym, and structure of the flavonoid from each class. The basic flavonoid structure is the flavan core, which consists of a 15 carbon skeleton (C6-C3-C6), arranged in three rings labeled as A, B, and C in Figure 1(a) [34]. The various classes of flavonoids differ in the level of oxidation and pattern of substitution of the C ring, including flavonols (quercetin and kaempherol), flavanols (the catechins), flavones (apigenin), and isoflavones (genistein). Individual compounds within a class differ in the pattern of substitution of the A and B rings. Figure 2 represents kinetic profiles of α-synuclein fibril formation in the absence (circles) or presence of different flavanoids, as monitored by changes in the ThT fluorescence. Different flavonoids affected the α-synuclein fibrillation to a different extend. Some flavonoids, such as 22-357 and 22-344 ( Figure 2, inverted triangles), slightly inhibited the fibrillation of α-synuclein, increasing the lag time 2-to 3-fold in comparison with that of the control, and were classified as weak (+) inhibitors. Other flavonoids, such as quercetin and G-500 ( Figure 2, squares), significantly inhibited α-synuclein fibrillation, with the lag time 4-to 5-fold longer than that of the control, and were classified as good (++) inhibitors.
In the presence of 6-HP ( Figure 2, diamonds), αsynuclein fibrillation was completely inhibited, as indicated by the negligible changes in the ThT fluorescence over the time course. More importantly, no detectable α-synuclein fibrillation was observed for about 3 days or longer. The inhibitory effect of 6-HP on α-synuclein fibrillation was as good as that from baicalein ( Figure 2), which was reported earlier by Zhu et al. [36]. Besides 6-HP, several other new flavonoids from different classes including eriodictoyl, 22-324, myricetin, EGCG, T-415, 22-340/tricetin, and 22-341, also exhibited strong inhibitory effects on α-synuclein fibrillation. Flavonoids that completely inhibited the fibril formation of α-synuclein under the current experimental conditions were classified as strong inhibitors (designated as +++). The inhibitory activities of each flavonoid analyzed in this study are listed in Table S1 (see Supplementary  Table S1).
In general, the intensity of ThT fluorescence was proportional to the amount of fibril forms. For instance, in the presence of the inhibitory flavonoids, the fluorescence intensity at the end of reaction was in the decreasing order: control (no flavanoid) > 22-257 > quercetin > 6-HP. EM analysis revealed that the detectable amount of fibrils formed at the end of the reaction was decreasing in the same order. In the plate assays, however, most of the tested compounds gave artificially low ThT signal. For example, in the presence of some flavonoids, such as wogonin, diosmetin, and tamarixetin (Figure 3), the ThT fluorescence intensity was much lower than the control, whereas the kinetics of αsynuclein fibrillation still exhibited a typical sigmoidal shape, with the lag time similar to the control. Since the amount of fibrils formed at the end of reaction was found to be similar to that of the control, as indicated by the EM imaging, the lower ThT fluorescence intensity for samples containing these flavonoids suggested that flavanoids either quenched the ThT fluorescence or competed with ThT at the binding site on the protein fibrils. Therefore, no matter how much the flavonoids diminished the ThT fluorescence, as long as they did not prolong the lag time of α-synuclein fibrillation, such as 19-612, chrysoeriol, wogonin, and tamarixetin, and they were all classified as noninhibitors (designated as −).
The quenching/competition was verified by titrations of fibril-ThT complex with various flavonoids. The increasing concentrations of flavonoids from different classes were added to a certain amount of existing fibrils in the presence of 10 μM ThT solution. The intensity of ThT fluorescence in the presence of fibrils decreased as a function of flavanoid added and approached a certain saturation level when the concentration of the flavonoid was around 50 μM ( Figure 4). The decrement degree was dependent on the number and the arrangement of hydroxyl groups for flavonoids from the same class (e.g., flavanones: homoeriodictyol, hesperetin, and eriodicyol) and on the level of saturation of Ring C for flavonoids from different classes but with comparable structures (e.g., flavanones with 2, 3 double bond in Ring C compared with the corresponding flavones).    To avoid interference of flavonoids on ThT fluorescence, manual ThT assays were employed. The final ratio of flavonoid to ThT in the manual assay was lower (about 1 : 10) compared with that in the plate assay; therefore the quenching by flavonoids was almost completely eliminated. Figure 5 compares the results of the plate assay and the manual assay for the fibrillation of α-synuclein in the presence of one of the non-inhibitory flavonoids, tamarixetin. In the plate assay, ThT fluorescence intensity in the presence of 50 μM tamarixetin was much lower (∼10-fold) than that in the absence of the flavonoid, whereas in the manual assay, the addition of 50 μM tamarixetin did not affect ThT fluorescence intensity ( Figure 5). Similar results were obtained when manual ThT assays were performed with other non-inhibitory flavonoids (data not shown). All potential inhibitory flavonoids identified in highthroughput plate-reader assays were tested in subsequent manual assays. In addition, the presence of fibrils was evaluated by EM and quantitatively estimated from SDS PAGE of supernatant fractions (described below).

