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Journal of Antimicrobial Chemotherapy (2002) 49, 641-649
© 2002 The British Society for Antimicrobial Chemotherapy

In vitro action of carboxyfullerene

Nina Tsaoa, Tien-Yau Luhb, Chen-Kung Chouc, Tsuey-Yu Changd, Jiunn-Jong Wue, Ching-Chuan Liuf and Huan-Yao Leia,*

a Departments of Microbiology and Immunology, d Parasitology , e Medical Technology and f Pediatrics, College of Medicine, National Cheng Kung University, Tainan; b Department of Chemistry, National Taiwan University; c Department of Medical Research, Veteran General Hospital, Taipei, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fullerene compounds have avid reactivity with free radicals and are regarded as ‘radical sponges’. The trimalonic acid derivative of fullerene is one of the water-soluble compounds that has been synthesized and found to be an effective antioxidant both in vivo and in vitro. Carboxyfullerene has been shown to be effective in the treatment of both Gram-positive and -negative infections, although its mode of action is poorly understood. We determined the MIC and minimal bactericidal concentration of carboxyfullerene for 20 isolates, including Staphylococcus spp., Streptococcus spp., Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Klebsiella pneumoniae. We further investigated the action of carboxyfullerene using transmission electron microscopy (TEM), anticarboxyfullerene antibody binding assay and a membrane perturbation assay. All Grampositive species were inhibited by 50 mg/L of carboxyfullerene, whereas Gram-negative species were not inhibited, even at 500 mg/L carboxyfullerene. Bactericidal activity was demonstrated only for Gram-positive species, particularly for Streptococcus pyogenes A-20, which was killed rapidly. Intercalation of carboxyfullerene into the cell wall of staphylococci and streptococci was demonstrated by TEM and anti-carboxyfullerene binding assay. Damage to the cell membrane in Gram-positive, but not Gram-negative, bacteria was confirmed by the membrane perturbation assay. These findings indicate that the action of carboxyfullerene on Gram-positive bacteria is achieved by insertion into the cell wall and destruction of membrane integrity.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The pure carbon spheres of C60 (buckminsterfullerene) have attracted considerable attention in different fields of science and engineering since their discovery, and investigation of the chemical and physical properties of fullerenes has yielded much information. Fullerene compounds have unique cage structures that allow them to interact with biomolecules and have avid reactivity with free radicals; therefore, they are regarded as ‘radical sponges’.1 These properties of fullerene have generated interest in biomedical research.1,2 However, because native fullerene is not soluble in water, it is necessary to convert hydrophobic fullerene into water-soluble derivatives before they can be used as free radical scavengers and antioxidants in medical applications. Several strategies have been used to enhance water solubility, and such forms are reported to have protective effects in various systems.3–8 The trimalonic acid derivative of fullerene, carboxyfullerene [C63(COOH)6], is one of the water soluble compounds that has been synthesized and found to be an effective neuroprotective antioxidant both in vitro and in vivo.9,10 Furthermore, carboxyfullerene also prevents cells from undergoing apoptosis in various systems.11,12

In our previous studies, we found that carboxyfullerene could inhibit Escherichia coli-induced meningitis by decreasing the damage caused by infiltrating neutrophils on the blood–brain barrier,13 but not by direct inhibition of the growth of E. coli.14 Moreover, we also demonstrated that group A streptococcal infection could be inhibited by carboxyfullerene, and that carboxyfullerene not only enhanced the bactericidal activity of infiltrating neutrophils, but also inhibited the growth of Streptococcus pyogenes A-20.15 Since carboxyfullerene had differential effects on E. coli and S. pyogenes A-20, the efficacy of carboxyfullerene on different Gram-positive and Gram-negative bacteria was studied further. In this study, we demonstrate that carboxyfullerene has bactericidal activity on Gram-positive staphylococci and streptococci, and that its lethal action was achieved by insertion into the cell walls of bacteria and disruption of their structure.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Carboxyfullerene

The water-soluble carboxylic acid C60 derivative with C3 symmetry, an effective free-radical scavenger, was synthesized as described previously.9 In this study, C63(COOH)6 (referred to as C3) dissolved in phosphate-buffered saline (PBS; 10 mg/mL) was used throughout.

