Mechanisms of Bacteriophage Inactivation via Singlet Oxygen

Jul 28, 2009 - The phage titer was first determined in terms of plaque forming units (PFU). ... This spectrometer had a wavelength range of 200−1100...
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Environ. Sci. Technol. 2009, 43, 6639–6645

Mechanisms of Bacteriophage Inactivation via Singlet Oxygen Generation in UV Illuminated Fullerol Suspensions ERNEST M. HOTZE,† APPALA RAJU BADIREDDY,‡ S H A N K A R A R A M A N C H E L L A M , * ,‡,§ A N D MARK R. WIESNER† Department of Civil and Environmental Engineering, Duke University, Durham, North Carolina 27708-0287, Department of Civil and Environmental Engineering, University of Houston, Houston, Texas 77204-4003, and Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204-4004

Received April 12, 2009. Revised manuscript received June 17, 2009. Accepted July 10, 2009.

Nonenveloped viruses are shown to be inactivated by singlet oxygen (1O2) produced in UVA photosensitized aqueous suspensions of a polyhydroxylated fullerene (C60(OH)22-24; fullerol, 40 µM). Experiments were performed with MS2, a ssRNA bacteriophage, as well as two dsDNA phages: PRD1, which has an internal lipid membrane, and T7, which entirely lacks lipids. MS2 was highly susceptible to inactivation, having a rate constant of 0.034 min-1 with UVA alone, which increased to 0.102 min-1 with photoactivated fullerol. PRD1 and T7 were not susceptible to UVA alone but were photoinactivated by fullerol with rate constants of 0.026 and 0.035 min-1, respectively. The role of 1O2 was demonstrated by three independent observations: (i) viruses that were insensitive to UVA alone were photoinactivated by rose bengal in the absence of fullerol, (ii) β-carotene reduced (but did not eliminate) photoinactivation rates, and (iii) singlet oxygen sensor green fluorescence spectroscopy directly detected 1O2 in UVA illuminated fullerol suspensions. Qualitative evidence is also presented that fullerol aggregates were closely associated with viruses allowing efficient transfer of 1O2 to their capsids. Fourier transform infrared spectroscopy revealed significant oxidative modifications to capsid proteins but comparatively minor changes to the DNA and (phospho)lipids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggested 1O2 induced crosslinking of proteins. Hence, phage inactivation by photoactivated fullerol nanoparticles appears to be caused by cross-linking of capsid protein secondary structures by exogenous 1O2 and consequent impairment of their ability to bind to surface receptors of their bacterial hosts (loss of infectivity) rather than by direct reactions with fullerol.

* Corresponding author phone: (713) 743-4265; fax: (713) 7434260; e-mail: [email protected]. † Department of Civil and Environmental Engineering, Duke University. ‡ Department of Civil and Environmental Engineering, University of Houston. § Department of Chemical and Biomolecular Engineering, University of Houston. 10.1021/es901110m CCC: $40.75

