Alteration of virulence phenotype of the <i>psm-mec</i>-transformed <i>S. haemolyticus</i>.

<p>(A) AgrA expression in <i>S. haemolyticus</i> strain transformed with pND50 as an empty vector, pF carrying intact <i>psm-mec</i>, pC1 carrying a stop codon-mutated <i>psm-mec</i>, or pM1 carrying a promoter-deficient <i>psm-mec</i> was examined. Protein (2.8 µg) was electrophoresed in each lane and subjected to Western blotting using anti-AgrA IgG. A representative result from two independent experiments is shown. (B) Hemolysin production of <i>S. haemolyticus</i> strain transformed with pND50, pF, pC1, pr pM1 was measured on tryptic soy agar plates containing 5% sheep erythrocytes. A color-changed region around the colonies reflects the lysis of erythrocytes. (C) PSMs in <i>S. haemolyticus</i> strains transformed with pND50 or pF were detected by reversed-phase HPLC. Respective PSM species were identified by LC/ESI-MS. (D, E, F) The amounts of PSMβ3, PSMβ2, and PSMβ1 in the <i>S. epidermidis</i> strain transformed with pND50, pF, pC1, or pM1 were measured by reversed-phase HPLC. The vertical axis represents the relative value to the amount of PSMs in the pND50-transformed strain. Data shown are means ± standard deviations from three independent experiments. Asterisks indicate a Student’s t-test p value less than 0.05 between the pND50-transformed stain and the others. (G) Biofilm formation of the <i>S. haemolyticus</i> strain that was transformed with pND50, pF, pC1, or pM1 was examined. Data shown are means ± standard deviations from three independent experiments.</p

Alteration of virulence phenotype in the <i>psm-mec</i>-transformed <i>S. epidermidis</i>.

<p>(A) Schematic representation of the <i>psm-mec</i> mutations in pC1, pC2, pC3, pM1, and pM2 is shown. (B) The amount of PSM-mec protein was measured by reversed-phase HPLC in the <i>S. epidermidis</i> strains transformed with plasmids listed in (A). Data are means ± standard deviations from three independent experiments. Asterisks indicate Student’s t-test p value less than 0.05 between the pF-transformed stain and the others. ND means not detected. (C) The amount of <i>psm-mec</i> RNA was measured by quantitative RT-PCR in the <i>S. epidermidis</i> strains transformed with plasmids listed in (A). Data shown are means ± standard deviations from three independent experiments. Asterisk indicates Student’s t-test p value less than 0.05 between the pF-transformed stain and the others. ND means not detected. (D) Extracellular proteins of overnight culture of <i>S. epidermidis</i> ATCC12228 strain, which was transformed with pND50 as an empty vector, pF carrying intact <i>psm-mec</i>, or plasmids carrying mutated <i>psm-mec</i> (pC1, pC2, pC3, pM1, or pM2) was electrophoresed in a 10% SDS polyacrylamide gel and stained by Coomassie brilliant blue. A representative result from three independent experiments is shown. (E) Expression of PSMs in the <i>psm-mec</i>-transformed strain (pF, magenta line) or empty vector-transformed strain (pND50, black line) was analyzed by reversed-phase HPLC. The PSM species were identified by LC/ESI-MS. (F, G, H, I) The amounts of PSMα + PSMδ, PSMγ, PSMβ1, and PSMβ2 in the <i>S. epidermidis</i> strain that was transformed with pND50 as an empty vector, pF carrying intact <i>psm-mec</i>, or plasmids carrying mutated <i>psm-mec</i> (pC1, pC2, pC3, pM1, or pM2) were measured by reversed-phase HPLC. The vertical axis represents the relative value to the amount of PSMs in the pND50-transformed strain. Data are shown as means ± standard deviations from three independent experiments. Asterisks indicate Student’s t-test with a p value less than 0.05 between the pND50-transformed stain and others. (J) Biofilm formation of the <i>S. epidermidis</i> strains transformed with pND50 as an empty vector, pF carrying intact <i>psm-mec</i>, or plasmids carrying mutated <i>psm-mec</i> (pC1, pC2, pC3, pM1, or pM2) was measured. Data shown are means ± standard deviations from three independent experiments. Asterisks indicate Student’s t-test with a p value less than 0.05 between the pF-transformed stain and the others.</p

<i>psm-mec</i> RNA inhibits <i>agrA</i> translation.

