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Cellular Microbiology (2014) 16(4), 473–481
doi:10.1111/cmi.12266 First published online 14 February 2014
Microreview Hsp90-dependent regulatory circuitry controlling temperature-dependent fungal development and virulence Teresa R. O’Meara and Leah E. Cowen* Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Summary The pathogenic fungi Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans are an increasing cause of human mortality, especially in immunocompromised populations. During colonization and adaptation to various host environments, these fungi undergo morphogenetic alterations that allow for survival within the host. One key environmental cue driving morphological changes is external temperature. The Hsp90 chaperone protein provides one mechanism to link temperature with the signalling cascades that regulate morphogenesis, fungal development and virulence. Candida albicans is a model system for understanding the connections between morphogenesis and Hsp90. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy.
Introduction Fungal pathogens are an increasing public health burden, especially due to the growing population of immunocompromised individuals. Candida albicans accounts for 9–12% of all nosocomial bloodstream infections, with an attributable mortality rate of 38% despite significant
Received 25 November, 2013; revised 9 January, 2014; accepted 13 January, 2014. *For correspondence. E-mail leah.cowen@ utoronto.ca; Tel. (+41) 6978 4085; Fax (+41) 6978 6885.
advances in diagnosis and increased use of antifungal therapies (Gudlaugsson et al., 2003; Wisplinghoff et al., 2003; Horn et al., 2009). Invasive aspergillosis due to Aspergillus fumigatus has a mortality rate of 40–50%, even with treatment (Herbrecht et al., 2002). Additionally, there are approximately one million new cases of cryptococcal meningitis every year, and more than 600 000 deaths (Park et al., 2009). One of the key aspects of fungal pathogenesis is the ability of the microbe to perform morphological changes in response to environmental cues. In this review, we will focus on the three major fungal pathogens of humans, C. albicans, A. fumigatus and Cryptococcus neoformans, and discuss aspects of fungal development in relation to temperature sensing, Hsp90 and pathogenesis. Candida albicans is an opportunistic pathogen that is also a member of the normal human microbiota (Rosenbach et al., 2010). C. albicans exists in multiple niches in the human host, including the oral and vaginal mucosa, the gastrointestinal tract, and the skin. However, when C. albicans enters the bloodstream, it can cause systemic disease and invasion of internal organs. One of the key virulence factors in C. albicans pathogenesis is the ability to perform a reversible transition to filamentous growth. Mutants that are locked in either the yeast or hyphal form have defects in causing disease. Additionally, C. albicans can form biofilms that are composed of a mixture of yeast, pseudohyphae and hyphal cells. The biofilms are important for persistence within the host, especially in the context of implanted medical devices, as they provide a reservoir for dissemination. Aspergillus fumigatus is a saprophytic fungus that normally resides in decaying matter in the soil (Latge, 2001). However, A. fumigatus can also cause severe disease within immunocompromised individuals. As part of its life cycle, A. fumigatus produces conidia, which can easily become airborne and inhaled. In immunocompromised individuals, the conidia are able to emerge from dormancy and grow as filaments. The filaments can then proliferate into the host lung tissue and set up infections, leading to invasive aspergillosis (Latge, 2001). The ability of the
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cellular microbiology
474 T. R. O’Meara and L. E. Cowen A. fumigatus conidia to germinate and develop into filaments is key for the capacity of this fungus to cause disease. Cryptococcus neoformans is another opportunistic human fungal pathogen that infects immunocompromised individuals. C. neoformans grows primarily as budding yeasts, both in the environment and within the human host. However, fungal development is important in the production of spores, which are infectious propagules (Giles et al., 2009). Additionally, during lung infections, a certain percentage of C. neoformans cells become titan cells, which are extremely large and resistant to phagocytosis by macrophages (Okagaki et al., 2010; Zaragoza et al., 2010). These titan cells are important in modulating the