Bacteria

Sixteen clinical isolates from National Cheng Kung University Hospital consisting of two oxacillin-resistant Staphylococcus aureus, two Staphylococcus epidermidis, three group B streptococci, four vancomycin-resistant Enterococcus faecalis (nos 002–005), three vancomycin-susceptible E. faecalis (nos 474, 487, 496) and two Streptococcus pneumoniae (serotypes 6 and 14) were used. As well as the strains mentioned above, S. pyogenes clinical isolate A-20 and its isogenic mutant S. pyogenes SW507, which was deficient in the speB gene, and another S. pyogenes NZ-131 and its speB- isogenic mutant, S. pyogenes SW510, were also used in this study.16 In our previous studies, the in vivo protective effect of carboxyfullerene on S. pyogenes SW507-induced infection was more effective than the S. pyogenes A-20-induced one (unpublished observation), so the in vitro effect of carboxyfullerene on these bacteria was studied further. Furthermore, Gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, which were obtained from the Culture Collection and Research Center (Food Industry Research & Development Institute, Hsinchu, Taiwan), and a clinical isolate of each of Salmonella typhi and Klebsiella pneumoniae were used for comparison.

Growth curves

The growth curves for bacteria after treatment with carboxyfullerene were determined. Staphylococcus spp. and Streptococcus spp., except S. pneumoniae, were cultured in brain–heart infusion broth and TSBY broth [tryptic soy broth (Difco Laboratories, Detroit, MI, USA) containing yeast extract 0.5% (w/v)], at 37°C for 12 h, respectively. S. pneumoniae was cultured in Todd–Hewitt broth (Difco Laboratories) at 37°C for 12 h in 5% CO2 in air. Gramnegative bacteria, including E. coli, P. aeruginosa, S. typhi and K. pneumoniae, were cultured in Luria–Bertani (LB) broth [1% (w/v) NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract]. After 12 h, the bacteria were subcultured (1/50 volume) into fresh broth for a further 12 h. During subculture, different concentrations (5–200 mg/L) of carboxyfullerene were added to the bacterial suspensions, and the growth curves of bacteria at different times were determined using a spectrophotometer (Beckman Instruments, Somerset, NJ, USA) by measuring the optical density at 600 nm. For quantification of viable bacteria, bacterial suspensions collected at different times were diluted 105-107 and 0.1 mL plated on to blood or LB agar plates. The number of colony-forming units (cfu) of bacteria was counted and was expressed as cfu/mL. These experiments were repeated three times.

MICs and MBCs

MICs and minimal bactericidal concentrations (MBCs) were determined according to NCCLS methodology.17 Bacteria were cultured for 12 h and then subcultured into fresh broth; the concentration of bacteria was adjusted to 5 x 105 cfu/mL in 2 mL broth. Different concentrations (5–500 mg/L) of carboxyfullerene were added to the bacterial suspension, which was incubated overnight. The minimal concentration of carboxyfullerene in non-turbid tubes represented the MIC. Non-turbid tubes were subcultured (0.1 mL) on to agar plates and incubated overnight. The surviving cfu/mL of bacteria were calculated from the number of colonies grown. The minimal concentration of carboxyfullerene on agar plates, where the number of cfu/mL was <0.1% of the initial concentration of bacteria, was taken to represent the MBC.

Direct bactericidal assay

As well as the MBC described above, direct bactericidal activity of carboxyfullerene after a short period (<3 h incubation) was determined. High concentrations of S. pyogenes A-20 (1 x 109 cfu/mL) were obtained from exponential phase cultures and incubated with various concentrations of carboxyfullerene (10–100 mg/L) in PBS. At various intervals, the concentration of bacteria was determined by plating on to blood agar (Becton Dickinson, Cockeysville, MD, USA) overnight. To avoid the carry-over effect of carboxyfullerene remnant in culture medium, the suspension of bacteria collected at various intervals was diluted with PBS at least 10 times; therefore, the lower limit of detection for 0.1 mL samples was 1 x 102 cfu/mL. PBS was used to prevent bacteria from continued growth, but it will also cause a slight decrease in bacterial viability during the 3 h incubation. These experiments were repeated three times. The result was expressed as the mean cfu ± s.d./mL.

