Cryptococcus neoformans phosphoinositide-dependent kinase 1 (PDK1) ortholog is required for stress tolerance and survival in murine phagocytes

Cryptococcus neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied in S. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show that PKH2-02 but not PKH2-01 is required for C. neoformans to tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion of PKH2-02 leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress. PKH2-02 function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using a Galleria mellonella model of low-temperature cryptococcosis, we found that PKH2-02 is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of the pkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion of PKH2-02 affects the interaction between C. neoformans and phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling in C. neoformans is crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence

A Genome-Wide Screen of Deletion Mutants in the Filamentous Saccharomyces cerevisiae Background Identifies Ergosterol as a Direct Trigger of Macrophage Pyroptosis

Phagocytic cells such as macrophages play an important role in the host defense mechanisms mounted in response to the common human fungal pathogen Candida albicans. In vitro, C. albicans triggers macrophage NLRP3-Casp1/11-mediated pyroptosis, an inflammatory programmed cell death pathway. Here, we provide evidence that Casp1/11-dependent pyroptosis occurs in the kidney of infected mice during the early stages of infection. We have also used a genome-wide screen of nonessential Σ1278b Saccharomyces cerevisiae genes to identify genes required for yeast-triggered macrophage pyroptosis. The set of genes identified by this screen was enriched for those with functions in lipid and sterol homeostasis and trafficking. These observations led us to discover that cell surface localization and/or total levels of ergosterol correlate with the ability of S. cerevisiae, C. albicans, and Cryptococcus neoformans to trigger pyroptosis. Since the mammalian sterol cholesterol triggers NLRP3-mediated pyroptosis, we hypothesized that ergosterol may also do so. Consistent with that hypothesis, ergosterol-containing liposomes but not ergosterol-free liposomes induce pyroptosis. Cell wall mannoproteins directly bind ergosterol, and we found that Dan1, an ergosterol receptor mannoprotein, as well as specific mannosyltransferases, is required for pyroptosis, suggesting that cell wall-associated ergosterol may mediate the process. Taken together, these data indicate that ergosterol, like mammalian cholesterol, plays a direct role in yeast-mediated pyroptosis.Innate immune cells such as macrophages are key components of the host response to the human fungal pathogen Candida albicans. Macrophages undergo pyroptosis, an inflammatory, programmed cell death, in response to some species of pathogenic yeast. Prior to the work described in this report, yeast-triggered pyroptosis has been observed only in vitro; here, we show that pyroptosis occurs in the initial stages of murine kidney infection, suggesting that it plays an important role in the initial response of the innate immune system to invasive yeast infection. We also show that a key component of the fungal plasma membrane, ergosterol, directly triggers pyroptosis. Ergosterol is also present in the fungal cell wall, most likely associated with mannoproteins, and is increased in hyphal cells compared to yeast cells. Our data indicate that specific mannoproteins are required for pyroptosis. This is consistent with a potential mechanism whereby ergosterol present in the outer mannoprotein layer of the cell wall is accessible to the macrophage-mediated process. Taken together, our data provide the first evidence that ergosterol plays a direct role in the host-pathogen interactions of fungi

Genetic analysis of the <i>Candida albicans</i> biofilm transcription factor network using simple and complex haploinsufficiency

<div><p>Biofilm formation by <i>Candida albicans</i> is a key aspect of its pathobiology and is regulated by an integrated network of transcription factors (Bcr1, Brg1, Efg1, Ndt80, Rob1, and Tec1). To understand the details of how the transcription factors function together to regulate biofilm formation, we used a systematic genetic interaction approach based on generating all possible double heterozygous mutants of the network genes and quantitatively analyzing the genetic interactions between them. Overall, the network is highly susceptible to genetic perturbation with the six network heterozygous mutants all showing alterations in biofilm formation (haploinsufficiency). In addition, many double heterozygous mutants are as severely affected as homozygous deletions. As a result, the network shows properties of a highly interdependent ‘small-world’ network that is highly efficient but not robust. In addition, these genetic interaction data indicate that <i>TEC1</i> represents a network component whose expression is highly sensitive to small perturbations in the function of other networks TFs. We have also found that expression of <i>ROB1</i> is dependent on both auto-regulation and cooperative interactions with other network TFs. Finally, the heterozygous <i>NDT80</i> deletion mutant is hyperfilamentous under both biofilm and hyphae-inducing conditions in a <i>TEC1</i>-dependent manner. Taken together, genetic interaction analysis of this network has provided new insights into the functions of individual TFs as well as into the role of the overall network topology in its function.</p></div

Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target

The <i>ndt80</i>Δ/<i>NDT80</i> and <i>bcr1</i>Δ/<i>BCR1</i> show altered biofilm architecture.