Analysis of α-Synuclein Species Formed in the Presence of Flavonoids.
During storage or incubation, monomeric αsynuclein tends to self-associate into complexes of various degrees including dimers, trimers, oligomers of various size and shapes, protofibrils, and fibrils. Based on EM, DLS, SEC, and SDS-PAGE, the typical distribution of αsynuclein species after 3 days of incubation was 75%-85% of fibrils, 5%-10% of protofibrils, and 5%-20% of oligomeric species and amorphous aggregates. The noninhibitory flavonoids did not affect the distribution and  morphology of fibrils (Figure 6(a)). Typical fibrils were 10 ± 1 nm in width and up to about 2 μm in length. Flavonoids classified as weak or good inhibitors decreased fibril amounts. Remained fibrils were shorter in length but similar in width (Figure 6(b)). When α-synuclein was incubated with strong inhibitory flavonoids only oligomers and a few protofibrils instead of fibrils were detected (Figure 6(c)). Flavonoid-induced protofibles were 5-10 times shorter in length and twice smaller in width compared with the typical α-synuclein fibrils. Flavonoid-induced oligomers appeared as round-or oval-shaped species with a 10-20 nm diameter. Their morphology was similar to the oligomeric species observed in the absence of flavonoids; however, the detailed structure and assembly could be different. Unfortunately, this is beyond the detection limits of AFM and EM. Inhibitory flavonoids greatly affected the distribution of aggregated αsynuclein species apparently by stabilizing monomeric and oligomeric species.
The distribution and stability of soluble species of αsynuclein were analyzed by SEC and SDS-PAGE. In addition, SDS PAGE was employed to quantitatively estimate the amount of protein in the supernatant and pellet fractions because Lowry and other spectrophotometric methods could not be used due to flavonoid interference with the absorbance. Figure 7(a) shows SDS-PAGE of supernatant fractions of α-synuclein samples incubated in the absence or presence of the strong inhibitory flavonoids. In the control lane, a weak band of monomeric α-synuclein was observed indicating that most of the protein was insoluble. No dimers or oligomers were detected. In the presence of non-inhibitory flavonoids such as H-114 and 22-323, there was also a relatively weak band of monomeric α-synuclein in the gel (Figure 7(b)), similar to that of the control further confirming that these flavonoids did not affect α-synuclein fibrillation. On the other hand in the presence of strong inhibitory flavonoids such as 6-HP and 22-324, essentially all of the protein was present in the supernatant. The monomer band was much intense than that of the control. Importantly, higher molecular weight species were also detected on SDS gel. They correspond to dimers (35 kDa band), trimers (50 kDa band), and oligomers (a band on the top of the gel). The oligomeric species and dimer/trimers were resistant to treatment with SDS at 100 • C for 20 minutes. They were also extremely stable in 8 M urea and 6 M GdmHCl (data not shown). The stability of these oligomers and dimers indicates that they are different from the similar species formed in the absence of flavonoids, which are unstable in the mentioned conditions. The extreme stability can be due to very strong noncovalent interactions and/or covalent binding.
The soluble forms of α-synuclein stabilized by the flavonoids were further analyzed by size exclusion HPLC. The elution profile of α-synuclein control sample showed only one peak at 11.5 mL corresponding to a trace amount of monomers ( Figure 8, black curves). These results were rather unexpected as EM, AFM, and DLS analyses revealed that oligomers constituted the majority of the supernatant fractions (data not shown). This contradiction can be explained by the instability of oligomers under HPLC condition and by their fast dissociation upon dilution during chromatographic process. On the contrary, SEC analysis revealed that soluble fractions of α-synuclein incubated with flavonoids contained significant amounts of oligomers. Oligomeric fractions of αsynuclein were obtained by running FPLC, which required much more protein for analysis and was used only for some flavonoid-protein samples (data not shown). The elution profiles of α-synuclein incubated in the presence of noninhibitory flavonoids such as wogonin and kaempferol were similar to the control profile (Figure 8(b)). Peaks eluted after the monomer peak (13.5 and 14 mL) corresponded to polymerized forms of flavonoids that occurred during incubation [67]. The flavonoid nature of the peaks was experimentally confirmed by the same elution times and absorbance spectra of incubated flavonoid alone and by the absence of protein band on SDS gel for the concentrated fraction sample from SEC-HPLC (data not shown). The elution profiles of α-synuclein incubated in the presence of inhibitory flavonoids had six major peaks. Besides the monomer and flavonoid peaks, peaks corresponding to dimer (11 mL), trimer (10.3 mL), and small (7.8 mL) and large oligomer (6.2 mL) were detected (Figure 8). Because of the flavonoid interference with the protein spectra, the protein nature of the peaks and the amount of protein eluted was analyzed by SDS-PAGE. The increased intensity of the monomer peak was mostly attributed to the elevated amount of monomers and partially to the absorbance of flavonoid tightly bound to the monomer. Absorbance intensity of dimer and oligomer peaks also partially corresponded to the flavonoid absorbance and indicated the stability of flavonoid-protein complexes. The observation of oligomeric species on HPLC confirms that flavonoidinduced oligomers are more stable and thus different from αsynuclein oligomers obtained without flavonoids that easily dissociate and are not detected by HPLC.
Interestingly, Figure 8 also shows that the distribution of oligomeric forms and monomers varied depending on the nature of flavonoid. For example, eriodyctiol mostly  stabilized dimers and monomers, G-500 mostly monomers, and baicalein mostly oligomers and monomers.
All inhibitory flavonoids affected α-synuclein aggregation and lead to the inhibition of fibril formation, accumulation of oligomeric species, and stabilization of monomers and oligomers. Therefore, at the next stage, we analyzed the conformational properties of flavonoid-stabilized αsynuclein species. To this end, attenuated total reflectance FTIR was applied to detect the secondary structure of flavonoid-stabilized soluble species of α-synuclein. The FTIR spectrum of fresh α-synuclein with baicalein exhibits a typical unfolded structure, similar to fresh α-synuclein alone, with the peak centered at 1652 cm −1 (Figure 9, solid lines). After 3 days of incubation, the spectrum of the protein alone changed into a structure with significant β-sheet, with the peak at 1630 cm −1 , indicative of fibril formation (Figure 9, black lines), whereas the spectrum of the protein with the compound did not exhibit significant structural changes ( Figure 10, red lines), reflecting that baicalein inhibits αsynuclein fibrillation by stabilizing the natively unfolded conformation of the protein.

Analysis of Flavonoid Inhibitory Effects on Various Stages of
Fibrillogenesis. The inhibition of α-synuclein fibrillogenesis by flavonoids may involve two different events, the inhibition of nucleus formation occurring during lag time or the inhibition of fibril elongation corresponding to the linear increase in the ThT fluorescence intensity. To resolve it, one of the strong inhibitory flavonoids, 6-HP, was added at various times during incubation of α-synuclein. As shown on Figure 10 of elongation, sample L3). Addition of 6-HP at any time during nucleation resulted in the complete inhibition of fibril formation. This indicated that 6-HP could act on the late steps of nucleation such as formation of oligomers. When 6-HP was added during linear growth of fibrils or at the end of fibrillation, the decrease in ThT intensity and disaggregation of formed fibrils were observed. The initial drop was mostly due to the quenching of ThT fluorescence by the flavonoid, and the subsequent decrease in ThT signal was due to disaggregation of the existing fibrils. This was confirmed by EM and SDS-PAGE. The rates of disaggregation were similar and did not depend on time of flavonoid addition. It was consistent with explanation that the fibrillar species were similar in structure and mechanism of disaggregation was the same.
SDS-PAGE of α-synuclein supernatant fractions with 6-HP added at various times of incubation was run to verify the results of ThT assays (Figure 10(b)). SDS-PAGE analysis demonstrated the considerable build-up of monomers, dimers, trimers, and higher MW oligomers as long as the addition of 6-HP was prior to nucleation (samples L0, L1). This indicated that the most efficient inhibition took place during, or prior to, nucleation. When 6-HP was added after the nucleation in the middle of or at the end of fibril elongation (L2 and L3), less amount of monomers and oligomers was detectable.
Similar results were observed for α-synuclein with other strong inhibitory flavonoids such as baicalein, EGCG, and T-415. It can be concluded that the inhibitory flavonoids stopped the progress of fibrillation at any stage. Importantly, the addition of flavonoids to the preformed fibrils resulted in almost complete fibril disaggregation. However, the most efficient inhibition of α-synuclein fibrillation by the flavonoids took place on or prior to nucleation through formation of the flavonoids-stabilized monomeric and oligomeric forms of the protein.