Transmission electron microscopic examination

The bacteria collected from the exponential phase were cultured with carboxyfullerene (50 mg/L) for 30 min at 37°C, then washed several times with PBS. Subsequently, the bacteria were fixed with 1.2% glutaldehyde in PBS containing 1 g/L of ruthenium red for the staining of the capsule at 4°C for 1 h.18 After several washes, the bacteria were osmicated in 1% osmium tetraoxide (Electron Microscopy Science, Washington, PA, USA) for 1 h, and then were dehydrated in a graded series of ethanol, cleared in propylene oxide and flat-embedded in EPON plastic [30% (w/w) dodecenyl succinic anhydride, 51% (w/w) EM bed-812, 18% (w/w) dadic methyl anhydride, 1% (w/w) 2,4,6-tri(dimethylaminomethyl) phenol] (Electron Microscopy Science). Thin sections (80 µm) were examined using an electron microscope (JEOL JEM-1200EX) at 75 kV.

Anti-carboxyfullerene antibody binding assay

The anti-carboxyfullerene antibody binding assay was performed using protein G/A–Sepharose beads coated with anti-carboxyfullerene antibodies, which were generated by immunizing BALB/c mice with 50 µg carboxyfullerene in Freund's complete adjuvant (Sigma Chemical Co., St Louis, MO, USA) five times.19 In order to remove antibacterial antibodies present in sera, the sera from naive and carboxyfullerene-primed mice were pre-absorbed with S. aureus, S. pyogenes, E. coli or P. aeruginosa. The pre-absorbed sera were incubated at 4°C overnight with Sepharose beads containing protein A and protein G (Amersham Pharmacia Biotech, Uppsala, Sweden), which are known to have high affinity for the Fc portion of immunoglobulin G.20,21 Bacteria collected from the exponential phase were cultured with carboxyfullerene (50 mg/L) for 30 min at 37°C, then washed several times with PBS. The treated bacteria were incubated with Sepharose beads that had conjugated with anti-carboxyfullerene antibodies for 1 h at room temperature, and non-binding bacteria were washed off with PBS containing 0.5% (v/v) Tween-20. The bacteria bound to Sepharose beads were visualized using Gram's stain.

Membrane perturbation assay

The membrane perturbing assay was performed using propidium iodide (PI) fluorescence detection by flow cytometry.22 The bacteria (2 x 107 cfu/mL) collected from the exponential phase were cultured with different concentrations (5–200 mg/L) of carboxyfullerene at 37°C for various times. At various intervals, the bacteria were collected and stained with PI (400 mg/L) for 10 min, and dye penetration was measured by the presence of fluorescence emission at 560 nm.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
To examine the inhibitory activity of carboxyfullerene in different bacteria, we selected Gram-positive bacteria (S. aureus, S. epidermidis, groups A and B streptococci, Enterococcus spp. and S. pneumoniae) and Gram-negative bacteria (E. coli, P. aeruginosa, S. typhi and K. pneumoniae) to perform the in vitro inhibition assays. As shown in Figure 1Go, the growth of all Gram-positive cocci selected for these assays was inhibited by carboxyfullerene at 50 mg/L, except S. pyogenes A-20, which was inhibited at 5 mg/L. However, carboxyfullerene had no effect on the growth of Gram-negative bacteria, even at 200 mg/L (Figure 2Go). The MICs and MBCs of carboxyfullerene determined by standard protocols17 are shown in the TableGo.



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  		Figure 1. . Growth inhibition of Gram-positive cocci by carboxyfullerene. The growth curve of Gram-positive cocci after treatment with carboxyfullerene was determined as described in Materials and methods. Results shown are from one representative experiment. {square}, PBS; {diamondsuit}, carboxyfullerene 5 mg/L; {circ}, carboxyfullerene 50 mg/L; {triangleup}, carboxyfullerene 200 mg/L.

 


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  		Figure 2. . Carboxyfullerene has no effect on the growth of Gram-negative bacteria. The growth curve of Gram-negative bacteria after treatment with carboxyfullerene was determined as described in Materials and methods. Results shown are from one representative experiment. {square}, PBS; {diamondsuit}, carboxyfullerene 5 mg/L; {circ}, carboxyfullerene 50 mg/L; {triangleup}, carboxyfullerene 200 mg/L.

