Analysis of CYP expression in skin by immunoblotting.

<p>Samples of human whole skin microsomal fraction (75 µg) prepared from 5 donors were separated by SDS-PAGE, transferred to nitrocellulose filters and the presence of CYP1A1, CYP1A2, CYP2E1 and CYP3A4 detected using antibodies specific to each form. The lane on the left hand side contained either 25 µg lymphoblast cell microsomes containing recombinant human CYP1A1 (∼2 pmol), or a sample of human liver loaded with 25, 35 or 5 µg microsomal fraction for detection of CYP1A2, CYP2E1, or CYP3A4, respectively. Immunoreactive bands were developed using goat anti-rabbit-horseradish peroxidase and ECL detection.</p

Flow diagram summarizing the methodology.

<p>Flow diagram summarizing the methodology.</p

Identification of unique serotype M18 specific mutations in regulators RocA and CovR.

<p>RocA and CovR sequence comparison of isolates representing major GAS serotypes. Strains sequenced from each serotype are outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003842#ppat-1003842-t001" target="_blank">Table 1</a>; at least one clinical isolate was tested for each serotype. Ten serotype M18 strains were sequenced including two obtained from patients in 1934. (A) <i>rocA</i> gene sequence of different M types. Codons are shown by alternating bold text. The premature stop codon in <i>rocA</i>_M18 is highlighted by red font. (B) The subsequent truncation in RocA_M18 protein is demonstrated schematically in comparison with RocA_M89. (C) CovR amino acid sequence in different M types. Highlighted residues indicate amino acid change in the M18 CovR protein resulting from non-synonymous mutations in the gene sequence compared with other M types. RocA and CovR sequences were also evaluated from genome sequenced isolates submitted to NCBI, representing serotypes M1, M2, M3, M4, M5, M6, M12, M18, M28 and M49.</p

Proposed mechanism of RocA mediated activity.

<p>CovR (black circle) is phosphorylated directly by CovS (blue ellipse), which induces CovR dimerization (green circles) and transcriptional repression of <i>hasA</i> and <i>spyCEP</i>, as well as auto-repression of <i>covR</i>. RocA (red ellipse) regulates expression of a number of gene products (solid red lines). RocA promotes transcription of <i>covR</i> potentially via an intermediate regulator (red dashed circle). RocA also promotes expression of proteins that are not part of the CovR regulon, whilst inhibiting many others. Such regulation may also require kinase action and an intermediate regulator.</p

Comparison of XME profiles from <i>in vitro</i> skin models and whole skin.

<p>The relative amount of each protein is represented by the number of different tryptic peptides specific to each protein or protein family that were detected. Details of the protein accession numbers and their subcellular location are shown in Table S1. Shading indicates different enzyme classes: oxidoreductase (black), hydrolase (magenta), transferase (red), antioxidant (green), and other (blue).</p

RocA regulation of capsule synthesis modulates bacterial colony structure.

<p>Imaging of serotype M18 strains (A) GAS-M18, (B) GAS-M18_rocAM89 and (C) GAS-M18_hasko following overnight culture on Todd-Hewitt agar. i) Macroscopic imaging. ii)–iv)Scanning EM performed on individual colonies, each one imaged at three magnifications: ii) 3 k (white line = 2 µm), iii) 10 k (white line = 1 µm) and iv) 35 k (white line = 0.5 µm) respectively.</p

Stability of Epiderm-200 XME expression profiles in culture.

<p>Epiderm-200 cultures were derived from either donor 254 or 1188 and were maintained for up to 3 days. The relative amount of each protein is indicated by the number of different tryptic peptides specific to each protein or protein family that were detected with adjacent bars representing results from 0, 1, 2 and 3 days, respectively. Details of the protein accession numbers and their subcellular location are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041721#pone.0041721.s003" target="_blank">Table S2</a>. Shading indicates different enzyme classes: oxidoreductase (black), transferase (red), antioxidant (green), and other (blue).</p

Serotype M18 GAS display a unique hyper-encapsulation phenotype which mediates enhanced nasopharyngeal carriage longevity.

<p>Characterization of serotype M18 GAS by comparison with representatives of each of six major serotypes (n = 5 isolates/group). (A) Inter-serotype comparison of absolute copy number for <i>hasA</i> standardized to housekeeping gene <i>gyrA</i>. Line shows median (Kruskal-Wallis; *** = p<0.001). (B) Inter-serotype comparison of capsular HA expression across all serotypes by an ELISA-based assay. Line shows median (Kruskal-Wallis; *** = p<0.001). (C) Inter-serotype comparison of absolute transcript copy number for <i>covR</i> and <i>covS</i> standardized to house-keeping gene <i>gyrA</i>. Line shows median (Kruskal-Wallis; p = NS). (D) Inter-serotype comparison of duration of nasopharyngeal shedding of pharyngitis-associated serotypes M4, M6, M12 and M18 with isogenic capsule disruptant mutant GAS-M18_hasko (n = 8 mice/group). Data represent percentage of mice shedding each strain for 21 days following intra-nasal challenge (LogRank; ** = p<0.01).</p

Detection of CYP proteins in skin and liver microsomal fraction by LC-MS/MS.

<p>Samples of skin microsomal fraction were spiked with a range of quantities of either recombinant CYP1A1 (expressed in lymphoblast cells) or with human liver microsomal fraction that contains known amounts of CYP1A2, CYP2E1, CYP3A4, and CYP3A5. The normal proteomics workflow was followed to identify peptides corresponding to the CYP proteins. Limits of detection based on the use of at least 2 tryptic peptides were established and based on these values the minimum level detectable by this technique was calculated for skin and compared with the mean level measured in liver. From these values the minimum comparative level in skin was calculated. CYP1A1 was not detected in either skin or liver making any comparison redundant (n/a; not applicable).</p

XMEs detected in whole skin. Protein identification was based on the presence of ≥2 different tryptic peptides in at least two donors.

<p>The proteins identified have been classified into functional groups as indicated. The corresponding NCBI numbers are indicated for each protein and for all members of groups of related proteins. The sub-cellular fraction in which each protein was principally detected is shown. The proportion of donor samples (skin n = 10, liver n = 5) in which each protein was identified is indicated. Fold difference was calculated by summing the intensity values of all detected peptides for a protein and comparing the values obtained for skin and liver. Where no peptides were detected, an intensity value equivalent to the limit of detection was used. Statistical significance was assessed using the Mann-Whitney U test.</p