Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureusPanton-Valentine leukocidin for Diagnostic and Therapeutic Applications
© Okolie et al.; licensee BioMed Central Ltd. 2013
Received: 9 September 2013
Accepted: 31 October 2013
Published: 19 November 2013
Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts.
Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively.
The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL.
KeywordsStaphylococcus aureus Panton-Valentine leukocidin Leukocytolytic exotoxin Chromatography Mass spectrometry Immuno-therapy
Among two-component synergo-hymenotrpoic toxins associated with the genus Staphylococcus, leukocidin (Luk), gamma-hemolysin (Hlg), and Panton-Valentine leukocidin (PVL) have been well documented [1–3]. The synergo-hymenotrpoic toxins are encoded by distinct genetic loci . Staphylococcus aureus PVL is encoded by two co-transcribed genes, lukS-PV and lukF-PV which reside in the genomes of several bacteriophages associated with various different strains of S. aureus[3, 5, 6]. The active form of PVL requires the assembly of the two corresponding polypeptides, respectively LukS-PV and LukF-PV. The prophage harbouring PVL-encoding genes in S. aureus strain USA400 (also known as S. aureus strain MW2) is phiSa2MW[7, 8].
Originally described in association with pus formation, carbuncles and furuncles , PVL did not receive much attention in scientific literature because the pathogenicity of S. aureus was formerly perceived to be restricted to secondary infections . PVL-positive S. aureus (PPSA) was first associated with primary skin infections in France . Following a report associating PVL with primary skin infections and pneumonia , reports of the prevalence of primary human diseases associated with PPSA, including highly contagious therapy-refractory skin infections and life-threatening haemoptysis, have been on the increase all over the world [13, 14].
In Germany, Jung and colleagues reported the case of a 23-year-old apparently healthy female patient without any typical predisposing findings who developed severe sepsis with necrotizing pneumonia and multiple abscesses following incision of a Bartholin's abscess . Methicillin-sensitive S. aureus harbouring PVL genes were cultured from the abscess fluid, multiple blood cultures and a postoperative wound swab. Aggressive antibiotic therapy with flucloxacillin, rifampicin and clindamycin, drainage and intensive supportive care lead finally to recovery. In their concluding recommendation, PPSA strains, should be considered when a young, immunocompetent person develops a fulminant necrotizing pneumonia as minor infections, such as Bartholin's abscess, can precede this life-threating syndrome. That was probably the first use of the word “SYNDROME” in the description of PVL-associated clinical conditions. Other signs and symptoms of the PVL-syndrome include multiple organ failure preceding acute respiratory distress syndrome (ARDS) such that the victim deteriorate or die rapidly (average: <72 hours, reports of <24 hours have been documented) from overwhelming toxicity [15–18]. Other reports from Germany [13, 16], Denmark [17, 18], France , Trinidad & Tobago  underline the existence of a "Staphylococcus aureus PVL-syndrome" independent of susceptibility to meticillin.
Furthermore, the body organs/systems involved including the skin, brain, respiratory and musculo-skeletal organs [21–23], points to PVL as a new worldwide public health threat. In Spain, a 34-year-old man was admitted for right parietal brain abscess and thickened dura mater in close proximity to a lytic bone lesion. The abscess culture yielded PVL-positive community-acquired meticillin susceptible S. aureus (PPCAMSSA). The patient survived after surgery and antibiotic treatment. This was the first reported case of a brain abscess due to PPSA . In Egypt, a 50-year-old man was diagnosed with brain abscess associated with PVL-positive community acquired-MRSA (PPCAMRSA) in April 2007; following two intensive care admissions, his death was reported at the end of July 2007 . Survivors of PVL-syndrome often go through rigorous intensive care [15, 23, 25, 26]. Other than the 50-year old Egyptian man, the victims of PVL syndrome are often young healthy persons, including school children (mean age = 20) [14, 15, 17].
Molecular characterization have shown that PVL-positive S. aureus (PPSA) belong to different genotypes  and could be acquired from the community [16, 25, 27] as well as from the hospital . Conventionally optimal medication and management is not effectual and no therapeutic preparation is particularly successful [26, 28] as gold standard treatment is lacking [29, 30]. Treatment is further complicated because the outcomes of PVL-associated conditions are independent of S. aureus meticillin susceptibility, while expert opinion signals bleakness “Until the toxins and inflammatory intermediaries responsible for the necrosis can be neutralised earlier,” [29, 30].
The use of immune bodies for the treatment of toxic S. aureus infection has been reported, including septicaemia which was successfully treated with immune blood . Therefore there is hope for immunoglobulin therapy. Human polyclonal immunoglobulin containing anti-PVL antibodies (TégélinR) was proposed as a potential treatment for PVL necrotizing pneumonia ; but, not much was reported subsequently of Tegelin. Thus the need for rapid and specific therapy against PVL syndrome, and the attendant rapidity of victim deterioration, remains to be met.
