Ydj1 governs fungal morphogenesis and stress response, and facilitates mitochondrial protein import via Mas1 and Mas2

We thank Zhen-Yuan Lin for help in the preparation of the AP-MS samples, and Cathy Collins for technical assistance. MDL is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072), LEC is supported by a Canada Research Chair in Microbial Genomics and Infectious Disease and by Cana-dian Institutes of Health Research (CIHR) Grants MOP-119520 and MOP-86452. OK is supported by National Insti-tutes of Health grant 5R01GM108975. A-CG is supported by a CIHR Foundation Grant (FDN143301), Genome Cana-da Genomics Innovation Network (GIN) Node and Tech-nical Development Grants, and a Canada Research Chair in Functional Proteomics. J-PL was supported by a TD Bank Health Research Fellowship at the Lunenfeld-Tanenbaum Research Institute and by a Scholarship for the Next Gen-eration of Scientists from the Cancer Research Society. JLX is supported by a CIHR – Frederick Banting and Charles Best Canada Graduate Scholarship. The funding agencies had no role in the study design, data collection and inter-pretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs

The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation

We thank Cowen lab members for helpful discussions. We also thank David Rogers (University of Tennessee) for sharing microarray analysis of the CAS5 homozygous mutant, and Li Ang (University of Macau) for assistance in optimizing the ChIP-Seq experiments. J.L.X. is supported by a Canadian Institutes of Health Research Doctoral award and M.D.L. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072). B.T.G. holds an Ontario Graduate Scholarship. C.B. and B.J.A. are supported by the Canadian Institutes of Health Research Foundation Grants (FDN-143264 and -143265). D.J.K. is supported by a National Institute of Allergy and Infectious Diseases grant (1R01AI098450) and J.D.L.C.D. is supported by the University of Rochester School of Dentistry and Medicine PREP program (R25 GM064133). A.S. is supported by the Creighton University and the Nebraska Department of Health and Human Services (LB506-2017-55). K.H.W. is supported by the Science and Technology Development Fund of Macau S.A.R. (FDCT; 085/2014/A2). L.E.C. is supported by the Canadian Institutes of Health Research Operating Grants (MOP-86452 and MOP-119520), the Natural Sciences and Engineering Council (NSERC) of Canada Discovery Grants (06261 and 462167), and an NSERC E.W.R. Steacie Memorial Fellowship (477598).Peer reviewedPublisher PD

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential

Functional Genomic Screening Reveals Core Modulators of Echinocandin Stress Responses in Candida albicans

Summary: Candida albicans is a leading cause of death due to fungal infection. Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis. Resistance is readily acquired via mutations in the drug target gene, FKS1. Both basal tolerance and resistance to echinocandins require cellular stress responses. We performed a systematic analysis of 3,030 C. albicans mutants to define circuitry governing cellular responses to echinocandins. We identified 16 genes for which deletion or transcriptional repression enhanced echinocandin susceptibility, including components of the Pkc1-MAPK signaling cascade. We discovered that the molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1, establishing key mechanisms through which Hsp90 mediates echinocandin resistance. We also discovered that perturbation of the CCT chaperonin complex causes enhanced echinocandin sensitivity, altered cell wall architecture, and aberrant septin localization. Thus, we provide insights into the mechanisms by which cellular chaperones enable crucial responses to echinocandin-induced stress. : Caplan et al. screen 3,030 Candida albicans mutants to define circuitry governing cellular responses to echinocandins, the first-line therapy for systemic candidiasis. They reveal that the molecular chaperone Hsp90 is required for stability of Pkc1 and Bck1 and that the CCT chaperonin complex is a key modulator of echinocandin susceptibility. Keywords: fungal pathogen, Candida albicans, echinocandins, Hsp90, Pkc1, CCT complex, client protein, stress response, functional genomic screen, drug resistanc

Functional Genomic Analysis of <i>Candida albicans</i> Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation

