Monocarbonyl Curcuminoids with Improved Stability as Antibacterial

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Article Cite This: ACS Omega 2019, 4, 675−687

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Monocarbonyl Curcuminoids with Improved Stability as Antibacterial Agents against Staphylococcus aureus and Their Mechanistic Studies Prince Kumar,† Shamseer Kulangara Kandi,‡ Sunny Manohar,‡,§ Kasturi Mukhopadhyay,*,† and Diwan S. Rawat*,‡ †

Antimicrobial Research Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India Department of Chemistry, University of Delhi, Delhi 110007, India § Department of Chemistry, Deen Dayal Upadhyaya College, University of Delhi, Delhi 110078, India

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‡

S Supporting Information *

ABSTRACT: Curcumin has been known to possess diverse pharmacological effects at relatively nontoxic doses; however, its therapeutic potential is severely restricted because of its low aqueous solubility and poor stability under physiological conditions. To overcome its limitations, we had previously designed several monocarbonyl curcuminoids by modifying the central β-diketone moiety of curcumin. In this study, the antibacterial activity of 33 curcuminoids from this designed library has been screened, six of which displayed potent antibacterial activity against clinically relevant Staphylococcus aureus. These curcuminoids were found to be very stable at physiological conditions and did not cause any toxicity toward mammalian cells. Mechanistically, out of these six curcuminoids, five caused instant membrane depolarization and were able to permeabilize the bacterial membrane, which could be the reason for their potent bactericidal activity and the sixth one killed staphylococcal cells without damaging the bacterial membrane. Overall, the present work established the staphylocidal potency of six water-soluble, nontoxic curcuminoids, thereby providing an impetus for the development of these lead curcuminoids for therapeutic use against S. aureus.

1. INTRODUCTION Staphylococcus aureus (S. aureus) is an opportunistic pathogen and a commensal colonizer of nasal carriage and skin in approximately 30% of the world population.1 Its carriers are at higher risk of infection, as colonization provides a reservoir for S. aureus and when host defenses are breached, it becomes a clinically significant pathogen and causes a wide spectrum of diseases, from skin and soft tissue infections, such as abscesses, carbuncles, and cellulitis to life-threatening conditions, such as bacteraemia, osteomyelitis, meningitis, pneumonia, endocarditis, and urinary tract infections.2,3 Due to frequent and indiscriminate use of antibiotics, the frequency of both hospital-acquired and community-acquired infections has increased significantly in last few decades.4 This increased prevalence of infections has been associated with the emergence of resistant strains, mainly, vancomycin-resistant S. aureus strains and methicillin-resistant S. aureus.5,6 Many clinically isolated S. aureus are now considered as multiple drug-resistant (MDR) bacteria as they showed resistance toward many available drugs.7 These resistant strains continue to parade higher mortality and morbidity in many developing as well as developed countries.8,9 There is a major deficit of effective treatment against its infection and demands an urgent © 2019 American Chemical Society

need to search for alternatives. Since a long time, plant-based active compounds (such as phenols, quinones, alkaloids, and other secondary metabolites) have been explored for discovering new compounds and still gain much attention.10 Among such natural compounds, curcumin is one of the most studied polyphenolic compounds, which targets various metabolic pathways in the biological system.11 Curcumin is a yellow polyphenolic compound obtained from the rhizomes of the perennial plant turmeric (Curcuma longa). Besides its well-known use as food coloring spice, it has also been used for the treatment of wound healing, burns, common cold, fever, arthritis, and liver disorders since ancient times in many Asian countries.12 Numerous studies in the last few decades have explored the diverse pharmacological properties of curcumin, which include antioxidant, antiinflammatory,13 antiviral,14 and antibacterial activities15 and have shown promising therapeutic utility against various neurodegenerative diseases.16 The excellent potency of curcumin was associated with its safe toxicity profile and it Received: October 2, 2018 Accepted: December 21, 2018 Published: January 9, 2019 675

DOI: 10.1021/acsomega.8b02625 ACS Omega 2019, 4, 675−687

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Table 1. Curcuminoids Tested for Antibacterial Activity

symmetrical molecule exists as a tautomeric mixture of ketoenol forms in solution because of the central β-diketone moiety. The diketone moiety can easily be converted to enolic form under mild basic condition. This enolic form is responsible for its rapid degradation. The pharmacokinetic study revealed that the metabolic action of liver aldoketo reductases on β-diketone moiety might be the reason for the rapid metabolic degradation of curcumin in vivo.21 Another

was found to be nontoxic even at high oral doses of 12 g/day in phase I clinical trial.17 Despite its efficacy and potential, its therapeutic usage is severely restricted due to its low aqueous solubility and poor stability under physiological conditions.18 It undergoes rapid degradation in physiological buffer, in presence of light and reducing conditions.19,20 These shortcomings result in its poor bioavailability, which limits its achievable concentration in the biological system. This 676


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Scheme 1. General Synthetic Scheme for the Preparation of the Curcuminoids

Table 2. Minimum Inhibitory Concentration (MIC) of Curcuminoids against Staphylococcal Strains minimum inhibitory concentration (MIC) in μg/mL name of test drugs

MSSAa (ATCC 29213)

MRSAb (ATCC 33591)

MRSAc (S-28)

MRSAc (S-30)

MRSAc (S-33)

MRSAc (S-34)

MRSAc (S-37)