<p>(<b>A</b>) Cell extracts of overnight cultures of Newman strain (WT) and the <i>agr-</i>null mutant (Δ<i>agr</i>) were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. One gel was stained with Coomassie Brilliant Blue (Left panel). Proteins in another gel were transferred to a membrane and used for Western blotting by anti-AgrA IgG (Right panel). (<b>B</b>) Cell extracts of 24 h-cultures of Newman strains transformed with empty vector (pND50), a plasmid carrying wild-type <i>psm-mec</i> (pF), a plasmid carrying <i>psm-mec</i> with a stop-codon (pC1), and a plasmid carrying <i>psm-mec</i> with the -7T>C promoter mutation (pM1) were subjected to Western blotting by anti-AgrA IgG. Each lane contains 3.5 µg proteins of cell extracts. (<b>C</b>) Cell extracts of 24 h-cultures of Newman, MW2 (USA400), and FRP3757 (USA300) strains that were transformed with pF carrying <i>psm-mec</i> (multi-copy), or integrated with <i>psm-mec</i> into the chromosome (single-copy) were subjected to Western blotting by anti-AgrA IgG (Upper panel). Each lane contains 3 µg proteins of cell extracts. Band intensities of AgrA were measured and are presented in the lower graph. The vertical axis represents the relative value against the AgrA band intensity of the parent strain in each Newman, MW2, and FRP3757 genetic background. Means ± standard deviations from four independent experiments are presented. Student t-test P-values between the parent strain and the <i>psm-mec</i>-introduced strain in each genetic background are presented. (<b>D</b>) The <i>agr</i> null mutant of Newman transformed with pMNS-agrBDCA carrying IPTG-inducible <i>agrBDCA</i> and pKE516 (empty vector), or pMNS-agrBDCA and pKE516-F carrying wild-type <i>psm-mec</i> was cultured in the presence or absence of IPTG. Cell extracts of 24-h cultures were subjected to Western blotting by anti-AgrA IgG. Each lane contains 6 µg proteins of cell extracts. (<b>E</b>) Schematic representation of <i>luc-</i>fusions of the <i>recF</i> promoter, <i>agrA</i> SD, the <i>agrA</i> ORF, and the <i>luc</i> ORF. Bold gray lines represent the plasmid construct. Horizontal dotted lines represent the regions deleted from the plasmids. Putative binding region means the region predicted to bind to the <i>psm-mec</i> RNA by <i>in silico</i> analysis. SD means Shine-Dalgarno sequence of <i>agrA</i>. (<b>F</b>) Luciferase activities of Newman strains that were transformed with the <i>luc-</i>fusion plasmids with <i>psm-mec</i> (+F) or without <i>psm-mec</i> (−F) were measured. The vertical axis represents the luciferase activity. Student t-test P-values between +F and −F are presented. NS, P>0.05. (<b>G</b>) Newman strain, which was integrated with <i>psm-mec</i> or without <i>psm-mec</i>, was transformed with the <i>luc-</i>fusion plasmids. Luciferase activities of the strains were measured. The vertical axis represents the relative luciferase activity of the <i>psm-mec</i>-integrated Newman [+F (single-copy)] against that of the Newman strain (−F). Student t-test P-values between +F and −F are presented. NS, P>0.05.</p

Typing of SCC<i>mec</i> of MRSA clinical isolates.

<p><i>ccr</i> genes and <i>mec</i> gene complex were identified by multiplex PCRs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003269#ppat.1003269-Kondo1" target="_blank">[48]</a>. All isolates were <i>mecA</i> positive. SCC<i>mec</i> types, I, II, and IV were assigned by the combination of types of <i>ccr</i> gene and <i>mec</i> gene complex. Abbreviations are as follows:</p>1<p>n.a., SCC<i>mec</i> type could not be assigned from the experiments;</p>2<p>Total, total number of isolates;</p>3<p>NT, non-typed, since DNA fragment was not amplified by PCR identifying either <i>ccr</i> genes or <i>mec</i> gene complex. ‘2+5’ in <i>ccr</i> type means that both type 2 and type 5 <i>ccr</i> were identified, indicating that 48 strains (25%) carry type II SCC<i>mec</i> and SCC carrying <i>ccrC</i>. ‘2+4’ in <i>ccr</i> type indicates that 2 strains (1%) carry type II or type VIII SCC<i>mec</i>. The combination of type 2 <i>ccr</i> and class C2 <i>mec</i> gene complex suggests that it might be a novel SCC<i>mec</i> element. Since it was out of scope of this paper, we classified it in the group of not assigned.</p

MRSA clinical isolates harboring a <i>psm-mec</i> mutation produce high amounts of PSMα3.

