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A latex agglutination assay for specific detection of Panton–Valentine leukocidin
Journal of Microbiological Methods 75 (2008) 411–415
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
A latex agglutination assay for specific detection of Panton–Valentine leukocidin Kanenari Oishi a, Tadashi Baba b, Yasuo Nakatomi a, Teruyo Ito b, Keiichi Hiramatsu b,⁎ a b
Bacterial Diagnostics Production Department, Denka Seiken Co., Ltd., Gosen, Niigata, 959-1695, Japan Department of Bacteriology, Juntendo University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 8 April 2008 Received in revised form 7 July 2008 Accepted 17 July 2008 Available online 22 July 2008 Keywords: Panton–Valentine leukocidin Staphylococcus aureus Reverse passive latex agglutination Toxin producer strain
a b s t r a c t Panton–Valentine leukocidin (PVL) is produced by some isolates of Staphylococcus aureus, and has been associated with the high pathogenicpotential of these strains. To rapidly detect the toxin producer strains, we developed a reverse passive latex agglutination (RPLA) reaction assay specific for PVL. By testing 64 S. aureus strains, the assay could detect the 35 pvl-gene-positive strains with 100% specificity and sensitivity. Furthermore, the assay revealed an extensive variation in the amount of PVL produced by the pvl-positive strains. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Community-acquired, methicillin-resistant Staphylococcus aureus is an emerging nosocomial pathogen. (Baba et al., 2002; Bohach and Foster, 2000; Hiramatsu et al., 2002; Torell et al., 2005). Recently, highly virulent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged and markedly increased worldwide (Hiramatsu et al., 2002; Naimi et al., 2001; Okuma et al., 2002; Vandenesch et al., 2005). Among these isolates, carriage of the genes encoding Panton–Valentine leukocidin, a toxin associated with increased (Diep et al., 2004; Etienne, 2005; Gillet et al., 2002; Ma et al., 2006; Torell et al., 2005). PVL toxin is likely to be involved in severe symptoms such as necrotic pneumonia and furunculosis (Gillet et al., 2002; Lina et al., 1999; Naimi et al., 2001; Panton and Valentine, 1932; Torell et al., 2005). On the other hands, there is a report that PVL is not critically involved in high virulence but small phenol soluble modulins (Wang et al., 2007). In order to clinch the controversy, methods to detect the toxins quantitatively and easily are urgently needed. The PVL, having cytolitic activity against human and rabbit monocytes and polymorph nuclear cells, was first reported by Panton and Valentine in 1932 (Panton and Valentine, 1932), purified in 1991 from S. aureus strain V8 (ATCC49775) isolated from a patient with chronic furunculosis (Finck-Barbancon et al., 1991). The toxin consists of two polypeptides, S- (slow-eluted) and F- (fast-eluted) components, ⁎ Corresponding author. Department Bacteriology, Juntendo University, Hongo 2-1-1, Bunkyo-ku, Tokyo, Japan 113-8421. Tel.: +81 5802 1040; fax: +81 3 5684 7830. E-mail address: [email protected] (K. Hiramatsu). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.07.013
based on their elution profiles through cation-exchange chromatography (Kaneko and Kamio, 2004; Noda and Kato, 1988; Woodin, 1960). The gene for the toxin was then cloned and sequenced in 1995 by Prevostet al., and was renamed as PVL (LukS-PV + LukF-PV) to distinguish it from other homologous leukotoxins with hemolytic activities (Prevost et al., 1995). The presence or absence of the PVL gene in clinical strains can be detected by PCR (Deurenberg et al., 2004; Johnsson et al., 2004; Lina et al., 1999; McClure et al., 2006; McDonald et al., 2005; Nakagawa et al., 2005). However, carriage of the genes for PVL toxin does not necessarily mean this toxin is expressed in these isolates, which is a potential limitation of PCRbased approaches to circumvent this limitation of PCR-based PVL gene detection, we developed an agglutination-based immunoassay for the PVL toxin. 2. Results 2.1. Preparation of recombinant PVL toxins Plasmid pGEX-4T3 (GE Healthcare Bio-Sciences KK) was employed to generate glutathione S-transferase (GST) fusion recombinant toxins. The fusion construction was done with PCR-amplified lukSPV, lukF-PV, lukE, lukD, and the three members of gamma toxin, hlgA, hlgC and hlgB of a CA-MRSA strain MW2 (Baba et al., 2002). The lukE, hlgA, hlgC are the homologs for lukS-PV; and lukD and hlgB are the homologs of lukF-PV, respectively (Clyne et al., 1992; Cooney et al., 1988, 1993; Dalla Serra et al., 2005; Gravet et al., 1998). Since the amino acid homologies among the S and F class peptides are quite high (HlgA, HlgC, and LukE are 65–77% identical to LukS-PV, and HlgB