MRSAc (S-41)

curcumin 34 36 52 55 58 63 tetracycline oxacillin vancomycin

≥256 16 8 32 16 32 32 0.5 0.5 0.5

≥256 16 16 32 32 32 32 32 64 1

ND 32 16 32 32 32 32 32 64 1

ND 32 16 32 64 32 32 64 64 1

ND 16 8 16 16 8 8 32 64 0.5

ND 32 16 32 32 32 32 64 128 1

ND 16 16 32 32 16 16 32 64 1

ND 32 16 64 64 32 32 32 64 1

a

Methicillin-sensitive S. aureus. bMethicillin-resistant S. aureus. cClinical isolates of MRSA, ND; not determined.

monocarbonyl analogs of curcumin was achieved by sequential nucleophilic substitution reactions followed by the Claisen− Schmidt reaction (Scheme 1). In the first step, the commercially available p-hydroxybenzaldehyde (1) or vanillin (2) was reacted with linear dibromoalkanes to obtain aldehydes (3−7) with free bromo group at the terminal position. These aldehydes (3−7) were subjected to nucleophilic substitution by various aliphatic and aromatic amines to obtain aldehydes (8−32). The aldehydes (8−32) were finally subjected to a Claisen−Schmidt condensation to get the desired curcuminoids (33−65) (Table 1). 2.2. Antibacterial Activity. To evaluate the efficacy of curcuminoids in the face of most threatening staphylococcal infections, we tested the antibacterial activity of both curcumin and curcuminoids against a panel of staphylococcal cells, including ATCC strains and clinical isolates of S. aureus. The antibacterial activity of curcuminoids is presented in Table 2 as their minimum inhibitory concentration (MIC), which is the lowest concentration of the test drug that inhibits the bacterial growth. Total 33 curcuminoids were screened against methicillinsensitive S. aureus (MSSA) ATCC 29213, and MIC could be determined only for 14 of the curcuminoids as the others were sparingly soluble in water. These 14 curcuminoids displayed MIC values in the range of 8−128 μg/mL (MIC of all 14 curcuminoids is given in Table S1 of the Supporting Information), whereas the parent molecule curcumin remained

study has shown that the curcumin derivatives with modified β-diketone moiety retained their biological activity.22 Keeping this in mind, we had modified β-diketone moiety of curcumin with mono-keto group; the resulting monocarbonyl curcuminoids had shown potent cytotoxicity against various cancer cell lines, with IC50 values less than 1 μM.23 Among these, a few of the curcuminoids were found to be more potent than the US FDA-approved drug, doxorubicin.24,25 Some of these curcuminoids also showed excellent antimalarial activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum.23,24 One of these curcuminoids was reported for its antibacterial activity by disk diffusion assay.26 Encouraged by these results, in the present study, we screened the antimicrobial efficacy of these curcuminoids against a panel of S. aureus strains and also studied their bactericidal kinetics against exponential and stationary-phase cells of S. aureus. Further, we assessed the stability of these curcuminoids under physiological conditions. Thereafter, we studied the toxicity of the curcuminoids toward mammalian cells. Finally, we investigated their mechanism of action using fluorescencebased assays and the finding was also confirmed by microscopy techniques.

2. RESULTS AND DISCUSSION 2.1. Synthetic Procedure. Synthesis and characterization of tested compounds (33−65, Table 1) have been reported in our previous publication.23 In brief, the synthesis of 677


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Figure 1. UV−visible absorption spectra of curcumin and curcuminoids in PBS buffer (pH 7.4): (i) Curcumin, (ii) 34, (iii) 36, (iv) 52, (v) 55, (vi) 58, and (vii) 63.

inactive even at the highest tested concentration of 256 μg/mL because of its poor aqueous solubility, as reported in previous studies.27 Six out of the 14 curcuminoids, viz., 34, 36, 52, 55, 58, and 63, were selected for further screening against methicillin-resistant S. aureus (MRSA) ATCC 33591 strain and clinical isolates of MRSA, on the basis of their lower MIC values compared to those of others (MIC value ranged from 8 to 32 μg/mL). The most potent antibacterial activity was observed for curcuminoid 36 among all, with the lowest MIC value of 8 μg/mL against MSSA, 16 μg/mL against MRSA, and 8−16 μg/mL against clinical isolates of MRSA (Table 2). Curcuminoids 34, 52, 55, 58, and 63 also displayed considerable activity against MSSA and MRSA with MIC values in the range of 16−32 μg/mL. Their MIC values against clinical strains were in the range of 8−64 μg/mL (Table 2). The activity of curcuminoids was also compared with that of conventional antibiotics, tetracycline (TET), oxacillin (OXA), and vancomycin (VAN), under similar tested conditions. TET and OXA showed potent activity against MSSA but were less effective against MRSA strains including clinical isolates, whereas VAN displayed excellent potency against all screened staphylococcal strains (Table 2). Overall, we found six water-soluble curcuminoids with potent antibacterial activity against S. aureus, including clinical isolates of MRSA. Among them, curcuminoid 36 showed the best antibacterial activity. Interestingly, MIC of all six curcuminoids was found to be comparable or even better than conventional antibiotics tetracycline and oxacillin against MRSA strains.

