Chemical Factors Influencing Adsorption of Bacteriophage MS2 to Membrane Filterst

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1982, p /82/ $02.00/0 Vol. 43, No. 3 Chemical Factors Influencing Adsorption of Bacteriophage MS2 to Membrane Filterst SAMUEL R. FARRAH Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida Received 28 August 1981/Accepted 2 November 1981 Antichaotropic salts, such as magnesium sulfate, and metal chelators, such as citrate ions, promoted adsorption of bacteriophage MS2 to membrane filters. In contrast, compounds that disrupt hydrophobic interactions, such as chaotropic salts, urea, Tween 80, and ethanol, did not promote adsorption of MS2 to membrane filters and counteracted the ability of magnesium sulfate to promote such adsorption. These results provide evidence that magnesium sulfate promotes the association of MS2 with membrane filters primarily by strengthening hydrophobic interactions between the virus and the filters. Studies on the association of viruses with solids have identified several factors that influence virus adsorption by different solids. The ph (8, 14, 18), the presence of organic compounds (8, 11, 14, 16, 18), and the concentration and type of salt (1-3, 7, 8, 10, 11, 14-18) all affect the association of viruses with solids. The presence of di- and trivalent cations has been found to promote virus adsorption by several types of solids, including membrane filters (2, 3, 7, 10, 14-18), magnetite (1), and clays (9, 11). Several mechanisms have been proposed to explain the observed ability of di- and trivalent cations to promote adsorption of viruses to membrane ifiters. It has been suggested that the cations promote electrostatic interactions between the viruses and the membrane ifiters by acting as salt bridges between negative charges on the virus and the filter surface (6, 8, 18), by altering the size of the layer of charged ions that surrounds charged particles in solutions (14), or by increasing the positive charge on the filter (6, 7, 15) Ṙecently, Farrah et al. found that certain salts promote retention of viruses by membrane filters by influencing hydrophobic rather than electrostatic the filters (2). Therefore, this study was undertaken to determine whether the ability of magnesium sulfate to promote adsorption of viruses to membrane ifiters was a result of its influence on hydrophobic or electrostatic interactions between the viruses and the filters. The results obtained in this study indicate that magnesium sulfate promotes adsorption of bacteriophage MS2 by nitrocellulose membrane filters primarit Journal paper 3224 from The Florida Agriculture Experiment Station, Gainesville. 659 ly by strengthening hydruphobic interactions between the virus and the filters. MATERIALS AND METHODS Viral and viral assays. Phage MS2 was determined as plaque-forming units by using Escherichia coli C-3000 as the host according to previously described procedures (13). Chemicals. The chemicals used in this work and their sources are as follows: trichloroacetic acid, sodium thiocyanate, sodium iodide, sodium fluoride, urea, Tween 80, tetrasodium EDTA, lysine, and imidazole were obtained from Sigma Chemical Co., St. Louis, Mo., and sodium citrate, sodium chloride, magnesium sulfate, sodium sulfate, hydrochloric acid, and sodium hydroxide were obtained from Fisher Scientific Co., Fair Lawn, N.J. Experimental procedures. Approximately 10' PFU of virus was added to 10 ml of each test solution described below. All test compounds were dissolved in M imidazole and adjusted to ph 6.0 by adding sodium hydroxide or hydrochloric acid as required. The sample with added virus was then passed through a membrane ifiter (pore size, 0.45,um; type HA; Millipore Corp., New Bedford, Mass.) contained in a 25-mm holder at a rate of approximately 1 ml/s. The virus in the initial sample and the virus in the filtrate were measured to determine the percentage of virus adsorbed by the filter. Next, 10 ml of 0.6 M trichloroacetic acid-0.1 M lysine that had been adjusted to ph 9 by adding 10 N NaOH was passed through each filter to remove the adsorbed virus. The virus eluted was expressed as a percentage of the virus present in the initial test solution. Values for percentages of unadsorbed virus and virus eluted by solutions of buffer and solutions of 0.2 M magnesium sulfate were obtained in several trials and represent the means of six determinations; the other values in Tables 1 through 3 represent the means of two to four determinations. The correlation coefficients and straight lines for the data in Fig. 1 were calculated by using a least-squares linear regression analysis and a Texas Instruments TI-55 calculator (Texas Instruments, Inc., Dallas, Tex.).

