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Biochem. J. (2008) 410, 147–155 (Printed in Great Britain) doi:10.1042/BJ20070891 147

Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity Dipti RAI*, Jay Kumar SINGH*, Nilanjan ROY† and Dulal PANDA*1 *School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, -400076, India, and †Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab-160062, India

The assembly and stability of FtsZ protofilaments have been on the assembly of FtsZ protofilaments. Curcumin inhibited the shown to play critical roles in bacterial cytokinesis. Recent assembly of FtsZ protofilaments and also increased the GTPase evidence suggests that FtsZ may be considered as an important activity of FtsZ. Electron microscopic analysis showed that antibacterial drug target. Curcumin, a dietary polyphenolic curcumin reduced the bundling of FtsZ protofilaments in vitro. compound, has been shown to have a potent antibacterial activity Further, curcumin was found to bind to FtsZ in vitro with a against a number of pathogenic bacteria including Staphylococcus dissociation constant of 7.3 −+ 1.8 µM and the agent also perturbed aureus, Staphylococcus epidermidis and Enterococcus. We found the secondary structure of FtsZ. The results indicate that the that curcumin induced filamentation in the Bacillus subtilis 168, perturbation of the GTPase activity of FtsZ assembly is lethal to suggesting that it inhibits bacterial cytokinesis. Further, curcumin bacteria and suggest that curcumin inhibits bacterial cell prolife- strongly inhibited the formation of the cytokinetic Z-ring in ration by inhibiting the assembly dynamics of FtsZ in the Z-ring. B. subtilis 168 without detectably affecting the segregation and organization of the nucleoids. Since the assembly dynamics of FtsZ protofilaments plays a major role in the formation and Key words: antibacterial activity, curcumin, filamentation, FtsZ functioning of the Z-ring, we analysed the effects of curcumin polymerization, GTPase activity, Z-ring.

INTRODUCTION combination with lactoferrin, N-acetylcysteine and pantoprazole has recently been shown to significantly reduce the symptoms FtsZ, a prokaryotic homologue of eukaryotic cytoskeletal protein caused by Helicobacter pylori infection in humans [20]. , polymerizes to form a Z-ring at the mid cell that Further, curcumin is shown to have a potent phototoxic orchestrates bacterial cell division [1–5]. FtsZ is shown to be effect against several bacteria such as Salmonella serotype essential for bacterial cell division and viability (reviewed in Typhimurium and Escherichia coli [21,22]. Curcumin also Biochemical Journal [5,6]). Previous studies have shown that the perturbation of FtsZ displays antigenotoxic activity against DNA-damaging agents functions by natural compounds and chemical agents leads to such as mutagens, UV rays, γ -radiation etc. [23–26]. Curcumin inhibition of bacterial proliferation [7–12]. Most recently, totarol, inhibits SOS induction in Salmonella serotype Typhimurium and a naturally occurring diterpenoid phenol, has been found to inhibit E. coli [24]. The DMSO extracts of turmeric are shown to protect bacterial cytokinesis by perturbing Z-ring formation [13]. Totarol DNA in E. coli cells against deleterious effects of X-rays [27]. has been shown to inhibit the assembly and GTPase activity of Although curcumin is known for its antimicrobial and anti- Mycobacterium tuberculosis FtsZ in vitro. Several studies have cancer activity for a long time, its therapeutic potential is severely suggested that inhibitors of FtsZ assembly may be developed limited because of its poor bioavailability. Understanding the into antibacterial drugs, including drugs against the multidrug- mechanism of the antibacterial activity of curcumin will greatly resistant strains of pathogenic bacteria [8,10,13,14]. assist in designing potent analogues of curcumin, a natural remedy Curcumin, a naturally occurring polyphenolic compound, is for several bacterial diseases. In the present study, curcumin extracted from the rhizomes of Curcuma longa (Figure 1). It has been found to induce filamentation in the B. subtilis 168, has been used as an important dietary component for a long suggesting that it inhibits bacterial cytokinesis. FtsZ plays an time. It exhibits various biological activities including anti- important role in orchestrating bacterial cytokinesis. Curcumin proliferative activity against various cancer cells, antioxidant binds to tubulin and inhibits polymerization [19]. activity, wound healing ability and antimicrobial activity [15– Since FtsZ is the prokaryotic homologue of tubulin, we sought to 19]. Recently, it has been found that curcumin binds to determine the effects of curcumin on the assembly dynamics of tubulin and perturbs microtubule polymerization [19]. Curcumin FtsZ. We found that curcumin increased the GTPase activity possesses antibacterial property against a number of Gram- of purified FtsZ and inhibited the assembly and bundling of FtsZ positive and Gram-negative bacteria [17]. It has been shown to kill protofilaments in vitro. To the best of our knowledge, curcumin several pathogenic Gram-positive bacteria such as Staphylococcus is the only inhibitor of FtsZ assembly found so far that increases aureus, Staphylococcus epidermidis and Enterococcus that cause the GTPase activity of FtsZ. We provide evidence suggesting infections such as skin diseases, pneumonia, meningitis and that curcumin perturbs the Z-ring formation and inhibits bacterial urinary tract infections in human beings. In addition, curcumin in cytokinesis by inhibiting FtsZ assembly.