<p>Confocal microscopy images of wild type, <i>ndt80Δ/NDT80</i>, <i>bcr1</i>Δ<i>/BCR1</i>, <i>tec1Δ/TEC1</i>, and <i>ndt80Δ/NDT80 tec1Δ/TEC1</i> strains grown in YETS for 48 hr. Cells were stained with concanavalin A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006948#sec011" target="_blank">Materials and methods</a> for full details. The top image is a three-dimensional rendering of the biofilm. The bottom image is a cross-sectional view of the same biofilm.</p

Epistasis analysis of the biofilm density for the biofilm TF network.

<p>(A) The nomarlized biofilm density (<i>NBD</i>) for each double heterozygote mutant was determined after 48 hr biofilm formation as described in Materials and Methods. The predicted NBD for each double heterozygous mutant was calculated by multiplying the <i>NBD</i> scores for each single mutant: NBD^<i>dh12</i> = NBD^<i>sh1</i> x NBD^<i>sh2</i>, where <i>dh</i>^<i>12</i> is double heterozygote of single heterozygotes <i>sh</i>^<i>1</i> and <i>sh</i>^<i>2</i>. The expected NBD^<i>dh12</i> is plotted against the observed NBD^<i>dh12</i>. NBD^<i>dh12</i> scores that plot on the diagonal (solid line with estimated error indicated by dotted line) indicate no genetic interaction (independence) between the single mutants and correspond to Ɛ = 0; NBD^<i>dh12</i> below the diagonal have Ɛ <0 indicating an cooperative interactions; NBD^<i>dh12</i> above the diagonal have Ɛ > 0 indicating a buffering interaction. The plot on the left is an inset showing the details of the area indicated. The full data set for biofilm formation of all strains used to generate this data and calculations are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006948#pgen.1006948.s002" target="_blank">S2 Table</a>. Strains with error that overlapped with the estimated error of the predicted NBD^<i>dh12</i> were considered to have no interaction. (B) Profile of cooperative and buffering interactions for each TF. (C) Correlation between number of buffering interactions and the number of network TF bound by that TF based on previously reported chromatin immunoprecipitation data. Pearson correlation coefficient is shown. Please note that <i>BRG1</i> and <i>TEC1</i> have identical numbers of buffering interactions and thus the data points overlap.</p

Properties of biofilm TF network.

<p>Properties of biofilm TF network.</p

The biofilm TF network contains feedforward loops that regulate <i>BCR1</i> expression.

<p>(A) A summary of the phenotypic genetic interaction network for the biofilm TFs. (B) Three network modules that show both phenotypic interactions and downstream target expression changes consistent feedforward loops. The numbers in parenthesis over each arrow indicate the fold change in expression of the upstream and downstream TF (upstream, downstream) in the corresponding double mutant as compared to each single mutant. The arrows indicate that the double mutant has reduced expression of the TF at the head of the arrow. The circular arrrows indicate auto-regulation. All indicated expression changes are statistically significant (<i>P</i> < 0.05) and the primary data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006948#pgen.1006948.s002" target="_blank">S2 Table</a>. (C) The higher level module that results in the combination of the three feed-forward loops.</p

Heterozygous deletion mutants of six core biofilm transcription factors show simple and complex hapoinsufficiency for biofilm density.

<p>(A) The density of the biofilms formed by the indicated wild type (SN250), heterozygous and homozygous deletion strains was determined by measuring the OD₆₀₀ after incubation for 48 hr as described in Materials and Methods (YETS medium). The raw data were normalized to wild type: Normalized Biofilm Density (NBD) = OD₆₀₀ mutant/OD₆₀₀ wild type. Data are representative of results obtained on at least three different days. Bars are the mean of three or four independent well replicates. Error bars are standard deviation. Red and blue bars indicate statistically significant reduction and increase from wild type, respectively (Student’s t test, <i>P</i><0.05). Grey bars indicate no change. (B) The interaction profile for the interactions of <i>tec1</i>Δ/<i>TEC1</i> with all five other network TFs is shown. Bars are the mean of three or four independent well replicates. Error bars are standard deviation. Red and blue bars indicate statistically significant reduction and increase from the <i>tec1</i>Δ/<i>TEC1</i> heterozygote, respectively (Student’s t test, <i>P</i><0.05). Grey bars indicate no change.</p