Structural Features of α-Synuclein Fibrillation Inhibitors.
Our results showed that a variety of flavonoids can inhibit the fibril formation of α-synuclein. Some flavonoids can also disaggregate existing fibrils. Among the 48 flavonoids tested, several compounds exhibited stronger inhibitory effects than others despite the overall structural similarity of the flavanoid family. The aim of this study was to eventually elucidate the relationship between the molecular structure of a series of structurally related flavonoids and their ability to inhibit α-synuclein fibrillation.
The molecular structural requirements that appear necessary to provide a flavonoid the ability to inhibit α-synuclein fibrillation were determined to be a vicinal dihydroxyphenyl moiety. In another words, flavonoids without any vicinal dihydroxyphenyl moieties have no inhibitory effect on αsynuclein fibrillation.
The substitution pattern, that is, the ring to locate in and the position on the ring, of the dihydroxyl groups could also affect the inhibitory activity but not necessarily. For example, comparisons were made among the following 5 flavones (Figure 11(a)). The 2 flavones, D-258 with the 3 ,4 -dihyhroxy substitution pattern on ring B, and D-406 with the 2 ,3 -dihyhroxyl group in ring B, have a similarly good inhibitory effect on α-synuclein fibrillation. Rather than the o-dihydroxy structure in the B ring, the other 2 flavones, D-112 and 22-357, have hydroxyl substituents in a catechol structure on the A ring at different positions. The 2 compounds also have a similar inhibitory effect on αsynuclein fibrillation, but weaker than the effect from D-258 and D-406. In contrast to the above 4 flavones, D-407 does not have a vicinal dihydroxyphenyl moiety, nor does it have any inhibitory effect on α-synuclein fibrillation (see activity in Supplementary Table S1).
Moreover, the difference in the number of vicinal dihydroxyl substituents and the number of individual hydroxyl groups also brings some difference in the inhibitory activities Flavonoids with three vicinal hydroxyl groups exhibited the enhanced inhibitory effects on α-synuclein fibrillation (structures in Figure 12). For example, 6-HP exhibited stronger inhibitory activity than 22-357 ( Figure 2); myricetin was stronger than quercetin, and tricetin was stronger than luteolin (data not shown). Interestingly, all the flavonoids (except 021037) with three vicinal hydroxyl groups from our screening were found to be strong inhibitors, that is, completely inhibited the fibril formation within 3 days of incubation (Figure 13(a)). The essentially weaker inhibitory effects of 021037 could result from the lack of 2,3-double bond in this flavonoid. On the other hand, only three flavonoids without three vicinal hydroxyl groups from this screening were found to exhibit strong inhibitory effect on α-synuclein fibrillation. They were as follows: T-415 an isoflavonoid, eriodictyol a flavanone, and 22-324 a flavone (Figure 13(b)).
Inversely, the flavonoids with structures exactly the same or very similar (the number and position of ring substitutions) to those of strong inhibitors, but with hydroxyl groups replaced by methoxyl groups, did not have any inhibitory effects on α-synuclein fibrillation, as exemplified by the following pairs of flavonoids H-114 versus 6-HP and 22-324 versus 22-323 ( Figure 14). The details of the molecular mechanisms underlying the flavanoid-induced inhibition of α-synuclein fibrillation were analyzed recently [68]. It was shown that the noncovalent binding of the inhibitory flavonoids to α-synuclein and the covalent modification α-synuclein (monomers and oligomers). All of these factors rather than a single one contribute to the inhibition of αsynuclein fibrillation induced by the flavonoid. The structural requirements for flavonoid to be a good inhibitor of αsynuclein fibrillation were vicinal dihydroxyphenyl moieties, irrespectively of their positions in the rings. Flavonoids with three vicinal hydroxyl groups exhibited enhanced inhibitory effects on α-synuclein fibrillation. The antioxidant activities of flavonoids were generally correlated with their in vitro inhibitory effects on α-synuclein fibrillation [68].

