This study was performed in accordance with the Australian Prevention of Cruelty to Animals Act 1986 and the Australian code of practice for the care and use of animals for scientific purposes. The protocol was approved by the Animal Ethics Committee of the University of Melbourne (Permit Number: 0911248.2).
Bacterial strains and culture
Bacterial strains used in this study are summarized in Table
1 (international clone collection) and Additional file
1 (ST93 strain collection), and include the ST93 reference strains JKD6159, USA300 strain FPR3757
, ST30 strain JKD6177, and the HA-MRSA ST239 clone JDK6009
, as well as 58 additional ST93 collected from around Australia and previously reported
. For all experiments except exotoxin expression bacteria were grown in brain heart infusion broth (BHI, Oxoid). For the mouse skin infection assay, S. aureus were harvested at the stationary phase of growth after 18 hours incubation (OD600 ~ 2.0), washed, diluted and resuspended in PBS. The bacterial inoculum (CFU) and viable counts were determined by plating onto BHI agar and colony enumeration.
For LukF-PV expression experiments, bacteria were grown in CCY media (3% yeast extract (Oxoid), 2% Bacto Casamino Acids (Difco), 2.3% sodium pyruvate (Sigma-Aldrich), 0.63% Na2HPO4, 0.041% KH2PO4, pH 6.7). For α-hemolysin (Hla) and PSMα3 expression experiments, bacteria were grown in tryptone soy broth (TSB, Oxoid). Overnight cultures were diluted 1:100 into fresh media and then incubated at 37°C with shaking (180 rpm) until stationary phase was achieved. For LukF-PV detection, isolates were cultured for 8 hours (OD600 ~ 1.8); for Hla detection, isolates were cultured for approximately 3 hours (OD600 ~ 1.8); and for PSMα3 detection, isolates were cultured for 24 hours (OD600 ~ 2.0). Culture supernatants were harvested by centrifugation and filter sterilized. These were performed in at least triplicate for each S. aureus strain.
Detection of LukF-PV and Hla by western blotting
Trichloroacetic acid was added to culture supernatants and incubated at 4°C overnight. Precipitates were then harvested by centrifugation, washed with acetone, air-dried and solubilized in a SDS and 2-mercaptoethanol containing sample buffer. The proteins were separated on 12% SDS-PAGE. A peptide sequence specific to LukF-PV, HWIGNNYKDENRATHT was synthesized and HRP conjugated polyclonal chicken IgY was raised against this peptide (Genscript). This antibody was used to detect LukF-PV with enhanced chemiluminescence. Images generated from the western blots were quantitated using GS800 Calibrated Densitometer (BioRad) and Image J
Hla was detected using a polyclonal rabbit anti-Hla (Sigma-Aldrich), in buffer containing 20 mM DEPC to inhibit non-specific protein A binding and HRP conjugated sheep anti-rabbit secondary antibody (Millipore) with enhanced chemiluminescence detection
. For comparison of JKD6159 versus international clone collection (Table
1), images generated from the Hla western blots were quantitated using GS800 Calibrated Densitometer (BioRad) and Image J
Subsequently, for comparison of JKD6159 and other ST93 strains (Table
1), detection of chemiluminescence was performed using the MF-ChemiBIS 3.2 platform (DNR Bioimaging systems). Quantitation was performed using Image J
Detection of PSMα3 expression
HPLC chromatography was performed on an Agilent Technology 1200 Series system with an analytical Agilent Eclipse XDB-C18 (4.6 mm × 150 mm) column. A water/acetonitrile gradient (0.1% trifluoroacetic acid) from 0 – 100% acetonitrile over 28 min at a flow rate of 1 mL/min was used. The total run time was 32 min, and peaks were quantified at a wavelength of 214 nm. The deformylated and formylated form of PSMα3 MEFVAKLFKFFKDLLGKFLGNN was identified in the S. aureus TSB culture supernatants by comparison of their retention times to a commercially synthesized PSMα3 standard (GenScript) and by spiking the samples with the synthesized standards. The identity of the deformylated peptide present in the samples was confirmed by analysing collected fractions by ESI-MS. There was only one peptide present in this fraction; the deformylated form of PSMα3. In contrast, other peptides were observed in the fractions of USA300, JKD6272, TPS3104, TPS3105r, and JKD6159_AraCr containing the N-formylated form of PSMα3. In these cases, the percentage of N-formylated PSMα3 peptide was determined using the total ion count of the major peaks in the ESI-MS and the peak area of the HPLC chromatogram was adjusted accordingly. The concentrations of the deformylated and formylated forms of PSMα3 were determined by comparison of their peak areas to those of the synthesized standards. The standard curves were constructed in the concentration range of 6.2 – 100 μg/ mL and were linear over this range.