Published on Web 07/28/2009

 2009 American Chemical Society

Introduction Virucidal effects of singlet oxygen (1O2) generated through sensitization of photoactive dyes (1-3) have been shown to depend on chemical composition and membrane organization with enveloped viruses being generally more susceptible to inactivation than their nonenveloped counterparts (1, 4). However, detailed information on specific modifications of biological targets during photodynamic therapy is still the subject of ongoing investigations (3, 5, 6). Similar to photosensitive dyes, UVA (315-400 nm) activated aqueous suspensions of fullerene nanoparticles and its derivatives also produce 1O2 since their conjugated pi bonding system and spherical structure allow for efficient energy transfer due to intersystem crossing (7, 8). Photosensitized fullerenes have been reported to exhibit virucidal properties (9-11) although the disinfection mechanisms still remain unclear, particularly given the short lifetime of 1O2 in solution. 1 O2 is known to react preferentially with protein side chains resulting in very high second order reaction rate constants at physiological pH values (O(107 M-1s-1)) whereas its reactions with lipids are 2-3 orders of magnitude lower (12, 13). Indeed, limited available data for nonenveloped bacteriophages show substantially lower photoinactivation rates for a lipid-containing phage (PRD1) compared with others that lack lipids (φX174 and T7) using the dye merocyanine (MC) 540 (4). Additionally, the extent of dye sorption is an important determinant of 1O2 transfer and antiviral effectiveness during photodynamic therapy (3-5). Analogously, we hypothesize that (i) aggregates of fullerol nanoparticles will associate closely with phages while producing exogenous 1O2 upon UVA illumination and (ii) presence of an internal (phospho)lipid membrane will significantly increase resistance of nonenveloped viruses to 1 O2 in nanoparticulate systems. The primary objective of this work is to elucidate virucidal mechanisms in photosensitized fullerol suspensions with particular emphasis on the role of any internal lipid components in reducing susceptibility to 1O2. We also examine whether viruses and fullerol aggregates associate closely in aqueous suspensions in a fashion that would facilitate efficient delivery of exogenous 1O2 to viruses. Understanding virucidal mechanisms and potential association of fullerol with viruses adds to the determination of possible environmental hazards related to widespread utilization of water-soluble fullerene materials. Moreover, valuable insight can be gained about the potential for employing these nanomaterials for water disinfection. In this manuscript, inactivation kinetics of three nonenveloped bacteriophages, MS2, PRD1, and T7, in UVA illuminated fullerol suspensions are compared with enumeration controls using a 1O2 sensitizer (rose bengal) and a 1 O2 quencher (β-carotene). Note that T7 and PRD1 differ significantly in their capsid chemistry (T7 lacks lipids whereas PRD1 has a double capsid containing 15% lipids in an internal layer) and both contain double-stranded (ds)DNA (14, 15). 1 O2 concentrations were also directly measured using the singlet oxygen sensor green (SOSG) procedure. Alterations to key chemical bonds in phages were identified through Fourier transform infrared (FTIR) spectroscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) provided additional evidence of protein cross-linkages leading to inactivation.

Materials and Methods Culture and Enumeration of Bacteriophages. Bacterial viruses were used without loss of generality since they are VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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accepted as good surrogates for animal viruses and are commonly used in environmental investigations (16, 17). Phages also simplify conduct of experiments by avoiding the advanced facilities and equipment necessary for propagating human pathogens and maintaining animal cell lines and minimize safety issues related to virus disposal. T7 (ATCC 11303-B7) was cultured using Escherichia coli (ATCC 11303) as the host and PRD1 (obtained from David Metge, United States Geological Survey, Boulder, CO) was cultured using Salmonella typhimurium LT2 (ATCC 19585). Limited experiments were also performed using a single stranded (ss)RNA phage viz. MS2 (ATCC 15597-B1) cultured with E. coli (ATCC 15597). The phage titer was first determined in terms of plaque forming units (PFU). Overnight cultures of S. typhimurium LT2 and E. coli grown in tryptic soy broth (TSB; Difco) were transferred to fresh TSB and cultured to midlog phase for 6 h at 37 °C. Next, bacterial suspension (0.9 mL) and MS2 and PRD1 dilution (0.1 mL) in phosphate-buffered saline (PBS, pH ) 7.3) were mixed in 3 mL of soft overlay agar and poured onto presolidified trypticase soy agar (TSA, 1.5% agar; Difco) Petri dishes. Sterile PBS (6 mL) was then added to Petri dishes that resulted in confluent plaques after incubation for 24 h at 37 °C. The PBS solution containing the phages was decanted and centrifuged at 5000g for 10 min to remove bacterial debris. Additional purification was achieved by filtering the supernatant through a 0.22 µm mixed cellulose ester (MCE) membrane and then ultracentrifuging the filtrate at 103,000g for 3 h. The phage pellet was resuspended in PBS and stored at 4 °C. T7 and its host were cultured in nutrient broth and plated in nutrient agar. Plaques numbering between 30-300 PFU were counted and the target initial concentration was 107 PFU/mL. The zeta potentials of the phages suspended in PBS were measured to be in the range -29 to -15 mV (Supporting Information (SI)Table S1). Fullerol Suspensions. Fullerol (C60(OH)22-24) powder, purchased from a commercial vendor (MER, Tucson, AZ, 99.9% purity) was suspended in autoclaved ultrapure water (7). All glassware was first washed thoroughly with distilled water and then autoclaved for 15 min. Suspensions were made by adding approximately 0.07 mg/mL powdered fullerol, as received from the manufacturer, in a volumetric flask and sonicated for 2 h. During this time, the gold color of the suspension gradually increased as more fullerol was dispersed. Next, the suspension was vacuum filtered through 0.45 µm MCE filters. The stock suspension was made only once since fullerol is known to remain stable with respect to size distribution and concentration over several months (18), a much longer duration than the one-month period during which all inactivation experiments were completed. Fullerol was highly polydispersed as measured by dynamic light scattering (DLS, Malvern Instruments ZetaSizer Nano ZS, Worcestershire, UK). The intensity-weighted average size and standard deviation at the end of 100 min of experimentation was 218 ( 33 nm, however number-weighted distributions and TEM imagery both indicate the presence of many aggregates that are considerably smaller than the intensityweighted mean. TEMs of PRD1 suspended with fullerol show many aggregates approximately 2-40 nm in diameter and in apparent close proximity with the viruses (Figure 1). Similar images for MS2 and T7 are given in SI Figure S1. The zeta potential of fullerol was also measured as -19.6 mV (SI Table S1). Suspensions were well stirred and maintained in equilibrium with the atmosphere at room temperature which yields a dissolved oxygen concentration near 9 mg/L. Irradiation and Inactivation Protocols. Low pressure (LP) UV irradiation was performed using two 15 W fluorescent bulbs (Philips TLD 15W/08). These bulbs have an output spectrum ranging from 315 to 400 nm with a peak at 365 nm in the UVA region. The total surface irradiance was 28.9 W/m2 6640