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and LukD are 71–82% identical to LukF-PV, respectively (Cooney et al., 1993; Gravet et al., 1998; Prevost et al., 1995)), they were cloned and expressed to produce recombinant toxins to be used for the absorption of anti-PVL antisera (see below). The specific primers used for the amplification of the lukS-PV and lukF-PV genes were S2F (5′-AAGGGATCCGATAACAATATTGAGAA-
TATTGGTG-3′) and S2R (5′-AAAGGCCGTCGACTCAATTATGTCCTTTCACTTTAATT-3′), and F2F (5′-AAGGGATCCCAACATATCACACCTGTAAGTG-3′) and F2R (5′-AAAGGCCGTCGACTTAGCTCATAGGATTTTTTTCCT-3′). They contained BamHI (S2F and F2F) or SalI (S2R and F2R) restriction sites (underlined). The PCR products were treated with the restriction enzymes, and cloned into the corresponding sites of pGEX-4T3 allowing
Fig. 1. A. Purified recombinant Panton–Valentine leukocidin and its homologous toxins. Purified toxin proteins were subjected to SDS-PAGE followed by Coomasie Brilliant Blue staining. Approximately 0.5 ng were applied to each lane. Lane 1, LukS-PV; lane 2, LukE; lane 3, HlgC; lane 4, HlgA; lane 5, LukF-PV; lane 6, LukD; lane 7, HlgB; MW, molecular weight markers. Note that lanes 1–4 are S-class components, whereas lanes 5–7 are F class ones. B. C. Specific detection of PVL with purified anti-LukS-PV antibody by immunoblotting. Purified PVL and homologous toxin components indicated in the panels were subjected to SDS-PAGE followed by electrotransfer to PVDF membrane. MW stands for molecular weight standards. Anti-LukS-PV rabbit polyclonal antibodies either before (B) or after affinity-purification (C) was used as detection antibodies. D. E. Specific detection of PVL with purified anti-LukF-PV antibody by immunoblotting. Experimental procedure was basically the same as panels B and C but indicated recombinant proteins were employed. Anti-LukF-PV rabbit polyclonal antibody (D) or affinity-purified anti-LukF-PV (E) was used.
K. Oishi et al. / Journal of Microbiological Methods 75 (2008) 411–415
in-frame fusion with glutathione S-transferase (GST). The resulting plasmids were introduced into E. coli JM109. Transformants were grown in LB-Medium (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1L) in the presence of 100 μg/ml ampicillin. After the O.D. at 550 nm of the culture reached 0.3, IPTG was added to 1 mM, and the cells were harvested after 2 h incubation. The recombinant toxins with GST-tags were purified from the supernatant of the ultrasonic-disrupted cells by affinity chromatography using glutathione-sepharose 4B beads (GE Healthcare BioSciences KK). Then the eluates from the beads were cleaved by thrombin protease (GE Healthcare Bio-Sciences KK), and the protein concentration was determined using the method by Bradford (Bradford, 1976). Purity of the protein was checked with Scion Image, version beta 4.0.2 image analysis software (Scion Corporation, Frederick, MD, USA) after Coomassie Blue staining of SDS-PAGE gels. 2.2. Preparation of specific anti-PVL antibodies Fifty micrograms each of the purified S-, and F-component recombinant proteins were mixed with Freund's complete adjuvant, and were injected in the skin of New Zealand white rabbits followed by four boosts at two-week interval. The antisera were then purified by ammonium sulfate precipitation, and their cross-reactivity was absorbed by passing them through the columns of 2-ml formyl-cellulofine gel (Seikagaku Corporation, Tokyo, Japan) coupled with either one of the purified recombinant proteins, LukE, HlgA, HlgC, LukD and HlgB. For the final purification, each absorbed antiserum was loaded onto the affinity column coupled with recombinant LukS-PV or LukF-PV. The antibodies recognizing either LukS-PV or LukF-PV were specifically eluted with 0.2 M glycine–HCl buffer pH 2.3, and neutralized with 0.4 M potassiumphosphate buffer. Fig. 1A is the result of Coomassie Blue staining of SDS-PAGE gel, which shows the purity of the recombinant proteins used to raise the antisera (LukS-PV and LukF-PV) and the toxin homologs used for the absorption. The recombinant proteins were subjected to SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes with a