Curr Microbiol (2014) 69:733–739 DOI 10.1007/s00284-014-0651-3
Differential Response of Candida albicans and Candida glabrata to Oxidative and Nitrosative Stresses Mayra Cue´llar-Cruz • Everardo Lo´pez-Romero Estela Ruiz-Baca • Roberto Zazueta-Sandoval
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Received: 15 February 2014 / Accepted: 17 May 2014 / Published online: 8 July 2014 Springer Science+Business Media New York 2014
Abstract Invasive candidiasis is associated with high mortality in immunocompromised and hospitalized patients. Candida albicans is the main pathological agent followed by Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. These pathogens colonize different host tissues in humans as they are able to neutralize the reactive species generated from nitrogen and oxygen during the respiratory burst. Among the enzymatic mechanisms that Candida species have developed to protect against free radicals are enzymes with antioxidant and immunodominant functions such as flavohemoglobins, catalases, superoxide dismutases, glutathione reductases, thioredoxins, peroxidases, heat-shock proteins, and enolases. These mechanisms are under transcriptional regulation by factors such as Cta4p, Cwt1p, Yap1p, Skn7p, Msn2p, and Msn4p. However, even though it has been proposed that all Candida species have similar enzymatic systems, it has been observed that they respond differentially to various types of stress. These differential responses may explain the colonization of different organs by each species. Here, we review the enzymatic mechanisms developed by C. albicans and C. glabrata species in response to oxidative and nitrosative stresses. Lack of experimental information for other pathogenic species limits a comparative approach among different organisms.
M. Cue´llar-Cruz (&) E. Lo´pez-Romero R. Zazueta-Sandoval Departamento de Biologı´a, Divisio´n de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta S/N, C.P. 36050 Guanajuato, Mexico e-mail: [email protected]; [email protected] E. Ruiz-Baca Facultad de Ciencias Quı´micas, Universidad Jua´rez del Estado de Durango, Durango, Mexico
Introduction Candida species are opportunistic fungal pathogens that are commonly part of the microbiota in healthy individuals [4]. However, when the host immune system is compromised, these pathogens are able to migrate to the bloodstream and colonize internal organs, causing invasive candidiasis (IC) [4, 45]. Death rates associated with IC in immunosuppressed patients are high, ranging from 30 to 40 % [45]. C. albicans is the primary species identified in these infections, followed by C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis [34, 35]. These species can colonize different organs in the human body, as they are able to adapt to the host responses and evade the immune system [28]. In particular, the success of these pathogens is based on their resistance to nitrosative and oxidative stresses, and other environmental attacks [3]. During the infection process, Candida species have to cope with the reactive species of nitrogen (RNS) and oxygen (ROS) generated during the respiratory burst of phagocytic cells [26]. The most important ROS and RNS generated inside the phagolysosome are nitric oxide (NO•), peroxynitrite (ONOO-), superoxide anion radical (O-• 2 ), and hydroxyl radical (•OH). Other agents that are not considered as free radicals but are important ROS precursors are hydrogen peroxide (H2O2) and singlet oxygen (1O2) [15]. In order to protect themselves from RNS and ROS, Candida species have developed enzymatic and non-enzymatic mechanisms to neutralize free radicals. One of these enzymes is Yhb1p which is a flavohemoglobin that allows Candida to detoxify NO• [18]. In response to oxidative stress (OSR), catalases (Cats), superoxide dismutases (Sods), and peroxidases (Pxs) are the main detoxifying enzymes in these organisms [6]. These
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enzymatic mechanisms are regulated by transcription factors that activate genes in response to these stresses. Nitrosative stress response (NSR) has not been extensively studied in Candida species, as Cta4p and Cwt1p transcription factors have been identified only in C. albicans as regulating genes against this stimulus [8, 52]. Yet, in other organisms such as Saccharomyces cerevisiae, enzymatic mechanisms against this stress have been reported. For instance, the yeast catalase not only is involved in ROS detoxification but also can regulate the concentration of NO• resulting from nitrosative stress [31]. Furthermore, it has been shown that transcriptional factors such as Yap1p, Msn2, and Msn4p, which are key regulators of responses to general and oxidative stresses, also participate as activity modulators of catalase and superoxide dismutase during the yeast response to nitrosative stress [2, 21, 30, 31]. These results suggest that transcriptional factors involved in the yeast RNS may also play an important role in the response to the same stress in Candida species. In support of this notion, it has been observed that S. cerevisiae, C. albicans, and C. glabrata adapt to ROS by upregulating the orthologous genes for Yap1p, Msn2p, and Msn4p [1, 9, 27, 48, 63, 64]. Thus, a future analysis of RNS in Candida may lead to the identification of the same factors described in S. cerevisiae. Recently, other enzymes have been identified that also protect Candida against ROS such as the heat-shock proteins Ssa2, Ssb, and enolases [11]. These observations indicate that successful response of Candida species to phagocytic cells depends on complex mechanisms that allow them to survive in the human host.