2 660 FARRAH APPL. ENVIRON. MICROBIOL. TABLE 1. Influence of salts on adsorption of MS2 by membrane filters % of virus in the % of virus eluted' Type of salt Salt' filter effluent' Buffer alone M imidazole Divalent cation M MgSO M MgSO M MgSO M MgSO M MgCl M CaCl Monovalent anion 0.2 M NaTCA M NaSCN M NaI M NaCl M NaF Multivalent anion 0.2 M Na2SO M tetrasodium EDTA M sodium citrate a All salts were dissolved in M imidazole buffer and were adjusted to ph 6. b Percentage of virus in the initial test solution. c Adsorbed virus was recovered by treating the filters with 10 ml of 0.6 M NaTCA-0.1 M lysine (ph 9). Values are expressed as percentages of the virus in the initial test solution. Adenine solubility. The abilities of solutions to solubilize adenine were determined as previously described (2) and were expressed as percentages of the adenine solubilized by the buffer (0.005 M imidazole, ph 6) alone. TABLE 2. RESULTS As Table 1 shows, viruses in buffer alone were not adsorbed efficiently by the filters. As the concentration of magnesium sulfate was increased, the percentage of virus adsorbed by the filters increased. At 0.2 M MgSO4, more than 80% of the virus was adsorbed by the filter. Solutions of salts containing multivalent cations or anions, such as MgCl2, CaC12, Na2SO4, sodium EDTA, and sodium citrate, also promoted adsorption of MS2 by membrane filters. When these salts were present in the solution, a minor portion of the virus was detected in the filter effluent, and a major portion of the virus added Influence of anions, a detergent, and urea on the ability of magnesium sulfate to promote adsorption of MS2 to membrane filters Solutiona % of virus in the filter % of virus elutedc effluentb Buffer aloned M MgSO4d M urea M urea M MgSO M urea M urea M MgSO M NaTCA M NaTCA M MgSO % Tween % Tween M MgSO M sodium citrate M sodium citrate M MgSO a All solutions contained M imidazole buffer and were adjusted to ph 6.0. b Percentage of virus in the initial test solution. c Adsorbed virus was recovered by treating the filters with 10 ml of 0.6 M NaTCA-0.1 M lysine (ph 9). Values are expressed as percentages of the virus in the initial test solution. d Values from Table 1.

3 VOL. 43, 1982 TABLE 3. Influence of ethanol on the ability of buffer or buffer containing 0.2 M MgSO4 to promote adsorption of MS2 by membrane filters %of virus % of virus in the filter CU~ Solutiona effluentb eluted' Buffer aloned %o Ethanol % Ethanol % Ethanol % Ethanol M MgSO4d M MgSO4 + 10%o ethanol M MgSO4 + 20%o ethanol M MgSO4 + 30% ethanol M MgSO4 + 40%o ethanol a All solutions contained M imidazole buffer and were adjusted to ph 6.0. b Percentage of virus in the initial test solution. ' Adsorbed virus was recovered by treating the filters with 10 ml of 0.6 M NaTCA-0.1 M lysine (ph 9). Values are expressed as percentages of the virus in the initial test solution. d Values from Table 1. to the initial solution was recovered by treating the filter with a solution containing 0.6 M sodium trichloroacetate (NaTCA) and 0.1 M lysine (ph 9). Different amounts of viruses in solutions of monovalent anions were adsorbed by the filters. Although most of the viruses in solutions of NaTCA and NaSCN passed through the filters, most of the viruses in solutions of NaF were adsorbed by the filters. Both NaCl and Nal were intermediate in ability to promote adsorption of MS2. Although viruses in buffer alone were adsorbed poorly by the filters, viruses in solutions of 0.2 M MgSO4 or MgCl2 were removed efficiently from the solutions and could be recovered from the filters with a subsequent elution step. However, mixing solutions of MgSO4 with urea, Tween 80, or NaTCA interfered with the ability of magnesium sulfate to promote adsorption of virus by the ifiters. The viruses in these solutions passed through the ifiters and were detected in the filter effluent. In contrast, adding a metal chelator, such as sodium citrate, to solutions of magnesium sulfate did not reduce the amounts of virus removed from the solutions. Most of the virus in the solutions of magnesium sulfate containing sodium citrate was retained by the filters and was recovered with the eluting solution (Table 2) The addition of increasing concentrations of ethanol to solutions of 0.2 M magnesium sulfate resulted in an increase in the number of viruses which passed through the filters and were detected in the filter effluent (Table 3). The viruses ADSORPTION OF BACTERIOPHAGE MS2 661 in solutions of buffer alone or butfer containing ethanol passed through the filters and could be detected in the filter effluent. The abilities of solutions of monovalent anions or solutions containing 0.2 M magnesium sulfate and increasing amounts of ethanol to promote adsorption of viruses were related to their abilities to solubilize adenine (Fig. 1). Solutions that solubilized relatively low amounts of adenine promoted adsorption of most of the virus in the solutions to membrane filters. In contrast, solutions that solubilized relatively high amounts of adenine permitted little adsorption of virus to