Stress tolerance in fungi — to kill a spoilage yeast Gertien J Smits1 and Stanley Brul1,2 The fungal spoilage of ingredients of food manufacture is an economic problem, often causes product loss and may constitute a health hazard. To effectively combat fungal food spoilage, a mechanistic understanding of tolerance for, and adaptation to, the preservation method used is crucial. Both are dependent on the genetic make-up and growth history of the organism. In the post-genomic era we are arriving at a situation in which, in the model organism Saccharomyces cerevisiae, physiological data, classical molecular biology and whole-genome responses can be combined to obtain explanatory and predictive models for growth. For food spoilage fungi we have not yet reached such a level of understanding, but we may use the knowledge gained for S. cerevisiae for the prevention of spoilage. Addresses 1 Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands 2 Department of Microbiological Control, Food Research Center, Unilever Research & Development, Olivier van Noortlaan 120, 3133 AT, Vlaardingen, The Netherlands Corresponding author: Brul, Stanley ([email protected])

Current Opinion in Biotechnology 2005, 16:225–230 This review comes from a themed issue on Food biotechnology Edited by Willem M de Vos Available online 2nd March 2005 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.02.005

Introduction Many different types of foods are prone to microbial spoilage and in many cases this spoilage is caused by the unwanted growth of fungi. In addition to their involvement in food spoilage, fungi are indoor surface contaminants and important human pathogens. Volatile compounds from fungi and fungal mycotoxins have been shown to provoke allergic reactions [1]. In food manufacture, the fungal spoilage of products causes severe economic loss and is a potential health hazard, owing to the presence of mycotoxins [2,3]. Fungal food spoilage can affect a variety of processed foods, including bread, cereals, spices, dairy products such as cheese, spreads (margarine), dressings, fondant, chocolate, fermented sauces (soy), soft drinks, fruits, jams, and high-sugar fruit syrups (Table 1). Most food spoilage www.sciencedirect.com

fungi belong to the ascomycetes and are thus closely related to the unicellular model fungus Saccharomyces cerevisiae. The complete genome sequence of this extremely well characterized microorganism is available and ‘omics’ tools are now being employed to analyze its physiological response to environmental conditions at the molecular level. Here, we review our current understanding of stress resistance and stress adaptation mechanisms in S. cerevisiae and other yeasts, and consider how this knowledge can be applied to the treatment of food-spoilage fungi.

The ecology of food-borne yeasts In many cases, the first indication of yeast spoilage is the visible growth of colonies on (processed) foods. Overt growth is observed in situations where bacterial growth is significantly suppressed (e.g. by the presence of high levels of weak organic acids). In packaged foods, blowing of cans may occur in non-alcoholic beverages and jams. Zygosaccharomyces bailii is well known for its high resistance to lipophilic weak organic acid preservatives, mediated primarily through the catabolism of these compounds (discussed in [4]). A remarkable new ecological niche for food spoilage yeasts was recently identified. Stratford et al. [5] indicated that wasps might be the origin of a yeast strain able to withstand pH 1.4 and moderate levels of weak organic acids. This strain — named Candida davenporti — was isolated from a soft drink. In addition to high levels of weak organic acids and low pH values, yeasts can be tolerant to low water activity. For this reason, products like fondant, chocolate and soy sauce are well known for their susceptibility to infection with Zygosaccharomyces rouxii (e.g. [6]). Remarkably, in many instances, the temperature resistance of S. cerevisiae isolates from spoiled processed foods is much higher than the temperature resistance of regular strains (e.g. [7]). It should be noted that similar stress resistance is often required in microorganisms (and thus also in yeasts) that successfully thrive as spoilage organisms in the food chain and in those that are used actively for food production. A classical example of the latter is Kluyveromyces lactis, which grows extremely well in dairy products such as cheeses and milk (e.g. [8]). In the following paragraphs we consider our current knowledge of the molecular basis of the resistance of well-characterized S. cerevisiae laboratory strains and food spoilage fungi to environmental stress, particularly in the context of conditions common to the natural environment of food spoilage yeast. Current Opinion in Biotechnology 2005, 16:225–230