Abbreviations used: Cy3, indocarbocyanine; DAPI, 4 ,6-diamidino-2-phenylindole; DIC, differential interference contrast; IPTG, isopropyl β-D- thiogalactoside; LB, Luria–Bertani; MIC, minimum inhibitory concentration. 1 To whom correspondence should be addressed (email [email protected]).

c The Authors Journal compilation c 2008 Biochemical Society 148 D. Rai and others

dilutions of bacterial culture were spread on to the LB agar plates and incubated for an additional 12 h at 37 ◦C. The number of colony-forming units was calculated by counting the colonies on each plate. The concentration of curcumin at which no colony was detected in plates was considered as the MIC. The experiment was performed three times.

Visualization of the morphology of bacteria Figure 1 Structure of curcumin [1,7-bis-(4-hydroxy-3-methoxy- phenyl)hepta-1,6-diene-3,5-dione] B. subtilis 168 was grown in the absence or presence of different concentrations of curcumin for 4 h in LB broth supplemented with MATERIALS AND METHODS 1 mM ascorbic acid. The cells were fixed in 2.8% formaldehyde and 0.04% glutaraldehyde at 25◦C. The bacterial cells were Materials finally collected, washed and resuspended in PBS (pH 7.4). IPTG (isopropyl β-D-thiogalactoside), Pipes, curcumin, glutam- The cell morphology was examined under a DIC (differential ate, GTP, ascorbic acid, DAPI (4,6-diamidino-2-phenylindole) interference contrast) microscope (Nikon ECLIPSE TE2000-U) × and Cy3 (indocarbocyanine)-conjugated goat anti-rabbit second- at 40 magnification. The images were captured using a Cool ary antibody were purchased from Sigma (St. Louis, MO, U.S.A.). SNAP-Pro camera. The cell length was measured using Image-Pro Primary polyclonal anti-FtsZ antibody developed in rabbit against Plus software (Media Cybernetics, Silver Spring, MD, U.S.A.). E. coli FtsZ was purchased from Bangalore Genei. All other chemicals used were of analytical grade. B. subtilis 168 (trpC2), Immunofluorescence microscopy E. coli K12 MG1655 and E. coli BL21 (DE3) are laboratory The immunostaining of the Z-ring was performed using FtsZ collection strains. antibody as described earlier [11]. Briefly, the B. subtilis 168 cells were grown for 4 h in LB broth containing 1 mM ascorbic acid without or with different concentrations of curcumin (25 and Methods 50 µM). The cells were fixed in 2.8% formaldehyde and 0.04% ◦ Curcumin stocks were prepared in DMSO and then diluted in glutaraldehyde for 45 min at 25 C. The cells were permeabilized aqueous buffer solution keeping the final concentration of DMSO and then incubated with a polyclonal FtsZ antibody developed below 1% (v/v) in all reaction milieu. The stability of curcumin in rabbit, followed by staining with a Cy3-conjugated goat anti- in aqueous buffers changes with various pH conditions. Curcumin rabbit secondary antibody. The nucleoids were stained with is highly unstable at neutral–basic pH conditions [28,29]. It has 1 µg/ml DAPI. The cells were observed under a fluorescence been found that curcumin is quite stable at pH 6.5. Therefore all microscope (Nikon ECLIPSE TE2000-U) at ×40 magnification. in vitro experiments were performed at pH 6.5. It is also reported The images were analysed using Image-Pro Plus software. that ascorbic acid helps to maintain curcumin stability [30]; therefore we incubated the bacterial culture with curcumin Purification of FtsZ supplemented with 1 mM ascorbic acid. Recombinant E. coli FtsZ was overexpressed and purified from E. coli BL21 strain as described previously [31]. The purity of Determination of antibacterial activity of curcumin the protein was analysed by SDS/10% PAGE and found to be The bacteria (B. subtilis 168, E. coli BL21 and E. coli K12 >98%. The concentration of purified FtsZ was determined by MG1655) were inoculated in LB (Luria–Bertani) broth (10 g/l the method of Bradford [32] using BSA as a standard. The protein ◦ casein enzymic hydrolysate, 5 g/l yeast extract and 5 g/l sodium was aliquoted and stored at –80 C. chloride) containing 1 mM ascorbic acid without or with different concentrations of curcumin. The inhibition of bacterial growth Light scattering assay was determined by monitoring D600, the attenuance at 600 nm, at µ different time (0, 30, 60, 90, 120, 150, 180, 240 and 300 min) FtsZ (12.5 M) was incubated without or with different concentrations (10–40 µM) of curcumin in 25 mM Pipes buffer intervals in the presence of different concentrations (20–120 µM) ◦ of curcumin. The D of respective blanks having only curcumin (pH 6.5) at 25 C for 10 min. Then, 50 mM KCl, 10 mM MgCl2 600 and 1 mM GTP were added to the reaction mixture. The kinetics was subtracted to give the final D600. The percentage inhibition of ◦ bacterial growth was calculated by using the equation: of the assembly of FtsZ was monitored by 90 light scattering (550 nm) at 37 ◦C using a JASCO 6500 spectrofluorimeter equipped with a constant temperature water circulating bath. A % Inhibition = [1 − (X D /CD )] × 100 600 600 0.3 cm path length cuvette was used for all the measurements. The appropriate blank was subtracted from all experimental data. µ where X D600 represents the final D600 value of X M curcumin- treated culture at a particular time. CD600 represents the D600 of the control culture at the same time. The percentage inhibition Electron microscopic analysis of bacterial growth was determined 4 h after the addition of cur- FtsZ (12 µM) was incubated in 25 mM Pipes buffer (pH 6.5) cumin to the bacterial culture. The IC50 of curcumin was deter- without or with different concentrations (10–50 µM) of curcumin ◦ mined by plotting the percentage inhibition of cell proliferation at 25 C for 10 min. Then, 50 mM KCl, 5 mM MgCl2 and 1 mM against curcumin concentration. MIC (minimum inhibitory GTP were added to reaction mixtures and polymerization was concentration) of curcumin for B. subtilis 168 cells was deter- carried out at 37 ◦C. After 15 min of polymerization, the protein mined by counting bacterial colonies as described earlier [13]. polymers were transferred to Formvar-carbon-coated copper Briefly, B. subtilis 168 cells were grown for 4 h in the absence grids (300 meshes) for 45 s. The sample-containing grids were and presence of different concentrations of curcumin. Appropriate stained with 1% uranyl acetate for 35 s, air-dried and observed