To provide the tools for the establishment of therapeutic monoclonal antibodies as indicated by expert opinion, we cloned the two genes (lukS-PV and lukF-PV) coding for LukS-PV and LukF-PV respectively from S. aureus USA400 into two separate expression E. coli BL21(DE3)-pLysS host systems from which we sub-cloned each of them into two separate expression E. coli host systems. Following expression of the constructs, which we confirmed using Coomassie stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, the pro-toxins were purified by chromatography. The molecular masses of the purified peptides were determined by electrospray ionization–time-of-flight (ESI–TOF) mass spectrometry.
Gene cloning and expression protocols and reagents
Except otherwise stated, gene cloning and protein expression protocols were based on standard molecular cloning textbook  with few modifications.
Bacterial strains and plasmids used for Recombination
Bacterial isolates and plasmids used for genetic engineering
S. aureus MW2 (USA400)
Network on antimicrobial resistance in Staphylococcus aureus (NARSA)
pGEM®-T Easy Vector
Intermediate plasmid vector
Lab stock maintained by Dr. Philip Bardelang 
Escherichia coli DH-5α
Intermediate E. coli host
Protein expression plasmid vector
E. coli BL21(DE3)pLysS
Protein expression E. coli host
Cultivation of bacteria and extraction of bacterial genomic DNA
Due to the unascertained risk level of S. aureus strain USA400 (also called MW2), isolation and DNA extraction were performed in biosafety level 3 (BSL3) containment laboratory designated for work with pathogens suspected to be potentially hazardous. It is required by CDC and NIH guidelines that when uncertain of the risk level, a presumably higher protection level should be applied . Sterile disposable loop was used to touch frozen culture and plated out on brain heart infusion (BHI) agar. Following overnight incubation (18-28 hours) at 37°C, genomic DNA was extracted from the grown cultures by suspending four discrete colonies in 1.0 ml of molecular grade nuclease-free water (Sigma, UK) in a 1500 μL PCR grade eppendorf tube (Eppendorf, Germany). The tube was heated (98°C, 10 minutes) and then centrifuged (13000 g, 30 seconds) in an Eppendorf benchtop microcentrifuge (Eppendorf, Germany). The supernatant was taken out of the level 3 suite and used for PCR in the general (BSL2) laboratory.
PCR amplification of recombinant lukS-PV and lukF-PV from S. aureusUSA400
Suitable primer sequences were identified within the lukS-PV and lukF-PV genes of the PVL coding region of S. aureus strain MW2 (GenBank BA000033). The 5´ primers were chosen within the coding sequence of each gene, omitting the region predicted to encode the signal peptide as published for PVL  and verified online (http://www.cbs.dtu.dk/services/SignalP/). On account of previous success in our laboratory , we replaced the signal sequence of each pro-toxin subunit with NcoI sequence, which inserts a methionine start codon; we also replaced the stop codons with XhoI sequence, which enables the insertion of 6His-tag to allow for interactions between poly-histidine fusion tags and immobilized metal ions for protein purification. The 3' primers encompassed the stop codon of each gene. NcoI site (C/CATGG) was engineered into the primer sequence for the 5' primers (rlukS-PV-1 and rlukF-PV-1) and Xho1 site (C/TCGAG) was engineered into the primer sequence for the 3´ primers (rlukS-PV-2 and rlukF-PV-2). We used Pwo DNA polymerase for PCR high yield and fidelity.
Separate cloning of DNA fragments into intermediate E. colihost system
The two recombinant DNA fragments amplified from S. aureus MW2 were separately TA ligated with intermediate vector pGEM®-T Easy (Promega, Madison, USA). Since the DNA fragments were blunt-ended PCR products of high fidelity Pwo polymerase, Taq DNA polymerase was used for 3’-A tailing to allow for ligation with the complementary T projecting from the pGEM®-T Easy plasmid. To allow for 3´-A overhangs necessary for the TA cloning principle of the pGEM®-T Easy vector, 3’-A tailing by Taq DNA polymerase is often performed in our laboratory. Briefly, gel-purified PCR product (4 μL) was transferred into a 200 μL PCR tube maintained on ice and containing thermopol buffer (2 μL), 1.0 μM of d’ATP and 5 units of Taq polymerase (Roche, Germany) all in a total volume of 10 μL. The mixture was vortexed for thorough mixing and incubated on the cycler (72°C, 10 minutes) to enable 3´-A tail. The resulting 3´-A tailed reaction was placed on ice and used immediately for ligation reaction with pGEM®-T Easy plasmid. Aliquot (2 μL) of the ligation was transformed into Escherichia coli 5α and subsequently plated out on Luria-Bertany (LB) agar containing ampicillin (100 μg/mL) and X-gal (40 μg/mL) for easy distinguishing of recombinant (white) colonies from non-recombinant (blue) transformant colonies with further confirmation by PCR amplification.
