MRSA epidemic linked to a quickly spreading colonization and virulence determinant

The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood1. While a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer2, there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare3,4 mobile genetic element-encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, significantly enhancing nasal colonization, lung disease, and abscess formation, and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type (ST) 239 to invasive clones belonging to other STs. Our study identifies sasX as a quickly spreading critical determinant of MRSA pathogenic success and a promising target for therapeutic interference. Importantly, our results provide proof-of-principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.

The sequences of the PCR products were compared with the existing sequences available at the MLST website (http://www.mlst.net) for S. aureus, and the allelic number was determined for each sequence. Spa typing of the polymorphic repeat regions of protein A of S. aureus was performed as previously described 34 . The spa-type was determined and assigned through the spa-type database (http://www.ridom.de/spaserver).
Overexpression and purification of SasX. The sasX gene was cloned and overexpressed as glutathione-S-transferase (GST) fusion. Primers for the amplification of the sasX gene from HS770 genomic DNA are listed in Supplementary Table 3. The PCR product was purified, digested with BamHI and EcoRI, and cloned into the GST gene fusion vector pGEX-4T-1 (Amersham Biosciences). The recombinant vector pGEXsasX was maintained in E. coli strain BL21 (Amersham Biosciences) for overexpression. Unless otherwise noted, cultures of the BL21 (pGEXsasX) strain were grown at 30 °C with aeration to an OD 600 of 1.0. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was subsequently added to a final concentration of 0.5 mM, and the culture was incubated with aeration for an additional 5 h. Then, cells were harvested. The fusion protein was purified using a GST-Tag purification kit (Chemicon) according to the manufacturer's instructions. The purified fusion protein was concentrated in Centriprep-10 concentrators (Amicon) and dialysed against 10 mM sodium phosphate buffer (pH 7.5) using PD-10 desalting columns (Amersham Biosciences). The size of the GST-SasX fusion protein was confirmed by SDS-PAGE (Supplementary Fig. 3).
Production of anti-SasX antiserum. An aliquot containing 200 µg of the purified GST-SasX protein was mixed with 200 cleavage units of thrombin (Sigma) and incubated overnight at room temperature. To remove thrombin and the GST tag from the GST-SasX fusion protein, the protein solution was applied to affinity chromatography on a GST-Tag purification column after digestion. The degree of enzymatic cleavage was followed by SDS-PAGE. The purified SasX protein was used to immunize rabbits by Immune Biotech Ltd. according to a standard immunization program.

Western blot analysis.
For Western immuno-blot analysis, staphylococcal whole cell protein, surface protein, cytoplasmic protein and extracellular protein samples were prepared, separated by SDS-PAGE, and transferred to a nitrocellulose membrane (GenScript). The membranes were treated with rabbit anti-SasX (1:1000) and anti-rabbit immunoglobulin G (IgG)/HRP-linked second antibody (Cell Signaling, 1:2000), and reactions were made visible using diaminobenzidine dye detection.
Immunofluorescence microscopy. S. aureus strains were grown to mid-exponential growth phase, washed, and resuspended in sterile phosphate-buffered saline (PBS) at 10 6 colony-forming units CFU per µl. Aliquots (10 µl) were applied to glass slides. The slides were air-dried and the bacteria were fixed with 4% formalin (Sigma). The fixed cells were incubated with anti-SasX antiserum diluted 1:200 in PBS for 2 h at 37 °C in a humid chamber, washed four times with PBS, and then incubated with FITC-conjugated goat anti-rabbit IgG (Santa Cruz, USA) diluted 1:500 for 1 h at 37 °C in a humid chamber. The slides were washed three times with PBS, twice with double-distilled water, and examined using a fluorescence microscope (Nikon).
Bacterial adherence to nasal epithelial cells. The adherence assay was performed as described by O`Brien et al 35 . Briefly, human nasal epithelial cells were harvested from healthy donors, washed with PBS, and adjusted to 10 4 cells/ml. Informed consent was obtained from human subjects. Bacterial cells were washed with PBS and adjusted to 10 8 cells/ml. Equal volumes (2 ml) of bacterial cells and nasal epithelial cells were mixed and incubated at 37 °C for 2 h with occasional shaking. The unbound bacteria were removed with 6% dextran. Finally, the samples containing epithelial cells and bound bacteria were resuspended in 100 µl PBS, and 20-µl samples were applied to microscope slides. After Gram staining, the number of bacteria per 100 nasal epithelial cells was determined. To test for reproducibility, three batches of cells were tested in the assay.
For blocking experiments, the epithelial cells (10 4 cells ml -1 ) were pre-incubated with recombinant GST-SasX protein, denatured GST-SasX protein, or GST protein alone, to give final concentrations of 0.01, 0.1, 0.5, and 1.0 µM. Epithelial cells were pre-incubated for 30 min at 37 °C. Bacterial cells were then incubated with the treated epithelial cells for 2 h at 37 °C with occasional shaking and the adherence assay was continued as described above. Blocking experiments were performed in triplicate.