<p>Nucleotide sequences of <i>psm-mec</i> genes of 325 MRSA isolates were determined (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003269#ppat-1003269-t001" target="_blank"><b>Table 1</b></a>). MRSA strains harboring intact <i>psm-mec</i> (Intact), -7T>C-mutated <i>psm-mec</i> (-7T>C), or no <i>psm-mec</i> (Absence) were cultured for 15 h. The amounts of PSMα3 in the culture supernatants were measured. The vertical axis represents the relative amount of PSMα3 against that of Newman strain. Closed circles represent the amounts of PSMα3 of each MRSA strains, which are the means from two independent experiments. Magenta lines represent the averaged amount of PSMα3 of each MRSA groups. Cyan dotted line represents the amount of PSMα3 of CA-MRSA strain FRP3757 (USA300). Student t-test P-values are presented. ND, not detected.</p

Identification of mutations of the <i>psm-mec</i> gene from MRSA strains.

<p>Mutation of <i>psm-mec</i> is presented as a number of nucleotides from the transcription start site of <i>psm-mec</i> and nucleotide substitutions. T>C means that thymine was exchanged with cytosine. Expression of the respective mutated <i>psm-mec</i> gene in the Newman strain was examined (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003269#ppat.1003269.s003" target="_blank">Fig. S3</a></b>) and is presented in the column ‘Expression’. 1, DNA fragment of 2206 bp (GenBank, AB 729111). 2, DNA fragment of 1332 bp (GenBank, AB 729110).</p

Typing of <i>spa</i> of MRSA clinical isolates.

<p><i>spa</i> types were identified by sequencing short-sequence repeats (SSRs) of <i>spa</i> gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003269#ppat.1003269-Shopsin1" target="_blank">[47]</a>. ‘New’ means new <i>spa</i> types that were identified in this study. These <i>spa</i> types were assigned as <i>spa</i> types 1491, 1492, 1493, and 1494.</p

Bacterial strains and plasmids used.

a<p>Amp, ampicillin; Cm, chloramphenicol; Tet, tetracycline; Phleo, phleomycin; Kan, kanamycin; Spc, spectinomycin.</p

Deletion of <i>psm-mec</i> in MRSA clinical isolates increases virulence in mice.

<p>(<b>A</b>) Mouse skin infection experiments using NI-13, SR-1, NIR-34, and the respective <i>psm-mec</i>-deleted mutants were performed. Mice (HR-1, n = 5) were subcutaneously injected with <i>S. aureus</i> cells and the dermonecrosis area was measured. Means ± standard deviations from the dermonecrosis areas of five mice are shown. Injected CFUs were as follows; NI-13 and its <i>psm-mec</i>-deleted mutant, 4×10⁷ CFU; SR-1 and its <i>psm-mec</i>-deleted mutant, 8×10⁶ CFU; NIR-34 and its <i>psm-mec</i>-deleted mutant, 2×10⁷ CFU. Black stars indicate that Student's t-test P-values between the parent strain and the <i>psm-mec</i>-deleted mutant were less than 0.05. Upper right panel is a representative image of a mouse injected with NI-13 and the <i>psm-mec</i>-deleted mutant at 143 h after bacteria injection. (<b>B</b>) Mouse systemic infection experiments were performed. ICR mice (n = 10) were intravenously injected with <i>S. aureus</i> cells. Injected CFUs were as follows; NI-13 and its <i>psm-mec</i>-deleted mutant, 4×10⁸ CFU; NIR-34 and its <i>psm-mec</i>-deleted mutant, 4×10⁸ CFU. Log-rank test P-values between the parent strain and the <i>psm-mec-</i>deleted mutant in NI-13 and SR-1 are 0.0005 and <0.0001, respectively.</p

<i>psm-mec</i> RNA increased the amount of HutU, Spa, and Ddh in CA-MRSA FRP3757 (USA300).

<p>(<b>A</b>) The nucleotide sequence of the <i>psm-mec</i> ORF in pF, the stop-codon introduced sequence of <i>psm-mec</i> ORF in pC1, and the synonymous-codon substituted sequence of <i>psm-mec</i> ORF in pFP are shown. The substituted nucleotides are colored in red. The amino acid sequence of PSM-mec protein is shown below the respective nucleotide sequence. (<b>B</b>) Cell extract of FRP3757 strain that was transformed with empty vector (pND50), <i>psm-mec</i> (pF), mutated <i>psm-mec</i> harboring a stop codon (pC1), or mutated <i>psm-mec</i> harboring synonymous codon substitutions (pFP) was analyzed by two-dimensional electrophoresis. Proteins were stained with Coomassie Brilliant Blue. The protein spot was excised and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003269#ppat.1003269.s007" target="_blank">Table S1</a></b>).</p