Response Mechanisms to RNS by Candida Species Once Candida cells are phagocytosed, macrophages release RNS and nitrogen reactive intermediates (NRI) to attack them. NO• is generated from arginine by the enzyme nitric oxide synthase (iNOS). Subsequently, NO• reacts with O2--producing peroxynitrite anion (ONOO-), which is a strong oxidant with fungicidal activity [41]. NO• and NRI cause damage to the cell by reacting with DNA, proteins, and various classes of lipids [22]. It has also been shown that NO• regulates the catalytic activity of various enzymes by interacting with Fe–S clusters, oxidizing copper (Cu2?), heme groups, and tyrosol radicals [22]. Furthermore, this radical is reversibly bound to the Cu2? of the cytochrome c oxidase, causing a decrease of mitochondrial oxidative phosphorylation and cellular respiration [22]. Recently, it was shown that the ability of C. albicans to survive in the presence of nitrosative stress during the initial contact with the host immune system is important for
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colonization [52]. Consequently, this pathogen appears to have a robust mechanism for detoxifying RNS and repairing the damage caused by them. So it can withstand the NO• generated from nitrates and nitrites present in the diet of bacteria in the oral cavity [44], in the gut [57], or the NO• generated in the urine, skin, or stomach of the human host. A different situation occurs in non-pathogenic yeasts such as S. cerevisiae which, when exposed to endogenous NO• under hypoxia conditions, can use endogenous nitrite instead of oxygen as an electron acceptor, thereby generating NO• within the cell [60]. An enzyme used by C. albicans to withstand NO• is the Yhb1p flavohemoglobin which converts NO• to nitrate (NO3-) [18]. This enzyme belongs to the globin family of proteins, and one of its features is that it is composed of a single polypeptide with a single heme group in the N-terminal domain, and flavin adenine dinucleotide and NADPH binding sites in the C-terminal domain [46]. It has been shown that C. albicans cells lacking the YHB1 gene have defects in NO• consumption and a diminished virulence in the tail vein model of disseminated candidiasis [61]. Interestingly, another research group showed that YHB1 is not required in the transcriptional response of C. albicans to NO• and that strains lacking this gene are hypersensitive to NO• and hyperfilamentous [22]. This result is contradictory to the observation by Chiranand et al. (2008) that YHB1 transcription is induced by NO• [8]. However, even though the function of flavohemoglobin has been demonstrated in C. albicans and other fungi such as S. cerevisiae and Cryptococcus neoformans, bacteria and protozoa, so far it has not been studied in C. glabrata, C. krusei, C. parapsilosis, or C. tropicalis. So, it will be necessary to prove whether this enzyme has the same function in these species, enabling them to detoxify RNS and thus colonize different tissues in the human host.
Response Mechanisms to ROS by Candida Species ROS, unlike RNS, have been extensively studied in both pathogenic and non-pathogenic microorganisms. This interest is probably driven by the fact that ROS are not only produced within phagolysosomes, but also in normal aerobic metabolism. Specifically, the delicate equilibrium in the concentration of ROS is one of the differences which make Candida behave as a pathogen. Generally, ROS are mainly formed in the mitochondrial electron transport chain by the partial reduction of O2, by transfer of one, two, • or three electrons, generating O-• 2 , H2O2, and OH. ROS attack DNA, proteins, and lipids [36]. To mitigate and repair the damage caused by these free radicals, Candida species have developed antioxidant mechanisms like Sods, Cats, Trxs, Grxs, and Prxs [36]. Accordingly, superoxide
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dismutase is an antioxidant enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide [24]. The C. albicans genome includes a family of six SOD genes encoding superoxide dismutase [32]. Four out of these are copper–zinc (CuZn) dependent (Sod1p, Sod4p, Sod5p, and Sod6p) and two are manganese dependent (Sod2p and Sod3p) [5]. These enzymes are located in different organelles; accordingly, Sod1p and Sod3p are found in the cytoplasm, Sod2p in the mitochondria, and the rest on the cell surface. Among these, Sod1p and Sod5p have been shown to be involved in pathogenesis of C. albicans [24] and those cells lacking Sod1p are susceptible to menadione, a compound that generates O-• 2 , and are killed by macrophages in comparison with wild strains [24]. For SOD5, it was observed that the mutant sod5D/D decreases its expression in the presence of polymorphonuclear neutrophils compared with native C. albicans cells [16]. SOD5 is also upregulated under conditions of oxidative stress, as well as during the yeast to hyphae transition [32, 40]. Interestingly, the sod1D/D mutant is hypersensitive to killing by a macrophage cell line in vitro, which is not seen with the mutant sod5D/D [24, 32]. These results indicate that the contribution of Sod5p to virulence may be due to resistance to neutrophils but not to macrophages. Sod2p has been dismissed as a virulence factor. Yet, it protects the cell against superoxide produced intracellularly [23]. In one study where behavior of SODs mutants of C. albicans was analyzed in the presence of macrophages and dendritic cells in vitro, it was observed that viability of the sod4D/D mutant was lost in the presence of macrophages [17]. This indicates that Sod4p is involved in the virulence of C. albicans, as it is essential for its survival in macrophages. In contrast, the mutants sod2D/D, sod3D/D, and sod6D/D showed no change in ROS production [17], and apparently these superoxide dismutases are not involved in the protection of C. albicans against superoxide ions. Collectively, these data suggest that C. albicans has a large number of Sods because they help to neutralize the superoxide generated during the respiratory burst in several anatomical sites of the human body, thus allowing them to survive. Sequence analysis has shown that C. glabrata genome carries the genes SOD1 and SOD2, which encode for CgSod1p and CgSod2p superoxide dismutases, respectively. The Cgsod1D mutant is highly sensitive to superoxide, suggesting that CgSod1p is involved in its detoxification in C. glabrata [49]. The difference in the number of SOD genes present in C. albicans and C. glabrata suggests that each pathogen adapts to different environments to survive in the human body. Studies on superoxide dismutases involved in superoxide detoxification have not been conducted in C. krusei, C. parapsilosis, and C. tropicalis.
