Dataset Information

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.

ABSTRACT: BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

SUBMITTER: Okolie CE

PROVIDER: S-EPMC3870988 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.

Okolie Charles Emeka CE Cockayne Alan A Penfold Christopher C James Richard R

BMC biotechnology 20131119

<h4>Background</h4>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 ...[more]

PMID: 24252611

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Project description:Staphylococcus aureus necrotizing pneumonia is a severe disease caused by S. aureus strains carrying the Panton Valentine leukocidin (PVL) genes (lukS-PV & lukF-PV) encoded on various bacteriophages (such as phiSLT). Clinical PVL+ strains isolated from necrotizing pneumonia display an increased attachment to matrix molecules (type I and IV collagens and laminin), a phenotype that could play a role in bacterial adhesion to damaged airway epithelium during the early stages of necrotizing pneumonia (J Infect Dis 2004; 190: 1506-15). To investigate the basis of the observed adhesion of S. aureus PVL+ strains, we compared the ability of PVL+ and their isogenic PVL(-) strains to attach to various immobilized matrix molecules. The expression of recombinant fragments of the PVL subunits and the addition of synthetic peptides indicated that the processed LukS-PV signal peptide (LukS-PV SP) was sufficient to significantly enhance the ability of S. aureus to attach to extracellular matrix (ECM) components. Furthermore, we showed that adhesion to ECM components was inhibited by heparin and heparan sulfates (HS) suggesting that in vivo, HS could function as a molecular bridge between the matrix and S. aureus expressing the LukS-PV signal peptide. Site directed mutagenesis, biochemical and structural analyses of the LukS-PV signal peptide indicate that this peptide is present at the S. aureus surface, binds to HS in solid phase assay, and mediates the enhanced S. aureus matrix component adhesion. Our data suggests that after its cleavage by signal peptidase, the signal peptide is released from the membrane and associates to the cell wall through its unique C-terminus sequence, while its highly positively charged N-terminus is exposed on the bacterial surface, allowing its interaction with extracellular matrix-associated HS. This mechanism may provide a molecular bridge that enhances the attachment of the S. aureus PVL+ strains to ECM components exposed at damaged epithelial sites.

Project description:Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Dataset Information

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.

Publications

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.

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