Fungal quorum-sensing molecules and mechanisms.

<p>(A) Structure of farnesol; (B) structure of tyrosol; (C) sequence of mature Qsp1 peptide; (D) model for Qsp1 action in <i>C. neoformans var. neoformans</i>.</p

Approaching the Functional Annotation of Fungal Virulence Factors Using Cross-Species Genetic Interaction Profiling

<div><p>In many human fungal pathogens, genes required for disease remain largely unannotated, limiting the impact of virulence gene discovery efforts. We tested the utility of a cross-species genetic interaction profiling approach to obtain clues to the molecular function of unannotated pathogenicity factors in the human pathogen <em>Cryptococcus neoformans</em>. This approach involves expression of <em>C. neoformans</em> genes of interest in each member of the <em>Saccharomyces cerevisiae</em> gene deletion library, quantification of their impact on growth, and calculation of the cross-species genetic interaction profiles. To develop functional predictions, we computed and analyzed the correlations of these profiles with existing genetic interaction profiles of <em>S. cerevisiae</em> deletion mutants. For <em>C. neoformans LIV7</em>, which has no <em>S. cerevisiae</em> ortholog, this profiling approach predicted an unanticipated role in the Golgi apparatus. Validation studies in <em>C. neoformans</em> demonstrated that Liv7 is a functional Golgi factor where it promotes the suppression of the exposure of a specific immunostimulatory molecule, mannose, on the cell surface, thereby inhibiting phagocytosis. The genetic interaction profile of another pathogenicity gene that lacks an <em>S. cerevisiae</em> ortholog, <em>LIV6</em>, strongly predicted a role in endosome function. This prediction was also supported by studies of the corresponding <em>C. neoformans</em> null mutant. Our results demonstrate the utility of quantitative cross-species genetic interaction profiling for the functional annotation of fungal pathogenicity proteins of unknown function including, surprisingly, those that are not conserved in sequence across fungi.</p> </div

<i>liv7</i>Δ but not <i>trs33</i>Δ cells are defective in phagocytosis evasion.

<p>A) Phagocytosis assays. <i>C. neoformans</i> cells were treated either with 1× PBS (unopsonized, blue) or 100% fetal bovine serum (opsonized, yellow) for 30 min, then used to infect RAW264.6 macrophage-like cells at a multiplicity-of-infection of two <i>C. neoformans</i> cells to one macrophage. Data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. B) Phagocytosis assays. Association of unopsonized <i>C. neoformans</i> cells with the addition of control buffer (blue), with 250 µM mannose (yellow), or 250 µM laminarin (purple). Data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. C) Model. Together with our previous work, the data described in this paper suggests that there are three parallel mechanisms by which <i>C. neoformans</i> evades phagocytosis. First, Liv7 acts in a partially redundant fashion with Trs33 in vesicle transport, a function that prevents exposure of pathogen associated molecular patterns (PAMPs) that are recognized by the immune system and result in phagocytosis of <i>C. neoformans</i> by phagocytes. Liv7/Trs33 are not part of the Gat201-Gat204-Blp1 pathway because phagocytosis of <i>liv7</i>Δ cells can be competitively inhibited by mannose, whereas phagocytosis of <i>gat204</i>Δ cells cannot. The Liv7/Trs33 pathway does not act to suppress phagocytosis via capsule production since capsule-deficient mutants do not display PAMP exposure and are sensitive to opsonization.</p

Lectin staining of surface of <i>liv7</i>Δ <i>trs33</i>Δ cells reveals a role for Liv7/Trs33 in PAMP shielding.

<p>A) GXM (left) and chitin (middle) staining of <i>C. neoformans</i> strains grown under tissue culture conditions (DMEM, 5% CO₂, 37°C, without shaking). Scale bars are 5 µm. B) α-β-glucan staining patterns. C) Concanavalin A (conA) staining patterns. Note that <i>liv7Δtrs33Δ</i> and <i>vps52Δ</i> mutants display a massive increase in conA staining.</p

<i>C. neoformans</i> bait genes.

