Abstract
The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H2N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.
Pollution of water with pathogens has been documented worldwide (13, 16, 17, 25, 37, 41). In particular, chlorine-resistant pathogens, such as pathogenic viruses (18), Cryptosporidium parvum, and so forth, have raised concerns about outbreaks of waterborne infectious diseases in our society. Actually, pathogenic viruses have been important agents in outbreaks of waterborne diseases (3, 19, 20). Although disinfection processes using ozone and UV light in conventional water and wastewater treatment systems have contributed to inactivating pathogens, these processes do not always effectively inactivate pathogenic viruses because the inactivation efficiency depends on the virus species and turbidity level (8, 11, 36). In addition, membrane technologies, such as microfiltration and ultrafiltration, are available to remove pathogens (14, 24), but the operation of the membrane separation process has some disadvantages, such as large energy consumption and the nuisance of maintenance.
On the other hand, the activated sludge process in wastewater treatment has been well known for removing viruses (9, 23), though the removal efficiency depends on the species and type of viruses (10). Many pathogenic viruses, especially enteroviruses, have been observed to be easily captured by sewage sludge (10) and difficult to be eluted (29). Microorganisms in activated sludge yield various polymeric substances including polysaccharides, lipopolysaccharides, proteins, and nucleic acids (38), and these polymers play an important role in the virus adsorption. However, biopolymers in activated sludge contributing to the virus adsorption have not been elucidated.
The objective of this study was to discover virus-binding biopolymers from the bacterial culture derived from activated sludge with affinity chromatography. Proteins among biopolymers in activated sludge were arbitrarily regarded as the most promising material in this study, because they have a high diversity of configuration. At first, activated sludge bacteria were cultivated in nonselective media, and crude proteins were extracted from bacterial cells harvested. Virus-binding proteins (VBPs) were isolated from the crude proteins by means of their affinity with a viral capsid peptide. The virus-binding ability of VBPs was confirmed by enzyme-linked immunosorbent assay (ELISA) and a cultural method using HeLa cells. Then, the isolated VBPs were characterized in terms of molecular weight and net surface charges. Amino acid sequences of N termini of VBPs were analyzed, and homology searches for the amino acid sequences of VBPs were conducted.
MATERIALS AND METHODS
Culture development.
Mixed microbial cultures were taken from return sludges of two municipal wastewater treatment plants located in Sendai, Japan. One wastewater treatment plant (plant A) processes about 10,000 m3 of domestic wastewater per day, providing the service for a population of about 36,000 in the western sector of Sendai city. In plant A, the anaerobic primary process, followed by the aerobic activated sludge process, has been employed for the treatment of domestic wastewater. Return sludge was sampled about once a week between October and November of 2001 and 2002 from plant A. The other plant (plant B) processes about 300,000 m3 of domestic and industrial wastewater per day and employs the conventional activated sludge process. Return sludge was sampled once in 2 weeks between October and December 2000 from plant B. Sample sludge (100 ml) was centrifuged (1,000 × g, 10 min, 4°C), and activated sludge bacteria in the supernatant liquid were cultivated in a 2 liters of a nonselective nutrient broth (Kyokuto, Tokyo, Japan) for 24 h at 20 ± 5°C (absorbance at 600 nm was 0.09 ± 0.01 at the beginning of the cultivation). In order to obtain a sufficient dissolved oxygen concentration in the cultivation medium, filtered air (0.2-μm-pore-size polytetrafluoroethylene membrane, AstroPore Disk CAPSULE FL50; FUJI FILM, Kanagawa, Japan) was introduced into the medium through a diffuser at a rate of 1 liter/min.
Crude protein extraction.