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Figure 1

Table 1 Examples of food-spoilage fungi and typically affected productsa. Organism Saccharomyces cerevisiae Zygosaccharomyces rouxii Zygosaccharomyces bailii Candida davenporti Trichoderma harzianum, Penicillium commune Aspergillus niger Aspergillus versicolor Aspergillus flavusb Penicillium roqueforti Fusarium oxysporum Neosartorya fischeric

Products affected Soft drinks, dressings Soy sauce, fondants, chocolate Dressings, soft drinks Soft drinks Margarines Spices, spreads (margarines) Dairy products, bread Spices Meat, eggs, dairy products (cheeses) Fruit Pasteurised foods

a

Data taken from [55] and expanded with recent observations. Severe mycotoxin-producing species. cForms highly thermo-resistant ascospores.

b

Why yeasts die. . . To understand the development of stress tolerance in yeast, it is crucial to understand what causes their death. Death, the irreversible inability to grow and multiply, can occur at various stages during exposure to harmful conditions (Figure 1). The conditions can be so harsh that the cells are killed immediately; for example, if the plasma membrane is disrupted. The cells can also be severely damaged, but stay metabolically active while they try to repair the damage or adapt their physiology. If this is not possible, the cells die. Therefore, to survive, yeasts must not be killed immediately but instead need to repair the damage and to adapt their phenotype. This causes a lag in growth, in some cases because the cells actively arrest growth at a checkpoint [9], resulting in arrest at a specific phase of the cell cycle. In other cases, a lag phase can arise because the cells cannot generate the resources to continue growth, which in addition to growth arrest at various different stages of the cell cycle may cause arrest at the START checkpoint that monitors nutrient availability. Following adaptation and physiological changes, in principle, the yeast cells can grow again, albeit at a growth rate that is lower then before because each new cell has the adapted, non-ideal, physiology. Initial cell death can only be prevented by an innate tolerance. This tolerance depends on the genetic make-up of the yeast and its growth history before the stress. The phenotypic stress tolerance can vary from cell to cell within a population, causing heterogeneity in cellular stress resistance [10].

Determinants of initial stress resistance in Saccharomyces cerevisiae Fermenting yeast cells are generally less stress-resistant than non-fermenting cells (e.g. [11,12]), in which a high trehalose content is the main determinant for stress resistance [12]. Most of our mechanistic knowledge has been gained from studies of fermenting cells (i.e. yeast Current Opinion in Biotechnology 2005, 16:225–230

Stress 3

2

1 Survival

Adaptation

Adapted growth

Death

No adaptation

No growth

Death Current Opinion in Biotechnology

Schematic representation of the effects of stress on survival. 1) When cells encounter a stressful situation (symbolized by lightning), survival depends on initial stress resistance, which is determined by the genetic make-up and growth history of the cell. 2) If the cell survives it will alter its physiology and gene expression program to adapt. It will need to both repair damage and to adapt to decrease the effects of the (continued) stress. Whether or not this is successful depends on the genetic possibilities for adaptation and the energy that is available or that can be liberated. 3) After successful repair and adaptation, the cell will grow again if enough resources are still available. Energetic requirements are increased for permanently stress-adapted growth.

growing exponentially on a rich medium). The key determinants of stress tolerance in fermenting cells have been identified as the presence of chaperone proteins (notably Hsp104p [13,14]), the ability to synthesize trehalose [15], the ability to adapt to stress using the transcription factors Msn2p and Msn4p [16,17], and the presence of an, as yet, unidentified factor [18] that might be related to the ability to clear denatured proteins [19]. Chaperones, and particularly Hsp104p, are important for tolerance to many different stresses [13]. Hsp104p, in concert with the Hsp40 and Hsp70 family members that can prevent the aggregation of denatured proteins, can remodel and reactivate damaged proteins [20]. The disaccharide trehalose is important for the tolerance of yeast to various stresses, including heat stress, cold stress, oxidative stress, high ethanol concentrations, dehydration, osmotic and salt stress, and weak organic acids (e.g. for reviews see [21]). Trehalose is thought to stabilize proteins and possibly membranes [22,23], can function as a compatible solute [24], and might function as a radical scavenger [25]. In S. cerevisiae, trehalose is synthesized by the trehalose-6-phosphate synthase (TPS) complex and it is degraded by two trehalases, a cytosolic neutral and a vacuolar acid trehalase (for a review see [26]). The cycle of synthesis and degradation is important for the increased stress tolerance generated by trehalose, as mutation of the TPS genes as well as the trehalase genes causes a decrease in stress resistance [27,28]. Besides these mechanisms, the drastic increase of trehalose concentration in response to stress also results in a www.sciencedirect.com