 

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  		Table. . The MIC and MBC of carboxyfullerene for different bacteria
 
To confirm direct bactericidal activity of carboxyfullerene on S. pyogenes A-20, we exposed a high concentration of bacteria (1 x 109 cfu/mL) to carboxyfullerene incubated over a short period of time. The viable bacterial count is shown in Figure 3Go. Carboxyfullerene killed S. pyogenes A-20 effectively. The viable bacteria decreased from 1 x 109 to 5 x 105 or 1 x 103 cfu/mL at 10 or 100 mg/L of carboxyfullerene after incubation for 1 h. No viable bacteria were found after a 3 h incubation with carboxyfullerene 100 mg/L. The bactericidal effect of carboxyfullerene was also observed in other staphylococci and streptococci (data not shown). These results indicate that carboxyfullerene is bactericidal against Gram-positive cocci.



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  		Figure 3. . Direct bactericidal activity of carboxyfullerene on S. pyogenes A-20. Direct bactericidal activity of carboxyfullerene on S. pyogenes A-20 at short periods was determined as described in Materials and methods. Results shown are from one representative experiment. The results are expressed as mean cfu/mL ± s.d. of triplicate cultures. The detection limit was 100 cfu/mL. {square}, PBS; {circ}, carboxyfullerene 10 mg/L; {blacktriangleup}, carboxyfullerene 100 mg/L.

 
To further examine the effect of carboxyfullerene on Gram-positive cocci, S. pyogenes A-20 and S. aureus were incubated with carboxyfullerene 50 mg/L for 30 min, and the bacterial structure was examined using TEM. Utilizing ruthenium red staining, PBS-treated S. aureus were shown to have smooth capsules surrounding the cell wall (Figure 4aGo). However, carboxyfullerene-treated S. aureus had a cottony surface, indicating that the structure of the cell wall was damaged by carboxyfullerene (Figure 4bGo), and the degree of damage was more significant after incubation for 90 min (data not shown). The same result was found for carboxyfullerene-treated S. pyogenes A-20 (Figure 4dGo), but not for Gram-negative bacteria (E. coli and P. aeruginosa; Figure 4eGo–h). Many ‘tiny sticks’ were found on the cell wall of bacteria after treatment with carboxyfullerene, indicating that carboxyfullerene might insert into the cell wall. In order to confirm this hypothesis, we generated anticarboxyfullerene antibodies in order to carry out a binding assay. Carboxyfullerene-specific antibodies that bound to protein A/G–Sepharose were incubated with carboxyfullerene-treated S. pyogenes or S. aureus. As shown in Figure 5Go, Sepharose beads containing anti-carboxyfullerene antibodies could bind carboxyfullerene-treated Grampositive cocci (Figure 5d and eGo), but not Gram-negative bacteria (Figure 5c and fGo). These results indicate that carboxyfullerene could insert into the cell walls of Gram-positive cocci.



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  		Figure 4. . The examination of bacteria using TEM after treatment with carboxyfullerene in vitro. Bacteria treated with 50 mg/L carboxyfullerene for 30 min were examined by TEM as described in Materials and methods. (a) PBS-treated S. aureus; (b) carboxyfullerene-treated S. aureus; (c) PBS-treated S. pyogenes A-20; (d) carboxyfullerene-treated S. pyogenes A-20; (e) PBS-treated E. coli; (f) carboxyfullerene-treated E. coli; (g) PBS-treated P. aeruginosa; (h) carboxyfullerene-treated P. aeruginosa (60 000x). Arrows indicate the damaged cell wall.

 


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  		Figure 5. . Sepharose-binding assay of bacteria after treatment with carboxyfullerene. The bacteria were incubated with 50 mg/L of carboxyfullerene for 30 min, then with Sepharose beads, which were pre-treated with normal (NS) or anti-carboxyfullerene (CS) immune sera as described in Materials and methods. The bacteria bound to Sepharose beads were visualized using Gram's stain. (a) S. pyogenes, NS; (b) S. aureus, NS; (c) E. coli, CS; (d) S. pyogenes, CS; (e) S. aureus, CS; (f) P. aeruginosa, CS (400x). The various sized beads are Sepharose beads, and the arrows indicate bundles of bacteria.

 
The insertion of carboxyfullerene into the cell walls of Gram-positive cocci, causing bactericidal activity, was confirmed by a membrane perturbation assay using PI staining with flow cytometry. As shown in Figure 6Go (a and b), PI uptake percentage in both S. pyogenes A-20 and S. aureus increased with time after treatment with carboxyfullerene in a dose dependent manner. However, membrane leakage was not detected in Gram-negative bacteria after treatment with different concentrations of carboxyfullerene (Figure 6cGo), indicating that the bactericidal effect of carboxyfullerene on Gram-positive cocci was achieved by insertion into the cell walls and destruction of membrane integrity.