host immune system (Okagaki and Nielsen, 2012). Figure 1 demonstrates some of the morphological changes that occur in these fungi in response to hostrelevant environmental signals. Some of the common features of morphogenetic switches include alterations in cell wall composition and structure. Additionally, the development of filaments involves a switch to multicellularity, and thus careful regulation of cell cycle, nuclear division and cytokinesis. Some of the conserved pathways involved in morphological changes include the cAMP/Protein Kinase A (PKA) pathway, the Ras pathway, the calcineurin pathway and the Mitogen-Activated Protein Kinase (MAPK) cascade. Temperature sensing is a common feature driving morphological changes in these organisms, with inputs into many of the conserved signalling cascades regulating development. Increased temperatures are often coupled with entry into the host, and fungi that are unable to survive at human body temperatures are unable to cause disease (Robert and Casadevall, 2009). Even C. albicans, which is obligately associated with the host, can experience heat stress during pathogenic growth due to host fevers. However, the connections between these conserved signal transduction cascades with temperature sensing remain largely enigmatic. Recent work has demonstrated that the Hsp90 chaperone protein provides one mechanism to link temperature with morphogenesis and fungal development. Figure 2 presents a model of the connections between Hsp90 and signal transduction cascades that will be discussed in this review. Hsp90 is an ATP-dependent molecular chaperone that interacts with multiple proteins involved in adaptation to stress and high temperatures. By modulating protein stability, Hsp90 can alter the ratio of active to inactive protein, thus adding an additional layer of regulation to signal transduction cascades (Taipale et al., 2010). Hsp90 activity and availability is regulated on multiple levels, including transcriptional regulation, posttranscriptional regulation and post-translational regulation
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Fig. 1. Morphological states of human fungal pathogens in a range of environmental conditions. A. C. albicans cells incubated in the indicated conditions for 6 h to induce morphological changes. Scale bar is 10 microns. B. A. fumigatus spores germinating and forming hyphae in response to glucose minimal medium. Images image of hyphae courtesy of W. J. Steinbach (Duke University Medical Center), reproduced with permission. Scale bars are 10 microns. C. C. neoformans titan cells obtained from murine lung infections. (i) Lavaged Cryptococci and (ii) H+E-stained murine lung sections. Black arrows indicate titan cells, red arrows indicate normal cells. Scale bars are 20 microns.
(Zhao et al., 2005). Moreover, Hsp90 function is modulated by co-chaperones, which are thought to mediate recognition of client proteins, many of which cycle dynamically through complexes with Hsp90 until their activation (Johnson et al., 2007; Taipale et al., 2010). The levels and activity of the co-chaperone proteins can thus © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 473–481
Hsp90 and fungal development
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Fig. 2. Model for Hsp90 interactions affecting morphology, development and virulence via the cAMP/PKA pathway (A), cell cycle control (B), or the calcineurin pathway (C). In red are the proteins that are required for filamentation in response to Hsp90 inhibition.
also modulate Hsp90 function. The availability of Hsp90 to regulate a particular pathway is also dependent on the global needs for Hsp90. For example, while heat stress increases the overall levels of Hsp90, it also increases the need for Hsp90 in complexes with unfolded and damaged proteins, potentially altering the availability of Hsp90 in the cytoplasm (Leach et al., 2012). Hsp90 has emerged as an attractive target for novel antifungal therapies. Depletion of Hsp90 impairs the evolution of resistance to azoles and echinocandins, which target the cell membrane and cell wall respectively. By stabilizing signal transducers such as the protein phosphatase calcineurin or elements of the protein kinase C (PKC) pathway, Hsp90 enables the phenotypic effects of resistance mutations, and thus the development of drug resistance (Cowen and Lindquist, 2005; Cowen et al., 2006; Singh et al., 2009; Singh-Babak et al., 2012). Hsp90 also likely plays a role in many cellular adaptations to invasive growth within a host. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy.