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When C. albicans is exposed to hydrogen peroxide, the activities of catalase and glutathione reductase are increased [19]. C. albicans has a catalase (Cta1p) that confers resistance to H2O2 and disrupting of the CTA1 gene generates a mutant strain that is sensitive to this compound and exhibits an attenuated virulence [62]. These results show that Cta1p is important for virulence of C. albicans [39]. C. albicans, like C. glabrata, has a single catalase. The analysis of the cta1D mutant of C. glabrata in both the logarithmic and stationary phases showed that the strain is sensitive to H2O2 in both phases, suggesting that Cta1p participates in the hydrolysis of this oxidizing agent [9]. Interestingly, it was found that Cta1p is not required for virulence of C. glabrata in a mouse model test, suggesting the operation in this organism of an alternative mechanism to compensate for the absence of the Cta1p in vivo, such as glutathione reductase that neutralizes H2O2 [9]. Additionally, when analyzing whether the Cta1p is involved in the detoxification of H2O2 generated from other oxidizing agents such as menadione and cumene hydroperoxide, Cta1p was found to be indispensable in the presence of menadione in the logarithmic phase, but not in the stationary phase [10]. Yet, Cta1p is not required in the presence of cumene hydroperoxide [10]. These observations indicate that C. glabrata responds differentially with respect to C. albicans in OSR and that this difference may confer advantages to each species in colonizing different organs of the human body. So far, catalases of C. krusei, C. parapsilosis, and C. tropicalis have not been described in OSR. However, there are two reports that describe the catalase sequence in C. tropicalis, and this might be useful for studying OSR in this species [38, 43]. Other enzymes with a high affinity for hydrogen peroxide and organic hydroperoxides are Pxs. Some Pxs use glutathione as reducing agent while others function coupled to the thioredoxin system. C. albicans has a peroxidase (Px1p) and two putative peroxidases (encoded by orf19.87 and orf19.85) with high homology for Px1p. However, in the presence of H2O2, only Px1p is expressed [28]. Recently, a peroxidase with the ability to reduce H2O2 and tert-butyl hydroperoxide (t-BOOH) was characterized in C. albicans [59]. Deletion of the corresponding gene generates a mutant susceptible to t-BOOH but less sensitive to H2O2, diamide, or menadione [59], suggesting that each peroxidase plays a different role in detoxification of ROS induced by different chemical compounds. Unlike C. albicans, C. glabrata has neither Px1p nor Px3p. However, sequence analysis revealed that it has CgPx2p which is 74 % identical to one of peroxidases of S. cerevisiae (ScPx2p). In addition to catalases and peroxidases, the expression of glutaredoxins has been shown to increase in response to H2O2 [12]. Grxs are cytosolic enzymes that protect and repair the thiol groups of proteins, and are responsible for
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reducing the disulfide glutathione linkages [7]. Depending on their structure and catalytic activity, these enzymes are classified as monothiolic and dithiolic, which contain one and two cysteine residues in their catalytic domain. They also differ in their mechanism of reduction. Thus, whereas monothiolic species act on an intermediate formed between glutathione and the disulfide bridge of oxidized protein [47], dithiolic enzymes catalyze the reduction in two steps: first, the cysteine located toward the N-terminal end reduces a cysteine of the dithiol protein disulfide bridge forming an intermediate between this and glutaredoxin. Next, the second cysteine attacks the intermediate releasing the oxidized Grx and the reduced protein [47]. According to the Candida Genome Database (candidagenome.org), C. albicans has four genes, namely GRX1, GRX2, GRX3, and GRX5, encoding Grxs. Chaves et al. (2007) showed that a mutant lacking the GRX2 gene presented a deficiency in the formation of hyphae and was more susceptible to killing by polymorphonuclear macrophages [6]. Also, the virulence of the grx2D strain was attenuated after systemic infection in a murine model and was susceptible to menadione and resistant to diamide [6, 7]. These observations indicate that in C. albicans, Grx2p participates in the detoxification of ROS. On the other hand, C. glabrata has three monothiol Grxs, two of which exhibit a Trx–Grx structure. In contrast, C. albicans has only one nuclear monothiol Grx with the hybrid Trx–Grx structure [20]. However, the role of the Grxs remains to be defined in C. glabrata, C. krusei, C. tropicalis and C. parapsilosis. Other enzymes involved in protection against oxidative stress are thioredoxins (Trx) which are specific NADPHdependent oxidoreductases involved in reduction of disulfide bridges [53]. In C. albicans, the location of the thioredoxin-dependent peroxidase CaTsa1p depends on growth conditions. Accordingly, an immunocytochemical study showed that this enzyme is located in the cytoplasm in the yeast morphotype while it is translocated into the nucleolus during transition to hyphae [55]. Interestingly, in mutants lacking TSA1, the concentration of H2O2 increases by an order of magnitude, suggesting that the function of CaTsa1p is not redundant with other enzymes that neutralize this peroxide. However, CaTsa1p is indispensable in the yeast to hyphae transition under stress [55]. Also, using mass spectrometry analysis, it was found that Trxs are overexpressed in other cells that form biofilms [54]. In C. glabrata, CgTsa1p is overexpressed in cells that change phenotypically from white to dark brown in the presence of CuSO4 [58]. This change may be considered as a virulence factor and that CgTsa1p may be involved in non-obvious function of colonization and virulence [50]. Although Candida species have developed efficient enzymatic mechanisms to counteract the ROS, other proteins have recently been identified that also protect these