c The Authors Journal compilation c 2008 Biochemical Society Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 149 under a transmission electron microscope (FEI TECNAI G2 12) of five scans was taken for each spectrum. Deconvolution and [31]. statistical analysis of the CD spectra were performed using CDNN, SSE software from Jasco and OriginPro 7.5 software respectively. Measurement of GTPase activity of FtsZ The amount of P released during the assembly of FtsZ was i RESULTS measured using a standard Malachite Green/ammonium molyb- date assay as described earlier [33–35]. Briefly, FtsZ (12 µM) was Effect of curcumin on the proliferation of B. subtilis 168 and E. coli incubated without or with different concentrations (5, 10, 20 and K12 MG1655 cells 30 µM) of curcumin in 25 mM Pipes buffer (pH 6.5) containing Curcumin inhibited the growth of B. subtilis 168 in a concen- 50 mM KCl and 5 mM MgCl2 on ice for 10 min. Then, 1 mM GTP ◦ tration-dependent fashion (Figure 2A). The IC was found to was added to the reaction mixtures and incubated at 37 Ctostart 50 be 17 + 3 µMforB. subtilis 168 (Figure 2B). The MIC of cur- the hydrolysis reaction. After 5 min of incubation, 40 µl samples − cumin for B. subtilis 168 cells was determined to be 100 µM. Sim- were taken out from the reaction mixtures, and hydrolysis reaction ilarly, curcumin also inhibited the growth of E. coli K12 MG1655 was quenched by addition of 10% (v/v) 7 M HClO . The samples 4 (see Supplementary Figure 1A at http://www.BiochemJ.org/ were kept in ice for 2 min and then all the samples were incubated ◦ bj/410/bj4100147add.htm). The IC for E. coli K12 MG1655 at 25 C for 10 min. A 900 µl portion of freshly prepared filtered 50 was calculated to be 58 + 5 µM, and 100 µM curcumin inhibited Malachite Green/ammonium molybdate solution was added to the − ◦ the growth of E. coli K12 MG1655 cells by 80 (Supplementary samples, and incubated at 25 C for 30 min in the dark. The P % i Figure 1B). The IC for E. coli BL21 was found to be 56 + 8 µM release was determined by measuring the A at 650 nm. The 50 − (results not shown). background A was subtracted from all the readings. A phosphate standard curve was prepared using sodium phosphate. All the solutions were prepared in deionized water. Effect of curcumin on the morphology of B. subtilis 168 Measurement of the dissociation constant of curcumin–FtsZ Curcumin induced filamentation in B. subtilis 168 cells (see interaction Supplementary Figure 2 at http://www.BiochemJ.org/bj/410/ bj4100147add.htm). The average length of B. subtilis 168 was The increase in curcumin fluorescence upon binding to FtsZ was determined to be 3.3 −+ 1 µm. The average cell length increased used to determine the binding of curcumin to FtsZ [19]. FtsZ by 2.5-fold from 3.3 + 1to8.6+ 5.3 µm after 4 h of incubation µ − − (1 M) was incubated with different concentrations of curcumin with 25 µM curcumin. Also, the cell growth was inhibited by µ ◦ (1,2,3,4,5,7and10 M) in 25 mM Pipes buffer (pH 6.5) at 25 C 81 −+ 11% in the presence of 25 µM curcumin (Figure 2B). In for 30 min in the dark. The fluorescence spectra of the samples thepresenceof50µM curcumin, the average cell length of λ were recorded using a ex of 425 nm. The fluorescence intensities B. subtilis 168 was increased by 6-fold from 3.3 −+ 1to20−+ at 495 nm were used for determining the binding constant. 16 µm (Figure 2C) and the growth was inhibited by 90 −+ 9% The dissociation constant (Kd) of curcumin–FtsZ interaction (Figure 2B). was calculated by plotting Lbound/Lfree against Lbound,whereLbound Most of the curcumin-treated cells had strikingly larger cell represents the bound curcumin concentration and Lfree represents length than the control (vehicle-treated) cells (Figure 2C). In the the free curcumin concentration [36]. The Lbound was calculated case of control, 33, 66 and 1% of the cells had mean length in from the equation the range of 1–3, 3–5 and 5–7 µm respectively (Figure 2C). In the presence of 50 µM curcumin, 12, 4, 8 and 76% of = + / L bound F C Fmax the cells had mean length in the range of 3–5, 5–7, 7–9 and >9 µm respectively. The results together indicate that curcumin  µ where Fmax is the fluorescence intensity of 1 M fully bound inhibits bacterial proliferation by inhibiting cytokinesis. Further,  curcumin. Fmax was calculated by plotting 1/fluorescence curcumin did not perturb the membrane structure of B. subtilis intensity (495 nm) of curcumin against 1/[FtsZ]. For this, 1 µM 168. curcumin was incubated without or with different concentrations (1,2,4,6,8and12µM) of FtsZ in 25 mM Pipes buffer (pH 6.5) ◦ at 25 C for 30 min in the dark. F was calculated by subtracting Effect of curcumin on Z-ring formation and nucleoid segregation in F0 from F,whereF is the fluorescence intensity of the FtsZ– B. subtilis 168 curcumin complex at a particular curcumin concentration and F0 is the fluorescence intensity of only curcumin in the absence of B. subtilis 168 cells formed large filaments in the presence of FtsZ. curcumin. Therefore we sought to find the effects of curcumin on The fluorescence intensities were corrected for the inner the nucleoids and as well as on the Z-ring in B. subtilis 168. In the filter effects using the following equation: F =F × absence of curcumin, most of the B. subtilis 168 cells had a well- C defined Z-ring in between the two nucleoids (Figure 3). Curcumin antilog[(A425+A495)/2]. FC represents the corrected fluorescence intensity. strongly inhibited the formation of Z-rings (Figures 3D, 3F, 3G and 3I). For example, 63% of the control cells had well-defined Z-rings (Figures 3A and 3C), whereas only 20 and 7% of the Analysis of secondary structural changes curcumin-treated cells had Z-rings in the presence of 25 and FtsZ (4 µM) was incubated without or with different concen- 50 µM curcumin respectively (Table 1). Further, the frequency trations of curcumin (20, 30 and 50 µM) in 25 mM phosphate of occurrence of Z-rings/µm of the bacterial cell length was buffer (pH 6.5) for 30 min at 25 ◦C. The far-UV CD spectrum was reduced strongly. For instance, the frequency of occurrence of the monitored over a wavelength range of 200–250 nm using a JASCO Z-ring/µm of the bacterial cell length was found to be 0.18 −+ 0.15, J-810 spectropolarimeter equipped with a Peltier temperature 0.03 −+ 0.07 and 0.01 −+ 0.03 in the absence and presence of 25 and controller using a 0.1 cm path length quartz cuvette. An average 50 µM curcumin respectively.