Sub-cloning of separate insert DNA fragments from intermediate E. coli host system into expression E. colihost system
One white colony of the transformant from recombinant E. coli clones was subcultured in LB broth (Difco Laboratories, Detroit, MI) to obtain plasmid miniprep which was purified using Wizard® SV system (Promega, USA). Each separate fragment of insert DNA (S or F subunit) was released from the intermediate plasmid DNA (pGEM®-T Easy plasmid vector) by double-digest of the miniprep. The double-digest reaction (40 μL) contained 15 uL of the intermediate plasmid miniprep, 4 μL of X10 multicore buffer (promega, UK), 4 μL of X10 Bovine Serum Albumin (Promega, UK) and 0.5 μL of NcoI (NEB, UK) and 0.5 μL of XhoI (NEB, UK). The double-digests were incubated for 4 hours at 37°C. The expression plasmid vector pET-21d (+) (Novagen, USA), was gated and linearized by the same double-digest. Following digests, the restriction digest enzymes were inactivated by heating at 65°C for 15 minutes according to manufacturer’s instructions. Shrimp alkaline phosphatise (SAP), according to the manufacturer’s instruction (Promega, UK), was used to prevent the linearized vector plasmid from recircularizing. Briefly, 20 μL from the 40 μL doubly-digested pET-21d(+) was transferred to a fresh sterile 500 uL Eppendorf tube and made up to 25 μL of dephosphorylation mix by adding 3.0 μL of SAP 10X reaction buffer (equivalent to 50 mM Tris–HCl, pH 9.0 at 37°C, and 10 mM MgCl2) and then 1.0 μL equivalent to 1.0U of SAP. Dephosphorylation was incubated at 37°C for 1 hour. SAP was inactivated by heating at 65°C for 15 minutes. The inserts were then ligated separately with the expression plasmid vector using T4 DNA ligase (NEB, UK). Each ligation reaction for the expression contained 2 μL of the digested insert, 2 μL of pET cut with the same RDEs, 1.0 μL of X5 DNA buffer, 5.0 μL of X2 ligase buffer and 1.0 μL of T4 DNA ligase. The ligation reaction was incubated on the Eppendorf cycler at 22.5°C for 5 minutes. The contents of the ligation reaction tube were concentrated to the bottom of the tube by centrifugation (30,000RPM, 1 minute). A volume of 4 uL of the ligation reaction was used to transform one aliquot of expression E. coli BL21(DE3)-pLysS cells (Invitrogen, Paisley, UK). The transformation reaction was maintained on ice for 30 minutes and heat-shock at 42°C for 45 seconds and returned to ice for 2 minutes. Pre-warmed LB broth (400 μL at 37°C) was added to the transformation reaction which was then maintained at 37°C for 90 minutes in the shaker incubator. Aliquots (20 μL) were then plated out on LB agar supplemented with ampicillin (100 mg/L) and incubated overnight (24 hours). Growth on ampicillin was used as evidence of successful transformation. Transformant E. coli clones were further confirmed by PCR. The resulting plasmids were transformed into the expression E. coli BL21(DE3)pLysS and plated out on LB agar containing ampicillin (100 μg/mL). Growth on LB agar containing ampicillin (100 μg/mL) was was the phenotypic evidence of successful transformation.
Structural confirmation of fusion DNA and translated peptide sequences
Transformant expression E. coli clones were further confirmed by PCR amplification and sequencing of the appropriate DNA insert (rlukS-PV and rlukF-PV) from the respective expression E. coli hosts using the same primers engineered with NcoI and XhoI sequences with which the template DNA was amplified from S. aureus MW2 at the beginning of the cloning experiment. To confirm the structural integrity of each reading frame, including the insertion of the N-terminal T7-Tag® sequence and the C-terminal 6His-Tag® sequence, we sent the purified plasmids to Cambridge Geneservice (http://www.geneservice.co.uk/home/) for sequencing as we did not have the necessary sequencing primers in Nottingham at the time. We then used the sequence translation tool at the European Bioinformatics Institute (transeq) to translate the nucleotide sequences from Geneservice into peptide sequences.