The culture, which was in stationary phase after the 24-h incubation (absorbance at 600 nm was more than 1.80), was centrifuged (3,000 × g, 10 min, 4°C), and the cell pellet was washed twice with 20 mM Tris-HCl buffer (pH 8.0). The washed pellet was frozen at −80°C for more than 2 h and thawed in a water bath (Yamato, Tokyo, Japan) at 30°C in order to promote cell destruction. Then, crude proteins were extracted with urea. First, 1 ml of 1 M urea in 20 mM Tris-HCl buffer (pH 8.0) was added per g (wet) of the pellet and treated with a 50-W homogenizer (Taitec, Saitama, Japan) for 2 min to destroy bacterial cells. After centrifugation (20,000 × g, 30 min, 4°C), the liquid-phase material was collected and desalted by dialysis against 2 mM Tris-HCl buffer (pH 8.0) at 4°C for at least 12 h.
Affinity isolation of VBPs.
Extracted crude proteins were filtered with a 0.45-μm-pore-size membrane for low protein binding (Millex-HV, polyvinylidene difluoride, SLHV 025 LS; Millopore) and applied to the affinity column (HiTrap NN-hydroxysuccinimide-activated; Amersham Bioscience Corp., Piscataway, N.J.), in which a custom-made polypeptide of poliovirus capsid protein was immobilized as a ligand. The sequence of the immobilized peptide was H2N-DNPASTTNKDKL-COOH, which was produced by the Peptide Institute, Inc. (Osaka, Japan). This peptide is a protruding part of the VP 1 protein of the poliovirus type 1 (PV1) Mahoney strain (15) and is a part of the major neutralization antigenic sites (7). Affinity chromatography was performed with the AKTA Fast Protein liquid chromatography system (Amersham Bioscience Corp.) at room temperature (the room temperature is controlled at 23°C). The start buffer of the affinity chromatography was 2 mM Tris-HCl (pH 8.0), and the elution buffer contained 0.5 M NaCl and 6 M urea in 20 mM acetic acid buffer (pH 3.0). The flow rate was set at 1 ml/min, and 1 ml (each) of affinity chromatographic fraction was collected by a fraction collector (Frac-900; Amersham Bioscience Corp.). Affinity chromatographic fractions were then desalted by dialysis against 10 mM NH4HCO3 (pH 8.0) for at least 12 h, and VBPs in these fractions were concentrated as follows. Ten milliliters of dialyzed fractions was decreased to 500 μl with a vacuum and centrifugal dehydrator (CVE-100, EYELA; TOKYO RIKAKIKAI Co. Ltd., Tokyo, Japan), and then 1.5 ml of acetone (−20°C) was added and mixed vigorously. The mixture was left at −80°C for 1 h and then centrifuged (10, 000 × g, 10 min, 4°C). After decantation, the remaining acetone was evaporated with the vacuum and centrifugal dehydrator. VBPs in the pellet were suspended in 100 μl of double-autoclaved milliQ water, and water was evaporated with the dehydrator for washing. This washing step was repeated twice, and VBPs in the pellet were preserved at −20°C until further analysis.
Evaluation of virus-binding ability of VBPs with ELISA.
The concentrated VBPs from 10 ml of the affinity chromatographic fraction were dissolved in 350 μl of 50 mM sodium carbonate buffer (pH 9.0). Then, a 50-μl portion of the dissolved VBPs was added to each well of a microtiter and left for 2 h to coat the well. Triplicate wells were used for each sample. Then, the wells were washed twice with phosphate-buffered saline (PBS) (Nissui Pharmaceutical Corporation Limited, Tokyo, Japan) and blocked with 5% bovine serum albumin (BSA) in PBS. After incubation at 4°C overnight, the wells were washed twice with PBS, and about 105 PFU of PV1 Sabin 1 in 100 μl of PBS containing 5% BSA was applied to the wells. Plates were incubated at room temperature for 1 h and washed twice with PBS, and then mouse anti-PV1 immunoglobulin G (Funakoshi, Tokyo, Japan) in 100 μl of PBS containing 5% BSA was inoculated into each well. After incubation at room temperature for 1 h, the wells were washed twice with PBS. Rabbit anti-mouse antibody modified by horseradish peroxydase (Funakoshi, Tokyo, Japan) was diluted in PBS containing 5% BSA, and 50 μl of the diluted antibody was added to each well. After incubation at room temperature for 1 h and three washes with PBS, secondary antibody bound was measured by coloring with o-phenylenediamine (P-7288; Sigma Chemical Co., St. Louis, Mo.) and H2O2 in citrate-phosphate buffer for 30 min. The coloring reaction was stopped with 2 M H2SO4. The absorbance at 492 nm was determined with a plate reader (Multiskan MS; Labsystems, Helsinki, Finland).