Candida albicans Candida albicans is a model system for understanding the connections between morphogenesis, Hsp90 and © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 473–481
virulence. The yeast-hyphal transition has been highly studied because of its importance for C. albicans virulence. Many conserved signalling pathways regulate the ability of C. albicans to respond to various environmental cues with specific morphogenesis programmes (Braun and Johnson, 2000). Some of the cues that induce filamentation include alkaline pH, elevated carbon dioxide levels and serum, especially with concomitant increases in temperature. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. It is possible that Hsp90 may play a similar role in other pathogenic fungi as it does in C. albicans because many of the same signal transduction pathways are regulating these host-relevant morphological changes. In this review, we will focus on the connections between some of these signalling pathways and Hsp90.
cAMP/PKA signalling and Hsp90 Temperature is an important signal for C. albicans filamentation (Mitchell, 1998). Many host-specific environmental stimuli, including serum and alkaline pH, must be combined with an increase in temperature for full induction of filamentation (Mitchell, 1998; El Barkani et al., 2000). Additionally, the kinetics of germ tube formation are decreased at low temperatures, even in the presence of other inducing cues.
476 T. R. O’Meara and L. E. Cowen The first mechanistic connection between temperature, filamentation and Hsp90 was the observation that C. albicans cells grow as filaments in response to chemical or genetic inhibition of Hsp90 (Shapiro et al., 2009). Raising the temperature decreased the levels of Hsp90 inhibitor necessary to induce filamentation. Together, these data suggested that high temperatures reduce the levels of available Hsp90 and thus remove Hsp90 repression of filamentation. To determine the target of Hsp90, Shapiro et al. examined the ability of mutants in the conserved cAMP/ protein kinase A (PKA) pathway to filament in response to inhibition of Hsp90 (Shapiro et al., 2009). The cAMPPKA pathway is one of the major pathways for inducing filamentation in response to environmental cues, including serum. These cues signal through G-protein coupled receptors or Ras1 to activate the Cyr1 adenylyl cyclase, induce cAMP and release the catalytic subunits of PKA. The Cdc25, Ras1, Cyr1 and PKA proteins are all required for filamentation in response to Hsp90 inhibition (Shapiro et al., 2009). Further analysis revealed that Hsp90 and the Sgt1 co-chaperone physically interact with Cyr1 (Shapiro et al., 2012b). The current hypothesis is that Hsp90 holds Cyr1 in an inactive conformation, leading to repression of the cAMP/PKA signalling cascade (Shapiro et al., 2012b). This physical interaction between Hsp90, Sgt1 and the adenylyl cyclase has been observed in Saccharomyces cerevisiae and Schizosaccharomyces pombe, demonstrating the conservation of this layer of regulation in cAMP signalling (Dubacq et al., 2002; Alaamery and Hoffman, 2008; Flom et al., 2012). The Efg1 transcription factor is required for filamentation in response to cAMP/PKA activation under most conditions. However, the efg1Δ/Δ mutant still filaments when Hsp90 is inhibited, suggesting that there are additional transcription factors downstream of PKA that are responding to Hsp90 inhibition (Shapiro et al., 2009). Interestingly, filamentation during colonization of the mouse intestinal tract does not depend on Efg1 (White et al., 2007; Shapiro et al., 2012a). Potentially, filamentation within the host requires a transcription factor that responds specially to the cAMP/PKA pathway, temperature and Hsp90. Further analysis of the downstream effectors of Hsp90 and the cAMP/PKA pathway may reveal novel circuitry for regulating filamentation within the host. One potential downstream effector is the Stp2 transcription factor, which is required for filamentation in response to Hsp90 inhibition (Shapiro et al., 2012a). Stp2 contains a conserved PKA phosphorylation motif, but this connection to the cAMP pathway has yet to be tested (Martinez and Ljungdahl, 2005; Vylkova et al., 2011).