Implications for the Treatment of PD.
PD has a multifactorial etiology depending on genetic susceptibility and environmental factors. From epidemiological and environmental studies, oxidative stress, transition metal accumulation, and inflammation appear to play a role in the pathology of PD. However, the detailed molecular mechanisms of triggering the oxidative stress as well as targets of transition metal action are not known. The cause of inflammation is also unknown. From genetic etiological studies, α-synuclein misfolding and aggregation as well as impairment in the cellular folding machinery appear to be important in the early onset of PD. However, functions of α-synuclein, a cause of protein aggregation, and its role in the triggering of neurodegeneration are still unknown. Recently, it has been proposed that αsynuclein may interact with mitochondrial membranes and affects their structures. This interesting idea may consolidate genetic and environmental studies of PD etiology. α-Synuclein could be a key factor, upon which oxidative stress or transition metals act resulting in protein aggregation or oligomerization that in turn affect mitochondrial membranes leading to mitochondrial dysfunctions including elevated ROS production, leakage of cellular membrane compartments, cell death, and inflammation. Therefore, the prevention of protein aggregation and oligomerization is an attractive strategy in combating the neurodegeneration   because it acts on the very beginning of the proposed cellular pathway leading to cell death. In our study, we analyzed 48 flavonoids for their efficiency to inhibit αsynuclein aggregation in vitro by stabilizing nonpathogenic protein conformations. Majority of the flavonoids inhibit αsynuclein polymerization either delaying or completely abolishing fibril formation. The thermodynamic distributions of α-synuclein species are tremendously altered in the presence of strong flavonoid inhibitors. Flavonoids such as 6-HP, eriodictyol, G-500, and baicalein tightly bind to the protein and greatly stabilize its natively unfolded conformation. The major species existing in the thermodynamic equilibrium in the presence of flavonoids are oligomers and monomers. Flavonoid compounds show different ratios in distribution of monomers and oligomers indicating that they stabilize mostly either monomeric or oligomeric conformations. Most importantly, strong inhibitors disaggregate preformed fibrils. Substantial evidence reveals that flavonoids act in vivo as health-beneficial and neuroprotective agents. For example, baicalein, a typical flavonoid compound, is the main component of a traditional Chinese herbal medicine Scutellaria baicalensis. Recent studies have shown that baicalein protects rat cortical neurons from amyloid β-induced toxicity by its inhibition of lipoxygenase [69]. Natural medicines containing these compounds have been reported to have beneficial effects in treating memory loss and dementia [70,71]. Another flavonoid, (−)-epigallocatechin-3-gallate (EGCG), has been shown neuroprotective in an MPTPinduced animal model of dopaminergic degeneration [64]. Their data from the yeast model also demonstrated that flavonoids counteract the cytotoxic properties of α-synuclein toxicity directly in a cellular context [65]. Epidemiological studies suggest an inverse relation between flavonoid intake and incidence of chronic diseases, such as coronary heart disease [72][73][74], as well as dementia and other neurodegenerative diseases [63,70].
However, little is currently known about the mechanism of flavonoid action in vivo. There are two models that explain the flavonoid health beneficial effects. First model suggests that the antioxidant properties of flavonoids are important in combating oxidative stress by scavenging radicals and by metal chelation. However, even with very high flavonoid intakes, plasma and intracellular flavonoid concentrations in humans are likely to be 100-1,000 times lower than concentrations of other antioxidants, such as ascorbate or glutathione. Furthermore, most circulating flavonoids are actually flavonoid metabolites, some of which have lower antioxidant activity than the parent flavonoid. For these reasons, the relative contribution of dietary flavonoids to plasma and tissue antioxidant function is most likely to be relatively minor [75,76].
The second model postulates that many of the biological effects of flavonoids can be related to their ability to modulate cell-signaling pathways rather than to act as antioxidants. For instance, several signaling mechanisms were suggested to explain polyphenol-induced protection against amyloid cytotoxicity [77] and the neuroprotective effects of flavonoids [78]. The validity of the hypothesis is supported by the facts that intracellular concentrations of flavonoids required to affect cell signaling pathways are considerably lower than those required to impact cellular antioxidant capacity, and flavonoid metabolites may still retain their ability to interact with cell signaling proteins, even if their antioxidant activity is diminished.
Our results have demonstrated that flavonoids significantly affect α-synuclein propensity to aggregate in vitro through binding and stabilizing the certain protein conformations. This direct act of flavonoids on protein aggregation is a highly possible mechanism of flavonoid action in vivo. The apparent K d of flavonoid-α-synuclein complexes is in range of 200-800 nm, which is sufficient for binding of flavonoids in vivo [68]. Furthermore, there is a certain correlation between antioxidant activities of flavonoids and their in vitro inhibitory effects on α-synuclein fibrillation; thus it is possible that the antioxidant potential of flavonoids may relate not only to the scavenging of radicals and metals but also to binding to amyloidogenic proteins such as α-synuclein, locking the certain protein structures, and abolishing protein aggregation.
It has been suggested that the precursors of fibrils, the transient oligomers, and protofibrils on the aggregation pathway in neurodegenerative diseases might be more toxic than fibrils [21,22]. Hence, it can be argued that the flavonoid-induced oligomers from our study could be more cytotoxic than fibrils. Several studies have been done to test the cytotoxicity of one of the flavonoid-synuclein complexes, the baicalein-stabilized oligomers, and have demonstrated their benign effects on membrane permeability [38], cell culture toxicity [79], and neuron degeneration in worms and mice. Masuda et al. have reported that, unlike α-synuclein fibrils and protofibrils, flavonoid-induced soluble oligomeric species did not reduce the viability of human dopaminergic neuroblastoma SH-SY5Y cells [79]. These findings suggest that the soluble oligomers formed in the presence of inhibitory flavonoids may not be toxic to human neurons and that these flavonoids may therefore have therapeutic potential for PD and other amyloidoses.
Flavonoids are among the most secure and reliable substances available now for preventing various diseases including maladies related to the oxidative-stress and neurodegeneration. In this study, we have demonstrated that flavonoids can directly act on aggregation of α-synuclein, being able to inhibit fibrillation and disaggregate the preformed fibrils into monomers and nonpathogenic oligomers. This implies that flavonoids may have been already combating PD, and that diets rich in flavonoids may be effective in preventing and curing the disorder.

Supporting Information Available
Experimental details of the inhibitory activities of each flavonoid analyzed in this study are available; the figures illustrate instant changes in the intensity of ThT fluorescence upon adding 50 μM flavonoids directly to the existing αsynuclein fibril solution. This material is available free of charge via the Internet at http://pubs.acs.org.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Lipoprotein biosynthesis as a target for anti-Wolbachia treatment of filarial nematodes

Background Lymphatic filariasis and onchocerciasis are debilitating diseases caused by filarial nematodes. Disease pathogenesis is induced by inflammatory responses following the death of the parasite. Wolbachia endosymbionts of filariae are potent inducers of innate and adaptive inflammation and bacterial lipoproteins have been identified as the ligands that bind toll-like receptors (TLR) 2 and TLR6. Lipoproteins are important structural and functional components of bacteria and therefore enzymes involved in Wolbachia lipoprotein biosynthesis are potential chemotherapeutic targets. Results Globomycin, a signal peptidase II (LspA) inhibitor, has activity against Gram-negative bacteria and a putative lspA gene has been identified from the Wolbachia genome of Brugia malayi (wBm). The amino acids required for function are strictly conserved and functionality was verified by complementation tests in a temperature-sensitive Escherichia coli lspA mutant. Also, transformation of wild type E. coli with Wolbachia lspA conferred significant globomycin resistance. A cell-based screen has been developed utilizing a Wolbachia-containing Aedes albopictus cell line to assay novel compounds active against Wolbachia. Globomycin was screened using this assay, which resulted in a dose-dependent reduction in Wolbachia load. Furthermore, globomycin was also effective in reducing the motility and viability of adult B. malayi in vitro. Conclusions These studies validate lipoprotein biosynthesis as a target in an organism for which no genetic tools are available. Further studies to evaluate drugs targeting this pathway are underway as part of the A-WOL drug discovery and development program.

