Cowen, Leah E

Leach, Michelle D

PLoS Pathogens

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Aberdeen University Research

PearlsSurviving the Heat of the Moment: A Fungal PathogensPerspectiveMichelle D. Leach1,2*, Leah E. Cowen21Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom, 2Department ofMolecular Genetics, University of Toronto, Toronto, Ontario, CanadaIntroductionTemperature is a critical parameter continually monitored bymicroorganisms. The dynamic environments inhabited by micro-organisms evoke constant and effective environmental responsestrategies that have been elaborated over evolutionary time. Forexample, a significant rise or fall in ambient temperature initiates astress response in the organism, commonly known as heat-shock orcold-shock responses, respectively. The phenomenon of temper-ature sensing has long been studied in microorganisms such asbacteria [1], but these mechanisms are only recently beingtranslated to pathogenic fungi.Fungal pathogens inhabit a remarkable diversity of environ-ments and exhibit a dazzling repertoire of life cycles andenvironmentally contingent cellular processes. Take for exampleCryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumiga-tus. These fungi are found in diverse environments such as pigeonexcreta and soil, but retain a common denominator: the ability togrow at 37uC. Loss of genes necessary for high-temperaturegrowth in these pathogens results in attenuated virulence and attimes even death [2–4]. Therefore, high-temperature growth isessential for pathogenesis. A classic example is that Saccharomycescerevisiae clinical isolates are able to grow at higher temperatures(41uC) than laboratory strains, a characteristic important for theirsurvival in mice [5]. Besides governing virulence, temperatureregulates many distinct processes. Morphological transitions andgrowth temperature are linked in the dimorphic fungi, such as H.capsulatum, which grow as filamentous molds at ambient temper-ature and switch to a yeast form at elevated host temperature [6].Temperature also controls morphological transitions in the leadingfungal pathogen of humans, Candida albicans; though for thisorganism ambient temperature favors the yeast form, whileelevated temperature induces filamentous growth [7]. C. albicanscan also switch from a white to opaque cellular state in host nicheswith lower temperatures, such as skin, facilitating mating [8].Many genes are simply induced in response to elevatedtemperatures and do not serve to sense the external stimuli.Generally the consequences of a thermal upshift, as opposed to thetemperature itself, provide a signal the cell will react to. Thisarticle focuses on how fungi sense and adapt to changingenvironmental temperature, using S. cerevisiae as a model.The Unfolded-Protein ResponseThe heat-shock response has been universally conserved frombacteria to humans. Underpinning this phenomenon is that anincrease in temperature contributes to protein denaturation invivo. All living organisms respond by inducing a set of proteinsimportant for high-temperature growth called heat-shock proteins,which, in eukaryotes, are dependent on the heat-shock transcrip-tion factor (Hsf1) (Figure 1) [9–11]. However, 25 years after thediscovery of Hsf1 in S. cerevisiae, the specific trigger responsible forits activation has yet to be determined. Two recent studies on theunfolded-protein response in S. cerevisiae suggest that it is thelocation of unfolded proteins that determines activation of theheat-shock response. Using five transcriptional markers, Metzgerand colleagues demonstrate that three different cellular compart-ments elicit distinct transcriptional responses upon proteindenaturation [12]. Unfolded proteins within the endoplasmicreticulum (ER) of cells induce expression of KAR2 as well as HAC1splicing, both early hallmarks of ER stress. In contrast, unfoldedproteins within the cytosol lead to rapid upregulation of SSA4, themain Hsp70 chaperone in the cytosol, as well as STI1, a cytosoliccochaperone. This response is equivalent to that induced