2.3. Chemical Stability Analysis of Curcumin and Curcuminoids by UV−Visible Absorption Spectra. There are several reports that have established the chemical instability of curcumin in physiological buffer conditions.18 To compare the chemical stability of curcuminoids with curcumin, UV− visible absorption spectrum was recorded at different time points at intervals of 5 min in phosphate buffer saline (PBS: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4). The absorbance of curcumin decreased significantly with time, and it lost more than 50% of its original absorption intensity within 20 min of incubation. In comparison, there were no changes in absorbance intensity within 20 min for the studied curcuminoids (Figure 1). Their absorbance remained stable even after 60 min of incubation. As the environment of most of the cellular compartments is reducing in nature,28 we investigated the stability of curcumin and curcuminoids in presence of DTT, a strong reducing agent. DTT is one of the most commonly used reducing agents in biological buffers, and previous reports suggested that curcumin loses its biological functions upon treatment of DDT29 as it accelerates curcumin degradation. The absorption spectra showed that curcumin lost its absorbance peak at around 428 nm and 50% of its absorption intensity within 5 min of co-incubation with DTT, whereas there were no changes in the spectra of curcuminoids even after 20 min of incubation (see Figure S1, Supporting Information). As evident from Figure 1 and Figure S1, Supporting Information, these curcuminoids were found to be very stable in 678


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control, which showed 83.60 ± 5.38% cytotoxicity relative to untreated cells (Figure 3). Collectively, hemolytic assay and MTT assay thus suggested that the studied curcuminoids did not impart any detrimental effects on mammalian cells. As these curcuminoids exhibited negligible hemolysis and cytotoxicity even at much higher concentrations than the required concentration for their antistaphylococcal activity. 2.5. Killing Kinetics of Curcuminoids against Exponential and Stationary-Phase S. aureus. Most of the antibacterial compounds are only effective against actively dividing cells and display limited activity against nondividing cells. In many infections, bacteria seldom encounter conditions that are favorable for exponential growth and mostly remain in stationary phase. Stationary-phase cells of S. aureus exhibit significant tolerance to many antibiotics.30 Therefore, to determine the antibacterial efficacy of the curcuminoids, killing kinetics was performed against both exponential and stationary-phase MSSA at two concentrations of curcuminoids, i.e., 1× MIC and 5× MIC. The killing kinetics against exponential phase cells showed that all curcuminoids except 55, were able to cause >3 log reduction at 1× MIC upon 4 h of incubation and complete eradication within 6−24 h, as shown in Figure 4A. Among all tested curcuminoids, at 1× MIC, curcuminoid 36 showed the highest efficacy, causing complete eradication of staphylococcal cells (5 × 105 CFU/mL) within 4 h of incubation, whereas curcuminoid 55 was the least active, causing only ∼2.2 log reduction even after 24 h of incubation (Figure 4A).

physiological buffer as well as in reducing conditions unlike curcumin. 2.4. Hemolytic and Cytotoxicity Studies of Curcuminoids toward Mammalian Cells. To rationalize the therapeutic potential of curcuminoids, it is paramount to ascertain their toxicity toward mammalian cells. Thus, the hemolytic and cytotoxic potential of curcuminoids were evaluated against mouse red blood cells (RBCs) and 3T3 murine fibroblast cell line, respectively. Here, we chose two different concentrations of curcuminoids, i.e., 250 and 500 μg/ mL, which were ∼7.5 and ∼15 fold higher, respectively, compared to the highest MIC value (32 μg/mL) of curcuminoids against MSSA. As evident from Figure 2, all

Figure 2. Hemolytic activity of curcuminoids against mice RBCs. The percentage hemolysis was measured at two different concentrations of curcuminoids (250 and 500 μg/mL) upon 1 h of incubation. Hemolysis by 0.1% Triton X-100 was considered as 100%.

curcuminoids were found to be nonhemolytic up to 500 μg/ mL and caused only 2.58 ± 0.61 to 5.62 ± 0.69% hemolysis at 250 μg/mL and 5.39 ± 0.21 to 10.78 ± 3.6% hemolysis at 500 μg/mL of curcuminoids used. In addition to hemolytic assay, cytotoxicity of curcuminoids was also evaluated using MTT assay, which assesses cell viability in terms of the ability of living cells to reduce tetrazolium salt to formazan crystals by mitochondrial dehydrogenases. Result of MTT assay (Figure 3) confirmed that the curcuminoids did not impart any cytotoxic effects on mammalian cell line up to 500 μg/mL and resulted in only 6.74 ± 2.91 to 10.51 ± 2.30% cytotoxicity at 250 μg/mL and 11.26 ± 5.02 to 15.36 ± 4.72% cytotoxicity at 500 μg/mL concentration. Triton X-100 (2.0%) was used as a positive

Figure 3. Cytotoxicity of curcuminoids was determined by MTT assay against 3T3 murine fibroblast cell line following treatment with two different concentrations, i.e., 250 and 500 μg/mL. Each bar represents mean ± SD from three independent assays performed in triplicate.

Figure 4. Bacterial killing kinetics of curcuminoids against exponentially growing S. aureus ATCC 29213 (MSSA) in MHB medium (A) at 1× MIC and (B) at 5× MIC concentrations. The experiment was performed twice and similar data was obtained. Representative data is shown here. 679


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Figure 5. Membrane depolarization of S. aureus ATCC 29213 by curcuminoids using membrane potential-sensitive dye DiSC3(5). (A) The change in membrane potential induced by curcuminoids was measured in real time and expressed as % depolarization relative to 10 μM melittin (100%). Each data point represents mean ± SD from three independent experiments and the % depolarization difference between 1× MIC and 5× MIC is found to be statistically significant (p < 0.05) except for TET. (B) Representative data of depolarization kinetics observed at 5× MIC for 250 s is presented here.

Figure 6. Membrane permeabilization of S. aureus ATCC 29213 upon treatment with curcuminoids using calcein-AM. Percentage calcein leakage relative to untreated control upon incubation of dye-loaded cells for (A) 2 min and (B) 2 h with different concentrations (1× MIC and 5× MIC) of curcuminoids, TET, and 10 μg/mL gramicidin D (Gram D). Each bar represents mean ± SD from three independent experiments.