Background
Lymphatic filariasis and onchocerciasis are leading causes of global morbidity, with 150 million people afflicted and 1.5 billion people at risk. The filarial nematodes responsible for these diseases; Wuchereria bancrofti, Brugia malayi (lymphatic filariasis) and Onchocerca volvulus (onchocerciasis), have a mutualistic association with a bacterial endosymbiont, Wolbachia pipientis. The symbiotic relationship is essential for parasite growth, development, fecundity and survival [1].
Wolbachia have emerged as a novel target for antibiotic therapy to treat filariasis. Antibiotic studies using ex vivo and in vivo animal model systems (reviewed by [1]) and extensive field trials have demonstrated the effectiveness of antibiotics such as doxycycline in the treatment of filariasis [2][3][4][5][6][7][8][9][10][11][12][13][14]. Depletion of Wolbachia leads to longterm sterility and ultimately to the death of adult worms. Furthermore, Wolbachia-targeted treatment has also been shown to lead to a reduction in the severity and improvement of lymphoedema and hydrocoele pathology in lymphatic filariasis [3,5]. Despite these clear benefits of doxycycline therapy over conventional treatments, the extended period of treatment and contraindication in children under eight and pregnancy compromises their delivery through mass drug treatment programmes. This has driven the formation of the A-WOL (anti-Wolbachia) consortium to discover and develop new drugs active against Wolbachia for the treatment of filariasis that would be compatible with current control programme strategies.
Wolbachia lipoproteins have emerged as potent stimulators of the inflammatory pathogenesis of filarial disease [15]. Genomic analysis indicates that Wolbachia contain the lipoprotein biosynthesis genes lgt and lspA but not lnt, N-acyltransferase, which is required for the triacylation of apolipoproteins. This suggests that Wolbachia lipoproteins cannot be triacylated and accounts for the recognition by the diacyl-lipoprotein receptor complex TLR2/6 [15]. Bioinformatic and database searches consistently predicted the presence of only three lipoproteins in Wolbachia: Peptidoglycan-associated lipoprotein (PAL), a Type IV Secretion System protein (VirB6) and Small protein A [15].
Lipoproteins are important structural and functional components of bacteria and their biosynthesis is essential for bacterial viability. Globomycin, an inhibitor of lipoprotein signal peptidase, LspA [16], has previously been demonstrated to have potent anti-bacterial activity against Gram-negative bacteria [17][18][19][20]. We therefore sought to test whether globomycin was active against Wolbachia and to validate lipoprotein biosynthesis as a Wolbachia drug target.

Identification and verification of a functional wBm lspA gene
Putative lspA genes were identified from the available genome databases of wBm and Wolbachia endosymbionts of Drosophila species. The deduced amino acid sequence of the putative Wolbachia (including wBm) LspA proteins contain a predicted signal peptide with three transmembrane domains, which are evolutionarily conserved features in LspA proteins (Figure 1). High sequence identity (80-81%)/similarity (88%) was observed between B. malayi Wolbachia LspA and Drosophila Wolbachia LspA homologs, all containing the five conserved catalytic residues [21], although a low amino acid identity (25%)/similarity (49%) was revealed when compared to the Escherichia coli LspA homolog ( Figure 1). The conservation of the amino acids required for function suggests that the wBm LspA gene should be functional.
wBm LspA recombinant clone is active in a complementation test To confirm activity of LspA, the recombinant clone was tested using a complementation assay in an E. coli strain with a ts LspA mutant. The point mutation in the lspA gene of E. coli Y815 strain confers temperature sensitivity to cell growth. At the non-permissive temperature (42°C), cell growth is hindered by the accumulation of prolipoproteins due to inactivation of LspA enzyme [22]. The plasmids pET21-EcLspA.His (PC, positive control), pET21a-wBmLspA.His (T1: test 1), pGEX5.1-GST. wBmLspA.His (T2, test 2), pET21a (NC1, negative control 1) and pGEX5.1 (NC2, negative control 2) were transformed into E. coli strain Y815 for functional complementation tests of growth. NC1, NC2 and T2 transformants did not grow at 42°C, while both PC and T1 grew and formed visual colonies at the non-permissive temperature, confirming the wBm lspA gene (T1) is a functional LspA enzyme (Table 1).
Globomycin resistance assays confirm wBmLspA activity Globomycin resistance assays in E. coli were conducted to further test the function of the wBm lspA gene. LspA inhibition by globomycin leads to the accumulation of unprocessed prolipoproteins in the inner cell membrane and thus hinders cell growth [16,23,24], while the inhibition can be overcome by over-expression of a functional lspA gene from other bacterial sources, which confers globomycin resistance [21,[25][26][27]. To confirm this with the wBm lspA gene constructs, the plasmids NC1, PC, T1, NC2 and T2 in T7 Express E. coli were tested in globomycin resistance assays. With one exception, in the group without IPTG induction, all transformants grew well in the absence of globomycin and showed arrested growth when treated with 100 μg/ml globomycin. The exception was the PC transformant (E. coli transformed with pET21a-EcLspA.His), which conferred globomycin resistance in the absence of induction by IPTG ( Figure 2). The significant "leaky" expression of EcLspA.His was also detected by western blot analysis ( Figure 3). In the IPTG induction set, the cell growth in both PC and T2 transformants (E. coli transformed with pGEX5.1-GST.wBmLspA.His) was dramatically inhibited even without addition of globomycin, suggesting that a relatively high level of overexpression of EcLspA.His and GST.wBmLspA.His proteins alone could hinder normal cell growth. wBmLspA.His (T1) was expressed when induced with IPTG, but did not lead to the significant growth inhibition observed for PC and T2 transformants. In the IPTG induction group, when treated with 100 μg/ml globomycin, E. coli cells with overexpressed EcLspA.His (PC), wBmLspA.His (T1) or GST. wBmLspA.His (T2) conferred strong globomycin resistance compared to negative controls (NC1 and NC2) ( Figure 3). A synthetic wBm lspA gene was created for improving gene expression level in both E. coli Y815 and T7 Express E. coli. However, no detectable improvement was observed in both E. coli cell strains (data not shown).