by heatshock [12]. The third response occurs when membrane proteinsunfold. This does not lead to upregulation of any of the genesmentioned above, but instead induces a distinct class of genes.Following this study, Geiler-Samerotte and colleagues examinedthe proteomic response during chronic low-level expression of amisfolded YFP protein in S. cerevisiae [13]. They discovered that themajority of significantly induced proteins were cytosolic chaper-ones and cochaperones, including members of the Hsp70 family aswell as Sti1. Indeed, 20 differentially regulated proteins identifiedin this study upon expression of a misfolded YFP are bound byHsf1 [13]. Unfolded proteins are thus able to activate the classicheat-shock response, but only when found in the cytosol. Thissuggests that the cell may use multiple distinct mechanisms tosense temperature.Membrane Fluidity DynamicsAn alternative theory to unfolded proteins being the primarysensor of heat is changes in membrane structure and function. Anabrupt increase in temperature triggers a rapid decrease in themolecular order of cellular membranes (i.e., fluidity increases).Studies on the fungal pathogen H. capsulatum found that exposureto saturated fatty acids alongside a rise in temperature leads to anCitation: Leach MD, Cowen LE (2013) Surviving the Heat of the Moment: AFungal Pathogens Perspective. PLoS Pathog 9(3): e1003163. doi:10.1371/journal.ppat.1003163Editor: Joseph Heitman, Duke University Medical Center, United States ofAmericaPublished March 7, 2013Copyright:  2013 Leach, Cowen. This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.Funding: MDL is supported by a Sir Henry Wellcome Postdoctoral Fellowshipfrom the Wellcome Trust (096072). http://www.wellcome.ac.uk/Funding/Biomedical-science/Funding-schemes/Fellowships/Basic-biomedical-fellowships/wtx033549.htm. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interestsexist.* E-mail: michelle.leach@abdn.ac.ukPLOS Pathogens | www.plospathogens.org 1 March 2013 | Volume 9 | Issue 3 | e1003163increase in heat-shock gene transcription; conversely, addition ofunsaturated fatty acids reduced heat-shock gene transcriptionduring a temperature upshift [14]. These authors also comple-mented a S. cerevisiae ole1/ole1 mutant that lacks a fatty aciddesaturase using constructs with the native promoter or two H.capsulatum promoters, which express OLE1 at different levels [15].Complemented strains modified the saturated versus unsaturatedfatty acids ratio, changing the physical state of cellular membranesand demonstrating a different threshold temperature of heat-shockgene expression relative to the mutant [14]. Although no suchwork has been done in C. albicans or C. neoformans, transcriptional-profiling studies of C. neoformans during high-temperature growthidentified the transcriptional activator MGA2 [16]. In S. cerevisiae,Mga2 and its homologue Spt23 regulate transcription of OLE1[17], suggesting that C. neoformans may require cell-membraneremodelling to mitigate responses to temperature change. How-ever, the time scale over which changes in membrane fluidityoccur may not be rapid enough for this to serve as primary sensorof temperature.RNA ThermometersAn increase in temperature to 37uC signals to pathogenicmicroorganisms that they have successfully invaded a warm-blooded mammalian host, leading to expression of virulence genes.In bacteria, RNA structures are a key element of the heat-shockresponse. They can occlude RNA binding sites in mRNAs of keyregulators that melt during heat shock to enable protein synthesis[18]. These have been termed RNA thermometers. Indeed, thethree major classes of temperature-responsive genes, includingheat-shock genes, cold-shock genes, and genes necessary forvirulence, control temperature-dependent translation initiation byexploiting RNA thermometers [19]. Until very recently thenumber and location of RNA thermometers in eukaryotesremained unknown. However, a study by Wan and colleaguesidentified thousands of putative RNA thermometers by probing S.cerevisiae RNA structures at different