<p>Schematics of the Liv5, Liv6, Liv7, Liv13, Mep1, and Blp1 proteins. Detectable motifs are shown.</p

<i>S. cerevisiae</i> genes whose knockouts significantly correlate with <i>C. neoformans</i> bait genes.

<p><i>C. neoformans</i> bait gene (column 1), <i>S. cerevisiae</i> ORF that shows significant correlation (column 2), Z-score (column 3), correlation score (column 4), <i>S. cerevisiae</i> gene name (column 5), and <i>S. cerevisiae</i> gene function (from the <i>Saccharomyces Genome Database</i> at yeastgenome.org) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003168#pgen.1003168-Cherry1" target="_blank">[78]</a> (column 6).</p

Phenotypes of <i>C. neoformans liv6</i>Δ cells are consistent with the endosomal function predicted by cross-species genetic interaction mapping.

<p>A) Pearson correlations between the genome-wide genetic interaction profiles of <i>pGPD-LIV6</i> (blue) with the published genome-wide interaction profiles of <i>S. cerevisiae</i> knockout mutants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003168#pgen.1003168-Costanzo1" target="_blank">[19]</a>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003168#pgen-1003168-t001" target="_blank">Table 1</a>. B) Wild-type <i>C. neoformans</i> cells grown under yeast culture conditions (YNB, 30°C, with shaking) stained with LysoTracker Green. We hypothesize that the dark area surrounded by staining is the vacuole, as it is in <i>S. cerevisiae </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003168#pgen.1003168-Raymond1" target="_blank">[56]</a>. Fluorescent images were exposed for two seconds and the scale bar represents five microns. C) <i>liv6</i>Δ <i>C. neoformans</i> cells grown under yeast culture conditions (YNB, 30°C, with shaking) stained with LysoTracker Green. Fluorescent images were exposed for two seconds and the scale bar represents five microns. D) Quantification of the number of “vacuoles” per cell (one, two, or ≥3 putative vacuoles) in LysoTracker-staining <i>C. neoformans</i> cells. <i>liv6</i>Δ cells but not other mutants show an increase in the number of vacuoles per cell (p≤0.005). Data shown are the averages of three experiments. 200 cells were counted per sample. Error bars represent the standard deviation of three experiments and p-values were calculated using the Student's t-test. E) Growth analysis of wild-type and mutant <i>C. neoformans</i> cells on yeast medium (YNB) without drug, with 2.5 mM neomycin, or with 10 µg/ml fluconazole. Cells were spotted at 10⁷ cells/ml in the upper spot and diluted 5-fold in each subsequent spot. Plates were incubated 48 hours at 30°C.</p

Liv7 localizes to the ER/Golgi in <i>C. neoformans</i>.

<p>A) Localization analysis. Shown are mCherry signals of cells grown under tissue culture conditions (left) (DMEM, 5% CO₂, 37°C, without shaking). The untagged control population (blue) shows mCherry signal in less than 20% of cells, whereas mCherry signal is visible in ∼50% of Liv7-mCherry positive cells (yellow). We then stained these same strains with BODIPY-labeled fluorescent BFA (fBFA; green channel; localizes to the ER/Golgi <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003168#pgen.1003168-Deng1" target="_blank">[44]</a>). Experiments were performed three times, 100 cells counted per sample, and data shown are the averages of three experiments. Error bars represent that standard deviation and p-values were calculated using Student's t-test. Scale bars are 5 µm. B) Untagged control cells stained with fBFA. 50 ms exposure. C) Liv7-mCherry cells stained with fBFA. 50 ms exposure. D–G) fBFA-staining of wild-type, <i>liv7</i>Δ, <i>trs33</i>Δ, and <i>liv7</i>Δ <i>trs33</i>Δ cells.</p

Fitting-derived parameters.

<p>Fitting-derived parameters.</p

Halftimes of maximal protein or fluorescence accumulation.

<p>Halftimes of maximal protein or fluorescence accumulation.</p