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FIGURE 1. Transmission electron micrograph of PRD1 in the presence of fullerol. as measured by a multi channel spectrometer fitted with a 3648 element linear CCD array detector (Ocean Optics, USB4000, Dunedin, FL). This spectrometer had a wavelength range of 200-1100 nm with a 0.3 nm resolution and was calibrated using lamps with NIST traceable calibrated spectral irradiance. All experiments were carried out in Petri dishes with identical sample depths and surface areas allowing for consistent delivery of UV fluence, which reached a maximum value of 17.3 J/cm2 at 100 min. All experiments were conducted at 23 ( 1 °C controlled via cooling coils connected to a water bath. FTIR Spectroscopy. Midinfrared spectra in the range 1800-900 cm-1 at a resolution of 4 cm-1 were obtained using a FTIR spectrophotometer (Nicolet Instrument Corporation, Madison, WI) equipped with a tungsten-halogen source, XTKBr beam splitter, TE cooled DLaTGS detector with KBr window and OMNIC operating software. To minimize interferences from CO2 and water vapor, the spectrophotometer was evacuated and stabilized for 5 min prior to scanning. All scans were made on 100 µL samples placed on infrared transmissive ZnSe windows. Spectra shown in this manuscript are an average of five individual spectra collected for each sample and each spectrum per se was derived from 512 coadded scans collected in transmission mode. Singlet Oxygen Sensor Green Procedure. 1O2 concentrations in photoactivated fullerol suspensions were measured with SOSG (Invitrogen, Carlsbad, CA) in the presence and absence of phages (19). SOSG was first prediluted in 33 µL of methanol and ultrapure water to 15.6 µM as recommended by the manufacturer and then diluted 10× to a final concentration of 1.56 µM prior to measurement. Suspensions were placed in a 25 mL Petri dish with approximately 20 cm2 surface area. A 10× dilution of fullerol was necessary (40 µM to 4 µM) to avoid fluorescence saturation. One mL samples were taken at 0, 5, 10, 15, and 20 min. The initial fluorescence units were read as a background measurement (Modulus Single Tube 9200, Turner Biosystems, Sunnyvale, CA) and subtracted from readings at each succeeding time point. This difference was compared to a standard curve (obtained using rose bengal) to determine 1O2 concentrations. Since various samples had no significant differences in absorption at 365 nm, it can be inferred that UV fluence was delivered evenly in all cases. SDS-PAGE. Five mL samples were mixed with 15 mL of ice cold ethanol (99.5% purity) and incubated at -20 °C overnight to precipitate out phages. The virus pellet was harvested by centrifuging at 12,000g for 1 h at 4 °C. Next the residual ethanol was removed by vacuum drying for 1 h and the pellet was resuspended in 1 mL of the sample buffer. The standard Laemmli gel method was used to examine phage capsid proteins. Separation gels were prepared using 10% polyacrylamide and 0.267% bisacrylamide in 0.375 M TrisCl containing 0.1% SDS at pH 8.8. Stacking gels consisted of 4% acrylamide and 0.75% bisacrylamide in 0.125 M Tris-Cl at pH 6.8. Identical sample and electrode buffers were used,