semidry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Then the membranes were treated with 5% skimmed milk in PBS containing 0.25% Tween20 (PBS-T) for an hour at room temperature and subjected to the immunoblotting assay. The membranes were incubated in PBS-T with 1/500 volume of each antibody for an hour at room temperature. After washing the membranes three times for 10 min each with PBS-T, anti-mouse immunoglobulin alkaline phosphatase-conjugated rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in PBS-T was added and incubated for an hour at room temperature. The membranes were washed again and developed in alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories). Since whole molecules were employed as antigens, the antisera reacted not only with LukS-PV or LukF-PV, but also with the components of gammahemolysin and LukDE leukocidin (Fig. 1B and D). After cross absorption by affinity columns, however, the antisera became specific to LukS-PV (Fig. 1C) and LukF-PV (Fig. 1E). 2.3. Setting the reverse passive latex agglutination (RPLA) assay using specific antibodies and determination of its detection limit Using the specific antisera prepared above, we next prepared specific antibody-sensitized latex. Polystyrene beads (JSR Corporation, Tokyo, Japan) were sensitized as described previously (Igarashi et al., 1986), with an optimum concentration of the specific antibodies. The beads were suspended in PBS containing 0.5% bovine serum albumin. The detection limit of RPLA assay was evaluated using the solutions containing 100 ng/ml of recombinant LukS-PV or LukF-PV protein as standards. A 25 μl each of the toxin solutions was serially diluted with reaction buffer from 1:2 to 1:128 in a 96 well V-bottom microtiter plate. A 25 µl each of the sensitized latex suspension was added to the
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Table 1 The RPLA-based detection and titration of PVL toxin in the culture medium of clinical S. aureus strains Strains
C199800370 C199900529 C1999000193 C1999000459 (MW2) C2001001201 C2001000101 C200100818 JCSC5174 JCSC5182 JCSC5183 JCSC5184 JCSC5192 JCSC5391 JCSC5169 JCSC517O JCSC5171 JCSC5172 JCSC5189 JCSC1O8 JCSC2911 JCSC2912 JCSC2913 JCSC2914 JC5C4488 ATCC49775 01083 01102 A803355 A823549 A83 0528 B826559 D82 1552 E802537 F81 0539 I802552 MW2 ΔPVL N315 JC3C2952 JC3C2962 M9N M33T W1S C7N 00215 01093 810342 810508 810937 811238 911573 911575 911703 912118 912125 912145 912231 912572 912574 912619 912666 SAP26O SAP344 SAP411 WCH379
Carriage Titers of LukS-PV and LukF-PV in of luk CCY modified BHl medium S-F-PV medium LukSPV
Reference(s)
LukSPV
LukFPV
LukFPV
+ + + +
N1:128 N1:128 1:32 N1:128
N 1:128 N 1:128 1:64 N 1:128
1:64 1:64 1:64 1:32
N 1:128 N 1:128 N 1:128 1:64
Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002)
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 1:8 N1:128 N1:128 N1:128 N1:128 N1:128 1:16 N1:128 N1:128 1:32 1:32 1:64 1:32 1:64 1:64 1:32 1:16 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2
N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 1:16 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 1:32 N 1:128 N 1:128 1:128 1:128 N 1:128 1:128 N 1:128 N 1:128 1:128 1:32 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2
1:32 1:8 1:32 1:64 1:64 1:64 1:64 1:32 1:128 1:16 1:64 1:128 1:128 1:32 1:8 N1:128 1:16 1:16 1:16 N1:128 1:2 1:16 1:16 1:32 1:8 1:32 1:16 N1:128 1:16 1:4 1:4 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2
1:32 1:32 1:64 N 1:128 N 1:128 1:128 1:128 1:64 1:128 1:64 N 1:128 N 1:128 N 1:128 1:64 1:32 N 1:128 1:64 1:64 1:64 N 1:128 1:16 1:64 1:64 1:64 1:16 1:64 1:32 N 1:128 1:32 1:16 1:16 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2
Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Prevost et al. (1995) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) This study Okuma et al. (2002) Ma et al. (2006) Ma et al. (2006) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002)
diluted toxins and mixed by agitation. For agglutination reaction, the plate was kept overnight at room temperature under the moist condition to prevent the plate from drying. Thereafter, the end points