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hyperosmotic cytoplasm, which leads to the activation of the cell integrity pathway (FIC Mensonides et al. unpublished) causing increased cell-wall mechanical strength and resistance to lytic enzymes.

. . . And why they don’t: adaptation and tolerance acquisition It seems likely that adaptation to a specific stress has specific requirements. Many conditions, however, have pleiotropic effects on cellular metabolism, but it is clear that adaptation and adapted growth require expenditure of extra energy. In the past few years, whole-genome approaches have revealed the transient environmental stress response (ESR) in S. cerevisiae [29,30]. Central to the ESR are the induction of genes controlled by the stress response and heat shock elements and the downregulation of genes controlled by the promoter elements rRPE and PAC. The downregulated genes mainly function in ribosomal RNA transcription and processing, and their downregulation might explain the growth arrest and decreased growth. The genes might be downregulated in order to liberate energy and nucleotides for specific stress responses. Interestingly, large-scale flux analyses in yeast subjected to various stresses showed a strong correlation between growth rate and tricarboxylic acid cycle activity, suggesting that yeast alters its physiology from (almost) exclusive fermentation to partial respiration [31]. This correlation was not valid for growth at increased temperatures, but in these cases the glycolytic flux was drastically increased [32]. Again, this illustrates the importance of energy generation for stress-adapted growth and suggests that respiration might be important for stress adaptation. The upregulated genes in the ESR are mostly under the control of the transcription factors Msn2p and Msn4p and include, among others, those involved in trehalose synthesis and degradation, genes implicated in oxidative stress defence and intracellular redox homeostasis, genes encoding several chaperones, and genes involved in stress signaling [33]. Analyses of the full set of knockout mutants in yeast have revealed genes and processes actually involved in generating tolerance and adaptation to stress and have identified targets for antifungal compounds [34–37]. Generally, only a fraction of the transcriptionally regulated genes shows a parallel response at the protein level [38] or actually has a phenotypic effect [34] and vice versa. In all likelihood, the ESR represents a transient repertoire of possibilities, a selection of which is used for adaptation.

to various stresses [39], has been relatively well studied. Likewise, in Candida albicans the mobilization of trehalose has been shown to be important for pathogenicity [40] and is required for tolerance to hydrogen peroxide [41,42], but not to other stresses such as heat or high salt [43,44]. In Aspergillus nidulans, trehalose is important for hydrogen peroxide and thermo-tolerance [45]. The halophilic yeast Debaryomyces hansenii accumulates trehalose in response to exposure to high salt concentrations, much more so than S. cerevisiae [46]. Similarly, in the salt-tolerant and osmo-tolerant Z. rouxii the genes for trehalose synthesis are highly expressed under non-stress conditions [47], suggesting that trehalose might be important for extreme salt resistance. By contrast, trehalose concentrations do not rise in response to weak organic acids in the acid-resistant Z. bailii, whereas in S. cerevisiae they do [48]. This observation suggests that trehalose is not crucial for generic weak organic acid resistance; however, the acid resistance of Z. bailii is known not to depend on the acid anion efflux pump Pdr12p and the organism can metabolize many acids [4]. Therefore, these stress responses may have become unnecessary for Z. bailii, while they are still needed for the genetically less adapted S. cerevisiae. The role of the ESR in other yeasts is also unclear. Salt stress effects on gene expression in Z. rouxii were studied using cross-hybridization experiments with S. cerevisiae gene filters. Several genes involved in protein synthesis, cell division and transcription that are downregulated or unaffected in S. cerevisiae were found to be unchanged or upregulated, respectively, in Z. rouxii. This suggests that in Z. rouxii growth is optimized for high salt conditions and the organism does not regard this as a stress [49]. In A. nidulans, an ESR might exist and as in S. cerevisiae might be controlled by the nutrient-regulated protein kinase A. In contrast to S. cerevisiae, however, protein kinase A would turn on the response in A. nidulans [50,51]. In S. pombe, the mitogen-activated protein kinase Sty1 mediates a common stress response [52]. Similarly, in C. albicans no ESR-like gene expression program for heat, oxidative and osmotic stress could be detected by microarray analyses [44] and the Msn2pand Msn4p-like transcription factors do not play a role in stress tolerance [53]. Instead, a common stress response program, optimized for survival in the human host, is coordinated by the Hog1p kinase [54].