Expression of recombinant proteins
Transformant E. coli clone (1 discrete colony), confirmed by PCR and sequencing, was picked into 200 mL of 2x YT broth in a 2 Litre flask and shaker-incubated at 35°C for 5 hours to mid-log phase (OD600nM = 0.7). Uninduced control (1.0 mL) was removed for SDS-PAGE before adding isopropyl--D-thiogalactopyranoside (IPTG) stock of 839 mM (Fisher, UK) to achieve a 1.0 mM final concentration. The induction was incubated at 30°C with shaking at 200 rpm for 4 hours in a Model G25 controlled environment incubator shaker (New Brunswick Scientific, USA). An aliquot (10 μl) diluted in equal volume of double strength SDS loading buffer was tested on SDS-PAGE to confirm induction using prestained protein marker as a guide of protein size. Protein expression E. coli cells were spun down (13,000RPM, 1 minute) using Eppendorf bench top centrifuge. After discarding the supernatant, the cell pellet was dissolved in 100 uL of 2x SDS-PAGE loading buffer and heated on the cycler for complete lysis (98°C, 3 minutes) followed by another spin at 13,000RPM for 2 minutes. Using pre-stained protein marker to ascertain protein size, an aliquot of the protein rich supernatant (10 μL) was loaded onto a 10% SDS resolving gel through a 6% stacking gel. The gel was run first at 150 V for 15 minutes settling the samples at the interphase between the stacking gel and the resolving gel and then at 175 V for further 30 minutes. The gel was stained with Coomassie stain for 2 hours and then transferred to fast decolorizer for 30 minutes and finally transferred to and left overnight in slow destain. After overnight slow destain, the images were viewed under the white light (UVP, Cambridge, UK).
Bioinformatics predictions prior to purification of S and F proteins
Bioinformatics tools were used to predict the theoretical iso-electric point (pI) and molecular weight (Mw) of both proteins. The direct strand of the nucleotide sequence for each protein subunit including the six histidine moieties were translated into protein using the nucleotide-protein translation tool (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html) provided by the European Bioinformatics Institute (EBI). To infer the structural bioinformatics image of the two pro-toxins, we entered the sequences obtained from transeq into the protein structure prediction server (PS)2 (http://ps2.life.nctu.edu.tw/) provided online by the National Chiao Tung University Molecular Bioinformatics Centre. PS2 is an automated homology modelling server. The method uses an effective consensus strategy by combining PSI-BLAST, IMPALA, and T-Coffee in both template selection and target-template alignment. The final three dimensional structure is built using the modeling package MODELLER .
Preparation of his-tagged pvl protein subunits for chromatographic purification
Following transeq translation, the theoretical pI and Mw of the protein subunits were computed by entering the sequences of single letter protein codes into the pI analysis tool at EXPASY (http://www.expasy.org/tools/pi_tool.html). Preparation of His-tagged PVL protein subunits followed a protocol used in our laboratory for other protein work [34, 37], with modifications. Briefly, the bulk of the E. coli cells expressing the fusion proteins pelleted and saved at -80°C were gently resuspended in 30 ml cold charge buffer (20 mM Imidazole pH 7.0, 50 mM NaCl, 10% (v/v) glycerol, 1 mM PMSF) and then sonicated on ice for 20 minutes (intermittent 15 seconds bursts of 10 microns) using Soniprep 150 Plus Ultrasonic Disintegrator (MSE, London, UK). Two successive 30 minutes centrifugations at 18000 g were used to clear the cell lysate of insoluble cell debris ready for purification by Nickel affinity (Ni++-affinity) chromatography.
Purification of his-tagged pvl protein subunits by nickel-affinity chromatography
Nickel-affinity chromatography (NAC) was performed using fast protein liquid chromatography (FPLC) system (BioLogic LP: Bio-Rad, USA). A communal protocol used for Ni++-affinity FPLC in our laboratory , was used for this study, with some modifications. The schema of the FPLC programme is detailed in Table 1. Briefly, a 5 mL HiTrap chelating HP column, Code Number: 17-0409-01 (GE Healthcare, Amersham Biosciences, Buckinghamshire, UK) was charged with Nickel using a single 20 ml injection of 50 mM NiCl2, equilibrated in buffer A (25 mM NaH2PO4 pH 7.0, 0.5 M NaCl, 5% (v/v) glycerol, 2 mM imidazole), then buffer B (25 mM NaH2PO4 pH 7.0, 0.5 M NaCl, 5% (v/v) glycerol, 1 M imidazole), finally buffer A again. Solid NaCl was added to the cleared cell lysate to a final concentration of 0.5 M and the sample applied to the column at a flow-rate of 1 ml/min. The loaded column was washed in 5% buffer B (15 mL), which is 95% buffer A, until no unbound proteins eluted from the column which is monitored in real-time via the chromatogram generated by the BioLogic LP software. Bound proteins were eluted from the column using a linear gradient of 5-85% buffer B in a total volume of 40 ml. The purity and relative Mw of the fusion proteins were assessed by SDS-PAGE (10%).