c The Authors Journal compilation c 2008 Biochemical Society 150 D. Rai and others

Figure 2 Inhibition of growth of B. subtilis 168 cells by curcumin

B. subtilis 168 cells were grown in LB media supplemented with 1 mM ascorbic acid without or with different concentrations of curcumin. Growth curves for B. subtilis 168 cells in the absence () and presence of 20 µM(♦), 30 µM(᭜), 60 µM(᭿) and 100 µM(ᮀ) curcumin are shown in (A). The results are the average of four independent experiments. (B) The Figure shows the inhibition + of growth of B. subtilis 168 in the presence of different concentrations of curcumin after 4 h of growth. The results are the means − S.D. for eight experiments. The effects of 25 and 50 µMcurcumin on the length of B. subtilis 168 cells are shown in (C). B. subtilis 168 cells were grown without or with different concentrations of curcumin supplemented with 1 mM ascorbic acid for 4 h, and the cells were imaged using a DIC microscope.

Curcumin increased the number of nucleoids per cell, but the the presence of 25 µM curcumin. None of the 50 µM curcumin- frequency of occurrence of nucleoids/µm of cell length was treated cells with two nucleoids had a Z-ring, indicating that similar to that of the control cells. For example, the percentage curcumin strongly inhibited the Z-ring formation. Taken together, of control cells having 1, 2 and 4 nucleoids per cell was 21, 75 the results showed that curcumin inhibited the formation of the and 4% respectively. In the case of 25 and 50 µM curcumin- Z-ring without affecting the nucleoid segregation detectably. treated cells, the number of cells with 1 or 2 nucleoids reduced considerably and the number of cells with 4 and 8 or more nucleoids increased significantly. For example, 5, 37, 33 and Effect of curcumin on FtsZ assembly and bundling in vitro 25% of the 25 µM curcumin-treated cells had 1, 2, 4 and 8 Since curcumin strongly induced filamentation in B. subtilis 168, or more nucleoids respectively. Further, in the case of 50 µM and inhibited the formation of the Z-ring in the bacteria, we curcumin-treated cells, the percentage of cells with 1, 2, 4 and 8 wanted to find out whether these effects are due to its interaction or more nucleoids was found to be 4, 14, 18 and 64% respectively. with FtsZ. Therefore we polymerized FtsZ (12.5 µM) in 25 mM µ The frequency of occurrence of nucleoids/ m of cell length was Pipes buffer containing 50 mM KCl, 10 mM MgCl2 and found to be similar without or with curcumin. For example, 1 mM GTP without or with different concentrations of curcumin the frequency of occurrence of nucleoids/µm of cell length was in vitro (Figure 4). FtsZ monomers assembled at a much determined to be 0.58 −+ 0.19, 0.52 −+ 0.16 and 0.53 −+ 0.11 in the higher rate in the absence of curcumin than in its presence. absence and presence of 25 and 50 µM curcumin respectively. Further, curcumin reduced the extent of FtsZ polymerization The results showed that curcumin had no detectable effect on the in a concentration-dependent fashion. For example, the light- nucleoid segregation. scattering intensity of FtsZ assembly was reduced by ∼17% and Out of the total control cells, 21% of cells had a single nucleoid 70% in the presence of 10 and 40 µM curcumin respectively with no Z-ring having a cell length of 2.5 −+ 0.5 µm. This popu- compared with that of the control, indicating that curcumin lation might reflect the neonate cells. The percentage of cells inhibits the assembly of FtsZ (Figure 4). The IC50 of FtsZ assembly with a single nucleoid decreased strongly in curcumin-treated occurred in the presence of 30 µM curcumin. cells, indicating that curcumin inhibited cell division. Further, the The effect of curcumin on the assembly of FtsZ was also percentage of cells with two nucleoids reduced from 75 to 14% analysed by transmission electron microscopy (Figure 5). The in the case of 50 µM curcumin. Although approx. 80% of the control showed thick bundles of FtsZ polymers with an average control cells containing two nucleoids had a Z-ring, only a small width of 71 −+ 36 nm (Figure 5A). The size and thickness of FtsZ population (27%) of the cells with two nucleoids had a Z-ring in polymers reduced extensively with increasing concentrations