which were prepared using 0.05 M Tris-glycine containing 0.2% SDS at pH 8.3. 100 µL aliquots of the virus sample was added to 100 µL of 2x Tris-glycine sample buffer (with 10% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue) and incubated in a water bath at 85 °C for 2 min and then loaded directly onto the gels. Electrophoresis was performed at 4 mA for approximately 15 h (i.e., overnight) after which the gels were fixed overnight using freshly prepared 50% trichloro-acetic acid and stained with 0.1% Coomassie brilliant blue R250 solution for 1 h. All gels were run in triplicate. Control Experiments. Dark experiments were performed to determine any potential effects of added chemicals on viability of phages and their bacterial hosts. First, rose bengal, β-carotene, and fullerol were added to bacterial hosts (E. coli and S. typhimurium), and colonies were measured after overnight incubation in the dark at 37 °C. Second, PRD1 and T7 concentrations were measured after Petri dishes containing phages and chemicals were placed in the UV chamber for 100 min but shielded from irradiation by wrapping them in aluminum foil. Third, increased T7 and PRD1 inactivation was measured in UVA illuminated rose bengal solutions as a positive control. Fourth, reduced inactivation was established by irradiating fullerol along with β-carotene as a negative control. Fifth, the entire set of PRD1 inactivation measurements (UVA alone, UVA+fullerol, UVA+rose bengal, UVA+fullerol+β-carotene) was duplicated on different dates. Sixth, UVA inactivation rate of MS2 was measured. Finally, absorbances of all phages at 525 and 504 nm were measured since these excitation and emission wavelengths correspond to the SOSG method.

Results and Discussion Control experiments demonstrated that phages and their hosts contacted with fullerol, rose bengal, or β-carotene in the dark produced negligible inactivation in all cases with an inactivation rate constant close to zero (SI Tables S2 and S3). There were no statistically significant differences (at 95% confidence) in all PRD1 inactivation rates measured under varying conditions on different days (SI Table S4). Additionally, the pseudo-first-order MS2 inactivation rate constant by UVA irradiation alone measured during this study (0.034 ( 0.001 min-1) was statistically indistinguishable from the recently reported value of 0.038 ( 0.004 min-1 (11). These controls validate our laboratory protocols, demonstrate a high degree of reproducibility, and allow comparison of data obtained over the duration of experimentation. PRD1 and T7 are More Resistant to 1O2 than MS2. Initial experiments revealed no inactivation of PRD1 for 20 and 30 µM fullerol suspensions illuminated for 300 and 150 min, respectively, (SI Table S5) indicating either that 1O2 produced by e30 µM fullerol was insufficient to inactivate PRD1 in the time frame of the experiments and/or that the concentration of fullerol was inadequate to promote sufficient association between PRD1 and fullerol aggregates to efficiently deliver 1 O2. We recently reported that photosensitization of just 1 µM fullerol in identical conditions to those used currently for PRD1 resulted in a significant inactivation of MS2 phages (11). Hence, it would appear that there was sufficient probability of fullerol contact and association with PRD1 especially since fullerol concentrations were higher. We therefore attribute the observed differences in inactivation kinetics of PRD1 and MS2 to dissimilarities in their structure and composition. In any case, a higher fullerol concentration was necessary to induce inactivation and consequently a 40 µM value was chosen. Figure 2 quantitatively demonstrates that susceptibility to photosensitized fullerol was in the order MS2 > T7 > PRD1 with pseudo-first-order inactivation rate constants of 0.102 ( 0.004, 0.035 ( 0.003, and 0.026 ( 0.002 min-1, respectively.

