The proposed view of Sap-induced inflammasome activation and inflammasome-dependent cytokine production.

<p>Sap2 and Sap6 activate the NLRP3 inflammasome pathway through an early cascade of events, causing upstream NLRP3 inflammasome activation and downstream caspase-1-mediated cytokine production. Late events depend on Sap endocytosis inducing the translocation of NF-κB (p50/p65) into the nucleus, pro-IL-1β and pro-IL-18 synthesis, then (through type I IFN production) caspase-11 activation that cooperates with the NLRP3 inflammasome in triggering downstream caspase-1-mediated cytokine production. For details about this proposed scheme of Sap/inflammasome/caspases activation, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref038" target="_blank">38</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref039" target="_blank">39</a>], and [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref042" target="_blank">42</a>].</p

Aspartyl Proteinases of Eukaryotic Microbial Pathogens: From Eating to Heating - Fig 1

<p><b>Left</b>. The molecular ribbon-like structure of Sap2, a major AP of <i>Candida albicans</i>. Note the flexible flaps that control the access to the central region faced and delimited by the two active sites DTGS and DSGT and accommodating an enzyme inhibitor. N-ter is the N-terminus and C-ter the C-terminus of the amino-acid sequence. <b>Right</b>. Sequences of Sap2 and plasmepsin II of <i>Plasmodium falciparum</i>, which is most similar to Sap2 among the APs of eukaryotic microbial pathogens, showing two regions of high similarity (highlighted in red). The identity of the two whole sequences is 28.2% and their similarity 57.4% (FASTA; MBL Swiss-Prot).</p

The <i>SAP</i> family of <i>C</i>. <i>albicans</i> contains at least ten proteins with a signal peptide and are secreted, except Sap9 and Sap10, which remain bound to the cell wall.

<p>They are characterized by broad spectrum proteolytic ability and virulence properties that are reported to be differentially expressed at different stages and forms of fungus growth and disease. Sap2 (alike Sap1 and Sap3) is active at acidic pH and is dominantly associated with yeast form of growth while Sap6 (alike Sap4 and Sap5) is more active at neutral to slightly alkaline pH Together with the dominant Sap5, Sap6 has been associated with hyphal growth. For details, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref007" target="_blank">7</a>] and [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref028" target="_blank">28</a>].</p

Effect of halofuginone on vaginal infection.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl or 10 µg/100 µl of halofuginone solution or diluent of halofuginone and, in selected experiments, were treated intravaginally with 10 pg of mouse rIL-17. (A) Evaluation of IL-17 concentration by ELISA in supernatants of vaginal fluids obtained at different days after vaginal <i>Candida</i> infection and halofuginone treatment. Results are expressed as mean±SD (n = 9 mice, 3 mice for each of three separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice). At day 4, 14 and 25 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC) and treated with halofuginone or diluent. Images are representative of 5 out of 10 mice in two different experiments. (C) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes of mice (n = 10) treated with halofuginone or diluent. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice).</p

IL-17 and IL-23 concentration in murine vaginal washes of mice infected with <i>Candida albicans</i>.

<p>Evaluation of IL-17 (A–C) and IL-23 (D) concentration by ELISA test on supernatants of vaginal fluids obtained at different times after vaginal infection with different doses of <i>Candida albicans</i> gLUC59 (A–B) or CA1399 (D). Results are expressed as mean±SD (n = 12 mice, 4 mice for each of three separate experiments). * <i>p</i><0.05, (infected mice vs non infected mice).</p

Model of murine vaginal infection and monitoring.

<p>(A) Timeline of vaginal infection model. CD1 mice are resistant to <i>Candida</i> vaginal infection unless they are treated with estradiol valerate. CD1 mice were treated subcutaneously with estradiol valerate and infected for two consecutive days with 10 µl of 10⁹/ml suspension of <i>Candida albicans</i> cells (gLUC) into vaginal lumen. Two days before and every two days after challenge mice were treated intraperitoneally with halofuginone or diluent of halofuginone and, in selected experiments, intravaginally with 10 pg of recombinant mouse IL-17. After 4, 8, 12, 14, 18, 20, 25, 30 days post infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anaesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline using mechanical pipette. The fungal burden of vaginal fluids was evaluated by colony forming units (CFU) assay. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC). Images are representative of 5 out of 10 mice for each experiment. C) Dot plots of total photon emission from the infected vaginal regions and corresponding CFU in vaginal washes of infected mice (n = 10). The statistical analysis was performed by non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one of two independent experiments with similar results. D) The correlation between the Total Photons emitted and CFU count in the vaginal wash was assessed using the Pearson's correlation statistics, and the correlation coefficients are shown for each time point. * <i>p</i><0.05, ** <i>p</i><0.01, (Log Photons Total Flux or Log CFU/ml of mice infected after 8, 12,14,18,20,25,30 days vs Log Photons Total Flux or Log CFU/ml of mice infected after 4 days).</p

Effect of <i>Candida albicans</i> infection on draining lumbar lymph nodes.