Globomycin affects Wolbachia growth in a cell-based assay
We investigated whether globomycin affected Wolbachia growth in a cell-based assay using Wolbachia-infected insect cells (C6/36Wp; [28]). Cultures were treated with globomycin in a 96-well plate format and Wolbachia growth was determined by qPCR targeting the Wolbachia 16S rRNA gene. As shown in Figure 4A, Wolbachia 16S copy numbers were significantly reduced when compared to the DMSO (1%) control (P < 0.01). This reduction was dose-dependent and equated to log reductions of 1.33, 1.75 and 1.97 for 50, 80 and 100 μg/ml concentrations respectively at day 10 after initiation of treatment, which increased to between 1.88 and 2.48 by day 16. The reduction in 16S gene copy numbers achieved with globomycin at 20 μg/ml, although significant (P < 0.05), did not extend beyond a 0.55 log-drop throughout the test period. A. albopictus 18S rRNA gene copy numbers were also analyzed by qPCR in order to check for any effect of globomycin on cell growth ( Figure 4B) and to normalize the 16S data by producing ratios of 16S to 18S gene copy numbers ( Figure 4C). Globomycin had no significant effect on cell growth at concentrations up to and including 80 μg/ml ( Figure 4B), although 100 μg/ml globomycin did have a small but significant effect on cell growth (P < 0.05). The normalization of 16S copy numbers to 18S copy numbers did not affect the overall results ( Figure 4C).

Globomycin affects lipoprotein processing
As globomycin targets the enzyme signal peptidase II in the lipoprotein biosynthesis pathway [16], any inhibition that occurs should prevent the cleavage of the signal peptide and thus result in an accumulation of the prolipoprotein. Cells treated with globomycin were used to assess the effect of this drug on lipoprotein processing in Wolbachia. A western blot using antibody raised against recombinant wBm PAL was conducted on lysates of cells treated with globomycin or vehicle-control for 24 hours. As shown in Figure 5, globomycin treatment inhibited processing of the precursor of the Wolbachia lipoprotein PAL in a dose-dependent manner. When compared to  Actively growing transformants were plated onto LB agar (100 μg/ml ampicillin, 10 μg/ml tetracycline and 0.6 mM IPTG) and incubated at both the permissive (30°C) and the nonpermissive (42°C) temperature for 3 days for colony formation as described [21]. '+' means growth, '-' means no growth.
the vehicle control which shows a band of approximately 15 kDa representing the mature lipoprotein, the globomycin treated cells also showed an additional larger band of approximately 18 kDa. This suggests that the target of globomycin in Wolbachia is LspA and indicates that the resulting inhibition of growth and death of bacteria is due to the accumulation of prolipoprotein in the cytoplasmic membrane [16,23,24].
The effect of globomycin on the motility and viability of B. malayi in vitro The effect of this compound on nematode motility was examined by incubating B. malayi adult females with globomycin in vitro. Motility was assessed daily using a method described by Rao and Weil [29]. Over the test period of 10 days, globomycin reduced motility in a dose-dependent manner ( Figure 6A). The highest concentration of globomycin (100 μg/ml) rendered the majority of worms inactive by day 3. Globomycin used at a concentration of 50 μg/ml was also found to render the majority of the nematodes immotile by day 7 ( Figure 6A). A similar dose dependent reduction in motility was observed in male worms (data not shown). At the end of the motility test period, worms were collected for use in an MTT assay to assess viability. Figure 6B shows reduced viability of those exposed to the higher concentrations of globomycin, which at a concentration of 100 μg/ml globomycin was significantly reduced compared to vehicle-treated controls at 10 days post-treatment (P < 0.05). Furthermore, the MTT result for this treated group was not significantly different from that obtained from dead worm controls (P = 0. 29) suggesting that globomycin at this concentration is able to kill adult B. malayi in vitro.

Discussion
One of the objectives of the A-WOL consortium is to identify and validate novel drug targets of Wolbachia to  develop high throughput target based drug screening assays. Validating a potential drug target in Wolbachia, however, is problematic as these bacteria are not amenable to genetic manipulation. We have therefore used complementation assays of mutant E. coli to validate enzymatic function and a pharmacological inhibitor of lipoprotein biosynthesis, globomycin, to determine the effect of inhibition of lipoprotein biosynthesis on Wolbachia in a whole organism cell-based drug screening assay.
wBm contains two genes encoding enzymes of the lipoprotein biosynthesis pathway [15,30]: lgt, prolipoprotein diacylglyceryl transferase and lspA, lipoprotein signal peptidase. The third enzyme in the pathway responsible for triacylation, typically found in other bacteria, appears to be absent from Wolbachia. Although Wolbachia is intractable to genetic manipulation we were able to confirm that Wolbachia LspA is functional in E. coli, despite having only 25% identity and 49% amino acid similarity to its E. coli homolog. wBm LspA has retained the conserved catalytic residues required for function and can genetically complement deficient growth of an lspA ts E. coli mutant. Globomycin is a known inhibitor of LspA and overexpression of Wolbachia LspA in wild-type E. coli conferred resistance to this compound allowing us to conclude that the expressed gene was functional.
We used a ts E. coli LspA mutant to confirm the functionality of the wBmLspA gene ( Table 1). As expected, the N-terminal GST-fused full-length wBmLspA.His (T2) cannot rescue the ts mutant (Table 1); likely due to the loss of signal peptide at its N-terminus, indicating that proper subcellular localization of LspA is crucial for its functional activity. However, the misplaced GST. wBmLspA.His still conferred strong globomycin resistance, although its overexpression appeared to be toxic to the E. coli cell even without addition of globomycin ( Figure 2). This may imply that the mis-localized GST. Figure 4 Dynamics of Wolbachia and cell response to globomycin over 16 days. Wolbachia growth was measured by qPCR targeting the 16S rRNA gene (A), C6/36Wp cell growth was measured by targeting the 18S rRNA gene (B) and data was normalized by calculating the ratio of 16S copies to 18S copies (C). Error bars represent standard deviations from triplicate cultures. wBmLspA.His (T2) still maintains a structural specificity for globomycin binding. However, after removal of the signal peptide, GST-fused mature wBmLspA.His totally lost resistance to globomycin (data not shown).
The leaky expression of EcLspA.His (PC) conferred strong resistance to globomycin without significantly reducing E. coli cell growth, while its excessive overexpression, induced by IPTG, although still conferring globomycin resistance, led to striking cell growth inhibition ( Figure 2). This might suggest that the expression of LspA is required and at the same time strictly regulated for achieving normal cell growth, which is in line with the fact that codon-optimized wBm LspA gene did not improve its expression in E. coli.
Lipoproteins perform multiple essential structural and functional roles in bacteria. For example, the lipoprotein PAL is not only involved in the maintenance of outer membrane integrity [31] but has also been demonstrated to be involved in the uptake of nutrients across the membrane of Pseudomonas putida [32] and required for virulence in Haemophilus ducreyi infection of humans [33]. Therefore the inhibition of lipoprotein biosynthesis is likely to affect multiple functions in bacteria. Globomycin is a 19-membered depsipeptide antibiotic isolated from actinomycetes strains [18][19][20], which targets LspA.
Although not commercially available for use as an antibiotic, it has been widely used as a research tool to determine the requirement of lipoprotein biosynthesis for a range of biological features of bacteria [34,35]. In addition to using globomycin to assess the functionality of the Wolbachia LspA enzyme in transformed E. coli, we used this compound to assess the importance of the lipoprotein biosynthesis pathway in a Wolbachia cell line (C6/36Wp) and in B. malayi adult nematodes cultured in vitro.
The C6/36Wp system is currently the primary screen used in the A-WOL drug discovery program for Wolbachia http://www.a-wol.com, and aims to discover novel compounds with anti-Wolbachia activity. In this study, globomycin was shown to have a dose-dependent anti-Wolbachia effect on the C6/36Wp cell line, demonstrating a new class of antibiotic active against Wolbachia.
Globomycin acts by an inhibition of a prolipoprotein processing enzyme, resulting in an accumulation of the prolipoprotein in the cell envelope [16,23,24] and this inhibition occurs through the non-competitive binding of globomycin to the lipoprotein signal peptidase and not by binding to the substrate [16]. Treatment of Wolbachia cell cultures with globomycin resulted in the accumulation of prolipoprotein suggesting a similar mode of action occurs in Wolbachia. Furthermore, globomycin affected both the processing of OmpA in Ehrlichia chaffeensis, an obligate intracellular bacterium closely related to Wolbachia pipientis, and its ability to infect HL-60 cells [35].
Globomycin was also shown to have adverse effects on the motility and viability of adult female and male B. malayi nematodes in vitro. QPCR analysis did not, however, demonstrate any differences in Wolbachia load between treated groups (data not shown) suggesting that the Wolbachia qPCR assay may not have sufficient sensitivity to detect effects on Wolbachia load over this time frame in nematodes, perhaps due to the slower growth rate of nematode Wolbachia compared to insect Wolbachia in cell culture. Notably doxycycline also fails to produce a reduction in Wolbachia load over this time frame. An alternative explanation is that inhibition of Wolbachia lipoprotein biosynthesis is sufficient to affect nematode motility and viability independent of or prior to any effect on Wolbachia load. We also cannot rule out a direct effect of globomycin on nematode motility and viability and alternative mechanisms of action have been suggested in Mycobacterium tuberculosis [36], Figure 6 The effect of globomycin on B. malayi motility and viability. The motility of worms cultured in the presence of globomycin was scored daily and is presented as percentages of the control (1% DMSO) motility (A). At the end of the test period worms were used in an MTT assay to assess effects on nematode viability (B). Remaining worms were used for qPCR analysis of Wolbachia numbers per worm (data not shown).
where globomycin was found to be active against this bacterium independently of its effect on LspA.