Cell cycle and filamentation Cell cycle control is an integral part of the decision to filament in C. albicans (reviewed in Berman, 2006). Recent work demonstrated that Hsp90 regulates filamentation in C. albicans via multiple elements of the cell cycle control pathways, although the extent of the connections between Hsp90, cell cycle control and morphogenesis in C. albicans have not been fully explored. Filaments induced by chemical inhibition of Hsp90 strongly resemble those that are induced by cell cycle arrest at the S, G2 and M phases (Bachewich et al., 2005; Bensen et al., 2005). These filaments display constrictions between the yeast and filament but no subsequent constrictions at cell junctions (Senn et al., 2012). One potential mechanism for this phenotype is contingent upon interactions between Hsp90 and the cyclindependent kinase (CDK), Cdc28. In S. cerevisiae, Hsp90 interacts with Cdc28 in conjunction with the Cdc37 co-chaperone protein, and this interaction allows for activation and stability of CDK-cyclin complexes and normal progression through the cell cycle (Farrell and Morgan, 2000). In C. albicans, depletion of Cdc28 results in a mixture of filaments, pseudohyphae and elongated cells (Umeyama et al., 2006). Recently, Senn et al. demonstrated that the C. albicans Cdc28 protein physically interacts with Hsp90, and that Hsp90 is required for stabilizing Cdc28 protein levels (Senn et al., 2012). However, there was still residual Cdc28 after inhibition of Hsp90, potentially explaining the difference in morphology. It is likely that the interaction between Hsp90 and Cdc28 has pleiotropic effects on the cell cycle and filamentation. To further examine the role of cell cycle arrest and Hsp90 in filamentation, Senn et al. examined the Bub2 mitotic exit checkpoint protein (Senn et al., 2012). Activation of Bub2 prevents progression of the cell cycle, leading to increased filamentation. The bub2Δ/Δ mutant strain does not filament in response to Hsp90 inhibition. This suggests that Hsp90 is involved in the activation of Bub2, potentially by stabilizing the activating kinase. In S. cerevisiae, this kinase is Cdc5; however, Cdc5 levels were not decreased upon Hsp90 inhibition in C. albicans, suggesting that an additional, as yet unidentified kinase is the Hsp90 client protein that is required for regulating mitotic exit. Additional connections between Hsp90 and the cell cycle include the Clb4 cyclin; when Hsp90 is inhibited, the levels of Clb4 are decreased (Senn et al., 2012). As the clb4Δ/Δ mutant strain is constitutively pseudohyphal, Senn et al. hypothesized that the decrease in Clb4 levels while Clb2 levels are maintained result in the observed delays in mitotic completion and thus increased filamentation (Bensen et al., 2005). © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 473–481
Hsp90 and fungal development The Pho85 cyclin-dependent kinase is another protein involved in regulating mitotic exit and morphogenesis in S. cerevisiae (Measday et al., 1997; Moffat et al., 2000). In S. cerevisiae, the Pho85 protein forms a complex with the Pcl1 cyclin and the Hms1 transcription factor. Recent work has demonstrated that all three components are required for C. albicans filamentation in response to Hsp90 inhibition or elevated temperature, suggesting that they act in a similar manner in this fungal pathogen (Shapiro et al., 2012a). Interestingly, Hms1 is not required for C. albicans filamentation in response to other inducing conditions, suggesting that it specifically responds to temperature stress (Shapiro et al., 2012a). Currently, the direct mechanism of Hsp90 interactions with the Pcl1–Pho85–Hms1 complex remains enigmatic. Hsp90 may target the Pho85 kinase to stabilize the complex and allow for phosphorylation and activation of Hms1; however, the upstream signals for this complex have not been elucidated in C. albicans, leaving open the possibility for Hsp90 involvement earlier in the signalling cascade. The mechanism by which Hms1 regulates filamentation is also unclear. However, analysis of the downstream targets of Hms1 by genome-wide chromatin immunoprecipitation revealed that Cph2 is a direct target (Perez et al., 2013). The cph2Δ/Δ mutant strain is also unable to filament in response to Hsp90 inhibition (Shapiro et al., 2012a). Cph2 regulates the transcription of several hyphal-specific genes via the Tec1 transcription factor, and it is required for colonization and filamentation in the mouse gut (Lane et al., 2001; Rosenbach et al., 2010). Calcineurin Calcineurin is a calcium-activated protein phosphatase that is important for regulating responses to stresses in many organisms (Kraus and Heitman, 2003). In C. albicans, calcineurin is required for