Preparation of rLukS-PV for further purification by ion exchange chromatography
The protein was dialysed overnight in buffer A (10 mM Tris, 50 mM NaCl, pH 7.0) using 8000 pore membrane (Millipore, UK). All buffers and solutions intended to run through the system, including water, were degassed before they getting in contact with the AKTA Explorer system.
Purification by ion exchange chromatography
Ion exchange chromatography (IEC) was performed using ÄKTA™ Explorer 10 FPLC (Amersham Pharmacia Biotech, USA) driven by UNICORN 4 software. The buffers and protocol are similar to those described elsewhere [38, 39]. Briefly, the contents of the tubes bearing the S-subunit were pooled together and loaded into the AKTA Explorer FPLC system. The S-loop was filled with degassed double distilled water and attached to the system. The IEC was programmed to bind protein in low salt buffer A (same buffer A used for NAC) and elute in high salt gradient IEC buffer B (10 mM Tris, 1.0 M NaCl, pH 7.0). Buffer A was the donor of positive charge, while buffer B is the ion exchange gradient buffer. Use of Mono S column for IEC had been described elsewhere . Briefly, following a system wash, the protein was injected into the sample inlet of the ÄKTA™ explorer while buffers A and B were loaded into inlets A11 and B1 respectively. The Mono S column was attached to the nozzle between valves V2 and V3 when V2 and V3 were in position 2. The alarm monitors were set to a limit of 3.2 MPa and enabled. The flow-path for the column position was set to position 2. Degassed double distilled water was allowed to flow through the column at a rate of 1 ml/minute for 5 minutes. Another system wash was performed. Water was replaced with buffer A into inlet A11 and buffer B into inlet B1. The protein in buffer A was loaded into the S-loop. The chromatography was performed by allowing the contents of the S-loop to load unto the column at a rate of 1.0 ml/minute.
Further Purification of rLukS-PV by size exclusion (gel filtration) chromatography
Except for the few differences in instrumentation mentioned below, the protocol and reagents used for size exclusion chromatography (SEC) and for the IEC were the same. We replaced the Mono S column used for IEC with a HisPrep™ Superdex 30 column previously used in our laboratory for SEC of protein sizes similar to the 6His-LukS-PV [34, 37]. HisPrep™ columns are designed for scaling up the purification of His-tagged proteins and have 1/16" fittings for convenient use on the ÄKTA™ Explorer FPLC system.
Results and discussion
Recombinant lukS-PV and lukF-PV generated from S. aureus MW2
Sub-cloning of insert rlukS-PV and rlukF-PV from intermediate system into expression system confirmed by culture and PCR
Primers designed in this study and used for recombination
Primer sequence in 5′ → 3′ directionb
(Size in bp)
rlukS-PV (868 bp)
rlukF-PV (919 bp)
Nucleotide sequencing confirms the structural integrity of the reading frames
Nucleotide sequence of the recombinant DNA amplified from the expression E. coli genome (Additional file 1 and Additional file 2) and the translated protein products for respective fusion proteins are presented (Additional file 3 and Additional file 4). At this stage of the experimentation, establishment of clones with perfect (100%) identity, were very encouraging for us to rule out mutations which could generate aberrant proteins. Ranging from the stringency of the Pwo used for initial amplification of the recombinant DNA, through every step of the cloning, we are pleased with the outcome. The accuracy in size and structure of the sequences are good rewards for all the effort.
SDS-PAGE shows over-expression and relative sizes of S and F protein subunits
Protein purification by nickel-affinity chromatography shows dissimilar yields
Schema of the Ni++-NTA fast protein liquid chromatography programmea,b
00 - 20
20 - 40
40 - 60
60 – 90
E = Protein
90 – 115
115 – 130
Mix 5% B
→ 120 Collect 2 mL
130 - 180
Gradient of 5 to 100% B
180 - 200
→ Divert to waste
End of method, washing up of the machine, and log off
Ion exchange chromatography removes some impurity from fusion LukS-PV
Characteristics of mono S column used for ion exchange chromatography a
50 × 5 mm
750 psi (5 MPa)
Approximate separation time
Size exclusion chromatography purified fusion LukS-PV ultimately
Molecular weight (Mw) of peptides determined by concordance between bioinformatics prediction, SDS-PAGE and mass spectrometry
Our aim to provide biological tools for possible translation into anti-PVL therapy was driven by expert opinion on the menacing PVL syndrome. As the risk level of PVL-positive S. aureus is yet to be ascertained, manipulations of the S. aureus substrain MW2 (USA400) from NARSA were carried out in the BSL3. Given the several life-threatening conditions associated with USA400 especially, we would rather apply higher safety precautions than actually required to guarantee the safety of our laboratory users. This step was very important to us and the outcome was quite rewarding as there has been no record of our laboratory users/visitors, both the BSL3 and the BSL2, including the adjacent offices, coming down with PVL-syndrome, even long after completion of our experiments.