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pathogens from a stress such as heat-shock proteins and enolases [11]. It has been shown that enolase and the heatshock proteins Hsp90 and Hsp70 in C. albicans are the major immunodominant proteins detected in patients with IC [25, 29]. Recently, it was determined that proteins induced by OSR in C. albicans are heat-shock proteins, while in C. glabrata and C. krusei these are enolases. These observations suggest that these immunomodulatory proteins are seemingly the primary ROS-protecting factors in these species [11]. In addition, other mechanisms that allow C. albicans to rapidly adapt to ROS inside the phagosome have been identified. These include changes in the expression of proteins involved in different metabolic pathways such as the downregulation of the carbon metabolism, and the upregulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, which indicates that yeast shifts to a starvation mode [14]. The differences in OSR in Candida species probably help each of these microorganisms to avoid the immediate, early immune response allowing them to colonize different tissues and survive in the human host. It is thus important to elucidate the mechanisms by which each of the Candida species are capable of withstanding the reactive species generated during the respiratory burst.
Transcription Factors Regulating the Response to Oxidative Stress Nitrosative stress response regulation in Candida species has not been extensively studied. The transcriptional response to RNS in C. albicans is dependent on the transcription factor Cta4p [8] that activates the nitrosative YHB1 stress gene induced in the presence of NO• [26]. Another transcription factor recently identified in NSR is Cwt1p. This factor was first characterized as a regulator of cell wall damage and morphogenesis in C. albicans [37]. By the combination of expression analysis and genomewide location, it was shown that Cwt1p is essential for the proper suppression of Yhb1p [52]. Several transcription factors have been described in Candida that regulate the expression of genes encoding enzymes involved in OSR. For instance, the transcription factor Yap1p controls gene activation in OSR. In C. albicans, the ortholog gene of YAP1 is CAP1 and deletion of this gene results in hypersensitivity to hydrogen peroxide, indicating that Cap1p is involved in OSR in C. albicans [1]. In C. glabrata, the ortholog gene of YAP1 responds to oxidative stress and compounds such as benomyl, diamide, and menadione. In oxidative stress tests, the mutant strain yap1D was susceptible to H2O2, indicating that Yap1p is required in C. glabrata for activating genes in OSR [9].
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Skn7p is another transcription factor involved in OSR [27] and is part of a two-component system. This two-component signal transduction system consists of a histidine kinase located in the cell membrane which senses the signal and phosphorylates a histidine residue in the second component which is the regulating response. In C. albicans, Skn7 participates in OSR since the skn7D mutant is susceptible to H2O2 [56]. As in C. albicans, the mutant skn7D of C. glabrata is sensitive to H2O2 in both the logarithmic and stationary phases [9]. Collectively, these data show that in both pathogenic species, Skn7p is required to activate transcription factor genes in OSR. Msn2p and Msn4p are transcription factors that were identified in S. cerevisiae as involved in general stress response [33]. Msn2p/Msn4p respond to many types of stress including heat stress, lack of nitrogen and glucose and oxidative, ethanol, and osmotic stresses [13, 51]. C. albicans mutants, msn4D/D and mnl1D/D (for Msn2- and Msn4-like), were constructed to evaluate the role of these transcription factors, and it was observed that their deletion had no effect on the resistance to different types of stress [42]. This suggests that Mnl1p and CaMsn4p do not participate in the stress response in C. albicans [42]. Unlike C. albicans, an evaluation of the simple mutants msn2D and msn4D of C. glabrata in the logarithmic and stationary phases demonstrated that adaptation to H2O2 in the logarithmic phase partly depends on Msn4p and Msn2p. On the other hand, in the stationary phase, simple mutants showed no susceptibility to H2O2. Yet, in the stationary phase, the double msn2Dmsn4D mutant was more susceptible to an oxidizing agent in comparison with the control strain. These data indicate that Msn2p and Msn4p are partially required in conjunction with OSR in the stationary phase [9]. Unfortunately, OSR for C. krusei, C. tropicalis, and C. parapsilosis has not been analyzed and consequently the transcription factors that regulate their response to this stress have not been identified.