c The Authors Journal compilation c 2008 Biochemical Society Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 151

Figure 3 Curcumin inhibited Z-ring formation without affecting nucleoid segregation in B. subtilis 168

Bacterial cells were grown with different concentrations of curcumin supplemented with 1 mM ascorbic acid. The Z-rings were stained with polyclonal antibody developed in rabbit against FtsZ followed by Cy3-conjugated goat anti-rabbit secondary antibody (red fluorescence). The nucleoids were stained with 1 µg/ml DAPI (blue fluorescence). The cells were observed under a fluorescence microscope. Shown are control cells [(A), (B) and overlay (C)] and 25 µM[(D), (E) and overlay (F)] and 50 µM[(G), (H) and overlay (I)] curcumin-treated cells. Scale bar, 10 µm(A–I).

Table 1 Effect of curcumin on the Z-ring and nucleoids of B. subtilis 168 of curcumin, the length of the FtsZ polymers was considerably The B. subtilis 168 cells were grown without or with different concentrations of curcumin (25 and smaller compared with that of the control. For example, in 50 µM) and incubated with polyclonal FtsZ antibody followed by staining with Cy3-conjugated the presence of 25 µM curcumin, the average length of FtsZ goat anti-rabbit secondary antibody. The nucleoids were stained with DAPI. The cells were polymers was 0.7 −+ 0.23 µm, which was at least 75% smaller observed under a fluorescence microscope. Images were analysed using Image-Pro Plus than the control FtsZ polymers, which were larger than the software. As many as 100 cells were examined for the control as well as for each of the field of view (3 × 4 µm). Under the polymerization conditions, concentrations of curcumin. high concentrations of curcumin induced aggregation of FtsZ monomers (Figures 5B–5D, arrows). Curcumin Description Control 25 µM50µM Effect of curcumin on the GTPase activity of FtsZ + + + Cell length (µm) 3.3 − 18.6− 5.3 20 − 16 The assembly dynamics of FtsZ is thought to be regulated by the Proportion of cells with a Z-ring (%) 63 20 7 + + + GTPase activity of FtsZ [37,38]. Curcumin was found to reduce Frequency of occurrence of the 0.18 − 0.15 0.03 − 0.07 0.01 − 0.03 the assembly and bundling of FtsZ protofilaments (Figures 4 and Z-ring/µm of cell length + + + 5). Therefore the effect of curcumin on the GTPase activity of Frequency of occurrence of 0.58 − 0.19 0.52 − 0.16 0.53 − 0.11 nucleoids/µm of cell length FtsZ was determined. Curcumin increased the GTPase activity Proportion of cells having two 75 37 14 of FtsZ in a concentration-dependent manner (Figure 6A). For nucleoids (%) example, when compared with the control, the GTPase activity of Proportion of cells with two 80 27 None FtsZ was increased by 35% in the presence of 30 µMcurcumin nucleoids having a Z-ring (%) (P < 0.0001), indicating that curcumin can perturb FtsZ assembly dynamics. GTP-bound FtsZ polymers are thought to be more stable than of curcumin. For example, curcumin (10 µM) reduced the the GDP-bound polymers [2,4,6,37,38]. Under challenged GTP thickness of the bundles of FtsZ protofilaments by 60% from conditions, FtsZ polymers are expected to disassemble once all 71 −+ 36 to 28 −+ 17 nm (Figure 5B). At a higher concentration available GTP molecules are exhausted. Curcumin increased the