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  		Figure 6. . Membrane perturbation assay by PI uptake after treatment with carboxyfullerene. The bacteria [(a) S. pyogenes A-20; (b) S. aureus; (c) E. coli] were incubated with different concentrations of carboxyfullerene. At various intervals, the bacteria were harvested and stained with PI. The PI uptake percentage was expressed as the mean ± s.d. {square}, PBS; {diamondsuit}, C3 5 mg/L; {circ}, C3 50 mg/L; {triangleup}, C3 200 mg/L.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Since the discovery of fullerene, biomedical scientists have been interested in its application, and several strategies have been developed to convert hydrophobic fullerene into water-soluble compounds by adding different groups of side chains to the fullerene molecules. Many derivatives of fullerene have been synthesized and used as antioxidants,9–12,23–25 anti-tumour agents,26 or carriers for DNA and other drugs.27,28 As a ‘free radical scavenger’, the protective effects of fullerene derivatives have been demonstrated in various systems, including decreasing the injury on the ischaemia–reperfusion intestine,25 protecting several cell types from undergoing apoptosis,11,12,23 decreasing the free radical level in organ perfusate24 and having a neuroprotective effect.9,10 However, the effect of fullerene on microbial infection is not clearly understood. In our previous studies, we found that carboxyfullerene could inhibit both E. coli-induced meningitis14 and group A streptococcal infection15 in murine models. The modulation of immune system, especially the function of neutrophils, was found in both systems, but the effect of carboxyfullerene on the bacteria was different.

How carboxyfullerene kills staphylococci and streptococci is intriguing and worth further investigation. Carboxyfullerene can insert into the cell walls of staphylococci and streptococci within 30 min, as demonstrated by our anti-carboxyfullerene antibody binding assay. This only occurred in Gram-positive and not Gram-negative bacteria. The differential effects of carboxyfullerene on Gram-positive and Gram-negative bacteria might be a result of the fact that there are different components in Grampositive and Gram-negative cell walls. The cell wall of Gram-positive bacteria consists of many layers of peptidoglycan and teichoic acid, which is absent in Gram-negative bacteria, whereas that of Gram-negative bacteria contains one or very few layers of peptidoglycan, but its outer membrane consists of lipoprotein, lipopolysaccharides and phospholipids.29 Once carboxyfullerene is intercalated into the Gram-positive bacterial cell wall, it might disrupt the structure of the cell wall and cause bacterial death, as suggested by the membrane perturbation assay. Huang et al.12 reported that carboxyfullerene can interact with lipid bilayers; the binding efficiency between water-soluble fullerenes and microbes might determine its activity. Mashino et al.30 reported that cationic ammonium fullerene derivatives, C60-bis(N,N-dimethylpyrrolidinium iodide) and C60-bis(N-methylpiperazinium iodide) can suppress the growth of E. coli, indicating the importance of the side chain.

The MICs of carboxyfullerene in Gram-positive cocci were 5–50 mg/L. This dosage is lower than the LD50 (250 mg/L) in mammalian cells such as lymphocytes, neutrophils, RAW 264.7 and A549 cells (unpublished observations). The LD50 of fullerenol in mice by intraperitoneal injection is 1000 mg/kg.31 Addition of mouse sera to as much as 5% did not affect the bactericidal effect of carboxyfullerene (unpublished observations). Carboxyfullerene inhibited group A streptococcal infection by modulating the function of neutrophils and suppressing the growth of S. pyogenes A-20 directly in vivo.15 In this study, we demonstrated that carboxyfullerene inhibits the growth of Gram-positive cocci through intercalation into the cell walls, disrupting the structure and causing death. This suggests that carboxyfullerene could be considered as a new antimicrobial agent against Gram-positive cocci.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by grant NHRI-GT-EX89B717L from the National Health Research Institute of the Department of Health of the Republic of China.


   Notes
 
* Correspondence address. Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan, Republic of China. Tel: +886-6-2353535, ext. 5643; Fax: +886-6-2097825; E-mail: hylei{at}mail.ncku.edu.tw Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 18 July 2001; returned 22 October 2001; revised 29 November 2001; accepted 20 December 2001


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