Dendrogram of the draining lymph node histopathological profiles.

<p>Each label indicates (in order): infecting species (P for <i>Y. pestis</i>, T for <i>Y. pseudotuberculosis</i>), delay between mouse infection and lymph node collection (in days), amount of injected cfu. Top scale = % similarity</p

Examples of the three histopathological types of the draining lymph nodes.

<p>Lymph nodes were taken 24 h (type 1) and 72 h (type 2 and 3) after inoculation of ∼4500 cfu of <i>Y. pseudotuberculosis</i> (types 1 and 2) or <i>Y. pestis</i> (type 3). Sections were stained by Hematoxylin-Eosin (HE), and by antibodies specific to <i>Y. pseudotuberculosis</i> or to <i>Y. pestis</i> (Bact), to PMNs and to B lymphocytes (BL). Immunostainings give a brownish color. Arrowheads on the HE-stained sections indicate the border of the inflammatory front. Bacteria (arrows) are present as tiny bacterial foci at the periphery (type 1), well delimited patches (type 2) or flame-like formations (type 3). The BL staining shows that the overall structure of the type 1, but not of the type 3, lymph node is preserved, while, in the type 2 lymph node, the inflammatory reaction forced B lymphocytes to the central part of the organ. Bar = 100 µm.</p

Illustrations of some of the criteria used for scoring.

<p>Panels A–E, G, H and J–L display HE stained sections, panels F and I show sections immunostained with <i>Y. pestis</i> specific antibodies. In the text below, the criteria are referred to according to the numbering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001688#pone-0001688-t001" target="_blank">Table 1</a>. A: Wedge shaped abscess (criterion 4) indicated by arrows. Within the abscess bacterial colonies are visible as pink patches. B: Peripheral layer of PMNs containing bacterial foci (criterion 9), with a blunt demarcation (arrows) from the lymph node tissue (criterion 11). Inset: higher magnification to show the characteristic horseshoe shaped nuclei of the PMNs, and a bacterial focus. C: Layer of PMNs bordering a peripheral band of bacteria, cell debris and PMNs (criterion 10). The inset shows the typical PMN morphology of the cells within the layer. Behind this layer, bacterial aggregates are seen as pink areas (arrowheads) containing purple dots that are, as seen as higher magnification (not shown here) PMNs and cell debris. D: Patch of densely packed bacteria, bordered by PMNs (criterion14). Packed bacteria form a pink 8-shaped area at the centre of the picture (star). E: Atypical bacterial patch (criterion 15). At the center of the picture an aggregate of bacterial rods, not as densely packed as the preceding one, is loosely surrounded by inflammatory cells. F: A large bacterial zone (criterion 17), stained brownish on the preparation. G: Isolated host cells within a bacterial zone (criterion 19). Isolated host cells and cell remnants are seen amid a sea of bacteria which gives a “ground glass” appearance to this part of the LN section. H, <i>left</i>: bacterial infiltration around host cells (criterion 20). A bacterial strand (arrow), that seems to originate from a nearby colony (star), passes between host cells. H, <i>right</i>: Free floating bacterium, indicated by an arrow (criterion 21). I: Zone of reduced tissular density (arrows) outside bacterial areas, which are brownish on this preparation (criterion 26). This image also shows flame-like inward bacterial projections (criterion 18). J: Area of reduced host cell density with a reticular pattern (criterion 27) and containing numerous pycnotic cells (criterion 32). K: Moth eaten appearance (criterion 33) of a lymph node with areas of contrasting tissular densities. L: Vascular congestion (criterion 40), showing bright red on this preparation. <i>Magnifications</i>: Panels A–C, F, I–L : bar = 200 µm. Insets of panels B and C: bar = 50 µm. Panels D, E, G and H: bar = 10 µm.</p

Criteria used to score sections of lymph nodes infected with <i>Y. pestis</i> or <i>Y. pseudotuberculosis</i>, and their discriminating indexes.

<p>Discriminating indexes are the test values in the right column. Only significant test values (i.e. >1.96 or <−1.96) are given. Positive and negative test-values are distinctive of, respectively, <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i> infections.</p

Draining lymph node gross pathology.

<p>Macroscopical aspect of lymph nodes two days after inoculation of either saline, ∼5500 <i>Y. pseudotuberculosis</i> cfu or ∼5500 <i>Y. pestis</i> cfu. Lymph node from <i>Y. pseudotuberculosis</i>-infected mice is purulent whereas <i>Y. pestis</i>-infected lymph node is reddish and adherent to neighboring tissues. Bar = 2 mm.</p

Bacterial loads at injection site and in draining lymph node.

<p>Data are means of log₁₀ of cfu numbers recovered from 77 mice infected over six independent experiments. Bars = Standard Errors. Stars indicate that the data are significantly different between the two <i>Yersinia</i> species (p≤0.0001). The dashed line shows the detection limit (10 cfu). The cross symbol indicates that most <i>Y. pestis</i>-infected mice died between days 2 and 3.</p

Validation of HR-specific interactions.

<p>(A) HeLa cells were transfected by pTK6E2BS-Luc reporter and HPV16 or HPV18 E2 expression plasmids. Where indicated, GTF2B was added. Fold activation is given relative to TK6E2BS-Luc in the absence of E2. (B) HeLa cells were transfected with a pool of four siRNA targeting GTF2B or control siRNA (Scramble). 48 h post silencing, pTK6E2BS reporter plasmid was transfected along with E2 expression plasmids. Results are given as a fold activation relative to TK6E2BS basal activity in the presence of the same siRNA. Experiments were performed in triplicate with each bar representing the mean ± SD. The stars (***) indicate a statistical significant difference between fold activation by 16E2 with a scramble siRNA or a GTF2B-directed siRNA directed (p-value<0,001) (C) HaCaT cells were co-transfected by GFP-E2 proteins from HPV16 or HPV18 and mCherry-VPS39. 24 h later, cells were fixed in 4% paraformaldehyde, stained with DAPI and subjected to fluorescence microscopy.</p

Characterization of E2 proteins expressed in HT-GPCA conditions.

<p>(A) Schematic representation of the HT-GPCA method. This assay is based on the reconstitution of a luciferase activity upon co-expression of interacting partners in fusion with two inactive fragments of the <i>Gaussia princeps</i> luciferase (designated GL1 and GL2). The reconstituted Luciferase activity is estimated from a Normalized Luminescence Ratio (NLR) (B) 293T cells were transfected with the pTK6E2BS-Luc reporter and the GL2-E2 expressing plasmids. Fold activation is given relative to TK6E2BS-Luc in absence of E2. (C) E2-Firefly luciferase fusion proteins were expressed in 293T cells and the firefly luciferase activity was determined 24 h post-transfection. The results are expressed as a percentage of the activity obtained with the firefly luciferase only.</p

HR-specific interactions of the E2 proteins from HPV16 and HPV18.

<p>List of the cellular proteins involved in interactions discriminating the E2 proteins of the genital HR-HPV 16 and 18 from the LR-HPV 6 and 11, based on the NLR profiles obtained by HT-GPCA. The asterisks (*) stand for cellular proteins generating lower NLR specifically with 16 E2 protein.</p

E2-targeted functional families.

<p>Cellular proteins (nodes) classified into enriched families based on the Gene Ontology annotations are colored according to the associated GO functions. Proteins shared by different families are bi-coloured. The network representation was generated by Cytoscape.</p