Evaluation of virus-binding ability of VBPs with a cultural method.
Wells of the ELISA plate were covered by VBPs and blocked by BSA. After two washes with PBS, PV1 Sabin 1 or adenovirus type 41 (AD41) in 50 μl of PBS containing 5% BSA was applied to each well. The most-probable number of cytopathogenic units (MPNCU) of the introduced test virus was determined by the most-probable-number method (three-dilution, five-tube approach) using HeLa cells. Plates were incubated at room temperature for 1 h. Then, the virus suspension was removed, and the amount of residual test virus in the removed mixture was determined by the most-probable-number method as well. Plates were washed twice with PBS, and the wash solutions were also applied to HeLa cells. Finally, the adsorption efficiency of the test virus to VBPs was calculated by the equation adsorption efficiency (%) = [(MPNCU0 − MPNCU1)/MPNCU0] × 100, where MPNCU0 is MPNCU of PV1 in the introduced mixture, and MPNCU1 is MPNCU of the test virus that did not adsorb to VBPs after the 1-h incubation.
Ion-exchange chromatography for evaluating net surface charges of VBPs.
In order to evaluate net surface charges of VBP molecules, affinity chromatographic fractions were processed for anion- and cation-exchange chromatography. RESOURCE Q (1 ml) and HiTrap SP (1 ml) (Amersham Bioscience Corp.) were used as the anion- and cation-exchange columns, respectively. The start buffer and the elution buffer in these ion-exchange chromatographies were 2 mM Tris-HCl (pH 8.0) and 1 M NaCl in 2 mM Tris-HCl (pH 8.0), respectively. The flow rate was set at 1 ml/min.
Two-dimensional (2D) electrophoresis.
The isolated VBPs were processed for 2D electrophoresis, which was outsourced to TAKARA BIO Inc. (Otsu, Shiga, Japan). At first, VBPs were dissolved in a protein lysis buffer (5.2 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 2% Zwittergent 3-14, 1% tributylphosphine, 0.2% Bio-Lyte 3-10) by stirring for 26 h at room temperature. Dissolved VBPs were processed for the isoelectric electrophoresis and then denatured by a buffer containing sodium dodecyl sulfate (SDS). Denatured VBPs were processed for SDS-polyacrylamide gel electrophoresis and stained by Coomassie brilliant blue (CBB).
Determination of amino acid sequences of N termini of VBPs.
The determination of amino acid sequences of N termini of VBPs was also outsourced to TAKARA BIO Inc. After the 2D electrophoresis, CBB-stained proteins were extracted from the 2D electrophoresis gel with 0.1% SDS in 0.1 M NH4HCO3 at 37°C overnight. Extracted VBP was adsorbed onto a membrane filter (pore size, 0.45 μm), and proteins were washed out twice with 50% acetonitrile (first wash) and 100% acetonitrile (second wash). The solvent of the VBP was changed from acetonitrile to milliQ water after evaporation. Then, the VBP was processed for a protein sequencer (HP G1005A protein sequencing system; Hewlett Packard).
Homology search for the N-terminal amino acid sequence of VBPs.
The protein-protein BLAST search programs (http://www.ncbi.nlm.nih.gov/BLAST/) were used for the homology search of the determined amino acid sequences of VBPs against all protein sequences in the NCBI database.