Conclusions What about food-borne fungi? The question arises as to whether the molecular determinants of stress tolerance are common for yeasts and fungi. For the described food-spoilage fungi, surprisingly little is known about molecular physiology and mechanisms of stress tolerance and adaptation. Schizosaccharomyces pombe, in which trehalose renders the cells tolerant www.sciencedirect.com

Work on S. cerevisiae has emphasized the importance of physiology in the stress response. The dramatic increase in stress resistance in non-growing or non-fermenting yeast that results from trehalose accumulation [12] and the change from fermentation to (partial) respiration in stressed cells has been clearly illustrated [31]. In most foods, oxygen availability is limited and fermentation Current Opinion in Biotechnology 2005, 16:225–230

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might be the only option for growth, which in S. cerevisiae would cause reduced tolerance. On the other hand, yeast will often be dormant in foods, which in S. cerevisiae correlates with increased stress resistance.

7. 

Recent studies in S. pombe, C. albicans and Z. bailii show that although general stress resistance mechanisms do exist in the various yeasts, these are not necessarily regulated in a fashion analogous to S. cerevisiae, or to each other, and the stresses by which they are activated have evolved in correspondence with the organism’s ecological niche [52,54]. This may be of particular relevance to food-spoilage yeasts, as these have evolved in niches very similar to the foods they spoil (e.g. [5]).

8.

Vasdinyei R, Deak T: Characterization of yeast isolates originating from Hungarian dairy products using traditional and molecular identification techniques. Int J Food Microbiol 2003, 86:123-130.

9.

Pearce AK, Humphrey TC: Integrating stress-response and cell-cycle checkpoint pathways. Trends Cell Biol 2001, 11:426-433.

It seems best to kill food-spoilage fungi with stresses they have not adapted to, either genetically through evolution or phenotypically. Understanding the interactions between the physiological conditions the cells encounter in food and the stress response is still crucial, and a better understanding of the stress response of quiescent yeast would be of great value. Also, many responses should be monitored in low oxygen conditions, as oxygen availability might affect stress adaptation. The combined knowledge of the stress response from classical physiological studies, microarray analyses, molecular biology, the diverse omics, and phenomenological observations in foods, can now give us a detailed understanding of the strengths and weaknesses of certain yeasts, and eventually filamentous fungi, in specific foods to help prevent spoilage.

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This paper uses a metabolic footprinting approach to analyze the effect of various mutations in the yeast deletion collection and shows that this approach can lead to the identification of the ‘defect’ in the mutant.