We are happy with the outcome of the DNA sequencing and BLAST which we used to confirm molecular identity at all necessary stages. The pET-21d(+) vector was chosen because it carries an N-terminal T7•Tag® sequence and a C-terminal 6His•Tag® sequence. The T7 and 6His-Tag motifs are very important for expression and for down-stream treatments including purification by Nickel-affinity chromatography. Therefore, it was necessary to send the DNA out of our facility to be sequenced by the Cambridge Geneservice as they had the T7 terminator primers which were not available in Nottingham at the time.
The reliability of the BioLogic-LP and AKTA systems to guide the progress of the purification cycle in real-time is commendable. The purification plots showed that the absorbance at any point during the elution correlated directly with the concentration of His-tagged protein in the eluate (Figures 4, 5 and 6). Also, the SDS-PAGE used to infer protein purity and molecular size showed that the relative intensity of the protein bands eluted from the system correlates with the AU observed during the run. This is one of the innovative and user-friendly features of modern chromatographic systems. The fact that the experimenter can readily identify the tube(s) containing the His-tagged proteins is very helpful as it reduces waste of time and materials. Though the initial cost of purchasing any of the modern chromatographic instruments might be perceived as high, in the end they are cost-effective.
Taken together, we engineered the lukS-PV and lukF-PV genes respectively encoding the S and F subunits of S. aureus PVL toxin from S. aureus stain USA400 into separate E. coli expression host systems. Having purified the pro-toxin subunits, we supplied them to the Nottingham Cancer Immunotherapy group who used them to establish anti-PVL monoclonal antibodies. We are happy that the pro-toxin subunits have served as useful input for the establishment of sequence-based diagnostic and therapeutic tools for rapid and specific diagnosis and abrogation of PVL-associated leukocytolysis and the attendant rapid deterioration of its victims. In keeping with the University of Nottingham policies on genetically modified organisms, we handed over the engineered E. coli clones expressing the PVL pro-toxin subunits, namely E. coli BL21(DE3)-pET-21d(+)-lukS-PV and E. coli BL21(DE3)-pET-21d(+)-lukF-PV, for LukS-PV and LukF-PV respectively, to the Biosafety Officer (AC) for safe-keeping.
The S. aureus MW2 used for this work was sub-cultured from NARSA staphylococcal strains collected by Dr. Richard Spence. The Escherichia coli, plasmids and the protocol for Nickel-affinity chromatography on BioLogicLP system were communal stuff maintained by Dr. Philip Bardelang (School of Molecular Medical Sciences, University of Nottingham). We thank Graham Coxhill (School of Chemistry, University of Nottingham) for assistance with Mass Spectrometry and Dr. Mireille Vankemmelbeke (School of Molecular Medical Sciences, University of Nottingham) for support with AKTA Explorer chromatography system. CEO gratefully thanks Dr. Harry Hoffmann Abruquah and his wife Mrs Nana Aquah Abruquah (Kwame Nkruma University of Science and Technology, Kumasi, Ghana) for proof-reading and making very helpful remarks on his PhD thesis and on the manuscript. For their support of his PhD, CEO thanks his friends and family as well as the University of Nottingham. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Supersac G, Prevost G, Piemont Y: Sequencing of leucocidin R from Staphylococcus aureus P83 suggests that staphylococcal leucocidins and gamma-hemolysin are members of a single, two-component family of toxins. Infect Immun. 1993, 61: 580-587.Google Scholar
- Prevost G, Bouakham T, Piemont Y, Monteil H: Characterisation of a synergohymenotropic toxin produced by Staphylococcus intermedius. FEBS Bp. 1995a, 376: 135-140.View ArticleGoogle Scholar
- Kaneko J, Kamio Y: Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes. Biosci Biotechnol Biochem. 2004, 68: 981-1003.View ArticleGoogle Scholar
- Prevost G, Cribier B, Couppie P, Petiau P, Supersac G, Finck-Barbancon V, et al: Panton-Valentine leucocidin and gamma-hemolysin from Staphylococcus aureus ATCC 49775 are encoded by distinct genetic loci and have different biological activities. Infect Immun. 1995b, 63: 4121-4129.Google Scholar
- Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, et al: Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet. 2006, 367 (9512): 731-739.View ArticleGoogle Scholar
- Ma XX, Ito T, Kondo Y, Cho M, Yoshizawa Y, Kaneko J, Katai A, Higashiide M, Li S, Hiramatsu K: Two different Panton-Valentine leukocidin phage lineages predominate in Japan. J Clin Microbiol. 2008, 46 (10): 3246-3258.View ArticleGoogle Scholar
- Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K-I, Oguchi A, et al: Genome and virulence determinants of high virulence community-acquired MRSA. The Lancet. 2002, 359: 1819-1827.View ArticleGoogle Scholar
- Hiramatsu K, Watanabe S, Takeuchi F, Ito T, Baba T: Genetic characterization of methicillin-resistant Staphylococcus aureus. Vaccine. 2004, 22 (1): S5-S8.View ArticleGoogle Scholar
- Panton PN, Valentine FCO: Staphylococcal toxin. The Lancet. 1932, 219: 506-508.View ArticleGoogle Scholar
- Hay RJ, Steer AC, Engelman D, Walton S: Scabies in the developing world–its prevalence, complications, and management. Clin Microbiol Infect. 2012, 18 (4): 313-323.View ArticleGoogle Scholar
- Prevost G, Couppie P, Prevost P, Gayet S, Petiau P, Cribier B, et al: Epidemiological data on Staphylococcus aureus strains producing synergohymenotropic toxins. J Med Microbiol. 1995, 42: 237-245.View ArticleGoogle Scholar
- Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter M-O, Gauduchon V, et al: Involvement of Panton-Valentine alentine leukocidin-producing staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999, 29: 1128-View ArticleGoogle Scholar
- Schleucher RD, Gaessler M, Knobloch J: Panton-Valentine leukocidin-producing methicillin-sensitive staphylococcus aureus as a cause for recurrent, contagious skin infections in young, healthy travelers returned from a tropical country: a new worldwide public health problem?. J Travel Med. 2008, 15: 137-139.View ArticleGoogle Scholar
- Muttaiyah S, Coombs G, Pandey S, Reed P, Ritchie S, Lennon D, et al: Incidence, risk factors, and outcomes of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus infections in Auckland, New Zealand. J Clin Microbiol. 2010, 48 (10): 3470-3474.View ArticleGoogle Scholar
- Jung N, Lehmann C, Hellmann M, Seifert H, Valter MM, Hallek M, et al: Necrotizing pneumonia caused by Panton-Valentine leucocidin-producing Staphylococcus aureus originating from a Bartholin's abscess. Infect Dis Obstet Gynecol. 2008, 2008: 491401-doi: 10.1155/2008/491401View ArticleGoogle Scholar
- Schefold JC, Esposito F, Storm C, Heuck D, Krüger A, Jörres A, et al: Therapy-refractory Panton Valentine Leukocidin-positive community-acquired methicillin-sensitive Staphylococcus aureus sepsis with progressive metastatic soft tissue infection: a case report. J. Med. Case Reports. 2007, 1: 165-View ArticleGoogle Scholar
- Skov R: Panton-Valentine-leukocidin producing Staphylococcus aureus as the cause of necrotizing pneumonia and death. Ugeskr Laeger. 2010, 172 (16): 1189-Google Scholar
- Rabol PH, Dessau RB, Bjerglund L: Fatal pneumonia with Panton-Valentine leucocidin-producing Staphylococcus aureus. Ugeskr Laeger. 2010, 172 (16): 1190-1191.Google Scholar
- Dubrous P, Cuguillère A, Gendrot A, Koeck JL: Panton-Valentine leukocidin-producing Staphylococcus aureus responsible for necrotizing pneumonia. Ann Biol Clin (Paris). 2007, 65 (3): 277-281.Google Scholar
- Akpaka PE, Monecke S, Swanston WH, Rao AC, Schulz R, Levett PN: Methicillin sensitive Staphylococcus aureus producing Panton-Valentine leukocidin toxin in Trinidad & Tobago: a case report. J Med Case Rep. 2011, 5: 157-View ArticleGoogle Scholar
- Al-Talib H, Hasan H, Yean CY, Al-Ashwal SM, Ravichandran M: Fatal necrotizing pneumonia caused by Panton-Valentine leukocidin-producing hospital-acquired Staphylococcus aureus: a case report. Jpn J Infect Dis. 2011, 64 (1): 58-60.Google Scholar
- Catena V, Baiocchi M, Lentini P, Badolati L, Baccarin M, Del Monte DD, Rubini A: Necrotizing pneumonia caused by Panton-Valentine leukocidin-producing methicillin-susceptible Staphylococcus aureus (MSSA). Infez Med. 2010, 20 (3): 205-210.Google Scholar