FIGURE 2. Inactivation of bacteriophages by 1O2 generated by photoactivating 40 µM fullerol nanoparticle suspension increases in the order MS2 > T7 > PRD1. The best-fit value of the slope and the standard error are shown. Initial phage concentration was O(107 PFU/mL). MS2 inactivation by UVA alone is shown in Figure S2 of the SI. Note that UVA irradiation alone caused negligible inactivation of PRD1 and T7 but inactivated MS2 with a rate constant of 0.034 min-1 (SI Figure S2). MS2 photoinactivation rate constant with 40 µM fullerol is 1.4 times higher than the value (0.071 min-1) we reported with 1 µM fullerol (11) indicating significantly higher 1O2 concentrations in the current study and/or closer association of fullerol aggregates with MS2 due to the higher concentrations employed herein. The high propensity for MS2 photoinactivation possibly arises from damage to its A-protein, necessary for infecting its host E. coli since it contains highly reactive amino acids such as Met, Cys, His, and Tyr (12, 20). Dissimilarities in phages’ composition resulted in differential resistance to 1O2, which were further explored using only PRD1 and T7 since PRD1 has a double capsid with an internal lipid membrane whereas T7 has a single proteinaceous capsid lacking lipids but both contain dsDNA with similar GC content (48% for T7 and 51% for PRD1). Inactivation of PRD1 and T7 by 1O2. As observed in Figure 3, photosensitizing rose bengal (40 µM, positive control) inactivated PRD1 and T7 with pseudo-first-order rate constants of 0.032 ( 0.002 and 0.045 ( 0.002 min-1, respectively (whereas UVA irradiation alone resulted in negligible inactivation). Irradiating fullerol suspensions in the presence of β-carotene (40 µM, negative control) reduced PRD1 and T7 inactivation rate constants to 0.013 ( 0.001 and 0.014 ( 0.001 min-1, respectively, demonstrating partial 1O2 quenching under these experimental conditions or that inactivation was due to a mechanism other than 1O2 production. However, MS2 inactivation was previously shown (11) to be completely quenched by β-carotene at a much lower concentration of fullerol (1 µM). One possible explanation for these results is that a higher concentration of fullerol favors fullerol-virus interactions that allow delivery of a limited amount of 1O2 directly to the virus before it can be quenched by β-carotene. 1 O2 has an extremely short inherent lifetime (4 µs in pure water and possibly lower in biological systems) resulting in a maximum diffusion radius over three lifetimes of only approximately 220 nm (21). This necessitates 1O2 to be generated near phages to react with their capsids before being quenched. Though not conclusive, ample amounts of dried fullerol aggregates visible alongside MS2, T7, and PRD1 in TEM images (Figures 1 and in SI) coupled to its short lifetime suggests that fullerol was closely associated with viruses in our experiments. A second explanation for the small degree VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Role of singlet oxygen in inactivating PRD1 (a) and T7 (b). The best-fit value of the slope and the corresponding standard error are shown.