the filters. The correlation coefficients between percentage of unadsorbed virus and percent adenine solubility for solutions of monovalent anions and solutions of magnesium sulfate containing ethanol were 0.84 and 0.96, respectively. DISCUSSION The factors that influence adsorption of viruses to solids in general and to membrane filters in particular have been studied both to understand virus-solid interactions better and to provide practical procedures for recovering the viruses present in the environment. It has been found that the presence of di- and trivalent cations promotes adsorption of viruses to a variety of solids, such as membrane filters (3, 7, 9-11, 14-18), clays (9, 11), and magnetite (1). These results have led to the development of procedures for recovering enteroviruses and adenoviruses from water by using magnesium ions or aluminum ions to promote adsorption of viruses to membrane filters (3, 7, 10, 17). The mechanism of cation enhancement of viral adsorption to membrane filters has been examined in several studies. It has been suggested that multivalent cations promote adsorption of viruses to membrane filters by altering the charge on the filter (6, 7, 18), by altering the thickness of the layer of charges surrounding the surface of the filter (14), or by forming salt bridges between the viruses and the filters (6, 8). The one common component of these explanations is the idea that the cations influence electrostatic interactions between the viruses and the membrane filters. Recent work has shown that certain ions promote the retention of viruses by membrane filters at ph 9.5 by strengthening hydrophobic the filters (2). The ions that promote retention of viruses by membrane filters are small monovalent ions, such as fluoride, or multivalent ions, such as magnesium or citrate ions. These ions are called antichaotropic ions and are thought to promote hydrophobic interactions by increasing the structure of water and therefore making the water less able to accommodate hydrophobic

4 662 FARRAH APPL. ENVIRON. MICROBIOL. 100 NaSCN 0 30 e / ~~~NaTCA / *40 >80// 6 Nal NaCI// 20 z 4 0 / * z 20_ O w 0 NaF hi 75 too PERCENT ADENINE SOLUBI LITY FIG. 1. Relationship between the ability of solutions to promote virus adsorption to membrane filters and the ability to solubilize adenine. Symbols: 0, solutions containing 0.2 M magnesium sulfate and the indicated percentages of ethanol (0 to 40%); 0, 0.2 M solutions of the indicated sodium salts of monovalent anions. All of the solutions were made in M imidazole buffer and adjusted to ph 6.0. groups (4, 5). In contrast, large singly charged ions, such as trichloroacetate ion, promote elution of viruses adsorbed to membrane filters (2). These ions are called chaotropic ions and are thought to disrupt the structure of water. With a decrease in organization, aqueous solutions are more able to accommodate hydrophobic groups, and the strength of hydrophobic interactions is reduced (4, 5). Since magnesium sulfate contains two multicharged ions that would likely be antichaotropic, the present study was undertaken to determine whether magnesium sulfate promotes virus adsorption to membrane filters by strengthening hydrophobic interactions. The ability of MgSO4, MgCl2, and Na2SO4 to promote adsorption of MS2 to membrane filters is consistent with previously published results with other viruses (3, 10, 15) and with suggestions that these salts influence electrostatic interactions between the viruses and the filters. However, the ability of EDTA and citrate ions to promote virus adsorption is at variance with the idea that the virus-membrane association is influenced mainly by electrostatic interactions. The possibility that electrostatic interactions are mainly responsible for virus-membrane filter associations has led to suggestions that chelating agents, such as EDTA and citrate ions, interfere with virus adsorption to membrane filters (6, 8, 18). The results of this and previous studies have shown that this is not the case (2, 16). Both EDTA and citrate ions have been found to be poor eluents for virus adsorbed to membrane filters. The failure of EDTA and citrate ions to elute virus and the ability of these ions to promote virus adsorption, as observed in this study, are consistent with their ability to act as antichaotropic agents (2, 5). The observation that a chaotropic salt (NaTCA) and un-ionized compounds (Tween 80 and urea) counteracted the ability of magnesium sulfate to promote virus adsorption by membrane filters is consistent with the idea that magnesium sulfate promotes hydrophobic interactions between the viruses and the membrane filters. Based on previous work (2, 4, 5), all three of these compounds would be expected to weaken hydrophobic interactions between the viruses and the filters. The observed relationship between the ability of solutions of monovalent salts to accommodate hydrophobic groups (as measured by adenine solubility) and the ability of these solutions to promote virus adsorption (Fig. 1) is consistent with the idea that the salts influence hydrophobic the filters. Solutions of the antichaotropic salt sodium fluoride promote virus adsorption to the filters and solubilize less adenine than buffer solutions alone, indicating that these solutions have relatively little ability to accommodate hydrophobic groups. In contrast, solutions of the chaotropic salt NaTCA do not promote virus adsorption to the filters and solubilize more adenine than buffer solutions, indicating that these solutions are able to accommodate hydrophobic groups. The strength of the electrostatic interactions