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of agglutination were determined visually. The detection limit for the recombinant PVL components was 1:64 dilutions for both anti-LukSPV and anti-LukF-PV beads, indicating that the detection limit of RPLA assay for PVL was both approximately 1 ng/ml. 2.4. Specificity and sensitivity of the RPLA assay To test the specificity and sensitivity of the RPLA assay to detect pvl-gene-carrying S. aureus strains, we tested a total of 64 S. aureus clinical and laboratory strains, whose carriage or non-carriage of pvl genes had been established by PCR experiments (Ma et al., 2005; Okuma et al., 2002). Strains ATCC49775 and MW2 were included as positive control strains with PVL gene, and strain MW2ΔPVL, a derivative strain of MW2 with its PVL genes replaced by a chloramphenicol acetyl-transferase gene (Schneewind et al., 1993), as a negative control strain. For the RPLA assay, two culture media were used: They were 1) CCY modified medium (Finck-Barbancon et al., 1991) and 2) BHI medium (Becton Dickinson and Company). The strains were grown in 3 ml of the media for 24 h with shaking at 140 rpm, and their supernatants were used for RPLA assay without any additional treatment. The results of RPLA assay perfectly correlated with the PCR-based detection of PVL gene (Table 1). All of the 35 pvlgene-positive strains were successfully detected by the RPFL assay, and none of the 29 pvl-gene-negative strains became false positive. Thus, the method demonstrated 100% sensitivity and 100% specificity in the detection of PCR-based pvl-gene-positive strains. In that sense, the assay was shown to be equivalent to PCR detection of PVL-genecarrying S. aureus. 2.5. Quantitation of the PVL produced by each clinical strain Besides identification of the PVL-producing strains, the quantitative nature of the RPLA assay was used to measure the amount of PVL produced by each of the strains. The results shown in Table 1 revealed the presence of significant difference in the amount of PVL produced by each strain (from 1:4 to N1:128; Table 1). This implies that the PVLgene carriage alone may not fully correlate with the degree of the strain's virulence if we assume that the amount of PVL toxin produced by the strain is associated with the degree of virulence of the strain. The role of PVL in the pathogenesis or virulence of S. aureus should be evaluated taking this quantitative difference in the PVL production among clinical S. aureus isolates. Table 1 also shows the influence of culture medium on the production of PVL toxin. The amount of produced toxin was generally greater in CCY medium than in BHI. Some strains such as C2001000101 produced at least 16 times more amount of LukS-PV in CCY than in BHI (Table 1). These data suggest that there is a factor that allows high PVL production in CCY medium, which is absent from BHI medium. Thus it is likely that the virulence level of PVL-producing S. aureus as well may be influenced by certain factors present in the environment. On the other hand, it was also noted that the production of PVL by strain 81/108 was not much influenced by the change of medium (Table 1). Thus, the toxin production may also be dependent on the genotype of the strain. 3. Discussion An ELISA assay to detect LukF-PV, one of the two components of the PVL toxin, was reported by Hamilton et al. (Hamilton et al., 2007). The LukF-PV-specific polyclonal rabbit antiserum used for their study was not evaluated for the cross-reactivity with the other toxin homologs such as HlgB or LukE. So, the antibodies are not suited for the rapid quantitation assay described in this study. They reported that the quantity of LukF-PV produced in vitro did not correlate with the severity of infection. However, our study revealed that the two components of the PVL toxin were produced in different quantity in some strains (see Table 1). Therefore, the quantitation of only the “F
component” of the toxin may not reflect the activity of the assembled composite PVL toxins. We consider that the use of a set of the highly specific anti-S and F-component antibodies would be needed in the correct analysis the role of PVL toxin in the high virulence of certain S. aureus clinical strains. In conclusion, the RPLA would be a useful, reliable and simple tool not only to detect PVL-producing S. aureus clinical strains but also to know the amount of toxin produced by the strains without the crossreactivity with other toxin homologs. The quantitative feature of the RPLA assay would add more information to the understanding of the pathogenesis of the PVL-producing S. aureus clinical strains. Acknowledgements This work was supported by a Grant-in-Aid for 21st Century COE Research from The Ministry of Education, Science, Sports, Culture and Technology of Japan. 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Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