TABLE 1. Net 1O2 Production Rates in Photoactivated Fullerol Suspensions in the Presence and Absence of Phages suspensiona fullerol fullerol fullerol fullerol

alone + T7 + PRD1 + MS2

net 1O2 production rate (µM/min) 0.039 0.028 0.016 0.017

a Fullerol conc. ) 4 µM, initial phage concentration O(107) PFU/mL. Net production measured by consumption by quencher over 20 min with sampling at 5 min intervals.

of inactivation observed is that the longer illumination times used for the PRD1 (100 min of UVA irradiation) might have degraded β-carotene and thus decreased its ability to quench 1 O2 (22). Although this has not been a limitation in previous work (11), β-carotene concentrations (measured as absorbance at 451 and 464.4 nm) in this work decreased substantially over the first 30 min of UV-A exposure revealing significant degradation (SI Figure S3). The β-carotene was added at the limit of its solubility to ensure that even with degradation, this 1O2 quencher would remain in the suspension. However, due to the higher concentrations of fullerol used in this work, the possibility that β-carotene was not present in excess cannot be ruled out. Higher inactivation rates of both PRD1 and T7 in the presence of rose bengal (Figure 3) compared with an identical dose of fullerol (Figure 2) can be attributed to (i) its higher 1 O2 quantum yield (reported to be 0.76 for rose bengal and 0.15 for fullerol (23), (24)), and/or (ii) better binding of rose bengal with phages resulting in higher local 1O2 concentrations (3, 4). This confirms the ability of 1O2 to inactivate these phages at rates that exceed those obtained with UVA illumination alone and diminishes the possibility that fullerol reacts directly (rather than through the 1O2 intermediary) with the viruses during 100 min of exposure. SOSG Measured Reduced Rates of 1O2 Production by Photosensitized Fullerol in the Presence of Phages. Table 1 summarizes 1O2 production rates with and without phages directly quantified using SOSG. 1O2 production rate by photoactivated fullerol in the absence of phages was measured as 0.039 µM/min which agrees closely with the rate recently obtained using electron paramagnetic resonance (0.032 µM/min) validating the SOSG technique (7). Decreased rate of 1O2 generation in the presence of phages indicates that viruses potentially (i) interfered with the photosensitization pathway, (ii) interfered with the SOSG 6642

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procedure, or (iii) reacted with 1O2 before SOSG detection. UV-vis scans (Hitachi U-2000 spectrophotometer) revealed no differences in the absorbance near the predominant wavelength of the irradiation system (365 nm) for fullerol suspensions in the presence and absence of phages demonstrating that phages did not demonstrably change the UVA flux (SI Figure S4). Additionally, dynamic light scattering measurements showed that phages did not significantly change the size of fullerol aggregates. Therefore, the first scenario can be ruled out since phages had no significant effect on UVA fluencies or in inducing fullerol aggregation and a consequent reduction in 1O2 generation. Phages did not absorb at 525 or 504 nm and therefore did not negatively interfere with SOSG fluorescence measurements ruling out the second scenario as well. Therefore, experimental evidence favors the third scenario in which phages had no effect on the absolute quantity of 1O2 generated by photoactivated fullerol but that 1O2 rapidly reacted with phages (undergoing quenching or oxidizing capsid components) before detection by SOSG. Capsid amino acids (and (phospho)lipids for PRD1) have 1O2 reaction rates that are several orders of magnitude below the diffusion limited rate of SOSG with 1O2 (12, 19, 25). Given the aforementioned kinetics, all 1O2 should have been detected by the SOSG before reactions with capsid if both viruses and fullerol were homogeneously distributed. Therefore, lower 1O2 production rates measured in the presence of phages compared with photoactivated fullerol alone lends further evidence for its accumulation near viruses resulting in preferential reactions of 1O2 with capsid material prior to SOSG detection. Furthermore, as described in the previous section, the short 1O2 lifetime also necessitates close proximity of fullerol aggregates to viruses to allow efficient delivery of 1 O2 to cause inactivation. Higher phage inactivation resulting from increased localized 1O2 concentration has also been reported in the presence of natural organic matter (16) and is a prerequisite for efficient photodynamic inactivation (1, 3, 5). We therefore attribute the differences in 1O2 generation rate summarized in Table 1 to differences in reactivity with viruses stemming from dissimilarities in the architecture and chemical composition of the phage capsids. However, this reactivity does not follow a consistent trend in inactivation. The hypothesis that phage inactivation was the consequence of oxidative damage of capsids by 1O2 was explored further. FTIR Spectra Reveal Capsid Protein Oxidation by 1O2. FTIR spectra (Figure 4) were interpreted to identify changes in the chemical composition and functionalities of T7 and

FIGURE 4. FTIR spectra of PRD1 (a) and T7 (b) after UVA irradiation for 100 min (top spectrum) and 100 min exposure to UVA photosensitized fullerol (bottom spectrum).