Conclusions
In summary, in the search for new anti-wolbachial drugs, we have identified and validated the lipoprotein biosynthesis pathway as a target for Wolbachia drug discovery and development and shown that globomycin, a drug targeting lipoprotein signal peptidase, is active against Wolbachia.

Globomycin
Globomycin was obtained as a kind gift from Professor Masatoshi Inukai of International University of Health and Welfare, Tochigi, Japan. A stock solution of 10 mg/ ml was made in dimethylsulphoxide (DMSO) and stored in single-use aliquots at -80°C. Before use, the stock solution was diluted in the appropriate culture medium to the appropriate concentrations.
LspA gene cloning B. malayi DNA (including Wolbachia DNA) was extracted with DNeasy extraction kit (Qiagen) from live adult worms purchased from TRS Labs, Athens, GA. Primers were designed with restriction endonuclease sites (Additional File 1; Table S1), based on the available wBm and E. coli lspA gene sequences (Genbank accession numbers AE017321 and NC_000913, respectively). The full-length lspA genes from B. malayi Wolbachia (wBm) and E. coli were amplified by PCR using Phusion polymerase (New England Biolabs, NEB), and were cloned into the expression vector pET21a+ with a C-terminal 6XHis tag (Novagen) after digestion with corresponding restriction endonucleases (NEB). The generated plasmids were named as pET21a-wBmLspA. His and pET21a-EcLspA.His, respectively. wBmlspA gene with an added C-terminal 6XHis tag was also cloned into the expression vector pGEX5.1 (GE Healthcare) with a N-terminal GST fusion, named as pGEX5.1-GST.wBmLspA.His. A codon-optimized version of wBmlspA was designed by DNAworks oligonucleotide designing software [37], synthesized using USER cloning methods [38] and cloned into pET21a+ vector for improvement of gene expression. The T7 Express competent E. coli strain 2566 (NEB) was used as a host for plasmid recipients. The sequences of the lspA gene inserts were verified by DNA sequencing.

Recombinant LspA protein expression in E. coli
Expression of C-terminally 6XHis tagged wBmLspA, EcL-spA, and GST.wBmLspA recombinant proteins in T7 Express E. coli strain with and without isopropyl-beta-D-thiogalactopyranoside (IPTG) inductions was detected by Mouse anti-His tag monoclonal antibody and subsequent secondary horseradish peroxidase (HRP) conjugated Rabbit anti-Mouse IgG antibody (Novagen) in combination with the use of LumiGLO (r) chemiluminescent reagent and peroxide (Cell Signaling Technology, CST).
ts E. coli lspA mutant E. coli strain Y815 is a type II lipoprotein signal peptidase [21] temperature sensitive (ts) mutant, containing a pHY001 vector, which carries a tetracycline resistance gene (tet R ) and a lpp gene encoding a major outer membrane lipoprotein inducible by IPTG [22].

Complementation assays in E. coli
The constructs pET21a-wBmLspA.His, pET21a-EcLspA. His, and pGEX5.1-GST.wBmLspA.His, along with the vector only controls, pET21a and pGEX5.1, were transformed into E. coli Y815 cells with appropriate antibiotic selection. The E. coli Y815 ts lspA mutant grows normally at 30°C, but not at 42°C for colony formation. The detailed protocol is described in [21].

Sequence analysis of Wolbachia lspA genes
The LspA protein sequences from different Wolbachia strains and other reference species (shown in Figure 1) were retrieved from NCBI Genbank database [39] via protein-protein BLAST similarity searches and were aligned using CLUSTALX 1.83 [40]. The sequences in the alignment were further analyzed by Genedoc 2.6 [41] for calculating percentage of amino acid identity/ similarity, TMHMM 2.0 [42] for prediction of transmembrane domains, and SignalP 3.0 [43] for predicting signal peptide.