<p>Lymph nodes, at different times after <i>Candida</i> infection, were aseptically recovered and mechanically homogenized. Cells were cultivated untreated or in presence of heat inactivated <i>C. albicans</i> for 72 hours. In the supernatant fluids of lymph node cell culture IL-17 (A) and IL-23 (B) were analyzed by ELISA. Results are expressed as mean±SD (n = 16 mice, 4 mice for each of four separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (Lymphocytes from infected mice vs Lymphocytes from non infected mice). # <i>p</i><0.05, ## <i>p</i><0.01, (Lymphocytes re-stimulated from infected mice vs Lymphocytes re-stimulated from non infected mice).</p

Enhanced Dectin-1 recognition is filament-specific and co-localizes with enhanced chitin deposition.

<p>Vaginal swab samples were stained with sDectin-1-Fc (sDectin-1; red), Calcofluor white (CFW; blue; outline of fungi for morphology and for identification of sites of increased chitin deposition), and Sytox Green (green; stains only extracellular DNA and DNA inside cells with compromised membranes). The EGFP-expressing strain KAH3-EGFP was spiked in before staining as a positive control. Several 40x fields (8–16 per sample) were imaged and scored for fungal morphology, sites of sDectin-1 staining, co-localization of sDectin-1 staining and increased CFW staining, and presence of extracellular DNA. (A) In samples from <i>C</i>. <i>albicans</i>-infected patients, only filaments were found with enhanced sDectin-1 staining. (B) In samples from patients infected with non-<i>albicans Candida</i> or a mix of <i>C</i>. <i>albicans</i> and non-<i>albicans Candida</i>, only filaments were stained with sDectin-1. The only sample with any sDectin-1+ cells was a mix of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>norvegensis</i> (Sample #SP-97366). The difference in frequency of staining is not significantly different between filaments and yeast (p >0.9999), because there was virtually no staining of either morphology. (C) Representative field from <i>C</i>. <i>albicans</i>-infection sample (#SP-66117). A single filament segment that is swollen (purple arrow) has high levels of sDectin-1 and CFW staining. The top image is a three-color overlay and the images below separate the sDectin-1 and CFW. (D) Representative field from a non-<i>albicans Candida</i> infection sample (#SP-12622). Most of the fungi are yeast from the infected patient without high levels of sDectin-1 or CFW staining (white arrows). In the upper right is a cluster of cells from the spiked-in control KAH3-EGFP (white arrowheads) with enhanced sDectin-1 and CFW staining. The CFW is overexposed to visualize the weakly-staining <i>C</i>. <i>krusei</i> cells from the patient, but the cell outlines can be clearly seen in the sDectin-1 staining image in red. (E) Schematic to illustrate that the majority of sites (58%) with enhanced sDectin-1 staining also had increased chitin deposition. Images are maximum projections of 6 slices (J) or 5 slices (K). Scalebar = 20 μm throughout. Statistics used for (H) and (I) were Mann-Whitney non-parametric tests. Significance throughout the figure is indicated with: n.s. p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.</p

Neutrophil infiltration and extracellular DNA are closely associated with enhanced sDectin-1 staining.

<p>Vaginal swab samples with <i>C</i>. <i>albicans</i> were scored by level of neutrophil infiltration, then stained and imaged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201436#pone.0201436.g001" target="_blank">Fig 1</a>. (A) Samples with high levels of neutrophil infiltration had sDectin-1-positive filaments, while those with low levels of infiltration had none. (B) Samples with high infiltration had a significantly higher level of extracellular DNA (eDNA), as imaged using Sytox Green. (C) Samples with high levels of eDNA also had high percentages of sDectin-1-positive cells. Samples with eDNA in every field were categorized as High eDNA, samples for which <100% of fields had eDNA were categorized as Low eDNA. (D) Representative field of a sample (#SP-14314) with no fields containing eDNA, no hyphae, and no sDectin-1-positive cells. Black arrowheads indicate nuclei from epithelial cells in the lavage. Image is maximum projection of 5 z-slices, created by ImageJ. (E) Representative field of a sample (#SP-12522) with high levels of eDNA, hyphae, and sDectin-1 positive cells. Black arrowheads indicate epithelial nuclei and white arrowheads indicate areas of diffuse Sytox Green staining of eDNA. Image is maximum projection of 10 z-slices, created by ImageJ. Scalebar = 20 μm. Statistics used in (A-C) were Mann-Whitney non-parametric tests. Significance throughout the figure is indicated with * p < 0.05, ** p < 0.01, *** p < 0.001.</p

<i>Lactobacilli</i> are found in most samples and are not associated with neutrophil infiltration or lack of sDectin-1+ cells.

<p><i>Lactobacilli</i> are found in most samples and are not associated with neutrophil infiltration or lack of sDectin-1+ cells.</p