A latex agglutination assay for specific detection of Panton–Valentine leukocidin Kanenari Oishi a, Tadashi Baba b, Yasuo Nakatomi a, Teruyo Ito b, Keiichi Hiramatsu b,⁎ a b
Bacterial Diagnostics Production Department, Denka Seiken Co., Ltd., Gosen, Niigata, 959-1695, Japan Department of Bacteriology, Juntendo University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 8 April 2008 Received in revised form 7 July 2008 Accepted 17 July 2008 Available online 22 July 2008 Keywords: Panton–Valentine leukocidin Staphylococcus aureus Reverse passive latex agglutination Toxin producer strain
a b s t r a c t Panton–Valentine leukocidin (PVL) is produced by some isolates of Staphylococcus aureus, and has been associated with the high pathogenicpotential of these strains. To rapidly detect the toxin producer strains, we developed a reverse passive latex agglutination (RPLA) reaction assay specific for PVL. By testing 64 S. aureus strains, the assay could detect the 35 pvl-gene-positive strains with 100% specificity and sensitivity. Furthermore, the assay revealed an extensive variation in the amount of PVL produced by the pvl-positive strains. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Community-acquired, methicillin-resistant Staphylococcus aureus is an emerging nosocomial pathogen. (Baba et al., 2002; Bohach and Foster, 2000; Hiramatsu et al., 2002; Torell et al., 2005). Recently, highly virulent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged and markedly increased worldwide (Hiramatsu et al., 2002; Naimi et al., 2001; Okuma et al., 2002; Vandenesch et al., 2005). Among these isolates, carriage of the genes encoding Panton–Valentine leukocidin, a toxin associated with increased (Diep et al., 2004; Etienne, 2005; Gillet et al., 2002; Ma et al., 2006; Torell et al., 2005). PVL toxin is likely to be involved in severe symptoms such as necrotic pneumonia and furunculosis (Gillet et al., 2002; Lina et al., 1999; Naimi et al., 2001; Panton and Valentine, 1932; Torell et al., 2005). On the other hands, there is a report that PVL is not critically involved in high virulence but small phenol soluble modulins (Wang et al., 2007). In order to clinch the controversy, methods to detect the toxins quantitatively and easily are urgently needed. The PVL, having cytolitic activity against human and rabbit monocytes and polymorph nuclear cells, was first reported by Panton and Valentine in 1932 (Panton and Valentine, 1932), purified in 1991 from S. aureus strain V8 (ATCC49775) isolated from a patient with chronic furunculosis (Finck-Barbancon et al., 1991). The toxin consists of two polypeptides, S- (slow-eluted) and F- (fast-eluted) components, ⁎ Corresponding author. Department Bacteriology, Juntendo University, Hongo 2-1-1, Bunkyo-ku, Tokyo, Japan 113-8421. Tel.: +81 5802 1040; fax: +81 3 5684 7830. E-mail address: [email protected] (K. Hiramatsu). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.07.013
based on their elution profiles through cation-exchange chromatography (Kaneko and Kamio, 2004; Noda and Kato, 1988; Woodin, 1960). The gene for the toxin was then cloned and sequenced in 1995 by Prevostet al., and was renamed as PVL (LukS-PV + LukF-PV) to distinguish it from other homologous leukotoxins with hemolytic activities (Prevost et al., 1995). The presence or absence of the PVL gene in clinical strains can be detected by PCR (Deurenberg et al., 2004; Johnsson et al., 2004; Lina et al., 1999; McClure et al., 2006; McDonald et al., 2005; Nakagawa et al., 2005). However, carriage of the genes for PVL toxin does not necessarily mean this toxin is expressed in these isolates, which is a potential limitation of PCRbased approaches to circumvent this limitation of PCR-based PVL gene detection, we developed an agglutination-based immunoassay for the PVL toxin. 2. Results 2.1. Preparation of recombinant PVL toxins Plasmid pGEX-4T3 (GE Healthcare Bio-Sciences KK) was employed to generate glutathione S-transferase (GST) fusion recombinant toxins. The fusion construction was done with PCR-amplified lukSPV, lukF-PV, lukE, lukD, and the three members of gamma toxin, hlgA, hlgC and hlgB of a CA-MRSA strain MW2 (Baba et al., 2002). The lukE, hlgA, hlgC are the homologs for lukS-PV; and lukD and hlgB are the homologs of lukF-PV, respectively (Clyne et al., 1992; Cooney et al., 1988, 1993; Dalla Serra et al., 2005; Gravet et al., 1998). Since the amino acid homologies among the S and F class peptides are quite high (HlgA, HlgC, and LukE are 65–77% identical to LukS-PV, and HlgB