cell wall integrity and antifungal drug tolerance (Singh et al., 2009). Although the role of calcineurin in hyphal development in C. albicans appears to be strain dependent (Sanglard et al., 2003; Bader et al., 2006), calcineurin plays a major role in the filamentation and thermotolerance of the closely related Candida dubliniensis, C. lusitaniae and C. glabrata species (Chen et al., 2011; 2012; SinghBabak et al., 2012; Zhang et al., 2012). In all these Candida species, calcineurin is required for survival in serum, infection in murine models and resistance to antifungal drugs, highlighting the importance of this protein phosphatase in virulence (Blankenship and Heitman, 2005; Chen et al., 2012; Zhang et al., 2012). In S. cerevisiae, Hsp90 physically interacts with calcineurin, stabilizing and sequestering the protein until the cell is exposed to stresses such as osmotic stress or © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 473–481
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drug treatment (Imai and Yahara, 2000). The interactions between C. albicans Hsp90 and calcineurin follow a similar pattern, with co-immunoprecipitation experiments revealing physical interactions between CaHsp90 and calcineurin (Singh et al., 2009). Additionally, calcineurin protein levels are depleted upon reduction of Hsp90 levels, confirming that Hsp90 is required for full calcineurin activity in the cell (Singh et al., 2009). The interaction between Hsp90 and calcineurin is modulated by post-translational regulation of Hsp90 function. Hsp90 activity is regulated by reversible acetylation events (Kovacs et al., 2005; Scroggins et al., 2007). Recently, Robbins et al. demonstrated that posttranslational modification of Hsp90 by lysine deacetylases is required for Hsp90 interactions with calcineurin, suggesting another potential avenue for combination antifungal treatment (Robbins et al., 2012). Aspergillus fumigatus To cause disease, A. fumigatus conidia must be able to germinate in the environment of the host lung and proliferate as invasive hyphae. There are many conserved signal transduction networks that regulate these processes, including the calcineurin, cAMP/PKA and the Hog/MAPK pathways. Currently, there is only evidence for connections between the calcineurin pathway and Hsp90. However, it is likely that further studies of the role of Hsp90 in A. fumigatus development and drug resistance will elaborate on these connections. Recent work has demonstrated that Hsp90 is required for A. fumigatus hyphal growth and proliferation (Lamoth et al., 2012; 2013). To determine the mechanism for Hsp90 regulation of A. fumigatus development, Lamoth et al. examined the effect of Hsp90 on antifungal drug resistance, and demonstrated that mutating the A. fumigatus calcineurin protein or repressing Hsp90 results in hypersensitivity to caspofungin (Lamoth et al., 2012). Hsp90 inhibition also increased sensitivity to the calcineurin inhibitor FK506 (Lamoth et al., 2012). Unlike C. albicans, there is no additive effect of Hsp90 inhibition with the other antifungal drugs nikkomycin Z, amphotericin B or voriconazole (Lamoth et al., 2012). Taken together, these results suggest a specific interaction between Hsp90 and calcineurin in A. fumigatus. Calcineurin is a major regulator of A. fumigatus filamentation and growth, and mutants have defects in hyphal morphology and conidiation (Steinbach et al., 2006; Cramer et al., 2008). To determine the extent of the connections between calcineurin and Hsp90 in A. fumigatus, Lamoth et al. examined calcineurin expression levels and found that they were unchanged in the Hsp90 overexpression strain (Lamoth et al., 2013). However, Hsp90 likely acts on calcineurin protein levels,
478 T. R. O’Meara and L. E. Cowen and so determination of calcineurin protein stability is required to evaluate the relationship between Hsp90 and calcineurin in A. fumigatus. Overall, the connections between Hsp90 and additional pathways in A. fumigatus have not been fully investigated. The conserved role of cAMP signalling in regulating A. fumigatus development suggests that there may be an additional pathway by which Hsp90 can regulate germination and polarized hyphal growth (Fuller et al., 2011). Cryptococcus neoformans The C. neoformans Hsp90 protein has not been fully characterized, but increased HSP90 transcript levels have been observed during murine lung infections and under host-mimicking in vitro conditions, suggesting the importance of this molecular chaperone during infection (Hu et al., 2008; O’Meara et al., 2013). To examine the role of Hsp90 in C. neoformans, we focus here on calcineurin, which is required for C. neoformans virulence in the host. Calcineurin is a