At 5× MIC, curcuminoids 34, 36, and 63 showed ∼1 log reduction instantaneously (5 min), indicating the rapid bactericidal potency of these curcuminoids. All curcuminoids except 55 were able to cause >3 log reduction in viable cell count within 2 h of incubation and complete eradication within 4−6 h of incubation. Curcuminoid 55 showed the bactericidal effect at 5× MIC after 24 h of incubation, which was slow compared with other curcuminoids (Figure 4B). Against stationary-phase cells, the curcuminoids exhibited equal effectiveness and showed an almost similar pattern of killing at every time point (see Figure S2, Supporting Information). This suggests that the antibacterial action of the curcuminoids does not depend on the growth phase of bacterial cells and is equally effective against both exponentially growing and stationary nondiving cells. 2.6. Mechanism of Action of the Curcuminoids. 2.6.1. Cytoplasmic Membrane Depolarization. Toward deciphering the mechanism of action, we examined whether the curcuminoids exert their antibacterial activities by disrupting the bacterial membrane integrity. For this, we evaluated their ability to depolarize bacterial membrane using DiSC3(5), a membrane potential-sensitive dye. The fluorescence of DiSC3(5) is self-quenched when it accumulates in cell interiors. If the membrane is depolarized in presence of a

depolarizing agent, the dye will be released from the cell, leading to a measurable increase in fluorescence. First, we evaluated the % depolarization by curcuminoids compared to that of 10 μM melittin (considered as 100%) at 1× MIC and 5× MIC in real time. The data showed concentration-dependent increase in depolarization, and they were found to be statistically significant (p < 0.05) except for TET, as at 1× MIC, the % depolarization was found to be 42.81 ± 2.93% for 34, 18.67 ± 4.67% for 36, 31.97 ± 4.34% for 52, 29.01 ± 2.46% for 55, 31.96 ± 1.24% for 58, 36.63 ± 6.20% for 63, and 6.06 ± 1.06% for TET. At 5× MIC, the % depolarization increased to 85.64 ± 10.66% for 34, 46.52 ± 8.82% for 36, 76.91 ± 8.33% for 52, 79.50 ± 6.32% for 55, 82.84 ± 7.28% for 58, 86.31 ± 4.58% for 63, and 11.37 ± 1.99% for TET. At both concentrations, curcuminoid 36 caused minimal depolarization. The depolarization trends observed for melittin and antibiotic TET were consistent with previous reports where it has been shown that melittin causes instant depolarization;31,32 in contrast, TET does not dissipate membrane potential.33,34 Further, kinetics of depolarization at 5× MIC was monitored for 250 s to examine the time-dependent increase in fluorescence and it was observed that depolarization was instant with the maximum increase within 2 min after addition of curcuminoids. (Figure 5B). Corresponding to the 680


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Figure 7. Epifluorescence microscopy assay for viability of S. aureus ATCC 29213 against 5× MIC concentration of curcuminoids. Fluorescence microscopy image of (A) untreated control cell, (B) 34-, (C) 36-, (D) 52-, (E) 55-, (F) 58-, and (G) 63-treated cells for 2 h after staining with fluorescent probes SYTO 9 and PI. The excitation/emission maxima is 480/500 nm for SYTO 9, corresponding to FITC (green) filter and 490/ 635 nm for PI, corresponding to CY-3 (red) filter.

mL), a well-known pore-forming peptide, caused an immediate 91.53 ± 3.29% dye leakage, whereas TET did not show membrane permeabilization. We further studied the time-dependent increase in membrane permeabilization. The results (Figure 6B) showed upon 2 h of incubation at 1× MIC all curcuminoids except 36 caused at least 80% permeabilization as the % leakage was 82.44 ± 4.08% for 34, 82.69 ± 6.03% for 52, 83.16 ± 3.89% for 55, 93.66 ± 4.57% for 58, and 98.17 ± 1.68% for 63. At 5× MIC, the permeabilization increased further as the % calcein leakage was observed to be 89.76 ± 2.44% for 34, 95.63 ± 1.93% for 52, 79.40 ± 9.86% for 55, 90.60 ± 6.99% for 58, and 98.00 ± 1.30% for 63 (Figure 6B). The % leakage was found to be 92.03 ± 1.89% for gramicidin D (10 μg/mL), whereas it was 8.02 ± 5.43% for tetracycline upon 2 h of incubation time, thus consistent with previous literature that gramicidin caused immediate membrane leakage by pore formation36 and tetracycline did not cause leakage.37 The histograms of calcein-loaded staphylococcal cells incubated with 1× MIC and 5× MIC concentrations of curcuminoids for 2 min and 2 h are provided in Figures S6−S9, Supporting Information. Overall, similar to membrane depolarization assay, all curcuminoids except 36, showed both time- as well as concentration-dependent increase in calcein leakage. The curcuminoid 36 did not show any membrane permeabilization, although the corresponding cell viability assay (Figure S5, Supporting Information) showed more than 4 log reduction in bacterial cell count. This suggests that the membrane may not be the primary target for curcuminoid 36. Like many other previously reported compounds,38,39 it might be penetrating the cell membrane and interfering with the vital functions intracellularly, other than membrane disruption. To find out its intracellular targets, we need a further study. Rest of the curcuminoids exhibited a membrane disrupting mode of