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(12.5 µM) were analysed in the presence of a limited amount (100 µM) of GTP. The light scattering signal of FtsZ assembly (control) decreased sharply after ∼300 s of assembly, indicating the disassembly of FtsZ protofilaments (Figure 6B). As expected, curcumin not only reduced the extent of FtsZ assembly but also strongly reduced the steady-state duration of the assembly (Figure 6B). Under the experimental conditions used, FtsZ proto- filaments disassembled more quickly (within 200 s of assembly) in the presence of curcumin than in the absence of curcumin (Figure 6B). The results indicated that curcumin-induced increase in the GTP hydrolysis rate might be the cause of the destabilization of FtsZ protofilaments.

Curcumin binds to FtsZ in vitro Curcumin (1 µM) was incubated in the absence and presence of different concentrations (1–12 µM) of FtsZ for 30 min. The Figure 4 Curcumin reduced the rate and extent of FtsZ polymerization fluorescence intensity of curcumin increased with increase in concentration of FtsZ, indicating that it binds to FtsZ (Figure 7A). FtsZ (12.5 µM) was polymerized in 25 mM Pipes buffer (pH 6.5) containing 50 mM KCl, 10 mM The increase in the curcumin fluorescence at 495 nm upon binding MgCl2 and 1 mM GTP without (᭜) and with 10 µM(᭿), 20 µM(), 30 µM(᭝) and 40 µM (ᮀ) of curcumin as described in the Materials and methods section. Appropriate blanks were to FtsZ was used to determine the binding of curcumin to subtracted from all the traces. The experiment was performed four times. FtsZ. FtsZ (1 µM) was incubated with different concentrations of curcumin for 30 min. The fluorescence intensity of the GTP hydrolysis rate of FtsZ (Figure 6A). Therefore we argued curcumin–FtsZ complex increased with increase in concentration that under the challenged GTP conditions, FtsZ protofilaments of curcumin (Figure 7B). The dissociation constant (Kd)ofthe would disassemble at a higher rate in the presence of curcumin curcumin–FtsZ interaction was determined to be 7.3 −+ 1.8 µM than in its absence. To examine this possibility, the effects of (Figure 7C). The number of binding sites of curcumin on FtsZ different concentrations of curcumin on the assembly of FtsZ was calculated to be 0.7 −+ 0.2 per FtsZ molecule.

Figure 5 Electron micrographs of FtsZ polymers

FtsZ (12 µM) was polymerized without or with different concentrations of curcumin at 37 ◦C as described in the Materials and methods section. The FtsZ polymers were transferred to Formvar-carbon-coated copper grids (300 meshes), negatively stained with 1% uranyl acetate and observed under a transmission electron microscope (FEI TECNAI G2 12) at ×20500 magnification. Shown are the electron micrographs of FtsZ polymers in the absence (A) and presence of 10 µM(B), 25 µM(C) and 50 µM(D) curcumin. The aggregates are shown by arrows (B–D). The thickness of a minimum of 100 polymers was measured in all cases. Scale bar, 2 µm(A–D).

c The Authors Journal compilation c 2008 Biochemical Society Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 153

DISCUSSION

In the present study, we have found that curcumin induced filamentation in B. subtilis 168, showing that it inhibits bacterial cytokinesis. Curcumin was found to bind to FtsZ in vitro;it increased the GTPase activity of FtsZ and inhibited the assembly and bundling of FtsZ protofilaments. Curcumin had no detectable effect on the segregation and organization of nucleoids in B. subtilis 168, indicating that it does not inhibit bacterial division by targeting DNA. We present evidence suggesting that curcumin inhibited cytokinesis in B. subtilis 168 by inhibiting the Z-ring formation through a direct interaction with FtsZ. The assembly dynamics of FtsZ is considered to be regulated by the GTPase activity of FtsZ [5,34,37–41]. Curcumin decreased the steady-state duration of FtsZ polymers (Figure 6B) and curcumin was also found to increase the GTPase activity of FtsZ (Figure 6A). GDP-bound FtsZ polymers are thought to be less stable than the GTP-bound polymers and they readily disassemble into FtsZ monomers [39,40]. In addition, GDP-FtsZ monomers do not assemble as effectively as GTP-FtsZ monomers [38]. The results indicated that curcumin decreases the stability of FtsZ protofilaments by increasing the GTP hydrolysis rate of FtsZ, which in turn inhibits FtsZ assembly. In addition, curcumin reduced the α-helix content, while it increased the random coil content of FtsZ, suggesting that the perturbation in the secondary structure of FtsZ might also be the cause of destabilization of FtsZ protofilaments. Curcumin is the first small molecule inhibitor of FtsZ assembly known to date that increases the GTPase activity of FtsZ. Most of the inhibitors of FtsZ assembly have been reported to suppress the rate of GTP hydrolysis [7,9,10,12,13]. For example, 100 µM SRI-3072 and SRI-7614 inhibited the GTPase activity by 20 and 25% respectively [7]. Similarly, PC58538 and its analogues inhibited the GTPase activity of FtsZ with an IC50 value of 136 µg/ml [12]. Zantrins inhibited GTP hydrolysis with