Globomycin resistance assays in E. coli
The experiments were devised and developed from a previous strategy [21]. T7 Express E. coli were transformed with plasmids pET21a-wBmLspA.His, pET21a-EcLspA.His, and pGEX5.1-GST.wBmLspA.His, along with the vector only controls pET21a and pGEX5.1. Transformants were grown to 0.6~1.0 OD 600 at 37°C in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin before being diluted to 0.1 OD 600 . The diluted samples were equally divided into IPTG (0.1 mM) induced and non-induced groups. In each group, the samples were further divided into globomycin treatment (100 μg/ml) and non-treatment subgroups. All samples were grown at 16°C for 16 hours with shaking. The samples with high cell density were diluted before measurement by spectrophotometer at 600 nm for accuracy. The samples had two replicates in each experiment and three independent experiments were carried out in total.
Wolbachia cell-based drug screening assay An Aedes albopictus cell line C6/36 (ATCC number CRL-1660) stably infected with Wolbachia pipientis wAlbB (C6/36Wp) was routinely cultured in Leibovitz-15 medium containing 2 mM L-glutamine, 5% foetal bovine serum, 1% non-essential amino acids and 2% tryptose phosphate broth at 26°C [28]. For drug assays, C6/36Wp cells were cultured in 96-well microtitre plates at a concentration of 10,000 cells per well overnight. Globomycin and vehicle (DMSO) controls were added in triplicate at the appropriate concentration, based on previous publications [17,19,21], the following day and replaced every 4 days. Samples were collected at appropriate timepoints by washing once in phosphate buffered saline (PBS) then adding 150 μl Wizard® SV Lysis Buffer (Promega) to each well. Lysates were stored at -80°C for processing at a later date.

Quantitative PCR (qPCR)
Genomic DNA was prepared from cell lysates using the Wizard (r) SV 96 Genomic DNA Purification System (Promega) according to the manufacturer's instructions and eluted in 100 μl water. qPCR targeting the 16S rRNA gene of Wolbachia and the 18S rRNA gene of the cell was conducted according to that described by [44] with minor modifications. Briefly, reaction mixtures consisted of a pair of primers (5'-TTGCTATTAGATGAGCCTATATTAG-3' and 5'-GTGTGGCTGATCATCCTCT-3' for 16S ribosomal DNA qPCR and 5'-CCGTGATGCCCTTA-GATGTT-3' and 5'-ATGCGCATTTAAGCGATTTC-3' for 18S ribosomal qPCR) at 200 nM each, 1 × SYBR Green reaction mix (Qiagen) and 2 μl DNA in the case of 16S qPCR or 1 μl DNA for 18S qPCR. Quantitative DNA standards were prepared as serial dilutions from stock singlestranded oligonucleotides representing the full-length amplicons (Sigma Genosys) and used at 5 × 10 6 to 5 × 10 0 copies [44], in duplicate reactions. Assays were performed on a DNA Engine PTC-200 thermocycler (MJ Research, GRI) with Chromo4 real-time PCR detection system (Bio-Rad) using the following conditions: denaturation at 95°C for 15 min followed by 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 15 sec. Melting curve analysis was performed between 50°C and 95°C to confirm specificity. Copy number was calculated from the standard curve by standard methods.

Western blot analysis
C6/36Wp cells were cultured in duplicate as described for the cell-based drug assays. Following 24 hours of globomycin treatment, cells were washed once in PBS then lysed by adding 25 μl of ice-cold RIPA buffer (Pierce) containing freshly added protease inhibitor solution (GE Healthcare) and incubating at room temperature for 5 min. Duplicate cultures were pooled and centrifuged at 16,000 × g for 15 min and the lysates were collected. Cell lysates were diluted in 2 × Laemmli sample buffer containing reducing agents (Sigma Aldrich) and incubated at 96°C for 10 min. 25 μl of samples were subjected to SDS-PAGE using 15% Tris-HCl gels (Bio-Rad) and protein bands were transferred onto a 0.45 μm PVDF membrane. After blocking for 2 h in 4% milk diluted in Tris-buffered saline (TBS) containing 0.1% Tween, membranes were incubated overnight at 4°C in affinity-purified anti-B. malayi Wolbachia PAL (wBmPAL) antibody [15] diluted 1 in 5,000 in blocking buffer. Membranes were washed at least four times in TBS 0.1% Tween then incubated with secondary antibody (goat-anti-rabbit-HRP, Perkin Elmer) diluted 1 in 5,000 for one hour at room temperature. Following washing, the blots were developed using the Supersignal West system (Pierce).

In vitro B. malayi assays
Adult B. malayi were obtained from TRS Laboratories, Athens, Georgia. Female and male adult worms were cultured in 12-well plates, five worms per well, three wells per group in 2.5 ml RPMI containing 10% foetal bovine serum and penicillin-streptomycin (Invitrogen, 200 U/ ml/200 μg/ml final concentration). Globomycin and vehicle controls were added the following day and motility was scored daily in a blinded manner using the scoring system described by [29]. The assay was terminated at day 10 and worms were used in an MTT assay to assess viability [45]. Worms were added singly to wells of a 96 well plate and washed with 200 μl PBS. 200 μl MTT was added at a final concentration of 0.5 mg/ml in PBS and the plate was incubated for two hours at 37°C with 5% CO 2 . MTT solution was removed and worms were washed twice with PBS then incubated in 200 μl of DMSO for one hour at 37°C, 5% CO 2 in order to solubilise the formazan product. The plate was read at 490 nm using DMSO alone as a blank. Frozen B. malayi were used as non-viable controls. Remaining worms were stored at -80°C for DNA extraction at a later date.

Statistical analysis
Differences between groups were assessed using Student's T test.

Additional material
Additional file 1: Table S1 -Wolbachia (wBm) and E. coli LspA gene specific primers used. Wolbachia (wBm) and E. coli LspA gene specific primers were used for PCR amplification of the full-length coding sequences for cloning into pET21a+ vector and pGEX5.1 vector. Primers were designed based on sequence information available in Genbank http:// www.ncbi.nlm.nih.gov. Restriction enzyme sites in primers are underlined. Abbreviations used: wBm: Wolbachia endosymbiont of Brugia malayi, Ec: E. coli, f: forward primer, r: reverse primer, LspA: type II lipoprotein signal peptidase.