depolarization, a cell viability assay was set up to evaluate if depolarization was a lethal event (see Figure S4, Supporting Information). Overall, all curcuminoids except 36 could rapidly dissipate the membrane potential in bacterial cells and caused at least 75% depolarization, suggesting membrane perturbation may be their primary mode of action. Notably, the curcuminoid 36, which caused more than 2 log reduction in cell count in the corresponding cell viability assay, showed the least membrane depolarization at both concentrations 1× MIC and 5× MIC. This suggests that staphylocidal activity of curcuminoid 36 was independent of bacterial membrane depolarization and membrane perturbation was a less probable mode of action. 2.6.2. Cytoplasmic Membrane Permeabilization by Calcein Leakage Assay. After studying instant membrane depolarization to have a better insight into membrane perturbation mode of action, next, we studied the ability of these curcuminoids to permeabilize the bacterial membrane using calcein-AM.35 It is a nonfluorescent and lipid soluble dye and as such can easily cross membranes. Once within the target cells, it is hydrolyzed by cytoplasmic esterases into membraneimpermeable calcein, which is fluorescent and cannot leak out of the cell unless the membrane is damaged. The results (Figure 6A) showed that after 2 min of exposure, all curcuminoids at 1× MIC, except 34 and 36, caused at least 40% permeabilization as the % leakage observed was 47.03 ± 12.76% for 52, 89.12 ± 2.74% for 55, 51.49 ± 8.0% for 58, and 40.45 ± 13.01% for 63. By increasing the concentration to 5× MIC, the % leakage increased to 58.75 ± 8.12 for 34, 81.51 ± 12.65% for 52, 95.78 ± 2.84% for 55, 84.81 ± 8.58% for 58, and 78.89 ± 9.29% for 63. This suggests that membrane permeabilization caused by curcuminoids was concentrationdependent except for 36, which did not cause any significant leakage (Figure 6A). In comparison, gramicidin D (10 μg/ 681


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Figure 8. Scanning electron microscopic images of S. aureus ATCC 29213 treated with 5× MIC of curcuminoids and 10 μg/mL gramicidin D for 4 h in 10% MHB medium. (A) Untreated control cells, (B) 34, (C) 36, (D) 52, (E) 55, (F) 58, (G) 63, and (H) gramicidin D.

Figure 9. Transmission electron microscopic images of S. aureus ATCC 29213 treated with 5× MIC of curcuminoids and 10 μg/mL gramicidin D for 4 h in 10% MHB medium. (A) Untreated control cells, (B) 34, (C) 36, (D) 52, (E) 55, (F) 58, (G) 63, and (H) gramicidin D.

36 appeared yellow (where both the green and red dye were retained within cells). This might be due to very minor damage to the cellular membrane that allows some of the PI to enter the cell and bind to DNA but is not sufficient enough to replace all of the SYTO 9 bound to DNA; thus, they appear yellow.40,41 2.6.4. Morphological Changes in S. aureus on Exposure to Curcuminoids Observed via Scanning Electron Microscopy (SEM). To further assess if curcuminoids can cause surface perturbations, alterations in surface integrity of MSSA were visualized using scanning electron microscopy (SEM) for 4 h after incubation with 5× MIC curcuminoids. As shown in Figure 8A, the untreated cells looked bright and round with a smooth intact surface. Upon treatment with all curcuminoids at 5× MIC for 4 h, the surface perturbation of cells was evident and resulted in bleb formation, rough-surfaced and irregularly shaped cells with dent, and depression on the surface (Figure

action, suggesting that the bacterial membrane could be the primary target for these curcuminoids. 2.6.3. Evaluation of Membrane Damage through Fluorescence Microscopy. The LIVE/DEAD kit was used to assess the viability and membrane integrity of MSSA cells (109 CFU/mL) treated with 5× MIC of each curcuminoid for 2 h. For comparison, the untreated cells in PBS buffer were taken as control (Figure 7). Fluorescence setting for FITC (green) and Cy-3 (red) filters followed by a bright field was used to view the sample, and merged (FITC+Cy-3) images of each sample are presented in Figure 7. The images of the untreated control samples show predominantly green cells, indicating that cells are viable and intact when untreated. However, the images of all curcuminoid-treated cells except 36-treated sample (Figure 7) were populated with red cells, indicating the loss of viability and membrane integrity of cells in the presence of curcuminoids. The cells treated with curcuminoid 682