IC50 values ranging from 4 to 100 µM [9]. Totarol suppressed the GTPase activity of M. tuberculosis FtsZ [13]. For example, Figure 6 Curcumin increased the GTPase activity of FtsZ 24% inhibition and 67% inhibition of GTP hydrolysis were found in the presence of 10 and 75 µM totarol respectively. Effects of curcumin on the GTPase activity of FtsZ (A). FtsZ (12 µM) was incubated without or Therefore curcumin perturbed the assembly dynamics of FtsZ with different concentrations (5, 10, 20 and 30 µM) of curcumin. Then, 1 mM GTP was added by a mechanism that is different from the other known assembly to the reaction mixtures and the hydrolysis reaction was initiated immediately by putting the ◦ inhibitors. reaction mixtures at 37 C. Samples were taken after 5 min of hydrolysis. The amount of Pi released was measured using a standard Malachite Green/ammonium molybdate assay. The Curcumin decreased the light scattering intensity of FtsZ results are the average of five experiments. Error bars represent S.D. The P values were from protofilaments, indicating that it reduced the polymerized mass <0.01 to <0.0001 for the different points. Curcumin reduced the steady-state duration of FtsZ of FtsZ protofilaments and/or decreased the bundling of FtsZ assembly (B). FtsZ (12.5 µM) was polymerized in 25 mM Pipes buffer (pH 6.5) containing protofilaments (Figure 4). Electron microscopic analysis of the µ ᭜ µ ᭛ µ  50 mM KCl, 10 mM MgCl2 and 100 M GTP without ( ) and with 10 M( ), 20 M( ), polymerization reaction showed that curcumin strongly reduced 30 µM(᭝) and 40 µM(᭿) curcumin as described in the Materials and methods section. Appropriate blanks were subtracted from all the traces. The experiment was performed three the length of FtsZ protofilaments and the extent of bundling times. of the protofilaments (Figure 5). The decrease in polymer length and bundling of FtsZ protofilaments may be due to the binding of bulky curcumin molecules on FtsZ. The presence of bulky curcumin in the protofilaments may cause steric hindrance that The effects of curcumin on the secondary structures of FtsZ inhibits the assembly and bundling of FtsZ. Curcumin perturbed were determined by monitoring far-UV CD spectra of FtsZ (Fig- the secondary structures of FtsZ (Figure 8). The decrease ure 8). Analysis of the CD spectra using deconvolution in the polymerization and bundling of FtsZ may be due to software indicated that the secondary structure of FtsZ contained the conformational changes induced by curcumin in the FtsZ + + + + + 31.4 − 0.8, 31.7 − 0.6, 9.4 − 0.3, 8.9 − 0.2 and 17.5 − 0.2% α- monomers. The IC50 of FtsZ assembly occurred in the presence helix, random coil, antiparallel, parallel and β-turn respectively. of 30 µM curcumin. Thus the inhibitory effects of curcumin Curcumin perturbed the secondary structures of FtsZ (Figure 8). on FtsZ polymerization were comparable with that of other For example, curcumin (50 µM) reduced the helical content by known inhibitors of FtsZ assembly [7,9,11,13]. For example, + + 9% from 31.4 − 0.8% to 28.4 − 0.5%, while it increased the the polymerization ID50 (infectious dose to 50% of exposed random coil content by 8.5% from 31.7 −+ 0.6% to 34.4 −+ 0.3%. individuals) values of SRI-3072 and SRI-7614 are shown to be The reduction in the α-helix (P < 0.0001) and the increase in the 52 −+ 12 and 60 −+ 0 µM respectively [7]. random coil (P < 0.00002) content of FtsZ in the presence of In the last few years, several natural and synthetic compounds 50 µM curcumin were found to be statistically significant. All were shown to inhibit bacterial growth by targeting the assembly other secondary structures did not change significantly. dynamics of FtsZ [7–14]. For example, viriditoxin inhibited the

c The Authors Journal compilation c 2008 Biochemical Society 154 D. Rai and others

