<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

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

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

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

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

Deletion of <i>psm-mec</i> in MRSA clinical isolates increases the PSMα production, <i>agrA</i> expression, and colony spreading, whereas decreases biofilm formation.

<p>(<b>A, B</b>) The amounts of PSMα3 (A) and Hld + PSMα1 (B) of 18 MRSA isolates and its <i>psm-mec-</i>deleted mutants were measured. White bar represents the clinical isolate used as the parent strain. Black bar represents the <i>psm-mec</i>-deleted mutant of the clinical isolate. The vertical axis represents the amount of PSMαs in arbitrary units based on A₂₁₅. Means ± standard deviations from three independent experiments are shown. Student t-test P-values between the parent strain and the <i>psm-mec</i>-deleted mutant are presented. NS, P>0.05. (<b>C</b>) Cell extracts (3.7 µg protein) of 15 h-cultures of clinical MRSA isolates and the <i>psm-mec</i>-deleted mutants were subjected to Western blotting by anti-AgrA IgG (Upper panel). Band intensities of AgrA were measured and are presented as relative values against that of the parent strain (Lower graph). Means ± standard deviations from three independent experiments are presented. Student t-test P-values between the parent strain and the <i>psm-mec</i>-deleted mutant are presented. NS, P>0.05. (<b>D</b>) Colony spreading abilities of clinical MRSA isolates and the <i>psm-mec</i>-deleted mutants were evaluated. Overnight cultures were spotted onto soft agar plates and incubated for 24 h at 37°C. The vertical axis represents diameters of giant colonies. Means ± standard deviations from three independent experiments are shown. Student t-test P-values between parent strain and the <i>psm-mec</i>-deleted mutant are presented. NS, P>0.05. (<b>E</b>) Biofilm formation of clinical MRSA isolates and <i>psm-mec</i>-deleted mutants were evaluated. Bacterial strains were grown on polystyrene plates for 3 days and the biofilm amounts were measured. White bar represents the clinical isolate used as the parent strain. Black bar represents the <i>psm-mec</i>-deleted mutant of the clinical isolate. Means ± standard deviations from four independent experiments are shown. NS, P>0.05.</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

<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

<i>psm-mec</i> RNA specifically binds <i>agrA</i> mRNA and inhibits its translation.

<p>(<b>A</b>) Hybridization between <i>psm-mec</i> RNA and <i>agrA</i> mRNA was predicted by an <i>in silico</i> program RNA hybrid. Black and gray lines represent strong and weak hydrogen bonds, respectively. (<b>B</b>) Binding between <i>psm-mec</i> RNA and <i>agrA</i> RNA (−20–717) was analyzed using a gel-retardation assay. Various amounts of nonlabeled <i>agrA</i> RNA were added to ³²P-labeled <i>psm-mec</i> RNA (0.13 pmol), and electrophoresed in 6% native polyacrylamide gel. In the right six lanes, nonlabeled <i>psm-mec</i> RNA or yeast tRNA was added to compete with the binding between <i>agrA</i> RNA and ³²P-labeled <i>psm-mec</i> RNA. (<b>C</b>) Binding experiment between <i>psm-mec</i> RNA and deletion mutants of <i>agrA</i> RNA. Various amounts of nonlabeled <i>agrA1</i> RNA (−20–267) or <i>agrA2</i> RNA (−20–198) were added to ³²P-labeled <i>psm-mec</i> RNA (0.13 pmol). (<b>D</b>) Nucleotide sequences of <i>psm-mec</i> RNA, a deletion mutant of <i>psm-mec</i> RNA (<i>psm-mec-</i>D), and a nucleotide-substituted <i>psm-mec</i> RNA (<i>psm-mec-</i>M) are presented. Red dotted line in <i>psm-mec</i>-D indicates the deleted region. Red letters in <i>psm-mec-</i>M indicate the substituted nucleotides that are not complementary to <i>agrA</i> RNA. (<b>E</b>) Various amounts of nonlabeled <i>psm-mec</i> RNA, <i>psm-mec-</i>D RNA, or <i>psm-mec-</i>M RNA were added to ³²P-labeled <i>agrA1</i> RNA (−20–267), and electrophoresed in 6% native polyacrylamide gel. (<b>F</b>) Luciferase activities of Newman strains that were transformed with pGP-agrA-luc carrying no <i>psm-mec</i> (−F), <i>psm-mec</i> (+F), <i>psm-mec</i>-D, or <i>psm-mec</i>-M were measured. The vertical axis represents the relative luciferase activity against that of pGP-agrA-luc carrying no <i>psm-mec</i>. Means ± standard deviations from three independent experiments are presented. Student t-test P-values are presented. (<b>G</b>) Cell extracts (3 µg protein) of 24 h-cultures of Newman strains transformed with pND50 (empty vector), pF carrying <i>psm-mec</i>, p-psm-mec-D carrying <i>psm-mec-</i>D, or p-psm-mec-M carrying <i>psm-mec-</i>M were subjected to Western blotting by anti-AgrA IgG (Left panel). Band intensities of AgrA were measured (Right graph). Means ± standard deviations from three independent experiments are presented. Student t-test P-values are presented. (<b>H</b>) Amounts of PSMα3 in the supernatants of 24 h-cultures of Newman strains transformed with <i>psm-mec</i>, <i>psm-mec-</i>D, or <i>psm-mec</i>-M were measured. The vertical axis represents the relative amount of PSMα3 against that of Newman strain transformed with pND50 (empty vector). Student t-test P-values are presented.</p