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and LukD are 71–82% identical to LukF-PV, respectively (Cooney et al., 1993; Gravet et al., 1998; Prevost et al., 1995)), they were cloned and expressed to produce recombinant toxins to be used for the absorption of anti-PVL antisera (see below). The specific primers used for the amplification of the lukS-PV and lukF-PV genes were S2F (5′-AAGGGATCCGATAACAATATTGAGAA-
TATTGGTG-3′) and S2R (5′-AAAGGCCGTCGACTCAATTATGTCCTTTCACTTTAATT-3′), and F2F (5′-AAGGGATCCCAACATATCACACCTGTAAGTG-3′) and F2R (5′-AAAGGCCGTCGACTTAGCTCATAGGATTTTTTTCCT-3′). They contained BamHI (S2F and F2F) or SalI (S2R and F2R) restriction sites (underlined). The PCR products were treated with the restriction enzymes, and cloned into the corresponding sites of pGEX-4T3 allowing
Fig. 1. A. Purified recombinant Panton–Valentine leukocidin and its homologous toxins. Purified toxin proteins were subjected to SDS-PAGE followed by Coomasie Brilliant Blue staining. Approximately 0.5 ng were applied to each lane. Lane 1, LukS-PV; lane 2, LukE; lane 3, HlgC; lane 4, HlgA; lane 5, LukF-PV; lane 6, LukD; lane 7, HlgB; MW, molecular weight markers. Note that lanes 1–4 are S-class components, whereas lanes 5–7 are F class ones. B. C. Specific detection of PVL with purified anti-LukS-PV antibody by immunoblotting. Purified PVL and homologous toxin components indicated in the panels were subjected to SDS-PAGE followed by electrotransfer to PVDF membrane. MW stands for molecular weight standards. Anti-LukS-PV rabbit polyclonal antibodies either before (B) or after affinity-purification (C) was used as detection antibodies. D. E. Specific detection of PVL with purified anti-LukF-PV antibody by immunoblotting. Experimental procedure was basically the same as panels B and C but indicated recombinant proteins were employed. Anti-LukF-PV rabbit polyclonal antibody (D) or affinity-purified anti-LukF-PV (E) was used.
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in-frame fusion with glutathione S-transferase (GST). The resulting plasmids were introduced into E. coli JM109. Transformants were grown in LB-Medium (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1L) in the presence of 100 μg/ml ampicillin. After the O.D. at 550 nm of the culture reached 0.3, IPTG was added to 1 mM, and the cells were harvested after 2 h incubation. The recombinant toxins with GST-tags were purified from the supernatant of the ultrasonic-disrupted cells by affinity chromatography using glutathione-sepharose 4B beads (GE Healthcare BioSciences KK). Then the eluates from the beads were cleaved by thrombin protease (GE Healthcare Bio-Sciences KK), and the protein concentration was determined using the method by Bradford (Bradford, 1976). Purity of the protein was checked with Scion Image, version beta 4.0.2 image analysis software (Scion Corporation, Frederick, MD, USA) after Coomassie Blue staining of SDS-PAGE gels. 2.2. Preparation of specific anti-PVL antibodies Fifty micrograms each of the purified S-, and F-component recombinant proteins were mixed with Freund's complete adjuvant, and were injected in the skin of New Zealand white rabbits followed by four boosts at two-week interval. The antisera were then purified by ammonium sulfate precipitation, and their cross-reactivity was absorbed by passing them through the columns of 2-ml formyl-cellulofine gel (Seikagaku Corporation, Tokyo, Japan) coupled with either one of the purified recombinant proteins, LukE, HlgA, HlgC, LukD and HlgB. For the final purification, each absorbed antiserum was loaded onto the affinity column coupled with recombinant LukS-PV or LukF-PV. The antibodies recognizing either LukS-PV or LukF-PV were specifically eluted with 0.2 M glycine–HCl buffer pH 2.3, and neutralized with 0.4 M potassiumphosphate buffer. Fig. 1A is the result of Coomassie Blue staining of SDS-PAGE gel, which shows the purity of the recombinant proteins used to raise the antisera (LukS-PV and LukF-PV) and the toxin homologs used for the absorption. The recombinant proteins were subjected to SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes with a semidry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Then the membranes were treated with 5% skimmed milk in PBS containing 0.25% Tween20 (PBS-T) for an hour at