DNA methods, molecular techniques and construction of mutants
DNA was extracted using the GenElute kit according to the manufacturer’s instructions (Sigma-Aldrich). A lukSF-PV knockout, hla knockout and a repaired agrA of TPS3105 were generated according to the published method
. For the knockouts, flanking sequences were amplified and ligated prior to cloning with pKOR1. For allelic replacement to generate TPS3105r, a PCR product of agrA from JKD6159 was cloned with pKOR1. For allelic replacement JKD6159_AraCr, a PCR product of this AraC regulator from TPS3106 was cloned with pKOR1. The deletion of the whole psmα locus in JKD6159, chromosomal restoration of psmα in JKD6159∆psmα and the restoration of Hla expression in JKD6159∆hla were conducted using the pIMAY protocol described by Monk et al.
. Knockout and restoration amplimers were cloned into pIMAY by SLIC
. The primers used are listed in Additional file
11. The knockout and restoration clones were confirmed by PCR and Sanger sequencing of the mutated locus. Mutations were further validated with functional assays of activity, which included sheep blood hemolysis, western blot, and/or HPLC.
Mouse skin infection assay
Mice were infected with S. aureus as previously described
. Briefly, six-week-old female BALB/c mice were infected by intradermal injection with 108 CFU of S. aureus. Mice were assessed and weighed daily for five days. Mice were culled on the 5th day and lesion size measured and CFU recovered from infected tissues by homogenization and colony enumeration on BHI. For each S. aureus strain, at least 10 mice were assessed.
Genome sequences for three ST93 strains (TPS3104, TPS3105, TPS3106) were obtained from an Illumina GAIIx analyzer using 100 bp paired-end chemistry with a mean fold coverage of 331×. Genome sequencing of the two laboratory-induced mutants JKD6159∆hla (TPS3265) and JKD6159_AraCr (TPS3268) was performed using Ion Torrent sequencing technology.
A read mapping approach was used to compare the sequences from all isolates used in this study, as previously described
[14, 37]. Briefly, the reads from all genomes were aligned to the JKD6159 reference using SHRiMP 2.0
. SNPs were identified using Nesoni v0.60 [
http://www.bioinformatics.net.au]. Using the whole genome sequence of JKD6159 as a reference, a global SNP analysis was performed, and allelic variability at any nucleotide position was tallied to generate a global SNP analysis for every genome compared to JKD6159.
Quantitative RT-PCR for RNAIII expression
To investigate activity of the agr locus (RNAIII) qRT-PCR was performed for RNAIII as previously described
. Briefly, RNA was prepared as previously described with two on-column DNase I digestion steps and cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Relative expression was determined as previously described and was normalised against gyrB. Results were obtained from 3 biological replicates each performed in triplicate.
Staphylococcus aureus strains JKD6159 and JKD6159_AraCr were grown to early stationary culture as described above. For RNA protection, 0.5 volumes of RNAlater® RNA stabilization reagent (Qiagen) was added immediately to the liquid culture and allowed to incubate with the bacterial suspension for 15 minutes at room temperature. Cells were pelleted at 5,000 × g for 5 minutes followed by RNA extraction using RNeasy mini kit (Qiagen) and two rounds of DNase I digestion (Qiagen) according to the manufacturer’s instruction. RNA concentration was quantified using Qubit® 2.0 Fluorometer and RNA quality assessed using Agilent 2100 Bioanalyzer. Ten μg of total RNA from the stationary growth phase with RNA intergrity number (RIN) greater than 7 was used in RNA-seq. Ribosomal depletion, cDNA library preparation and pair ended sequencing using HiSeq2000 sequencing platform was performed by Beijing Genome Institute (Hong Kong, China). RNAseq was performed on two biological samples for each strain.
RNAseq reads were mapped onto the JKD6159 reference genome
, using SHRiMP 2.2.2
. Alignment to CDS features from each biological replicate of each strain provided counts that were a measure of mRNA levels. Counts were normalized using the trimmed-mean normalization function in edgeR, part of the BioConductor package
. A heat map was created based on log2 transformed counts to identify consistent changes in expression profiles between strains. To be included in the heat map, genes were required to have at least 1000 counts, totaled over all samples, where and the standard deviation of the log2 expression levels had to exceed two.
Percentage mouse weight change at day 5, viable counts of S. aureus in mouse tissues and skin lesion area of each isolate, Hla, LukF-PV and PSMα3 expression versus JKD6159 were analyzed using an unpaired t test. A similar analysis was used to analyze virulence outcome measures and exotoxin expression between TPS3105 and TPS3105r. (There was no difference in results when Bonferonni analysis was performed). All analyses were performed using Prism 5 for Macintosh v5.0b (GraphPad Software Inc.).
Availability of supporting data
The data sets supporting the results of this article are in the NCBI Sequence Read Archive under study accession SRP004474.2 and the NCBI BioProject Archive under study accession PRJNA217697.