<div><p>Fungal biofilms are complex, structured communities that can form on surfaces such as catheters and other indwelling medical devices. Biofilms are of particular concern with <i>Candida albicans</i>, one of the leading opportunistic fungal pathogens of humans. <i>C</i>. <i>albicans</i> biofilms include yeast and filamentous cells that are surrounded by an extracellular matrix, and they are intrinsically resistant to antifungal drugs such that resolving biofilm infections often requires surgery to remove the contaminated device. <i>C</i>. <i>albicans</i> biofilms form through a regulated process of adhesion to surfaces, filamentation, maturation, and ultimately dispersion. To uncover new strategies to block the initial stages of biofilm formation, we utilized a functional genomic approach to identify genes that modulate <i>C</i>. <i>albicans</i> adherence. We screened a library of 1,481 double barcoded doxycycline-repressible conditional gene expression strains covering ~25% of the <i>C</i>. <i>albicans</i> genome. We identified five genes for which transcriptional repression impaired adherence, including: <i>ARC18</i>, <i>PMT1</i>, <i>MNN9</i>, <i>SPT7</i>, and <i>orf19</i>.<i>831</i>. The most severe adherence defect was observed upon transcriptional repression of <i>ARC18</i>, which encodes a member of the Arp2/3 complex that is involved in regulation of the actin cytoskeleton and endocytosis. Depletion of components of the Arp2/3 complex not only impaired adherence, but also caused reduced biofilm formation, increased cell surface hydrophobicity, and increased exposure of cell wall chitin and β-glucans. Reduced function of the Arp2/3 complex led to impaired cell wall integrity and activation of Rho1-mediated cell wall stress responses, thereby causing cell wall remodelling and reduced adherence. Thus, we identify important functional relationships between cell wall stress responses and a novel mechanism that controls adherence and biofilm formation, thereby illuminating novel strategies to cripple a leading fungal pathogen of humans.</p></div

The Arp2/3 complex plays a critical role in maintaining cell wall integrity.

<p><b>(A-B)</b> Osmotic stabilization with sorbitol rescues adherence defect of Arp2/3 complex mutants. Cells were grown in YPD ± 1M sorbitol at 30°C and adherence assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g001" target="_blank">Fig 1C</a>. Representative microscopy images of 96-well adherence assay are shown <b>(A)</b> and quantification of adherence is plotted in <b>(B)</b>. Error bars represent standard deviations of nine technical measurements. Sorbitol significantly restores the ability of the <i>arc40Δ/arc40Δ</i> mutant to adhere to a solid surface (<i>P</i><0.001, ANOVA, Bonferroni's Multiple Comparison Test). <b>(C-D)</b> Treatment with sorbitol restores chitin <b>(C)</b> and glucan <b>(D)</b> content in the cell wall of an <i>arc40Δ/arc40Δ</i> mutant to wild-type levels. Microscopy was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g003" target="_blank">Fig 3A</a>. Fluorescence intensity was quantified. Error bars represent standard deviations of five technical measurements. Significant differences in fluorescence intensity upon staining with Calcofluor White and Aniline Blue were observed in an <i>arc40Δ/arc40Δ</i> mutant in the absence and presence of sorbitol (<i>P</i><0.001, ANOVA, Bonferroni's Multiple Comparison Test).</p

The Arp2/3 complex plays a critical role in adherence, biofilm formation and cell wall physiology.