major regulator of the ability of C. neoformans to grow at the host-relevant temperatures of 37°C (Odom et al., 1997). Calcineurin also plays a role during mating and haploid fruiting. Calcineurin mutants are unable to form conjugation tubes, and calcineurin is required for hyphal elongation in diploids (Cruz et al., 2001). Additionally, in both C. neoformans and the sister species C. gattii, calcineurin mutants display altered morphology at 37°C due to altered vesicle formation and plasma membrane disruptions (Chen et al., 2013). A proteomic analysis of calcineurin-associated proteins identified Hsp90 and the Hsp90 co-chaperone Aha1 (Kozubowski et al., 2011), suggesting that the interaction between Hsp90 and calcineurin is conserved in C. neoformans as well. Hsp90 or calcineurin have yet to be implicated in an additional morphological change observed during host colonization, where C. neoformans forms titan cells, which are large (> 15 μm), polyploid cells that appear to be specifically induced during the context of infection (Okagaki et al., 2010; Zaragoza et al., 2010). These cells have thickened cell walls and dense, highly cross-linked polysaccharide capsules. An interesting feature is that although titan cells have increased DNA content (8–16 N), they are able to produce haploid daughter cells, suggesting careful regulation of cell cycle (Okagaki et al., 2010; Zaragoza et al., 2010). Currently, the signalling networks that control the production of titan cells have not been fully established. However, the cAMP/PKA pathway, the Ras pathway and the homologue to the Pcl1 G1-cyclin have all been implicated in titan cell formation (Okagaki et al., 2010; 2011; Zaragoza et al., 2010; O’Meara et al., 2013). At this point, it is unclear whether Hsp90 in C. neoformans regulates
cAMP or Ras signalling, or if there is a role for Hsp90 in C. neoformans cell cycle. Potentially, inhibition of Hsp90 would alter the ability of C. neoformans to form titan cells in the lung, thus altering the capacity of this fungus to evade the immune system. Thermal dimorphs Although C. albicans, A. fumigatus and C. neoformans are responsible for the majority of human fungal infections, there are many other human fungal pathogens that also use temperature as marker for the host and for morphogenetic switching. The ‘thermal dimorphs’, including Histoplasma capsulatum, Coccidioides spp., Blastomyces dermatitidis, Paracoccidiodes brasiliensis and Penicillium marneffei, typically grow as filaments in the environment and as yeasts in the infected hosts. The role of Hsp90 in governing this transition is still being explored. Both H. capsulatum and P. marneffei have increased Hsp90 levels during yeast-phase growth, and Hsp90 is required for adaptation to stress and infection in the host (Minchiotti et al., 1992; Xi et al., 2007; Nicola et al., 2008; Edwards et al., 2011). In P. brasiliensis, Hsp90 may act with calcineurin to promote the filament to yeast transition that is required for virulence (Matos et al., 2013). These examples highlight the importance of Hsp90 in the morphogenetic programmes of a diverse set of fungal pathogens. Conclusions To cause disease, microbes must sense and respond to the varying stresses of the host environment. A key regulator of both adaptation to host stresses and morphogenesis is Hsp90. C. albicans is a model system for understanding the connections between Hsp90, fungal development and pathogenesis. These connections raise intriguing questions about the role of Hsp90 in additional fungal pathogens, especially due to the importance of Hsp90 in regulating development in parasites such as Leishmania donovani or Toxoplasma gondii (Wiesgigl and Clos, 2001; Echeverria et al., 2005). Targeting Hsp90 has enormous potential for antifungal therapies, especially in combination therapies. Preventing developmental switches is likely to alter the ability of the microbe to cause disease in the host. The current challenges of targeting Hsp90 are due to the high degree of conservation between microbial and host Hsp90 proteins. Understanding the networks that interact with Hsp90 may reveal new targets for therapies that will be specific to the microbe. Acknowledgements We thank William Steinbach for providing images of Aspergillus germ tubes. LEC is supported by a Canada Research Chair in © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 473–481
Hsp90 and fungal development Microbial Genomics and Infectious Disease, by a Ministry of Research and Innovation Early Researcher Award, by Natural Sciences & Engineering Research Council Discovery Grant # 355965 and by Canadian Institutes of Health Research Grants MOP-86452 and MOP-119520.
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