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27. Singer MA, Lindquist S: Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. Trends Biotechnol 1998, 16:460-468. 28. Wera S, De Schrijver E, Geyskens I, Nwaka S, Thevelein JM: Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae. Biochem J 1999, 343:621-626. 29. Causton HC, Ren B, Koh SS, Harbison CT, Kanin E, Jennings EG, Lee TI, True HL, Lander ES, Young RA: Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell 2001, 12:323-337. 30. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO: Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 2000, 11:4241-4257. 31. Blank LM, Sauer U: TCA cycle activity in Saccharomyces  cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates. Microbiology 2004, 150:1085-1093. In large-scale flux analysis experiments the authors show a clear correlation of growth rate in yeast, altered by various stresses, with tricarboxylic acid cycle activity. This observation suggests an increased respiratory metabolism in stressed cells. 32. Mensonides FI, Schuurmans JM, Teixeira de Mattos MJ,  Hellingwerf KJ, Brul S: The metabolic response of Saccharomyces cerevisiae to continuous heat stress. Mol Biol Rep 2002, 29:103-106. Metabolic analysis of yeast shifted to various temperatures shows a transiently highly increased glycolytic flux during the lag phase, followed by a continuously increased flux during resumed growth. The temperature range from growth to no growth and death is extremely narrow, only 2 8C. 33. Gasch AP: The environmental stress response: a common yeast response to diverse environmental stresses. In Topics in Current Genetics: Yeast Stress Responses. Edited by Hohmann S, Mager WH: Springer-Verlag; 2003:11-70. 34. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, Dow S,  Lucau-Danila A, Anderson K, Andre B et al.: Functional profiling of the Saccharomyces cerevisiae genome. Nature 2002, 418:387-391. The first whole-genome parallel deletion mutant analysis reveals that only 1% of the genes transcriptionally induced by a stress leads to a clear growth effect under the same conditions. 35. Lum PY, Armour CD, Stepaniants SB, Cavet G, Wolf MK, Butler JS,  Hinshaw JC, Garnier P, Prestwich GD, Leonardson A et al.: Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 2004, 116:121-137. The use of heterozygous diploids in drug-effect analysis reveals specific targets for various compounds. 36. Mollapour M, Fong D, Balakrishnan K, Harris N, Thompson S,  Schuller C, Kuchler K, Piper PW: Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative. Yeast 2004, 21:927-946. The identification of pH- and sorbic-acid-sensitive and resistant mutants in the complete yeast deletion collection revealed that, in addition to the acid anion efflux pump Pdr12p, the vacuole, ergosterol synthesis, and redox balance all have a role in weak acid tolerance. 37. Allen J, Davey HM, Broadhurst D, Heald JK, Rowland JJ,  Oliver SG, Kell DB: High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat Biotechnol 2003, 21:692-696. www.sciencedirect.com

40. Van Dijck P, De Rop L, Szlufcik K, Van Ael E, Thevelein JM: Disruption of the Candida albicans TPS2 gene encoding trehalose-6-phosphate phosphatase decreases infectivity without affecting hypha formation. Infect Immun 2002, 70:1772-1782. 41. Alvarez-Peral FJ, Zaragoza O, Pedreno Y, Arguelles JC:  Protective role of trehalose during severe oxidative stress caused by hydrogen peroxide and the adaptive oxidative stress response in Candida albicans. Microbiology 2002, 148:2599-2606. The authors show that tolerance to hydrogen peroxide is mediated by trehalose and TPS1. Acquired tolerance by pre-incubation with low hydrogen peroxide or slightly increased temperature does not depend on trehalose, however. 42. Gonzalez-Parraga P, Hernandez JA, Arguelles JC: Role of  antioxidant enzymatic defences against oxidative stress H2O2 and the acquisition of oxidative tolerance in Candida albicans. Yeast 2003, 20:1161-1169. The authors show that acquired tolerance to hydrogen peroxide depends on the induction of the anti-oxidant enzyme catalase. In the absence of trehalose synthesis, superoxide dismutase and glutathione reductase are also induced, but this does not lead to increased stress tolerance. 43. Zaragoza O, Gonzalez-Parraga P, Pedreno Y, Alvarez-Peral FJ, Arguelles JC: Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans. Int Microbiol 2003, 6:121-125. 44. Enjalbert B, Nantel A, Whiteway M: Stress-induced gene  expression in Candida albicans: absence of a general stress response. Mol Biol Cell 2003, 14:1460-1467. Conditions that lead to the induction of the ESR in S. cerevisiae do not result in a common expression response in C. albicans. Similarly, no cross-tolerance is induced by these conditions. 45. Fillinger S, Chaveroche MK, van Dijck P, de Vries R, Ruijter G, Thevelein J, d’Enfert C: Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans. Microbiology 2001, 147:1851-1862. 46. Gonzalez-Hernandez JC, Jimenez-Estrada M, Pena A:  Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress. Extremophiles 2004 (Epub ahead of print) DOI: 10.1007/s00792-004-0415-2. D. hansenii has a high trehalose content even when not challenged with salt, much more so than S. cerevisiae. 47. Kwon HB, Yeo ET, Hahn SE, Bae SC, Kim DY, Byun MO: Cloning  and characterization of genes encoding trehalose-6phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii. FEMS Yeast Res 2003, 3:433-440. The authors clone the Z. rouxii genes for trehalose synthesis and show that they are highly expressed in the absence of salt. In fact, the genes are downregulated transiently in the presence of high salt concentrations or heat, suggesting a protective role for trehalose in initial tolerance, but not in adaptation. 48. Cheng L, Moghraby J, Piper PW: Weak organic acid treatment causes a trehalose accumulation in low-pH cultures of Saccharomyces cerevisiae, not displayed by the more preservative-resistant Zygosaccharomyces bailii. FEMS Microbiol Lett 1999, 170:89-95. 49. Schoondermark-Stolk SA, ter Schure EG, Verrips CT, Verkleij AJ,  Boonstra J: Identification of salt-induced genes of Current Opinion in Biotechnology 2005, 16:225–230