RESULTS
VBP isolation with affinity chromatography.
Urea solution was used for the extraction of crude proteins from a bacterial culture derived from activated sludge. In four trials, the average concentration of the crude protein obtained from plant A was 9,000 (standard deviation [SD], 1,600) mg/liter. On the other hand, the average concentration of crude proteins obtained from plant B was 8,300 (SD, 3,000) mg/liter in three trials. These crude proteins were applied to the affinity column to isolate VBPs. Figure 1 shows the affinity chromatographic profiles of crude proteins. Since the elution buffer contains 6 M urea, which can result in a denaturation of proteins (1), VBPs in the affinity column captured by specific interaction could be recovered in the elution step. The single peak around the buffer volume of 10 ml (concentration of the elution buffer was about 40%) in Fig. 1 indicates the successful isolation of VBPs. These VBPs were considered rare proteins because a large proportion of the extracted crude proteins were washed out between the buffer volume of 0 and 4 ml.
FIG. 1.
Affinity chromatographic profile of extracted crude proteins from bacterial culture derived from activated sludge. The capsid peptide of poliovirus type 1 Mahoney (H2N-DNPASTTNKDKL-COOH) was used as a ligand for the affinity chromatography. The start buffer and the elution buffer were 2 mM Tris-HCl (pH 8.0) and 0.5 M NaCl-6 M urea in 20 mM acetic acid buffer (pH 3.0), respectively. The flow rate was set at 1 ml/min. (a) Affinity chromatographic profile of VBPs extracted with 1 M urea in 20 mM Tris-HCl buffer (pH 8.0) from plant A in 2001. (b) Affinity chromatographic profile of VBPs extracted with 1 M urea in 20 mM Tris-HCl buffer (pH 8.0) from plant A in 2002. (c) Affinity chromatographic profile of VBPs extracted with 1 M urea in 20 mM Tris-HCl buffer (pH 8.0) from plant B in 2000. The vertical axes of these figures show milli-absorbance units.
The peak indicating the successful isolation of VBPs was repeatedly obtained from both plants throughout the experimental period (between October and November 2001 and 2002 for plant A, between October and December 2000 for plant B) at almost the same buffer volume (around 10 ml). The peak height in Fig. 1a was 90 ± 52 mAU (trial number is 22), that in Fig. 1b was 18 ± 7 (trial number is 40), and that in Fig. 1c was 19 ± 15 (trial number is 6). The large SDs and the difference in the mean values of the peak height between Fig. 1a, b, and c would be explained by the diversity and dynamics of microbial communities of each bacterial culture derived from activated sludge.
Since VBPs from plant A in 2001 (between October and November) were stably obtained in a larger quantity, VBPs in the peak in Fig. 1a were collected and processed for subsequent experiments on confirmation of virus-binding ability, evaluation of surface charge, and determination of N-terminal sequences of VBPs. Furthermore, the sequencing of N termini of VBPs in Fig. 1b was also conducted in order to investigate the existence of VBPs in the same plant in different years.
Confirmation of virus-binding ability.
Although the isolated VBPs had an affinity with the poliovirus capsid peptide, it is unclear whether they can bind intact virions (virus particles). Therefore, the virus-binding ability of the isolated VBPs was evaluated with ELISA, using PV1 Sabin 1, which is the attenuated strain of PV1 Mahoney (28). Each well of the microtiter plates was coated with the isolated VBPs or BSA, and PBS with PV1 Sabin 1 was applied to each well. If VBPs have the ability to adsorb PV1, these virions are captured by VBPs and remain in wells after washing. Two conditions (conditions A and B) were set to detect the specific interaction between PV1 and VBPs. For condition A, PV1 suspension was inoculated to VBPs-immobilized wells, while PV1 suspension was inoculated to BSA-immobilized wells for condition B.