Conclusions The differential response to nitrosative and oxidative stress observed for each Candida species is probably due to the fact that each pathogen confronts distinct environmental conditions characteristic of the specific tissue colonized. These responses enable them to adapt to adverse conditions and survive in the human host. Thus, understanding how each of these microorganisms responds to different types of stress should pave the way for the development of new therapeutic strategies to confront these mycoses. Acknowledgments The authors are thankful for the financial support provided by Grants: Proyecto-Institucional-UGTO-id202/2013
737 from the University of Guanajuato, Me´xico and PROMEP-UGTOPTC-328 (M Cue´llar-Cruz, respectively). Conflict of interest The authors declare that there were no conflicts of interest with any organization or entity with a financial interest or financial conflict with the material discussed in this review.
References 1. Alarco AM, Raymond M (1999) The bZip transcription factor Cap1p is involved in multidrug resistance and oxidative stress response in Candida albicans. J Bacteriol 181:700–708 2. Bhattacharjee A, Majumdar U, Maity D, Sarkar TS, Goswami AM, Sahoo R, Ghosh S (2010) Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae. Arch Biochem Biophys 496:109–116 3. Brown JP, Haynes K, Quinn J (2009) Nitrosative and oxidative stress responses in fungal pathogenicity. Curr Opin Microbiol 12:384–391 4. Calderone RA (2002) Candida and candidiasis. ASM Press, Washington, DC 5. Chauhan N, Latge JP, Calderone R (2006) Signalling and oxidant adaptation in Candida albicans and Aspergillus fumigatus. Nat Rev Microbiol 4:435–444 6. Chaves GM, Bates S, MacCallum DM, Odds FC (2007) Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model. Genet Mol Res 6:1051–1063 7. Chaves GM, da Silva WP (2012) Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide. Mem Inst Oswaldo Cruz 107:998–1005 8. Chiranand W, McLeod I, Zhou H, Lynn JJ, Vega LA, Myers H, Yates JR 3rd, Lorenz MC, Gustin MC (2008) CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans. Eukaryot Cell 7:268–278 9. Cue´llar-Cruz M, Briones-Martin-del-Campo M, Can˜as-Villamar I, Montalvo-Arredondo J, Riego-Ruiz L, Castan˜o I, De Las Pen˜as A (2008) High resistance to oxidative stress in the fungal pathogen Candida glabrata is mediated by a single catalase, Cta1p, and is controlled by the transcription factors Yap1p, Skn7p, Msn2p, and Msn4p. Eukaryot Cell 7:814–825 10. Cue´llar-Cruz M, Castan˜o I, Arroyo-Helguera O, De Las Pen˜as A (2009) Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata. Mem Inst Oswaldo Cruz 104:649–654 11. Cue´llar-Cruz M, Gutie´rrez-Sa´nchez G, Lo´pez-Romero E, RuizBaca E, Villago´mez-Castro JC, Rodrı´guez-Sifuentes L (2013) Identification of heat shock proteins in Candida albicans and enolases in Candida glabrata and Candida krusei involved in the response to oxidative stress. Cent Eur J Biol 8:337–345 12. Enjalbert B, Nantel A, Whiteway M (2003) Stress-induced gene expression in Candida albicans: absence of a general stress response. Mol Biol Cell 14:1460–1467 13. Estruch F, Carlson M (1993) Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13:3872–3881 14. Ferna´ndez-Arenas E, Cabezo´n V, Bermejo C, Arroyo J, Nombela C, Diez-Orejas R, Gil C (2007) Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction. Mol Cell Proteomics 6:460–478 15. Ferrari CK, Souto PC, Franc¸a EL, Honorio-Franc¸a AC (2011) Oxidative and nitrosative stress on phagocytes’ function: from
123
M. Cue´llar-Cruz et al.: Differential Response of Candida albicans
738
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
effective defense to immunity evasion mechanisms. Arch Immunol Ther Exp 59:441–448 Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B (2005) Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56:397–415 Frohner IE, Bourgeois C, Yatsyk K, Majer O, Kuchler K (2009) Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 71:240–252 Gardner PR, Gardner AM, Martin LA, Salzman AL (1998) Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci USA 95:10378–10383 ´ , Arguelles JC (2003) Role of Gonzalez-Parraga P, Hernandez JA antioxidant enzymatic defences against oxidative stress H(2)O(2) and the acquisition of oxidative tolerance in Candida albicans. Yeast 20:1161–1169 Herrero E (2005) Evolutionary relationships between Saccharomyces cerevisiae and other fungal species as determined from genome comparisons. Rev Iberoam Micol 22:217–222 Horan S, Bourges I, Meunier B (2006) Transcriptional response to nitrosative stress in