Histological features of trichodysplasia spinulosa.

<p>Left column illustrates H&E staining of a low power field of healthy skin (<b>A1</b>) and TS lesional skin (<b>B1</b>). High power fields of healthy (<b>A2</b>) and TS (<b>B2</b>) epidermis and hair follicles (<b>A3</b> and <b>B3</b>). Note the enlarged and dysmorphic hair follicle shown in <b>B3</b>, containing eosinophilic granular protein deposits in the cytoplasm of the cells (arrowheads in inset) with abrupt cornification in the center of the follicle. Bars depict 100 µm.</p

List of analyzed TS samples.

<p>*, Viral copies per cell measured as described (all) and reported (TS4-TS11) by Kazem <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108947#pone.0108947-Kazem1" target="_blank">[1]</a>.</p><p>**, TS13 concerned a kidney transplantation patient immunosuppressed with tacrolimus, mycophenolate, and prednisone. TS was diagnosed 10 months after the rash was noted. Symptoms improved after reduction of immunosuppression and remained absent ever since.</p><p>List of analyzed TS samples.</p

Polyomavirus-Associated Trichodysplasia Spinulosa Involves Hyperproliferation, pRB Phosphorylation and Upregulation of p16 and p21

<div><p>Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16^i<i>nk4a</i>, p21<i>^waf</i>, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16<i>^ink4a</i> and p21<i>^waf</i>. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.</p></div

A hypothetical scenario of TSPyV LT-antigen interference in cell cycle regulation.

<p>An oversimplified cell-cycling scenario is shown that envisions the TSPyV LT-antigen involvement in regulation of pRB pathway activity. In a normal physiological condition, hypophosphorylated pRB is complexed with transcription factor E2F during early G1 (rest) phase of the cell cycle. When pRB is hyperphosphorylated at specific residues by Cyclin-dependent kinases (CDK) coupled to Cyclin-D1, E2F is released that activates expression of growth stimulatory genes needed for the cells to enter the S (DNA synthesis) phase. pRB phosphorylation is under tight regulation of p16<i>^ink4a</i> and p21<i>^waf</i>. Hypothetically, through its conserved LXCXE motif TSPyV LT-antigen interacts with pRB/E2F complex to dissociate these proteins via pRB hyperphosphorylation, resulting into S phase entry and subsequent increased expression of p16<i>^ink4a</i> and p21<i>^waf</i> as a negative cell cycling feedback (red arrows).</p

Primary and secondary antibodies used for immunofluorescence.

<p>*, Rabbit immunized with two TSPyV LT-antigen-derived synthetic peptides FSSQHDVPTQDGRD (AA, 77–90) and NSRRRRAAPPEDSP (AA, 151–164).</p><p>**, Rabbit immunized with TSPyV VP1-antigen-derived synthetic peptide TGNYRTDYSANDKL (AA, 170–183) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108947#pone.0108947-Kazem1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108947#pone.0108947-Kanitakis1" target="_blank">[29]</a>.</p><p>Primary and secondary antibodies used for immunofluorescence.</p

Cell cycle regulation markers and TSPyV LT-antigen expression.

<p>Sections of healthy epidermis (<b>A1–A4</b>), healthy hair follicle (<b>B1–B4</b>), TS epidermis (<b>C1–C4</b>) and TS follicle (<b>D1–D4</b>) are stained for p16<i>^ink4a</i> (first panel), p21<i>^waf</i> (second panel), pRB (third panel) and TSPyV (fourth panel). Insets in the fourth panel depict the same region with Hoechst DNA staining (blue). Dotted lines indicate the dermoepidermal junction. Bar depicts 100 µm.</p

Phevalin (aureusimine B)Production by <em>Staphylococcus aureus</em> Biofilm and Impacts on Human Keratinocyte Gene Expression

<div><p><em>Staphylococcus aureus</em> biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. <em>S. aureus</em> contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in <em>S. aureus</em>, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that <em>S. aureus</em> biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of <em>S. aureus</em> as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from <em>S. aureus</em> spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, <em>S. aureus</em> biofilm-based infections.</p> </div

<i>S. aureus</i> biofilms produce more phevalin than their planktonic counterparts.

<p>(A) HPLC-MS analysis of organic extracts from <i>S. aureus</i> biofilm, planktonic, and growth medium control revealed that biofilms produce more phevalin (aureusimine B) than planktonic cultures (arrow). A compound that is likely tyrvalin (aureusimine A) was also present at higher levels in the biofilm (*). (B) Phevalin production was detected directly in samples without prior organic extraction. Samples were normalized to cell density (optical density, 600 nm, OD₆₀₀) in biofilm (OD₆₀₀ 0.9), resuspended biofilm (OD₆₀₀ 1.4), and planktonic cultures (OD₆₀₀ 0.66). Data represent means ± SEM, n = 3, ***p<0.001.</p

Conditioned medium from <i>S. aureus</i> cultures with or without additional phevalin induces differential gene expression in HKs.

<p>Significant (p<0.05) transcripts regulated ±2 fold in any one condition relative to controls. Transcripts shared between HKs treated with BCM, +PCM, and –PCM are shown at ±2 and ±5 fold change cutoffs (A and B, respectively). HKs treated with BCM shared more transcripts with +PCM treated HKs than –PCM treated HKs (arrows). Transcripts shared between –PCM and BCM had modest fold changes as no transcripts were shared above the ±5 FC cutoff. (C) The top 20 upregulated and downregulated genes (p<0.05) in +PCM treated HKs relative to –PCM treated HKs are listed. For a complete list of significantly regulated genes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040973#pone.0040973.s003" target="_blank">Table S1</a>. (D) Selected genes were confirmed by RT-qPCR. The fold change relative to a GAPDH normalizer is indicated (p<0.05 for all comparing DMSO to phevalin). (E) Functional annotation clustering of microarray data revealed significantly (Benjamini p<0.01) enriched biological processes in +PCM treated HKs.</p

Phevalin production in various strains of bacteria as detected by SRM HPLC-MS.

<p>Phevalin production in various strains of bacteria as detected by SRM HPLC-MS.</p