- Rafai M, Abouelalaa K, Skhsoukh Y, Balkhi H, Belyamani L, Dimou M: Evolution of a fatal septic arthritis caused by a Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus strain. Joint Bone Spine. 2013, 80 (5): 525-527.View ArticleGoogle Scholar
- Ramos A, Ley L, Muñez E, Videl A, Sánchez I: Brain abscess due to Panton-Valentine leukocidin-positive Staphylococcus aureus. Infection. 2009, 37 (4): 365-367.View ArticleGoogle Scholar
- Enany S, Higuchi W, Okubo T, Takano T, Enany M, Yamamoto T: Brain abscess caused by Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus in Egypt, April 2007. Euro Surveill. 2007, 12 (39):Google Scholar
- Noah MA, Dawrant M, Faulkner GM, Hill AM, Harvey C, Hussain A, Jenkins DR, Nichani S, Peek GJ, Sosnowski AW, Firmin RK: Panton-Valentine leukocidin expressing Staphylococcus aureus pneumonia managed with extracorporeal membrane oxygenation: experience and outcome. Crit Care Med. 2010, 38 (11): 2250-2253.View ArticleGoogle Scholar
- Enany S, Yaoita E, Yoshida Y, Enany M, Yamamoto T: Molecular characterization of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus isolates in Egypt. Microbiol Res. 2010, 165 (2): 152-162.View ArticleGoogle Scholar
- Gillet Y, Dumitrescu O, Tristan A, Dauwalder O, Javouhey E, Floret D, Vandenesch F, Etienne J, Lina G: Pragmatic management of Panton-Valentine leukocidin-associated staphylococcal diseases. Int J Antimicrob Agents. 2011, 38 (6): 457-464.View ArticleGoogle Scholar
- Morgan MS: Diagnosis and treatment of Panton-Valentine leukocidin (PVL)-associated staphylococcal pneumonia. Int J Antimicrob Agents. 2007, 30 (4): 289-296.View ArticleGoogle Scholar
- Morgan M: Staphylococcus aureus, Panton-Valentine leukocidin, and necrotising pneumonia. BMJ. 2005, 331: 793-794.View ArticleGoogle Scholar
- Hooker RS: The treatment of staphylococcus septicaemia by transfusion of immune blood. Ann Surg. 1917, 66: 513-521.View ArticleGoogle Scholar
- Gauduchon V, Cozon G, Vandenesch F, Genestier A-L, Eyssade N, Peyrol S, et al: Neutralization of staphylococcus aureus Panton valentine leukocidin by intravenous immunoglobulin in vitro. J Infect Dis. 2004, 189: 346-353.View ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: a Laboratory Manual. 1989, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2Google Scholar
- Bardelang P, Vankemmelbeke M, Zhang Y, Jarvis H, Antoniadou E, Rochette S, Thomas NR, Penfold CN, James R: Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin. Biochem J. 2009, 418 (3): 615-624.View ArticleGoogle Scholar
- Centers for Disease Control and Prevention and National Institutes of Health: Biosafety in Microbiological and Biomedical Laboratories. 2007, Washington: United States Government Printing Office, 5Google Scholar
- Chen CC, Hwang JK, Yang JM: (PS)2: Protein structure prediction server. Nuc Acids Res. 2006, 34 (suppl 2): W152-W157. Available at: http://ps2.life.nctu.edu.tw/View ArticleGoogle Scholar
- Warfield R, Bardelang P, Saunders H, Chan WC, Penfold C, James R, Thomas NR: Internally quenched peptides for the study of lysostaphin: an antimicrobial protease that kills Staphylococcus aureus. Org Biomol Chem. 2006, 4: 3626-3638.View ArticleGoogle Scholar
- Chang V, Ling-Yi C, Wang A, Yuan X: The effect of Lipopolysaccharide core structure defects on transformation efficiency in isogenic Escherichia coli BW25113 rfaG, rfaP, and rfaC mutants. J ExpMicrobiol Immunol (JEMI). 2010, 14: 101-107.Google Scholar
- Kuo WH, Chase HA: Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions. Biotechnol Lett. 2011, 33 (6): 1075-1084.View ArticleGoogle Scholar
- Kanade RP, Notani NK: Cloning and characterization of a DNA repair gene in Haemophilus influenzae. J Biosci. 1987, 12 (2): 115-123.View ArticleGoogle Scholar
- Liu YG, Nagaki K, Fujita M, Kawaura K, Uozumi M, Ogihara Y: Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector. Plant J. 2000, 23 (5): 687-695.View ArticleGoogle Scholar
- Graves SF, Kobayashi SD, Braughton KR, Diep BA, Chambers HF, Otto M, et al: Relative contribution of Panton-Valentine leukocidin to PMN plasma membrane permeability and lysis caused by USA300 and USA400 culture supernatants. Microbes Infect. 2010, 12 (6): 446-456.View ArticleGoogle Scholar
- Sriwanthana B, Island MD, Maneval D, Mobley HL: Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography. J Bacteriol. 1994, 176 (22): 6836-6841.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.