Figure 2 shows the results of the evaluation of poliovirus-binding ability of VBPs. The homoscedasticity between variances of the absorbance for conditions A and B was certified by F-test at a significant level of 0.01. The significant differences of the absorbance between conditions A and B were also confirmed by student t test at a significant level of 0.01. These results indicate that VBPs isolated with an affinity to capsid peptide of PV1 Mohoney have the ability to bind intact particles of PV1 Sabin 1.
FIG. 2.
Evaluation of the poliovirus-binding ability of the isolated VBPs obtained from plant A in 2001. Condition A, PV1 suspension was innoculated to VBP-immobilized wells; condition B, PV1 suspension was inoculated to BSA-immobilized wells. Absorbance value of each condition is the mean value of triplicate trials. The bars represent standard errors of the means.
The adsorption of PV1 to VBPs was compared with that of AD41 by the cell culture method. Table 1 shows the efficiencies of adsorption of PV1 and AD41 to VBPs and BSA after a 1-h incubation. The adsorption efficiencies of PV1 and AD41 to BSA were 47% (±5%) and 47% (±11%), respectively. These values corresponded to the background of this study. On the other hand, the mean efficiency of adsorption of PV1 to VBPs reached 94%, while that for AD41 was 62%. Although the possibility for inactivation of PV1 by VBPs in ELISA wells could not be excluded by the results in Table 1 alone, the combined interpretation of the contents in Fig. 2 and Table 1 demonstrated that the isolated VBPs had the ability to capture PV1, and the adsorption of PV1 to VBPs was obviously stronger than that of AD41.
TABLE 1.
Adsorption efficiency of PV1 and AD41 to VBPs and BSA
| Test virus | Immobilized protein | MPNCU of the test virus in introduced supernatant (95% confidence interval)a | MPNCU of PV1 that did not adsorb after 1-h incubation (95% confidence interval) | Adsorption efficiency (%) | Mean [% (SD)] |
|---|---|---|---|---|---|
| PV 1 | VBPs | 46 (15-150) | 2.6 (0.9-7.0) | 94 | |
| 46 (15-150) | 3.4 (1.6-9.0) | 93 | |||
| 80 (27-240) | 3.4 (1.6-9.0) | 96 | 94 (2) | ||
| BSA | 46 (15-150) | 22 (7.6-62) | 52 | ||
| 46 (15-150) | 26 (9.4-72) | 43 | |||
| 80 (27-240) | 44 (17-120) | 45 | 47 (5) | ||
| AD41 | VBPs | 220 (83-590) | 88 (33-228) | 60 | |
| 110 (38-310) | 44 (15-120) | 60 | |||
| 94 (32-280) | 31 (10-96) | 67 | 62 (4) | ||
| BSA | 220 (83-590) | 132 (48-364) | 40 | ||
| 110 (38-310) | 44 (15-120) | 60 | |||
| 94 (32-280) | 56 (20-148) | 40 | 47 (11) |
Each MPNCU given represents a separate trial.
Evaluation of the net surface charge of VBPs with ion-exchange chromatography.
In order to evaluate the net surface charge of VBP molecules, affinity fractions were processed to anion- and cation-exchange chromatographies. Figure 3a and b are anion- and cation-exchange chromatographic profiles of VBPs, respectively. The start buffer, temperature, and flow rate in these ion-exchange chromatographies were the same as those in the affinity chromatography. As shown in Fig. 3a, VBPs were obtained at the elution step in anion-exchange chromatography. Since VBPs in the anion-exchange column were easily eluted by increasing the concentration of NaCl in the buffer, the electrostatic attractive force must be the main factor for adsorbing VBPs in the anion-exchange gel. On the other hand, Fig. 3b shows that VBPs were washed out from the cation-exchange column with the start buffer. These results support that VBPs have a net negative charge under the condition of the start buffer of the affinity chromatography.
FIG. 3.