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4. EXPERIMENTAL SECTION 4.1. Chemicals. Curcumin, calcein-acetoxymethyl ester (calcein-AM), melittin, dimethylsulfoxide (DMSO), propidium iodide (PI), gramicidin D, Dulbecco’s modified Eagle’s medium (DMEM), dithiothreitol (DTT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3,3′dipropylthiadicarbocyanine iodide (DiSC3(5)), vancomycin (VAN), and oxacillin sodium salt monohydrate (OXA) were acquired from Sigma-Aldrich. Cation-adjusted Mueller Hinton broth (MHB) was procured from Difco. Live/Dead BacLight viability assay kit was purchased from Invitrogen (Eugene, OR). Sodium dihydrogen orthophosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen orthophosphate dihydrate (Na2HPO4·2H2O), and sodium chloride (NaCl) were purchased from Qualigens Fine Chemicals, India. Tetracycline hydrochloride (TET), agar powder, and brain heart infusion (BHI) medium were procured from Himedia Laboratories, India. Glutaraldehyde and Triton X-100 were purchased from Merck, Germany. 4.2. Bacterial Strains. S. aureus strain ATCC 29213 (MSSA), S. aureus strain ATCC 33591 (MRSA), and six clinical isolates of MRSA (S-28, S-30, S-33, S-34, S-37, and S41) were used in this study. All MRSA clinical isolates were kindly provided by Dr. Benu Dhawan, Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India. The bacterial strains were stored in 15% (v/v) glycerol stock at −80 °C till subcultured on nutrient agar plates for further use. 4.3. Methodology. 4.3.1. Antibacterial Activity under Planktonic Conditions. The antibacterial activity of the curcuminoids has been reported as their MIC and determined using classical broth micro dilution assay using Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) guidelines.46 In brief, S. aureus was allowed to grow overnight at 37 °C in MHB medium. The suspension from overnight grown bacteria was used to inoculate fresh MHB medium and incubated for 3−5 h to reach mid-log phase. The cells were resuspended in MHB medium to OD600 = 0.5 (∼108 CFU/ mL). Curcuminoids/antibiotics were diluted using 2-fold serial dilution in 100 μL of cation-adjusted MHB medium in the first 10 columns of a 96-well microtiter plate. Then, aliquots (100 μL) of exponential phase bacterial suspension was added to each well of the first 11 columns to get a final inoculum density of 5 × 105 CFU/mL. The culture without the test drug (in the 11th column) was used as growth control, and uninoculated MHB in the last column without cells and drugs was used as negative control. The microtiter plate was incubated at 37 °C for 16−18 h. After incubation, OD600 was recorded using a plate reader (Verioskan Flash Multiplate reader, Thermo Scientific). For comparisons, the standard antibiotics vancomycin (VAN), tetracycline (TET), and oxacillin (OXA) were also tested under similar conditions. The experiment was carried out in duplicate on three different days. 4.3.2. Chemical Stability Analysis of Curcumin and Curcuminoids by UV−Visible Absorption Spectra. To evaluate the stability of curcumin and curcuminoids under physiological buffer conditions, absorption spectra were recorded from 250 to 600 nm using a UV−vis spectrophotometer (UV-2450 Spectrophotometer, Shimadzu). For this, curcumin was dissolved in DMSO and curcuminoids were dissolved in water, as these curcuminoids were water soluble. A small volume (∼1 μL) of curcumin and curcuminoids was

8B−G). The loss of surface morphology and release of intracellular content as debris in curcuminoid-treated cells was comparable with 10 μg/mL gramicidin D-treated cells (Figure 8H). 2.6.5. Ultrastructural Changes in S. aureus on Exposure to Curcuminoids Observed via Transmission Electron Microscopy (TEM). Further, ultrastructural changes in the cell wall and membrane of MSSA treated with 5× MIC curcuminoids for 4 h were examined by TEM and compared to those of gramicidin D (10 μg/mL)-treated cells. As shown in Figure 9A, the untreated cells had an intact cell wall and cell membrane. After incubation with curcuminoids, surface integrity was largely compromised. Many cells were devoid of a cell wall (ghost cells) and lacked cytoplasmic content, whereas few cells had remnants of the cell membrane and cell wall through which the cytoplasmic content was leaking out (Figure 9B−H). The results were comparable with those of gramicidin D (10 μg/mL)-treated cells.

3. CONCLUSIONS In the present study, the antibacterial activities of 33 curcuminoids have been studied against a series of different S. aureus strains. Out of these curcuminoids, six compounds showed excellent potency in the range of 8−64 μg/mL. Killing kinetics in growth medium showed equal effectiveness of these curcuminoids against both exponentially dividing cells and stationary-phase nondiving cells. These curcuminoids were found to be nonhemolytic and nontoxic against mammalian cells even at a concentration much higher than the dose required for their antibacterial activity. To decipher the mechanism of the rapid bactericidal activity of these curcuminoids, we investigated the interaction of these curcuminoids with the bacterial membrane and carried out both membrane depolarization and permeabilization assays. All curcuminoids except compound 36 showed immediate concentration-dependent membrane depolarization and permeabilization. However, the most potent bactericidal curcuminoid 36 showed the least membrane depolarization and permeabilization at studied concentrations and time points. This suggests that the membrane may not be the primary target for this curcuminoid. To elucidate the mode of action of this curcuminoid 36, further studies need to be performed. Rest of the other curcuminoids exhibited membrane disrupting/depolarizing mode of action against S. aureus, suggesting that the bacterial membrane is the primary target for these curcuminoids. These findings were further corroborated by fluorescence and microscopy-based assays. Fluorescence microscopy studies also confirmed the excessive entry of PI stain in bacterial cells due to membrane damage by these curcuminoids. The SEM and TEM images of all curcuminoidtreated cells also showed surface perturbation including morphological changes; these effects were not present in untreated control cells. Overall, the present study affords water-soluble, stable under physiological conditions, nonhemolytic, nontoxic, membranetargeting curcuminoids and provides valuable information for further research for therapeutic use of these lead curcuminoids against S. aureus. Further, combination therapy combining these curcuminoids with conventional antibiotics represents a potential area for future investigation. As recent discoveries have shown, there is promise and potential of a combination therapy in the treatment of MDR bacterial infection42,43 and it might reverse the antibiotic resistance.44,45 683