5 VOL. 43, 1982 in a solution is inversely proportional to the dielectric constant of the solution (12). Since ethanol has a lower dielectric constant than water (19), the addition of increasing amounts of ethanol to solutions should decrease the dielectric constant and increase the strength of the electrostatic interactions in the solution. By making the solutions more able to accommodate hydrophobic groups, ethanol should have the opposite effect on hydrophobic interactions and should decrease hydrophobic interactions between viruses and membrane filters. Table 3 and Fig. 1 show that ethanol antagonizes the ability of magnesium sulfate to promote viral adsorption to membrane filters and that this antagonism is proportional to the ability of the solutions to accommodate hydrophobic groups, as measured by adenine solubility. These results would be expected if magnesium sulfate promoted hydrophobic rather than electrostatic interactions between the viruses and the filters. Previously published results have shown that ph influences virus adsorption to membrane filters (8, 18). In general, viruses adsorb better at ph values below their isoelectric points, where the viruses have a net positive charge and many filters have a net negative charge. The observed ph dependence of virus-membrane filter interactions suggests that electrostatic forces are also involved in such interactions. The fact that both electrostatic and hydrophobic interactions influence the association of viruses with membrane filters provides one possible interpretation for the different slopes of the lines in Fig. 1. According to this interpretation, increasing the concentration of ethanol weakens hydrophobic interactions but strengthens electrostatic the filters. Even though solutions containing 20%o ethanol and 0.2 M magnesium sulfate are better able to accommodate hydrophobic groups (as measured by adenine solubility) than 0.2 M solutions of NaTCA, the ethanol-magnesium sulfate solutions permit greater adsorption of viruses than the NaTCA solutions (Fig. 1). This would be expected if the ethanol promoted electrostatic the filters. If hydrophobic interactions alone controlled adsorption of MS2 to the filters, it is likely that solutions with relatively greater abilities to accommodate hydrophobic groups would permit relatively less adsorption of virus. The results presented in this paper indicate that magnesium sulfate promotes hydrophobic interactions between MS2 and membrane filters. Previous studies on the effect of ph on virus adsorption to membrane filters have shown that electrostatic interactions are factors which influence virus adsorption to membrane filters. Therefore, it is likely that both electrostatic and ADSORPTION OF BACTERIOPHAGE MS2 663 hydrophobic interactions influence the association of viruses with membrane filters. Determining the relative strengths of these interactions under different conditions may lead to a better understanding of virus-membrane filter interactions. ACKNOWLEDGMENT The support of the Center for Environmental and Natural Resource Programs, Institute of Food and Agricultural Sciences, University of Florida, is gratefully acknowledged. LITERATURE CITED 1. Bitton, G., 0. C. Pancorbo, and G. E. Gifford Factors affecting the adsorption of poliovirus to magnetite in water and wastewater. Water Res. 10: Farrah, S. R., D. 0. Shah, and L. 0. Ingram Effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed to membrane ifiters. Proc. Natl. Acad. Sci. U.S.A. 78: Flelds, H. A., and T. G. Metcalf Concentration of adenovirus from seawater. Water Res. 9: Hatefi, Y., and W. G. Hanstein Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc. Natl. Acad. Sci. U.S.A. 62: Hatefi, Y., and W. G. Hanstein Destabilization of membranes with chaotropic ions. Methods Enzymol. 31: Kessiclk, M. A., and R. A. Wagner Electrophoretic mobilities of virus adsorbing filter materials. Water Res. 12: Metcalf, T. D., C. Walls, and J. L. Melnick Environmental factors influencing isolation of enteroviruses from polluted surface waters. Appl. Microbiol. 27: Mix, T. W The physical chemistry of membranevirus interaction. Dev. Ind. Microbiol. 15: Moore, B. E., B. P. Sagik, and J. F. Malina, Jr Viral association with suspended solids. Water Res. 9: Rao, N. U., and N. A. Labzoffsky A simple method for the detection of low concentrations of viruses in large volumes of water by the membrane technique. Can. J. Microbiol. 15: Schaub, S. A., and B. P. 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