Ion exchange chromatographic profiles of the isolated (VBPs). The black line shows the ion exchange chromatographic profile of VBPs, and the gray line shows the conductivity. (a) Anion exchange chromatographic profile of VBPs. (b) Cation exchange chromatographic profile of VBPs. The start buffer and the elution buffer in these ion exchange chromatographies were 2 mM Tris-HCl (pH: 8.0) and 1M NaCl in 2 mM Tris-HCl (pH 8.0), respectively. The flow rate was set at 1 ml/min. The vertical axes of these figures give milli-absorbance units.
2D electrophoresis profiles for the isolated VBPs.
Fig. 4 and 5 show the 2D electrophoresis profiles for the isolated VBPs from plant A in 2001 and 2002, respectively. Each spot (indicated by a circle) in Fig. 4 and 5 means each single molecule of VBP, which was separated according to pI and molecular weight. The molecular weights of the isolated VBPs were widely distributed but smaller than 100,000. Although the profiles of 2D electrophoresis for the isolated VBPs were considerably variable, several VBPs stably emerged in each figure. Therefore, these stably emerged VBPs were extracted from the gels (spots 1 to 3 in Fig. 4 and spots 4 to 6 in Fig. 5 indicated by circles with thick lines), and N terminal sequence of each VBP was analyzed.
FIG. 4.
Profile of 2D electrophoresis for VBPs recovered from bacterial culture derived from activated sludge between October and November 2001. Spots for VBPs are indicated by circles with thick and thin lines. The gel was stained with CBB. The molecular masses of standard proteins are indicated on the right. Amino acid sequences of N termini of VBPs in circles with thick lines were analyzed.
FIG. 5.
Profile of 2D electrophoresis for VBPs recovered from bacterial culture derived from activated sludge between October and November 2002. Spots for VBPs were indicated by circles with thick and thin lines. The gel was stained with CBB. The molecular masses of standard proteins are indicated on the right. Amino acid sequences of N termini of VBPs in circles with thick lines were analyzed.
Determination of amino acid sequences of N termini of VBPs.
Tables 2 and 3 show the amino acid sequences of N termini of VBPs. Twenty-two consecutive amino acids in N termini of VBPs in spots 1 and 3 were determined, while only 12 amino acids was obtained in spot 2 (Table 2). On the other hand, at least 28 amino acids in N termini in spots 4, 5, and 6 in Fig. 5 could be determined (Table 3).
TABLE 2.
N termini of the isolated VBPs from activated sludge culture between October and November 2001
| Spot no.a | N terminus → Direction of analysis |
|---|---|
| 15101520 | |
| 1 | A V V Y D K D G T S F D I Y G R V Q A N Y Y |
| 2 | V D F H G Y F R P Q V G |
| 3 | A D Y S G D I H K N D Y K W F Q F N L M G T |
| 15101520 |
Spot number corresponds to that in Fig. 4.
TABLE 3.
N termini of the isolated VBPs from activated sludge culture between October and November 2002
| Spot no.a | N terminus → Direction of analysis |
|---|---|
| 151015202530 | |
| 4 | A V V G G G A T L P Q G L Y T T P G V L G A G F D T Y T |
| 5 | A D Y S G D I H K N D Y K W F Q F N V M H T I D Q L P Y A E |
| 6 | Q D A F S Y A K G S A T W A H T K S D Y V G G K D S N N R D F |
| 151015202530 |
Spot number corresponds to that in Fig. 5.
The homology search with VBPs against all protein sequences in the NCBI database was conducted by the BLAST (the homology search was done on 2 August 2003). The amino acid sequence of the VBP in spot 2 (Table 2) was not used in this homology search because of its short length. In results, the VBP in spot 6 (Fig. 5 and Table 3) did not have amino acid sequences homologous to sequences in the protein database. On the other hand, one or more proteins provoked high levels of homology with the other four VBPs (spots 1, 3, 4, and 5).