Saccharomyces cerevisiae. Yeast 23:519–535 Hromatka BS, Noble SM, Johnson AD (2005) Transcriptional response of Candida albicans to nitric oxide and the role of the YHB1 gene in nitrosative stress and virulence. Mol Biol Cell 16:4814–4826 Hwang CS, Baek YU, Yim HS, Kang SO (2003) Protective roles of mitochondrial manganese-containing superoxide dismutase against various stresses in Candida albicans. Yeast 20:929–941 Hwang CS, Rhie GE, Oh JH, Huh WK, Yim HS, Kang SO (2002) Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence. Microbiology 148:3705–3713 Jong AY, Chen SH, Stins MF, Kim KS, Tuan TL, Huang SH (2003) Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells. J Med Microbiol 52:615–622 Kaloriti D, Tillmann A, Cook E, Jacobsen M, You T, Lenardon M, Ames L, Barahona M, Chandrasekaran K, Coghill G, Goodman D, Gow NA, Grebogi C, Ho HL, Ingram P, McDonagh A, de Moura AP, Pang W, Puttnam M, Radmaneshfar E, Romano MC, Silk D, Stark J, Stumpf M, Thiel M, Thorne T, Usher J, Yin Z, Haynes K, Brown AJ (2012) Combinatorial stresses kill pathogenic Candida species. Med Mycol 50:699–709 Krems B, Charizanis C, Entian KD (1995) Mutants of Saccharomyces cerevisiae sensitive to oxidative and osmotic stress. Curr Genet 27:427–434 Kusch H, Engelmann S, Albrecht D, Morschha¨user J, Hecker M (2007) Proteomic analysis of the oxidative stress response in Candida albicans. Proteomics 7:686–697 Laı´n A, Elguezabal N, Amutio E, Ferna´ndez de Larrinoa I, Moragues MD, Ponto´n J (2008) Use of recombinant antigens for the diagnosis of invasive candidiasis. Clin Dev Immunol. doi:10. 1155/2008/721950 Lushchak OV, Inoue Y, Lushchak VI (2010) Regulatory protein Yap1 is involved in response of yeast Saccharomyces cerevisiae to nitrosative stress. Biochemistry (Mosc) 75:629–664 Lushchak OV, Lushchak VI (2008) Catalase modifies yeast Saccharomyces cerevisiae response towards S-nitrosoglutathione-induced stress. Redox Rep 13:283–291 Martchenko M, Alarco AM, Harcus D, Whiteway M (2004) Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal-induced SOD5 gene. Mol Biol Cell 15:456–467
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33. Martı´nez-Pastor MT, Marchler G, Schu¨ller C, Marchler-Bauer A, Ruis H, Estruch F (1996) The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15:2227–2235 34. Miceli MH, Diaz JA, Lee SA (2011) Emerging opportunistic yeast infections. Lancet Infect Dis 11:142–151 35. Miramo´n P, Kasper L, Hube B (2013) Thriving within the host: Candida spp. interactions with phagocytic cells. Med Microbiol Immunol. doi:10.1007/s00430-013-0288-z 36. Moradas-Ferreira P, Costa V (2000) Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death. Redox Rep 5:277–285 37. Moreno I, Martinez-Esparza M, Laforet LC, Sentandreu R, Ernst JF, Valentin E (2010) Dosage-dependent roles of the Cwt1 transcription factor for cell wall architecture, morphogenesis, drug sensitivity and virulence in Candida albicans. Yeast 27:77–87 38. Murray WW, Rachubinski RA (1989) Nucleotide sequence of peroxisomal catalase from the yeast Candida tropicalis pK233: identification of an upstream BamHlI site polymorphism. Nucleic Acids Res 17:3600 39. Nakagawa Y, Kanbe T, Mizuguchi I (2003) Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents. Microbiol Immunol 47:395–403 40. Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, Bouin AP, Sensen CW, Hogues H, van het Hoog M, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M (2002) Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13:3452–3465 41. Nathan C, Shiloh MU (2000) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA 97:8841–8848 42. Nicholls S, Straffon M, Enjalbert B, Nantel A, Macaskill S, Whiteway M, Brown AJ (2004) Msn2- and Msn4-like transcription factors play no obvious roles in the stress responses of the fungal pathogen Candida albicans. Eukaryot Cell 3:1111–1123 43. Okada H, Ueda M, Sugaya T, Atomi H, Mozaffar S, Hishida T, Teranishi Y, Okazaki K, Takechi T, Kamiryo T, Tanaka A (1987) Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources. Eur J Biochem 170:105–110 44. Palmerini CA, Palombari R, Perito S, Arienti G (2003) NO synthesis in human saliva. Free Radic Res 37:29–31 45. Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163 46. Poole RK, Hughes MN (2000) New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol Microbiol 36:775–783 47. Rodrı´guez-Manzaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E (1999) Grx5 Glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Mol Cell Biol 19:8180–8190 48. Roetzer A, Gregori C, Jennings AM, Quintin J, Ferrandon D, Butler G, Kuchler K, Ammerer G, Schu¨ller C (2008) Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol Microbiol 69:603–620 49. Roetzer A, Klopf E, Gratz N, Marcet-Houben M, Hiller E, Rupp S, Gabaldo´n T, Kovarik P, Schu¨ller C (2011) Regulation of Candida glabrata oxidative stress resistance is adapted to host environment. FEBS Lett 585:319–327