<p><b>(A)</b> Non-barcoded conditional expression strains for Arp2/3 complex components were grown overnight in the absence or presence of doxycycline. Adherence was measured as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g001" target="_blank">Fig 1C</a>. Error bars represent standard deviations of five technical replicates. Transcriptional repression of all target genes significantly impaired adherence (<i>P</i><0.001, ANOVA, Bonferroni's Multiple Comparison Test). <b>(B)</b> Transcriptional repression of Arp2/3 complex components reduces biofilm formation. Biofilms were grown and metabolic activity was measured as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g002" target="_blank">Fig 2a</a>. Error bars represent standard deviations of three technical replicates. Asterisks indicate significantly altered biofilm formation upon transcriptional repression of target genes (<i>P</i><0.05, ANOVA, Bonferroni's Multiple Comparison Test). <b>(C)</b> Transcriptional repression of Arp2/3 complex components alters cell wall physiology. Staining and visualization of cells was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g003" target="_blank">Fig 3A</a>. Scale bar represents 10 μm. <b>(D)</b> Transcriptional repression of Arp2/3 complex components alters cell surface hydrophobicity. Experiment was performed and analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g003" target="_blank">Fig 3B</a>. Error bars represent standard deviations of nine technical measurements. Transcriptional repression of Arp2/3 components significantly alters cell surface hydrophobicity (<i>P</i><0.001, ANOVA, Bonferroni's Multiple Comparison Test). <b>(E)</b> <i>ALS2</i> or <b>(F)</b> <i>HWP1</i> transcript levels were monitored after strains were sub-cultured overnight in the absence and presence of doxycycline, followed by a sub-culture and growth to log-phase in the absence and presence of doxycycline. The expression of <i>ALS2</i> and <i>HWP1</i> was normalized to a <i>GPD1</i> control, and is plotted relative to the wild-type strain in the absence of doxycycline. Error bars represent standard deviation of technical triplicates. * indicates significant differences between untreated and doxycycline treated conditions (<i>P</i><0.01, ANOVA, Bonferroni's Multiple Comparison Test). Experiment was performed in duplicate with a representative image shown.</p

Transcriptional repression of components of the Apr2/3 complex leads to impaired actin patch formation and enhanced rates of endocytosis.

<p><b>(A)</b> Microscopy images of Arp2/3 complex mutants grown in the absence or presence of doxycycline and then stained with Rhodamine-Phalloidin. <b>(B)</b> Microscopy images of Arp2/3 complex mutants grown in the absence or presence of doxycycline and then incubated with Lucifer Yellow. Scale bar represents 30 μm. <b>(C)</b> Quantification of endocytosis defect by counting the number of cells staining positive for Lucifer Yellow. Error bars represent standard deviations of nine technical measurements. Depletion of each component of the Arp2/3 complex significantly increases the rate of endocytosis (<i>P</i><0.01, ANOVA, Bonferroni's Multiple Comparison Test).</p

Loss of function of the Arp2/3 complex leads to hyperactivation of Rho1, altered cell wall physiology and impaired cell-to-surface adhesion.

<p><b>(A)</b><i>RHO1</i> transcript levels were monitored after strains were incubated for four hours in the presence and absence of doxycycline. The expression of <i>RHO1</i> was normalized to a <i>GPD1</i> control, and is plotted relative to the wild-type strain in the absence of doxycycline. Error bars represent standard deviation of technical triplicates. Experiment was performed in duplicate with a representative image shown. * indicates <i>P</i><0.05, (ANOVA, Bonferroni's Multiple Comparison Test). ** indicates <i>P</i><0.01. <b>(B)</b> Growth curves of strains cultured with or without doxycycline in YPD or YPD + 1M sorbitol. Growth was monitored by measuring optical density every 15 minutes. Experiment was performed in biological triplicate with representative plot shown. <b>(C</b>) Quantification of endocytosis defect by counting the number of cells staining positive for Lucifer Yellow, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g006" target="_blank">Fig 6C</a>. Error bars represent standard deviations of nine technical measurements. * represent significant adherence defects compared to a wild-type strain, and ** represents significant adherence defects between indicated strains (<i>P</i><0.01, ANOVA, Bonferroni's Multiple Comparison Test). <b>(D)</b> Quantification of Calcofluor White and Aniline Blue fluorescence intensity to estimate the amount of chitin and glucan respectively in the fungal cell wall. Error bars represent standard deviation of five technical replicates. * represent significant adherence defects compared to a wild-type strain, and ** represents significant adherence defects between indicated strains (<i>P</i><0.01, ANOVA, Bonferroni's Multiple Comparison Test). <b>(E)</b> Adherence was evaluated and measured as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006452#pgen.1006452.g001" target="_blank">Fig 1C</a>. Error bars represent standard deviations of five technical replicates. Asterisks represent significant adherence defects compared to a wild type strain (<i>P</i><0.001, ANOVA, Bonferroni's Multiple Comparison Test).</p