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added in 1 mL of PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) from the stock solution of 20 mM to achieve a final concentration of 25 μM. The absorbance spectra of this reaction mixture were recorded for a span of 20 min at 5 min intervals. Cytoplasmic environment is reducing in nature, so to check the stability of curcumin and curcuminoids under reducing conditions, the same experiment was repeated and absorption spectra were recorded in presence of 10 μM DTT, a strong reducing agent. 4.3.3. Hemolytic Assay. The assay was performed as described previously.47 Briefly, fresh blood drawn from mice in presence of an anticoagulant was centrifuged at 1500 rpm for 10 min to isolate red blood cells (RBC). The pellet of RBC was washed thrice and resuspended to 4% v/v in PBS buffer (35 mM sodium phosphate, 150 mM NaCl, pH 7.4). Next, 100 μL of 2-fold dilutions of curcuminoids were added to 100 μL of RBC suspension in a 96-well plate and incubated for 1 h at 37 °C. Untreated RBC suspension was used as negative control, and RBC suspension incubated with 0.1% Triton X100 was considered as positive control. After incubation, the plate was centrifuged and 20 μL of supernatant from each well was added to 80 μL of PBS buffer in a new microtiter plate. The absorbance of the released hemoglobin from lysed RBCs was measured at 414 nm using a plate reader (Verioskan Flash Multiplate reader, Thermo Scientific). Percentage hemolysis was calculated using the following equation

% cytotoxicity = [(OD of negative control − OD of sample) /(OD of negative control)] × 100

4.3.5. Bacterial Killing Kinetics Assay. The efficacy of curcuminoids against S. aureus ATCC 29213 was assessed against exponential and stationary-phase cells in MHB medium using a previously defined assay.48 Briefly, the bacterial cells were grown in MHB medium for 16−18 h till they reached a stationary phase. Next, fresh medium was inoculated with 1% of this suspension and grown for 4−6 h till mid-log phase. Both stationary and mid-log-phase cells were resuspended separately in MHB to achieve OD600 = 0.5, which is ∼108 CFU/mL. A final inoculum of approximately 5 × 105 CFU/mL was exposed to 1× MIC and 5× MIC concentrations of curcuminoids at 37 °C for 24 h. Afterward, at selected time points (0, 1, 2, 4, 6, and 24 h), 50 μL aliquots were withdrawn and subjected to dilution in PBS buffer and plated in triplicate on BHI agar plate. The agar plates were incubated overnight at 37 °C. Next morning, viable colonies were counted and represented as log10 CFU/mL. The bactericidal activity was defined as ≥3 log reduction in viable cell count compared to the untreated cells at the start of the experiment. For comparison, killing kinetics of standard antibiotic TET was performed along with curcuminoids. The experiment was carried out on three separate days independently. 4.3.6. Membrane Depolarization Assay. Membrane depolarization was monitored using the potential-sensitive fluorescent probe DiSC3(5), as reported in our previous publications.47,48 Briefly, MSSA cells were grown in MHB medium for 4−6 h till the exponential phase. The cells were harvested by centrifugation, washed, and resuspended to OD600 of 0.05 (2.5 × 106 CFU/mL) in respiration buffer (5 mM HEPES, 20 mM glucose, pH 7.4). Subsequently, cell suspensions were incubated with 3.6 μM DiSC3(5) for ∼30 min in dark till their fluorescence got quenched. The loaded cells were then placed in a cuvette to which 1× MIC and 5× MIC concentrations of curcuminoids were added and then the increase in fluorescence intensity was monitored in real time by a Shimadzu RF-5301 PC spectrofluorimeter (Japan). The emission spectra were recorded with a 3 nm slit using excitation and emission wavelengths of 620 and 670 nm, respectively. The percentage depolarization was calculated in comparison to 10 μM melittin, which triggers a rapid and complete dissipation of membrane potential (100% depolarization).31,48 For kinetics of depolarization, changes in fluorescence intensity were recorded from 0 to 250 s. The experiment was carried out on three different days. 4.3.7. Membrane Permeabilization Assay. The bacterial membrane permeabilization induced by curcuminoids was detected and quantified by calcein leakage assay using a flow cytometer (BD FACS verse, San Jose, CA), as described in our previous publications with slight modifications.49,50 Briefly, mid-logarithmic phase cells of S. aureus were adjusted to OD600 = 1.0 (∼109 CFU/mL) in PBS buffer, as previously described. Next, cells were incubated with 2 μg/mL calcein-AM for 2 h at 37 °C in the presence of 10% MHB to energize the cells. The loaded cells were then washed and resuspended in PBS buffer to OD600 = 0.5 (∼108 CFU/mL). The loaded cells were diluted to a final inoculum of approximately 2.5 × 106 CFU/mL and exposed to 1× MIC and 5× MIC concentrations of curcuminoids for 2 h in the

% hemolysis = [(OD of sample − OD of PBS) /(OD of 0.1% Triton‐X − OD of PBS)] × 100

Swiss albino mice were procured from the Animal House Facility, Jawaharlal Nehru University (JNU), New Delhi, India. The assay was performed according to the guidelines of Institutional Animal Ethics Committee (IAEC-02/2014) of JNU, New Delhi and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). 4.3.4. Cytotoxicity Assay. The potential cytotoxic effect of curcuminoids was determined against mouse fibroblast 3T3 cells (a kind gift from Prof. Rakesh K. Tyagi, SCMM, JNU) using MTT assay, as described in our previous reports.47,48 Briefly, cells were grown in 10% serum-supplemented DMEM medium at 37 °C in a 5% CO2 incubator until they reached around 75% confluency. Approximately, 0.8 × 105 cells were seeded in a 24-well plate and incubated for 24 h. Thereafter, used media was removed and cells were suspended in fresh media with 10% FBS containing different concentrations of curcuminoids. After 4 h of incubation, cells were washed with PBS buffer and 1 mL of MTT solution (0.1 mg/mL) was put into each well. The plate was incubated in the dark for 2 h additionally. Thereafter, the supernatant was discarded and 200 μL of DMSO was added to each well to solubilize formazan crystals and then the absorbance was recorded at 570 nm using a plate reader (Verioskan Flash Multiplate reader, Thermo Scientific). The absorbance for untreated control cells was considered as growth control, with 100% cell viability. Percentage cytotoxicity was determined using the following equation 684