M. Cue´llar-Cruz et al.: Differential Response of Candida albicans 50. Rogers PD, Vermitsky JP, Edlind TD, Hilliard GM (2006) Proteomic analysis of experimentally induced azole resistance in Candida glabrata. J Antimicrob Chemother 58:434–438 51. Schu¨ller C, Brewster JL, Alexander MR, Gustin MC, Ruis H (1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene. EMBO J 13:4382–4389 52. Sellam A, Tebbji F, Whiteway M, Nantel A (2012) A novel role for the transcription factor Cwt1p as a negative regulator of nitrosative stress in Candida albicans. PLoS ONE 7:e43956 53. Sen CK (1998) Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem Pharmacol 55:1747–1758 54. Seneviratne CJ, Wang Y, Jin L, Abiko Y, Samaranayake LP (2008) Candida albicans biofilm formation is associated with increased anti-oxidative capacities. Proteomics 8:2936–2947 55. Shin DH, Jung S, Park SJ, Kim YJ, Ahn JM, Kim W, Choi W (2005) Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans. Yeast 22:907–918 56. Singh P, Chauhan N, Ghosh A, Dixon F, Calderone R (2004) SKN7 of Candida albicans: mutant construction and phenotype analysis. Infect Immun 72:2390–2394 57. Sobko T, Reinders CI, Jansson E, Norin E, Midtvedt T, Lundberg JO (2005) Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide 13:272–278
739 58. Srikantha T, Zhao R, Daniels K, Radke J, Soll DR (2005) Phenotypic switching in Candida glabrata accompanied by changes in expression of genes with deduced functions in copper detoxification and stress. Eukaryot Cell 4:1434–1445 59. Srinivasa K, Kim NR, Kim J, Kim M, Bae JY, Jeong W, Kim W, Choi W (2012) Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans. Mol Cells 33:301–307 60. Tillmann A, Gow NAR, Brown AJP (2011) Nitric oxide and nitrosative stress tolerance in yeast. Biochem Soc Trans 39:219–223 61. Ullmann BD, Myers H, Chiranand W, Lazzell AL, Zhao Q, Vega LA, Lopez-Ribot JL, Gardner PR, Gustin MC (2004) Inducible defense mechanism against nitric oxide in Candida albicans. Eukaryot Cell 3:715–723 62. Wysong DR, Christin L, Sugar AM, Robbins PW, Diamond RD (1998) Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene. Infect Immun 66:1953–1961 63. Zhang X, De Micheli M, Coleman ST, Sanglard D, MoyeRowley WS (2000) Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p. Mol Microbiol 36:618–629 64. Znaidi S, Barker KS, Weber S, Alarco AM, Liu TT, Boucher G, Rogers PD, Raymond M (2009) Identification of the Candida albicans Cap1p regulon. Eukaryot Cell 8:806–820
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Differential Response of Candida albicans and Candida glabrata to Oxidative and Nitrosative Stresses Mayra Cue´llar-Cruz • Everardo Lo´pez-Romero Estela Ruiz-Baca • Roberto Zazueta-Sandoval
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Received: 15 February 2014 / Accepted: 17 May 2014 / Published online: 8 July 2014 Springer Science+Business Media New York 2014
Abstract Invasive candidiasis is associated with high mortality in immunocompromised and hospitalized patients. Candida albicans is the main pathological agent followed by Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. These pathogens colonize different host tissues in humans as they are able to neutralize the reactive species generated from nitrogen and oxygen during the respiratory burst. Among the enzymatic mechanisms that Candida species have developed to protect against free radicals are enzymes with antioxidant and immunodominant functions such as flavohemoglobins, catalases, superoxide dismutases, glutathione reductases, thioredoxins, peroxidases, heat-shock proteins, and enolases. These mechanisms are under transcriptional regulation by factors such as Cta4p, Cwt1p, Yap1p, Skn7p, Msn2p, and Msn4p. However, even though it has been proposed that all Candida species have similar enzymatic systems, it has been observed that they respond differentially to various types of stress. These differential responses may explain the colonization of different organs by each species. Here, we review the enzymatic mechanisms developed by C. albicans and C. glabrata species in response to oxidative and nitrosative stresses. Lack of experimental information for other pathogenic species limits a comparative approach among different organisms.