Two proteins provoked the highest homology (90%) for the VBP in spot 1. The Aeromonas hydrophila outer membrane protein (AAF87725) (Fig. 6a) was one of the proteins that showed the highest homology. There were two apparent amino acid differences between VBP in spot 1 and the A. hydrophila outer membrane protein.
FIG. 6.
Sequence alignments of VBPs and bacterial proteins that provoked the highest levels of identity. (a) Alignment of the VBP in spot 1 and the outer membrane protein of A. hydrophila (identity, 90%). (b) Alignment of the VBP in spot 3 and the outer membrane protein OmpK of V. cholerae accession (identity, 90%). (c) Alignment of the VBPs in spot 4 and the ABC-type phosphate transport system, periplasmic component of P. aeruginosa, identity is 75%). (d) Alignment of the VBP in spot 5 and the outer membrane protein OmpK of V. vulnificus (identity, is 78%). Solid line indicates identical amino acids. A dashed line indicates similar amino acids. A dash (−) is a space introduced to maximize the alignment.
Five bacterial outer membrane proteins were retrieved for the VBP in spots 3 and 5 according to the homology search. The outer membrane protein OmpK of Vibrio cholerae (NP_231936) (Fig. 6b, 90% identity) and OmpK of Vibrio vulnificus (NP_760691) (Fig. 6d, 78% identity) have the highest identity with these VBPs, respectively. Although the locations of these VBPs on the 2D electrophoresis gels were almost the same when compared with each other (Fig. 4 and 5), two amino acids were apparently different between the N termini of these VBPs.
On the other hand, only one bacterial protein was obtained by the high level of identity (75%) with the VBP in spot 5. This protein was the ABC-type phosphate transport system, periplasmic component of Pseudomonas aeruginosa (ZP_00138283) (Fig. 6c). Six amino acids are totally different between the VBP in spot 5 and the protein for the phosphate transport.
DISCUSSION
Some materials, such as membrane filters and polyethylene glycol, have been used for recovering and concentrating viruses in water (2, 4, 12, 31, 35, 42), because it is easy to release viruses being captured by these substances. On the contrary, it is known that viruses seeded in activated sludge are not well recovered (29). One of the reasons for the poor recovery of seeded viruses is an irreversible adsorption of viruses to sludge polymers (29). The recovery of adhered viruses from sludge flocs is impossible if virus particles are irreversibly captured by substances in activated sludge.
Based on the hypothesis that there might be virus-binding polymers in activated sludge, we surveyed the specific materials from a bacterial culture derived from activated sludge and discovered VBPs. The finding of VBPs was achieved by applying crude proteins to the affinity column in which a custom-made capsid peptide of PV1 Mahoney was immobilized as a ligand (Fig. 1). The protein extraction with urea was important for the isolation of VBPs from the bacterial culture. Urea competes for the NH and CO groups of a polypeptide. The action of acids or bases, which can protonate or deprotonate groups involved in hydrogen bonding or change the Coulombic interaction that determines the conformation of a protein, can result in denaturation (1). This conformational change brings about an increase in the hydrophilic property of proteins. Accordingly, urea increases the water solubility of hydrophobic proteins, including outer membrane proteins of activated sludge bacteria. This characteristic of urea was thought to contribute to the efficient extraction of crude protein and isolation of VBPs from bacterial cultures derived from activated sludge.