Semiquantitative biofilm assay.
Semiquantitative biofilm assays were performed as described elsewhere 36 . Subsequently, cells were fixed by Bouin's fixative. The fixative was removed after 1 h and wells were washed with PBS. Organisms in the wells were then stained with crystal violet, and the floating stain was washed off with slowly running water. After drying, the stained biofilm was read with a MicroELISA autoreader (BioRad) at 570 nm.
Primary attachment assay. S. aureus strains were grown to the early stationary phase, and cell suspensions were adjusted with TSB to an OD 600 of 1.0. The diluted cultures were pipetted into 96-well tissue culture plates and incubated at 37 °C for 1 h. The subsequent procedures were the same as those used for the semiquantitative biofilm assay.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR). For RNA isolation, overnight cultures were diluted 1:100 into 50 ml TSB and incubated at 37 °C with shaking at 180 rpm to different times of growth (4 h, 8 h, 12 h, 24 h). Complementary DNA (cDNA) was synthesized from total RNA using the QuantiTect reverse transcription system (Qiagen) according to the manufacturer's instructions. Oligonucleotide primers were designed using Primer Express and are listed in Supplementary Table 3. The resulting cDNA and negative control samples were amplified using the QuantiTect SYBR green PCR kit (Qiagen). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7500 Sequence Detector (Applied Biosystems). Standard curves were determined for each gene, using purified chromosomal DNA at concentrations of 0.005-50 ng ml -1 . All quantitative qRT-PCR experiments were performed in duplicate, with gyrB as control.
Phagocytosis and survival in blood assays. Human neutrophils were isolated from heparinized venous blood of healthy individuals with a standard method in accordance with a protocol approved by the ethics committee of Huashan Hospital, Fudan University, Shanghai. All individuals gave informed consent prior to donating blood. S. aureus strains were grown to mid-exponential growth phase, washed, and resuspended in sterile PBS at 10 5 CFU per µl. 10 7 CFU of S. aureus in 100 µl PBS were pipetted slowly into 10 ml heparinized blood, mixed gently for 30 s and incubated at 37 °C. Blood smears from each time point (5 min, 15 min, 30 min, 45 min, 60 min, and 90 min) were prepared and cells were stained with a modified Wright-Giemsa stain. The total number of S. aureus (bound, ingested and those within 10 µm distance of the neutrophils) was evaluated using 50 neutrophils per assay. Percent phagocytosis was calculated using the equation: number of ingested bacteria/Σ (PMN-associated bacteria, PMN-bound bacteria, ingested bacteria) × 100. To determine survival rates, 100 µl of heparinized blood was plated on TSA containing 5% sheep blood for S. aureus detection.
Neutrophil lysis assays. Lysis after phagocytosis was measured using a lactate dehydrogenase (LDH) cytotoxicity detection kit according to the manufacturer's protocol (Roche) essentially as described previously 37 . Bacteria were grown to mid-logarithmic growth phase and human neutrophils were used at a 100:1 (10 9 bacteria ml -1 :10 7 neutrophils ml -1 ) ratio. Samples were diluted 1:50 for the assays.