TABLE 2. Peak Area Reductions for PRD1 and T7 in the Mid-Infrared Region Obtained by FTIR Spectroscopy peak area reduction (%) wavenumber (cm-1)

assignment

PRD1

T7

1768, 1772 1730, 1733 1693, 1687 1651, 1649 1620 1570 1542 1505 1461

νCdO νCdO β-structures (amide I) R-helix (amide I) β-structures (amide I) amide II amide II amide II/ring stretch δasCH3, δ(CH), ν(CC), ν(CN), CH2 scissoring δ(NH), ν(CC), δ(COH) or δ(CO2H), γw(CH2), δ(COH), ν(C-O) γt(CH2), ν(C-O), PO2- symmetric stretching νsCO-O-C, γt(CH2) C-O-P ribose-phosphate skeletal motions

31 67 56 65 47 65 43 91 0

85 40 92 83 80 75 83 not applicable 91 interpreted as peak shift 77 0 5 2

1194 1086 1070 1040 975

PRD1 following 100-min fullerol photosensitization. Second derivative spectra were employed to better resolve peaks and shoulders present in the original spectra (SI Figures S5 and S6). Spectra of pure fullerol showed insignificant attenuation in the region of interest (1800-900 cm-1). Band assignments of spectral peaks were based on model biomolecules, organic compounds, and previously reported PRD1 and T7 capsid compositions (SI Table S6). Chemical modifications and decreasing peak areas upon fullerol photosensitization in both PRD1 and T7 are summarized in Table 2 and were attributed predominantly to protein oxidation by 1O2. Oxidation of protein side chains by 1O2 could potentially form carbonyl derivatives (12, 26). Reductions in amide I peaks were attributed to oxidative modifications to protein secondary structures (β-structures and R-helices), especially since they are known to be important structural capsid components of both PRD1 and T7 (14, 15). Table 2 demonstrates that nucleic acids in both PRD1 and T7 and (phospho)lipids in PRD1 were relatively less affected (e13% reductions in peak areas) compared with proteins, which were reduced significantly (30-92%). Alterations in phage capsids were predominantly attributed to 1O2-induced oxidation of Trp (forming N-formylkynurenine, 3R-hydroxypyrroloindoles, etc.), His (forming aspartic acid, urea, and several other products), and Tyr (forming an endoperoxide among other products and intermediates). Rate constants for the reactions of 1O2 with these amino acids are very high

44 2 0 13 6

contributors carbonyl groups carbonyl groups Arg, Gln Arg Tyr, Trp, Asn His, Asp Glu Trp Phe, Trp, Pro, PRD1 lipids His, Trp, Ser, Tyr, Asp, Glu Trp, Thr, PRD1 phospholipids PRD1 lipids, Trp PRD1 phospholipids, DNA DNA