temperatures [20]. In keepingwith transcript-profiling data, genes with low melting temperatureswere enriched for ribosomal proteins, whereas transcripts withFigure 1. Mechanisms of temperature sensing in fungi. Upon a thermal insult, cells utilize different mechanisms to sense changes in thesurrounding temperature for adaptation and survival. Hsf1 becomes phosphorylated, binding to heat-shock elements (HSEs) in target promoters,leading to the upregulation of chaperone proteins. These aid in the stabilization and refolding of denatured proteins, and Hsp90 and Hsp70 assist inthe downregulation of Hsf1 [25]. The cellular plasma membrane becomes more fluid in response to thermal insults (right inset box). Moreover,although little is known about RNA thermometers in fungi, there is evidence suggesting they are involved in RNA decay during heat shock (left insetbox) [20].doi:10.1371/journal.ppat.1003163.g001PLOS Pathogens | www.plospathogens.org 2 March 2013 | Volume 9 | Issue 3 | e1003163higher melting temperatures included key unfolded-proteinresponse activators such as HAC1 [20]. Strikingly, Wan andcolleagues showed that the most structurally stable mRNAs barelydecline during heat shock, whereas mRNAs with low meltingtemperatures displayed considerable and prolonged decreases intranscript levels. They concluded that RNA thermometers in S.cerevisiae play a role in RNA decay during heat shock [20].However, whether these mechanisms contribute to temperaturesensing in fungal pathogens remains to be determined. Despite thecentral importance of RNA thermometers for temperature-dependent gene regulation, it is clear that there are other keymechanisms as it is unlikely that RNA thermometers account foractivation of Hsf1, the transcription factor that commands theheat-shock response.Hsf1-Hsp90 Autoregulatory CircuitThe heat-shock response is governed by Hsf1, an essentialprotein in yeast, which is activated by phosphorylation upon athermal upshift (Figure 1) [10]. In C. albicans, Hsf1 is phosphor-ylated rapidly (within one minute) of a 30–42uC heat shock. It is arelatively transient response, downregulated upon cellular adap-tation [21]. The means of this downregulation have followed amodel whereby two classes of heat-shock proteins have beenimplicated in regulation of Hsf1 activity. The first class is Hsp70.In S. cerevisiae, deletion of the two cytosolic Hsp70 isoforms SSA1and SSA2 induces Hsf1 transcriptional activity [22]. Hsp70operates within the Hsp90 chaperone machine, which stabilizesa wide range of cellular clients [23]. Mutations that interfere withHsp90 function in S. cerevisiae cause increased levels of Hsf1 activity[24]. However, it is frequently argued that interfering with majorcytosolic chaperone proteins generates an unfolded-proteinresponse, which could be responsible for de-repressing Hsf1transcriptional activity. Until recently no physical interactionbetween Hsf1 and either Hsp90 or Hsp70 had been observed inyeast. Leach and colleagues utilized the fungal pathogen C. albicansto determine genetically and biochemically that Hsf1 interactswith Hsp90 under steady-state conditions, and that upon thermalinsults, this interaction is strengthened [25]. This stands in contrastto the model that Hsf1 activation requires release from Hsp90, butis consistent with a recent perspective on the impact oftemperature on the Hsp90 interactome [26]. Leach and colleaguesalso detected an interaction between Hsp90, Hsf1, and Hsp70when cells were exposed to a 60- or 120-minute heat shock at42uC, and that Hsp90 localised to the nucleus during elevatedtemperatures. Together, this provides the strongest evidence todate for the existence of the Hsf1-Hsp90 autoregulatory circuit.However, as Hsf1 did not dissociate from Hsp90 upon heat shock,it is unlikely that Hsp90 directly controls the rapid activation ofHsf1. Indeed, it is feasible that the kinase responsible for activatingHsf1 serves as the immediate sensor of temperature, yet theidentity of this kinase has remained elusive since the discovery thatHsf1 is activated by phosphorylation.Beyond