room temperature and subjected to the immunoblotting assay. The membranes were incubated in PBS-T with 1/500 volume of each antibody for an hour at room temperature. After washing the membranes three times for 10 min each with PBS-T, anti-mouse immunoglobulin alkaline phosphatase-conjugated rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in PBS-T was added and incubated for an hour at room temperature. The membranes were washed again and developed in alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories). Since whole molecules were employed as antigens, the antisera reacted not only with LukS-PV or LukF-PV, but also with the components of gammahemolysin and LukDE leukocidin (Fig. 1B and D). After cross absorption by affinity columns, however, the antisera became specific to LukS-PV (Fig. 1C) and LukF-PV (Fig. 1E). 2.3. Setting the reverse passive latex agglutination (RPLA) assay using specific antibodies and determination of its detection limit Using the specific antisera prepared above, we next prepared specific antibody-sensitized latex. Polystyrene beads (JSR Corporation, Tokyo, Japan) were sensitized as described previously (Igarashi et al., 1986), with an optimum concentration of the specific antibodies. The beads were suspended in PBS containing 0.5% bovine serum albumin. The detection limit of RPLA assay was evaluated using the solutions containing 100 ng/ml of recombinant LukS-PV or LukF-PV protein as standards. A 25 μl each of the toxin solutions was serially diluted with reaction buffer from 1:2 to 1:128 in a 96 well V-bottom microtiter plate. A 25 µl each of the sensitized latex suspension was added to the
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Table 1 The RPLA-based detection and titration of PVL toxin in the culture medium of clinical S. aureus strains Strains
C199800370 C199900529 C1999000193 C1999000459 (MW2) C2001001201 C2001000101 C200100818 JCSC5174 JCSC5182 JCSC5183 JCSC5184 JCSC5192 JCSC5391 JCSC5169 JCSC517O JCSC5171 JCSC5172 JCSC5189 JCSC1O8 JCSC2911 JCSC2912 JCSC2913 JCSC2914 JC5C4488 ATCC49775 01083 01102 A803355 A823549 A83 0528 B826559 D82 1552 E802537 F81 0539 I802552 MW2 ΔPVL N315 JC3C2952 JC3C2962 M9N M33T W1S C7N 00215 01093 810342 810508 810937 811238 911573 911575 911703 912118 912125 912145 912231 912572 912574 912619 912666 SAP26O SAP344 SAP411 WCH379
Carriage Titers of LukS-PV and LukF-PV in of luk CCY modified BHl medium S-F-PV medium LukSPV
Reference(s)
LukSPV
LukFPV
LukFPV
+ + + +
N1:128 N1:128 1:32 N1:128
N 1:128 N 1:128 1:64 N 1:128
1:64 1:64 1:64 1:32
N 1:128 N 1:128 N 1:128 1:64
Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002)
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 N1:128 1:8 N1:128 N1:128 N1:128 N1:128 N1:128 1:16 N1:128 N1:128 1:32 1:32 1:64 1:32 1:64 1:64 1:32 1:16 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2
N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 1:16 N 1:128 N 1:128 N 1:128 N 1:128 N 1:128 1:32 N 1:128 N 1:128 1:128 1:128 N 1:128 1:128 N 1:128 N 1:128 1:128 1:32 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2
1:32 1:8 1:32 1:64 1:64 1:64 1:64 1:32 1:128 1:16 1:64 1:128 1:128 1:32 1:8 N1:128 1:16 1:16 1:16 N1:128 1:2 1:16 1:16 1:32 1:8 1:32 1:16 N1:128 1:16 1:4 1:4 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2 b1:2
1:32 1:32 1:64 N 1:128 N 1:128 1:128 1:128 1:64 1:128 1:64 N 1:128 N 1:128 N 1:128 1:64 1:32 N 1:128 1:64 1:64 1:64 N 1:128 1:16 1:64 1:64 1:64 1:16 1:64 1:32 N 1:128 1:32 1:16 1:16 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2 b 1:2
Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2005) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Ma et al. (2006) Prevost et al. (1995) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) This study Okuma et al. (2002) Ma et al. (2006) Ma et al. (2006) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002) Okuma et al. (2002)
diluted toxins and mixed by agitation. For agglutination reaction, the plate was kept overnight at room temperature under the moist condition to prevent the plate from drying. Thereafter, the end points