The mechanism of adsorbing VBPs to the viral peptide in the affinity column cannot be explained by the simple electrostatic attraction and hydrophobic effect, although they have been reported as main factors in the adsorption of viruses to several substances, such as clay, sandy soil, estuarine sediments, and so on (5, 6, 21, 22, 26, 39). Under the condition of the start buffer (pH 8.0), the net charge of the viral peptide in the affinity column was expected to be negative, because the pI of the immobilized viral peptide was estimated to be 3.88 (34). Since the majority of the VBPs have net negative charges in the start buffer of the affinity chromatography (Fig. 3), it is thought that the electrostatic repulsive force is produced between the viral peptide and VBPs in the affinity column. This suggests that the adsorption of the VBPs to the viral peptide in the affinity column must occur with a strong attractive force able to overcome the electrostatic repulsive force. The hydrophobic effect is not sufficient to explain such a strong affinity of VBPs with the peptide, because 9 residues in the viral peptide (12 residues) are hydrophilic. Furthermore, four residues (two lysines and two aspartic acids) of the nine hydrophilic residues in the viral peptide are charging positively or negatively under the condition of the start buffer of the affinity chromatography. These residues provide the viral peptide with a high hydrophilicity. Consequently, it is doubtful that the hydrophobic effect sufficiently contributed to the VBP binding to the viral peptide in the affinity column.
Not hydrophobic and simple electrostatic interactions but the specific lock-and-key interaction might explain the binding of VBPs to the viral peptide. In the specific adsorption, strongly stable binding can be achieved by employing patterned multiple arrays of several bonds (27). Although it is impossible to fully verify the mechanism of VBP binding to the viral peptide in this study, the specific lock-and-key interaction would be one of the persuasive explanations for the successful isolation of VBPs from the bacterial culture derived from activated sludge. Elucidation of the mechanism of binding between VBPs and viruses will be the focus of further study.
These VBPs were repeatedly obtained in our study, but the amount and species of the isolated VBPs were considerably variable (Fig. 1, 4, and 5), which might be attributed to the water quality of influents, type of wastewater treatment, seasons, and so on. This implies that the isolated VBPs in this study represent just a fraction of VBPs in the bacterial culture derived from activated sludge. It seems difficult to explain the occurrence of proteins with a high affinity to human viruses in the bacterial culture derived from activated sludge, because bacteria definitely cannot play host to human viruses. Human enteric viruses have their own life cycles in the aquatic environment, where there are several factors inducing inactivation of viruses (30, 33). However, it is well known that viruses adsorbed to some kinds of substances can survive for a longer period than freely suspended viruses (8). We believe that one such substance might be the VBP, which could prolong viral lives in an aquatic environment.
No amino acid sequences in the protein database were perfectly matched with the sequences of VBPs in spot 1 to 5, except in spot 2, although one or more proteins were retrieved in the homology search against all protein sequences in the NCBI database. No amino acid sequence was retrieved in the homology search for the VBP in spot 6. These results indicate that VBPs in spots 1 and 3 to 6 are previously unknown proteins. It is of great interest to compare amino acid sequences of these VBPs with that of the human poliovirus receptor, because the poliovirus receptor definitely has the ability to bind poliovirus particles in the human body. However, no similarity between amino acid sequences of VBPs and those of the poliovirus receptor was observed. One reason why there were not any similarities could be that the amino acid sequences obtained in this study were just small parts of N termini of VBPs. When the whole sequences of VBPs are obtained in further study, it would be worthwhile to compare them with that of the poliovirus receptor, which could bring insight into mechanisms of binding between VBPs and poliovirus particles.
Since most of the retrieved proteins in the homology search were bacterial outer membrane proteins, it can be expected that the isolated VBPs are closely related to outer membrane proteins. Some researchers reported that no proteolysis was observed after intact bacteria were subjected to protease digestion (32, 40), which means that some outer membrane proteins of bacteria are protease resistant and stable in the environment. In this study, VBPs did not lose the ability to bind PV1 even after these VBPs were applied to serial treatments for the isolation (cultivation of bacteria, extraction of crude proteins, affinity chromatography, dialysis, and so on), and it was a fact that these VBPs existed in the bacterial culture derived from activated sludge. These results suggest that the recovered VBPs are stable proteins and might be available for the virus adsorbent in water and wastewater treatment.
Acknowledgments
This work was funded in part by the Grant-in-Aid for the Development of Innovative Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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