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dark at 37 °C. Thereafter, fluorescence of the released calcein was detected using excitation and emission wavelengths of 490 and 517 nm, respectively. For each analysis, a total of 10 000 cells were acquired. Cells showing 10 FL units were interpreted to have retained calcein, which suggests that the membrane remained intact. The test was reproduced on three separate days independently. 4.3.8. Microscopic Evaluation of the Effect of Curcuminoids (Membrane Damage) on Bacterial Viability. The membrane integrity and bacterial viability were assessed using the LIVE/DEAD BacLight Kit according to the manufacturer’s protocol.35,50 This epifluorescence staining method is based on a combination of two nucleic acid fluorescent dyes SYTO 9 and PI. SYTO 9 is a green fluorescent dye that stains all cells, and PI permeates only the cells with a damaged membrane. Since PI has a stronger affinity for DNA than SYTO 9, it replaces SYTO 9 from DNA, causing reduction in green fluorescence of SYTO 9.50 Briefly, a solution containing a mixture of SYTO 9 and PI (equal volume from each dye) was prepared. The bacterial suspensions (∼109 CFU/mL) were treated with 5× MIC curcuminoids for 2 h were incubated with the dye mixture of SYTO 9 and PI for 15 min in the dark. A 5 μL aliquot of the stained sample was kept on a clean glass slide and covered with a coverslip, and the images were acquired under a fluorescence microscope (Olympus Fluoview FV 1000 model) with filters that allow green and red light excitations. The experiment was performed on two different days. 4.3.9. Scanning Electron Microscopy (SEM). For SEM, samples were prepared as described previously in our publications.49,50 Briefly, mid-logarithmic-phase cells (109 CFU/mL) were treated with 5× MIC curcuminoids in 10% MHB, at 37 °C with shaking for 4 h. For comparison, untreated bacterial cells and gramicidin D-treated cells were used as controls. After incubation, PB buffer (10 mM sodium phosphate, pH 7.4) was used to wash the cells and fixed with 2.5% glutaraldehyde overnight at 4 °C. The next day, cells were again washed thrice with the same buffer and dehydrated sequentially with 30−100% ethanol for 10 min each and dried on a coverslip under vacuum in a desiccator. Finally, the cells were coated with gold and viewed via a scanning electron microscope (EVO 40, Carl Zeiss, Germany) available at AIRF, JNU. The experiment was performed twice independently on two separate days, and similar images were acquired. 4.3.10. Transmission Electron Microscopy (TEM). For TEM, samples were prepared as described previously in our publications.49 The sample preparation was identical to the SEM protocol until overnight fixation with 2.5% glutaraldehyde at 4 °C. Osmium tetroxide (1%) was used to postfix the cells. The cells were then dehydrated sequentially with 50− 100% acetone. Each sample was fixed in epoxy resin and finesectioned using microtome. The sections were placed onto a copper grid and stained with uranyl acetate followed by lead citrate. Samples were dried under vacuum in a desiccator after washing them twice in Milli-Q water. The transmission electron microscopy (Zeol-JEM 2100, Japan) available at AIRF, JNU was used to view the grid. For comparison, untreated bacterial cells and gramicidin D-treated (10 μg/mL) cells were used as control.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02625.

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MIC of all 14 curcuminoids against methicillin-sensitive S. aureus ATCC 29213; UV−visible absorption spectra of curcumin and curcuminoids in PBS buffer (pH 7.4) in the presence of 10 μM DTT; killing kinetics against stationary-phase S. aureus; fluorescence emission spectrum of DiSC3(5); cell viability assay corresponding to depolarization and permeabilization assay; histograms of calcein-loaded cells treated with different concentrations of the curcuminoids at different time points (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected]. Phone: 011-26704307, 09871955700 (K.M.). *E-mail: [email protected]. Phone: 27667501, 27667794 Ext 177 (D.S.R.). ORCID

Diwan S. Rawat: 0000-0002-5473-7476 Author Contributions

P.K. designed and performed the experiments, analyzed the data, and wrote the manuscript. S.K.K. and S.M. prepared curcuminoid compounds. K.M. and D.S.R. conceived and designed the experiments and wrote the manuscript. All authors reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the grants from DSTSERB, DST-PURSE (Phase-II), and UPE-II (UGC) to K.M. and DST-SERB [EMR/2014/001127] New Delhi, Govt. of India and DST-PURSE (Phase-II) to D.S.R. P.K. is grateful to UGC and S.M. and S.K.K. are grateful to CSIR for Senior Research fellowship, respectively. We acknowledge Ashok Sahu, Dr. Ruchita Pal, and Manu Vashistha, AIRF, JNU for their help in acquisition of confocal microscopy, SEM, and TEM images respectively. We thank Dr. Benu Dhawan, AIIMS for providing clinical isolates of MRSA and Prof. Rakesh K. Tyagi, SCMM, JNU for providing 3T3 mouse fibroblast cell line.

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ABBREVIATIONS S. aureus, Staphylococcus aureus; MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; DTT, dithiothreitol; PI, propidium iodide; VAN, vancomycin; TET, tetracycline; OXA, oxacillin

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REFERENCES

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