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of agglutination were determined visually. The detection limit for the recombinant PVL components was 1:64 dilutions for both anti-LukSPV and anti-LukF-PV beads, indicating that the detection limit of RPLA assay for PVL was both approximately 1 ng/ml. 2.4. Specificity and sensitivity of the RPLA assay To test the specificity and sensitivity of the RPLA assay to detect pvl-gene-carrying S. aureus strains, we tested a total of 64 S. aureus clinical and laboratory strains, whose carriage or non-carriage of pvl genes had been established by PCR experiments (Ma et al., 2005; Okuma et al., 2002). Strains ATCC49775 and MW2 were included as positive control strains with PVL gene, and strain MW2ΔPVL, a derivative strain of MW2 with its PVL genes replaced by a chloramphenicol acetyl-transferase gene (Schneewind et al., 1993), as a negative control strain. For the RPLA assay, two culture media were used: They were 1) CCY modified medium (Finck-Barbancon et al., 1991) and 2) BHI medium (Becton Dickinson and Company). The strains were grown in 3 ml of the media for 24 h with shaking at 140 rpm, and their supernatants were used for RPLA assay without any additional treatment. The results of RPLA assay perfectly correlated with the PCR-based detection of PVL gene (Table 1). All of the 35 pvlgene-positive strains were successfully detected by the RPFL assay, and none of the 29 pvl-gene-negative strains became false positive. Thus, the method demonstrated 100% sensitivity and 100% specificity in the detection of PCR-based pvl-gene-positive strains. In that sense, the assay was shown to be equivalent to PCR detection of PVL-genecarrying S. aureus. 2.5. Quantitation of the PVL produced by each clinical strain Besides identification of the PVL-producing strains, the quantitative nature of the RPLA assay was used to measure the amount of PVL produced by each of the strains. The results shown in Table 1 revealed the presence of significant difference in the amount of PVL produced by each strain (from 1:4 to N1:128; Table 1). This implies that the PVLgene carriage alone may not fully correlate with the degree of the strain's virulence if we assume that the amount of PVL toxin produced by the strain is associated with the degree of virulence of the strain. The role of PVL in the pathogenesis or virulence of S. aureus should be evaluated taking this quantitative difference in the PVL production among clinical S. aureus isolates. Table 1 also shows the influence of culture medium on the production of PVL toxin. The amount of produced toxin was generally greater in CCY medium than in BHI. Some strains such as C2001000101 produced at least 16 times more amount of LukS-PV in CCY than in BHI (Table 1). These data suggest that there is a factor that allows high PVL production in CCY medium, which is absent from BHI medium. Thus it is likely that the virulence level of PVL-producing S. aureus as well may be influenced by certain factors present in the environment. On the other hand, it was also noted that the production of PVL by strain 81/108 was not much influenced by the change of medium (Table 1). Thus, the toxin production may also be dependent on the genotype of the strain. 3. Discussion An ELISA assay to detect LukF-PV, one of the two components of the PVL toxin, was reported by Hamilton et al. (Hamilton et al., 2007). The LukF-PV-specific polyclonal rabbit antiserum used for their study was not evaluated for the cross-reactivity with the other toxin homologs such as HlgB or LukE. So, the antibodies are not suited for the rapid quantitation assay described in this study. They reported that the quantity of LukF-PV produced in vitro did not correlate with the severity of infection. However, our study revealed that the two components of the PVL toxin were produced in different quantity in some strains (see Table 1). Therefore, the quantitation of only the “F
component” of the toxin may not reflect the activity of the assembled composite PVL toxins. We consider that the use of a set of the highly specific anti-S and F-component antibodies would be needed in the correct analysis the role of PVL toxin in the high virulence of certain S. aureus clinical strains. In conclusion, the RPLA would be a useful, reliable and simple tool not only to detect PVL-producing S. aureus clinical strains but also to know the amount of toxin produced by the strains without the crossreactivity with other toxin homologs. The quantitative feature of the RPLA assay would add more information to the understanding of the pathogenesis of the PVL-producing S. aureus clinical strains. Acknowledgements This work was supported by a Grant-in-Aid for 21st Century COE Research from The Ministry of Education, Science, Sports, Culture and Technology of Japan. 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