Temperature SensingThere have been many advances in understanding temperatureregulation in eukaryotes over the years, although numerousenigmas remain to be resolved. Despite the high degree ofconservation of many components of the heat-shock response, thebiology of temperature regulation in fungi is remarkably distinctfrom mammalian systems. Although studies in S. cerevisiae haveprovided a foundation for understanding how cells sensetemperature shifts, whether this regulation is conserved inmedically relevant fungi needs to be addressed given theimportance of high temperatures for infection. The mostfundamental challenge is to understand how the cell sensestemperature and initiates the appropriate cellular response. Forexample, does an increase in membrane fluidity act as the primarysensor of temperature, or is it a response for adaptation? Howrapidly do these changes occur? Which kinase phosphorylatesHsf1? The rapid timescale of Hsf1 phosphorylation suggests thepossibility of a temperature-sensing kinase, whose activity might beexquisitely sensitive to temperature-dependent changes in struc-ture. Understanding the mechanisms of temperature sensing inpathogens remains a priority and could lead to novel drug targets.References1. Klinkert B, Narberhaus F (2009) Microbial thermosensors. Cell Mol Life Sci 66:2661–2676.2. Bhabhra R, Miley MD, Mylonakis E, Boettner D, Fortwendel J, et al. (2004)Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrAimpairs thermotolerant growth and reduces virulence. Infect Immun 72: 4731–4740.3. Lamoth F, Juvvadi PR, Fortwendel JR, Steinbach WJ (2012) Heat shock protein90 is required for conidiation and cell wall integrity in Aspergillus fumigatus.Eukaryot Cell 11: 1324–1332.4. Odom A, Muir S, Lim E, Toffaletti DL, Perfect J, et al. (1997) Calcineurin isrequired for virulence of Cryptococcus neoformans. EMBO J 16: 2576–2589.5. McCusker JH, Clemons KV, Stevens DA, Davis RW (1994) Saccharomycescerevisiae virulence phenotype as determined with CD-1 mice is associated withthe ability to grow at 42uC and form pseudohyphae. Infect Immun 62: 5447–5455.6. Maresca B, Kobayashi GS (1989) Dimorphism in Histoplasma capsulatum: A modelfor the study of cell differentiation in pathogenic fungi. Microbiol Rev 53: 186–209.7. Gow NA, Brown AJ, Odds FC (2002) Fungal morphogenesis and host invasion.Curr Opin Microbiol 5: 366–371.8. Lachke SA, Lockhart SR, Daniels KJ, Soll DR (2003) Skin facilitates Candidaalbicans mating. Infect Immun 71: 4970–4976.9. 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Nat Rev Microbiol 10: 255–265.20. Wan Y, Qu K, Ouyang Z, Kertesz M, Li J, et al. (2012) Genome-widemeasurement of RNA folding energies. Mol Cell 48: 169–181.21. Leach MD, Tyc KM, Brown AJP, Klipp E (2012) Modelling the regulation ofthermal adaptation in Candida albicans, a major fungal pathogen of humans.PLoS ONE 7: e32467. doi:10.1371/journal.pone.003246722. Craig EA, Jacobsen K (1984) Mutations of the heat inducible 70 kilodalton genesof yeast confer temperature sensitive growth. Cell 38: 841–849.PLOS Pathogens | www.plospathogens.org 3 March 2013 | Volume 9 | Issue 3 | e100316323. Taipale M, Krykbaeva I, Koeva M, Kayatekin C, Westover KD, et al. (2012)Quantitative analysis of Hsp90-client interactions reveals principles of substraterecognition. Cell 150: 987–1001.24. Duina AA, Kalton HM, Gaber RF (1998) Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J BiolChem 273: 18974–18978.25. Leach MD, Budge S, Walker L, Munro C, Cowen LE, et al. (2012) Hsp90orchestrates transcriptional regulation by Hsf1 and cell wall remodelling byMAPK signalling during thermal adaptation in a pathogenic yeast. PLoS Pathog8: e1003069. doi:10.1371/journal.ppat.100306926. Leach MD, Klipp E, Cowen LE, Brown AJ (2012) Fungal Hsp90: A biologicaltransistor that tunes cellular outputs to thermal inputs. Nat Rev Microbiol 10:693–704.PLOS Pathogens | www.plospathogens.org 4 March 2013 | Volume 9 | Issue 3 | e1003163

Surviving the heat of the moment : a fungal pathogens perspective

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