Figure 7 Binding of curcumin with FtsZ monitored by fluorescence spectroscopy

FtsZ (1 µM) was incubated with different concentrations of curcumin (1–10 µM) and the fluorescence spectra were monitored as described in the Materials and methods section. The λex and λem values were 425 and 495 nm respectively. (A) The Figure shows an increase in the fluorescence intensity of FtsZ–curcumin complex in the absence (᭺) and presence of 1 µM(᭹), 2 µM(᭝), 4 µM(), 6 µM(♦), 8 µM(᭜) and 12 µM(ᮀ) FtsZ at constant curcumin concentration (1 µM). (B) The Figure shows an increase in the fluorescence intensity of the FtsZ–curcumin complex in the absence (᭺) and presence of 1 µM(᭹), 2 µM(), 3 µM(), 4 µM(♦), 5 µM(᭜), 7 µM(ᮀ) and 10 µM(᭿) curcumin at constant FtsZ concentration (1 µM). (C) A plot of L bound/L free against L bound of the binding of curcumin to FtsZ. The experiment was performed six times.

Similarly, in a recent study, totarol has been shown to inhibit the proliferation of B. subtilis 168 cells with an MIC value of 2 µM [13]. Totarol is thought to inhibit the Z-ring formation in bacteria by inhibiting FtsZ assembly. Curcumin inhibited the proliferation of both Gram-positive (B. subtilis 168) and Gram- negative (E. coli K12 MG1655 and E. coli BL21) bacteria with an MIC of 100 µMforB. subtilis 168 and induced filamentation in B. subtilis 168 cells. The high MIC value for curcumin may be due to the poor bioavailability of curcumin [42]. Several bioconjugates of curcumin have been synthesized to enhance the cellular uptake and targeted delivery of curcumin, thereby increasing curcumin’s antibacterial potential, and they have shown improved antibacterial activity [17,43]. Curcumin inhibited Z-ring formation in B. subtilis 168. Cur- cumin is known to protect DNA against several DNA-damaging agents like UV rays, X-rays, γ -radiation and chemical mutagens [23–25,27]. Further, curcumin is found to inhibit SOS induction Figure 8 Effects of curcumin on the far-UV CD spectra of FtsZ in Salmonella serotype Typhimurium and E. coli [24]. In the present study, we found that curcumin did not affect the nucleoid FtsZ (4 µM) was incubated without or with different concentrations (20–50 µM) curcumin at ◦ segregation and organization, further supporting the idea that 25 C. The Figure shows the far-UV CD spectra of FtsZ in the absence (᭺) and presence of 20 µM(᭹), 30 µM(᭝) and 50 µM() curcumin. The experiment was repeated five times. curcumin does not induce DNA damage detectably. In summary, the results presented in this study suggest that curcumin inhibits bacterial cell division, apparently by perturbing E. coli wild-type cell division with an MIC of >64 µg/ml and the cytokinetic Z-ring through a direct interaction with FtsZ. induced filamentation in the bacterial cell [8]. Zantrins are found These findings may help to design potent curcumin analogues to inhibit the Z-ring formation in E. coli wild-type followed by with improved stability and bioavailability. cell death with MIC values from 20 to 80 µM or more, and only E. coli µ zantrin Z5 induced filamentation in [9]. Further, 10 M We thank Dr H.P. Erickson (Department of , Duke University Medical Center, sanguinarine is shown to completely inhibit the proliferation Durham, NC, U.S.A.) for providing us with the FtsZ clone, and SAIF (Sophisticated of B. subtilis 168 [11]. Sanguinarine induced filamentation in Analytical Instrument Facility) IIT Bombay, Mumbai, India, for use of the electron- bacteria and inhibited Z-ring formation in the B. subtilis 168. microscopy facility. We thank Dr T.K. Beuria, Dr M.K. Santra and Renu Mohan of the

c The Authors Journal compilation c 2008 Biochemical Society Curcumin inhibits bacterial cytokinesis by inhibiting FtsZ assembly 155

School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, 20 Di Mario, F., Cavallaro, L. G., Nouvenne, A., Stefani, N., Cavestro, G. M., Iori, V., Mumbai, India, for their assistance in several experiments. We thank Richa Jaiswal, also Maino, M., Comparato, G., Fanigliulo, L., Morana, E. et al. (2007) A curcumin-based of the School of Biosciences and Bioengineering, for a critical reading of the manuscript 1-week triple therapy for eradication of Helicobacter pylori infection: something to learn before its submission. This work was supported by a grant from the Department of Science from failure? Helicobacter 12, 238–243 and Technology, Government of India. D.P. is supported by a Swarnajayanti fellowship. 21 Tonnesen, H. H., de Vries, H., Karlsen, J. and Beijersbergen van Henegouwen, G. (1987) J.K.S.acknowledgessupportfromtheCSIR(CouncilofScientificandIndustrialResearch). Studies on curcumin and curcuminoids. 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Received 6 July 2007/11 October 2007; accepted 22 October 2007 Published as BJ Immediate Publication 23 October 2007, doi:10.1042/BJ20070891

c The Authors Journal compilation c 2008 Biochemical Society