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Zygosaccharomyces rouxii by using Saccharomyces cerevisiae GeneFilters((R)). FEMS Yeast Res 2002, 2:525-532. Using the limited information that can be obtained with arrays from another organism, the authors have obtained transcription data to show that Z. rouxii is optimized for growth at high salt concentrations. 50. Chang MH, Chae KS, Han DM, Jahng KY: The GanB Ga-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans. Genetics 2004, 167:1305-1315. 51. Han KH, Seo JA, Yu JH: Regulators of G-protein signalling in  Aspergillus nidulans: RgsA downregulates stress response and stimulates asexual sporulation through attenuation of GanB (Ga) signalling. Mol Microbiol 2004, 53:529-540. The authors show the role of RgsA in stress tolerance and reveal a regulatory mechanism in A. nidulans that functions contrary to that in S. cerevisiae. They do, however, also suggest the presence of a feedback loop involving a stress response element, and it is tempting to speculate about the existence of an ESR in this fungus. 52. Chen D, Toone WM, Mata J, Lyne R, Burns G, Kivinen K, Brazma A,  Jones N, Bahler J: Global transcriptional responses of fission yeast to environmental stress. Mol Biol Cell 2003, 14:214-229. The core ESR common to many stresses in S. pombe reveals a repertoire of up- and downregulated processes similar to that in S. cerevisiae,

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but the response is coordinated by a mitogen-activated protein kinase Sty1p. 53. Nicholls S, Straffon M, Enjalbert B, Nantel A, Macaskill S,  Whiteway M, Brown AJ: Msn2- and Msn4-like transcription factors play no obvious roles in the stress responses of the fungal pathogen Candida albicans. Eukaryot Cell 2004, 3:1111-1123. In C. albicans, Msn2p and Msn4p homologs exist but they play no role in gene expression responses to various stresses. Their disruption has no effect on stress resistance or adaptation. 54. Smith DA, Nicholls S, Morgan BA, Brown AJ, Quinn J: A  conserved stress-activated protein kinase regulates a core stress response in the human pathogen Candida albicans. Mol Biol Cell 2004, 15:4179-4190. A core stress response does exist in C. albicans, but it is not activated by the same stresses as S. cerevisiae. Conditions commonly encountered by the pathogen in the human host, such as high oxygen or high temperature, do not lead to a stress response. The core stress response is coordinated by a mitogen-activated protein kinase, Hog1p, similar to S. pombe. 55. Brul S, Klis FM: Mechanistic and mathematical inactivation studies of food spoilage fungi. Fungal Genet Biol 1999, 27:199-208.

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