Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway.

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an "a" mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1-Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans

Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein-protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules

<i>ERG5</i>, <i>STT4</i>, and <i>SNT1</i> are genetic interactors of <i>HSP90</i>.

<p>The fitness defect of the <i>tetO-HSP90/hsp90Δ</i> and <i>stt4Δ/Δ</i>, <i>erg5Δ/Δ</i>, or <i>snt1Δ/Δ</i> double mutant strains is greater than the product of the fitness defect of each individual mutant. The dotted line indicates the expected double mutant fitness defect. In the <i>tetO-HSP90/hsp90Δ</i> strains, <i>HSP90</i> depletion is achieved by transcriptional repression with 0.05 μg/mL doxycycline (DOX).</p

Stt4, Erg5, and Snt1 are required for <i>C</i>. <i>albicans</i> virulence.

<p>(A) Representative images of infections. ASC-mCherry macrophages were infected with the indicated strains and monitored for pyroptosis by foci of fluorescence Scale bar is 50 microns (B) Average percent pyroptosis, * indicates <i>p</i> < 0.05, error bars indicate standard deviation. At least 700 infected cells were counted per strain.</p

Perturbation of ergosterol biosynthesis causes hypersensitivity to Hsp90 inhibitors.

<p>(A) Mutants of ergosterol biosynthesis genes are hypersensitive to geldanamycin. Minimum inhibitory concentration (MIC) assays were performed in RPMI medium at 37°C for 48 hours and optical densities at 600 nm were averaged for two biological replicates with two technical replicates each. Percent growth is normalized to the no drug condition. To deplete target gene expression, the strains were incubated in 0.05 μg/mL doxycycline (DOX). (B) Chemical inhibition of the ergosterol biosynthetic pathway using fluconazole, fenpropimorph, or terbinafine leads to synergy with the Hsp90 inhibitor geldanamycin. Synergy is not observed with amphotericin B, which binds to ergosterol in the membrane. Dose response matrixes were performed with the wild-type strain in YPD and incubated for 24 hours. (C) Hypersensitivity to geldanamycin is not rescued by addition of ergosterol. Strains were incubated with either 50 μM ergosterol or with the vehicle (ethanol) in RPMI at 37°C for 48 hours.</p

Stt4 and Erg5 modulate the cellular requirements for Hsp90.

<p>(A) Hsp90 levels are not decreased in the <i>stt4Δ/Δ</i>, <i>erg5Δ/Δ</i>, or <i>snt1Δ/Δ</i> mutant strains compared with the wild type. Hsp90 levels were normalized to the tubulin loading control and the relative levels were compared with the wild-type strain. (B) Hog1 activation is decreased in the mutant strains. Strains were treated with or without 10 mM hydrogen peroxide to induce Hog1 phosphorylation. Protein levels were normalized to the H3 loading control and the levels of total Hog1 and pHog1 were determined relative to the wild-type strain under the same conditions. (C) Low doses of the Hsp90 inhibitor geldanamycin are sufficient to induce filamentation in the <i>stt4Δ/Δ</i> and <i>erg5Δ/Δ</i> mutant strains. Strains were incubated for 6 hours in YPD in the presence of 2.5 μM geldanamycin before imaging. Scale bar is 10 microns.</p

Mapping the <i>C</i>. <i>albicans</i> Hsp90 genetic interaction network.

<p>(A) Chemical genomic screening revealed 11 mutants that are hypersensitive to Hsp90 inhibition. Strains were screened at 3 μM geldanamycin in RPMI at 37°C, and percent growth is normalized to the no drug condition. * indicates <i>p</i> <0.05 compared to the wild-type strain using t-tests. (B) The <i>HSP90</i> genetic interaction network is environmentally contingent. The network is composed of 158 genetic interactors identified in five different growth conditions (grey boxes). Each <i>HSP90</i> genetic interactor is indicated by a box, with edges connecting it to the environmental conditions in which it interacts with <i>HSP90</i>. The color of the box reflects the number of conditions in which the mutant demonstrates hypersensitivity to Hsp90 inhibition, with the majority of interactions only occurring under a single environmental condition. FLU = fluconazole, C = caspofungin, T = tunicamycin. Thick black outline = screened at 1 μM geldanamycin except for <i>stt4Δ/Δ</i> which was screened at 0.375 μM geldanamycin.</p

Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis

<div><p>Fungal pathogens have evolved diverse strategies to sense host-relevant cues and coordinate cellular responses, which enable virulence and drug resistance. Defining circuitry controlling these traits opens new opportunities for chemical diversity in therapeutics, as the cognate inhibitors are rarely explored by conventional screening approaches. This has great potential to address the pressing need for new therapeutic strategies for invasive fungal infections, which have a staggering impact on human health. To explore this approach, we focused on a leading human fungal pathogen, <i>Candida albicans</i>, and screened 1,280 pharmacologically active compounds to identify those that potentiate the activity of echinocandins, which are front-line therapeutics that target fungal cell wall synthesis. We identified 19 compounds that enhance activity of the echinocandin caspofungin against an echinocandin-resistant clinical isolate, with the broad-spectrum chelator DTPA demonstrating the greatest synergistic activity. We found that DTPA increases susceptibility to echinocandins via chelation of magnesium. Whole genome sequencing of mutants resistant to the combination of DTPA and caspofungin identified mutations in the histidine kinase gene <i>NIK1</i> that confer resistance to the combination. Functional analyses demonstrated that DTPA activates the mitogen-activated protein kinase Hog1, and that <i>NIK1</i> mutations block Hog1 activation in response to both caspofungin and DTPA. The combination has therapeutic relevance as DTPA enhanced the efficacy of caspofungin in a mouse model of echinocandin-resistant candidiasis. We found that DTPA not only reduces drug resistance but also modulates morphogenesis, a key virulence trait that is normally regulated by environmental cues. DTPA induced filamentation via depletion of zinc, in a manner that is contingent upon Ras1-PKA signaling, as well as the transcription factors Brg1 and Rob1. Thus, we establish a new mechanism by which metal chelation modulates morphogenetic circuitry and echinocandin resistance, and illuminate a novel facet to metal homeostasis at the host-pathogen interface, with broad therapeutic potential.</p></div

Nrg1 protein levels are reduced in DTPA-induced filamentous cells even in the absence of Ubr1.

<p>Wild-type (SN250) or <i>ubr1</i>Δ<i>/ubr1</i>Δ cells with one allele of HA-tagged Nrg1 were inoculated into YPD medium at 30°C for 4.5 hours in the presence or absence of 100 μM DTPA. Western blot analysis was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006350#pgen.1006350.g005" target="_blank">Fig 5</a>, using α-HA to monitor total Nrg1 and α-tubulin as a loading control. In all lanes, 40 μg of protein was loaded.</p