(O(107) M-1s-1 near pH 7.3 of our experiments). Additionally, Arg, Pro, and Thr commonly undergo carbonyl modification. Further, 1O2 is known to cross-link, aggregate, or fragment several amino acid residues. Finally, a peak-shift from 1206 to 1189 cm-1 was observed for T7, which is attributed to changes in hydrogen bonding of Ser, Tyr, Asp, His, Trp, or Glu residues. These modifications indicate localized denaturation, conformational changes, and conjugation of capsid proteins possibly due to cross-linking (26). Negligible changes in nucleic acid peaks indicate that 1O2 was consumed due to reactions with the capsid surface before it could reach the phages’ dsDNA (especially since His and Trp are also efficient 1O2 quenchers). Hence, T7 inactivation by exogenous 1O2 in our experiments can be predominantly linked to oxidative damage of capsid proteins without necessarily loosening protein-DNA interactions as in the case of endogenous 1O2 (6, 27). The higher T7 inactivation rate shown in Figure 3 is consistent with greater damage to its proteinaceous capsid as evidenced by larger reductions in its peak areas (Table 2). Damages to membrane components suggest that inactivation resulted from the phages’ loss of ability to bind with receptors on the surface of their bacterial hosts and subsequent impairment of infectivity. SDS-PAGE Indicates Cross-Linking of Capsid Proteins by 1O2. Figure 5 depicts the SDS-PAGE analysis of PRD1 and T7 labeled as: (A) UVA alone, (B) fullerol alone in the dark, (C and F) UVA+fullerol, (D) UVA+rose bengal, and (E and G) dark (no fullerol and no UVA corresponding to ultrapure VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. PRD1 and T7 capsid proteins analyzed by SDS-PAGE showing evidence of oxidative damage following UVA irradiation of fullerol nanoparticles for 100 min. Lane A: UVA treated; lane B: fullerol in the dark; lanes C and F: UVA+fullerol; lane D: UVA+rose bengal; lanes E and G: phages alone in the dark. phages alone). For discussion purposes alone PRD1 protein bands were labeled as P1-P5; an extra band P6 was obtained for T7. Since the same number of bands was observed in all lanes for each phage, protein fragmentation does not appear to be a major consequence of 1O2 attack. Identical mobility of all bands in lanes A, B, and E (or G) demonstrates that capsid proteins of PRD1 and T7 were unaffected by UVA alone or fullerol alone in the dark, which is consistent with no inactivation observed under these conditions (Figure 3). In contrast, protein bands migrated abnormally under conditions where significant inactivation was observed, such as when phages were exposed to photoactivated fullerol or rose bengal (Figures 2 and 3). For example, P3 in lanes C (or F) and D for both phages as well as P4 in lane C (or F) for T7 nearly disappeared from their usual position in the gel. Mobility was altered for P2 in lane D for T7 indicating a change in its structure. Additionally, intensities were weakened for P3, P4, and P5 in lane C (or F) and P5 in lane D for PRD1 as well as P2 and P5 in lane C (or F) for T7. Differences in protein migration between lanes C (or F) and D were the consequence of dissimilar inactivation levels caused by rose bengal and fullerol, which in turn arose from differences in 1 O2 quantum yields. These observations lend further evidence that oxidative cross-linking of capsid proteins induced by exogenous 1O2 is the likely cause of phage inactivation. In summary, this work underscores that (i) virus inactivation in UVA-illuminated suspensions of fullerol is due to the production of 1O2 by nanoparticle aggregates that are closely associated with viral targets and (ii) the structure and composition of capsids, especially the presence of an internal lipid membrane, are important determinants of the resistance offered by nonenveloped viruses to exogenous 1O2. SOSG measurements of 1O2 production, partial inhibition of inactivation by β-carotene, and TEM imagery support the hypothesis of close association of fullerol aggregates and viruses and 1O2 production in these systems. Proximity of fullerol to viruses controls the amount of exogenous 1O2 delivered to the capsid. Moreover, these results suggest that there may be interesting trade-offs on design of fullerenes as virucides. Derivatization of fullerenes changes quantum yield for reactive oxygen species production while also changing fullerene surface chemistry that may affect the affinity of fullerenes for viral surfaces. Thus, it may be possible to optimize fullerene derivatization for viral inactivation. Future experiments are being designed to obtain stronger evidence for this mechanism including the direct detection 6644

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and identification of oxidative products to determine the specific damaged capsid proteins, e.g., ref 26.

Acknowledgments Authors A.R.B. and E.M.H. contributed equally to this work. This work was funded through a NSF CAREER award to S.C. (CBET 0134301), and by the NSF and the EPA under NSF Cooperative Agreement Number EF-0830093, Center for the Environmental Implications of NanoTechnology (CEINT). Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been subjected to EPA review and no official endorsement should be inferred.

Supporting Information Available Additional data related to quality control experiments and controls. This information is available free of charge via the Internet at http://pubs.acs.org/.

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