M. Cue´llar-Cruz (&) E. Lo´pez-Romero R. Zazueta-Sandoval Departamento de Biologı´a, Divisio´n de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta S/N, C.P. 36050 Guanajuato, Mexico e-mail: [email protected]; [email protected] E. Ruiz-Baca Facultad de Ciencias Quı´micas, Universidad Jua´rez del Estado de Durango, Durango, Mexico
Introduction Candida species are opportunistic fungal pathogens that are commonly part of the microbiota in healthy individuals [4]. However, when the host immune system is compromised, these pathogens are able to migrate to the bloodstream and colonize internal organs, causing invasive candidiasis (IC) [4, 45]. Death rates associated with IC in immunosuppressed patients are high, ranging from 30 to 40 % [45]. C. albicans is the primary species identified in these infections, followed by C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis [34, 35]. These species can colonize different organs in the human body, as they are able to adapt to the host responses and evade the immune system [28]. In particular, the success of these pathogens is based on their resistance to nitrosative and oxidative stresses, and other environmental attacks [3]. During the infection process, Candida species have to cope with the reactive species of nitrogen (RNS) and oxygen (ROS) generated during the respiratory burst of phagocytic cells [26]. The most important ROS and RNS generated inside the phagolysosome are nitric oxide (NO•), peroxynitrite (ONOO-), superoxide anion radical (O-• 2 ), and hydroxyl radical (•OH). Other agents that are not considered as free radicals but are important ROS precursors are hydrogen peroxide (H2O2) and singlet oxygen (1O2) [15]. In order to protect themselves from RNS and ROS, Candida species have developed enzymatic and non-enzymatic mechanisms to neutralize free radicals. One of these enzymes is Yhb1p which is a flavohemoglobin that allows Candida to detoxify NO• [18]. In response to oxidative stress (OSR), catalases (Cats), superoxide dismutases (Sods), and peroxidases (Pxs) are the main detoxifying enzymes in these organisms [6]. These
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enzymatic mechanisms are regulated by transcription factors that activate genes in response to these stresses. Nitrosative stress response (NSR) has not been extensively studied in Candida species, as Cta4p and Cwt1p transcription factors have been identified only in C. albicans as regulating genes against this stimulus [8, 52]. Yet, in other organisms such as Saccharomyces cerevisiae, enzymatic mechanisms against this stress have been reported. For instance, the yeast catalase not only is involved in ROS detoxification but also can regulate the concentration of NO• resulting from nitrosative stress [31]. Furthermore, it has been shown that transcriptional factors such as Yap1p, Msn2, and Msn4p, which are key regulators of responses to general and oxidative stresses, also participate as activity modulators of catalase and superoxide dismutase during the yeast response to nitrosative stress [2, 21, 30, 31]. These results suggest that transcriptional factors involved in the yeast RNS may also play an important role in the response to the same stress in Candida species. In support of this notion, it has been observed that S. cerevisiae, C. albicans, and C. glabrata adapt to ROS by upregulating the orthologous genes for Yap1p, Msn2p, and Msn4p [1, 9, 27, 48, 63, 64]. Thus, a future analysis of RNS in Candida may lead to the identification of the same factors described in S. cerevisiae. Recently, other enzymes have been identified that also protect Candida against ROS such as the heat-shock proteins Ssa2, Ssb, and enolases [11]. These observations indicate that successful response of Candida species to phagocytic cells depends on complex mechanisms that allow them to survive in the human host.
Response Mechanisms to RNS by Candida Species Once Candida cells are phagocytosed, macrophages release RNS and nitrogen reactive intermediates (NRI) to attack them. NO• is generated from arginine by the enzyme nitric oxide synthase (iNOS). Subsequently, NO• reacts with O2--producing peroxynitrite anion (ONOO-), which is a strong oxidant with fungicidal activity [41]. NO• and NRI cause damage to the cell by reacting with DNA, proteins, and various classes of lipids [22]. It has also been shown that NO• regulates the catalytic activity of various enzymes by interacting with Fe–S clusters, oxidizing copper (Cu2?), heme groups, and tyrosol radicals [22]. Furthermore, this radical is reversibly bound to the Cu2? of the cytochrome c oxidase, causing a decrease of mitochondrial oxidative phosphorylation and cellular respiration [22]. Recently, it was shown that the ability of C. albicans to survive in the presence of nitrosative stress during the initial contact with the host immune system is important for
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colonization [52]. Consequently, this pathogen appears to have a robust mechanism for detoxifying RNS and repairing the damage caused by them. So it can withstand the NO• generated from nitrates and nitrites present in the diet of bacteria in the oral cavity [44], in the gut [57], or the NO• generated in the urine, skin, or stomach of the human host. A different situation occurs in non-pathogenic yeasts such as S. cerevisiae which, when exposed to endogenous NO• under hypoxia conditions, can use endogenous nitrite instead of oxygen as an electron acceptor, thereby generating NO• within the cell [60]. An enzyme used by C. albicans to withstand NO• is the Yhb1p flavohemoglobin which converts NO• to nitrate (NO3-) [18]. This enzyme belongs to the globin family of proteins, and one of its features is that it is composed of a single polypeptide with a single heme group in the N-terminal domain, and flavin adenine dinucleotide and NADPH binding sites in the C-terminal domain [46]. It has been shown that C. albicans cells lacking the YHB1 gene have defects in NO• consumption and a diminished virulence in the tail vein model of disseminated candidiasis [61]. Interestingly, another research group showed that YHB1 is not required in the transcriptional response of C. albicans to NO• and that strains lacking this gene are hypersensitive to NO• and hyperfilamentous [22]. This result is contradictory to the observation by Chiranand et al. (2008) that YHB1 transcription is induced by NO• [8]. However, even though the function of flavohemoglobin has been demonstrated in C. albicans and other fungi such as S. cerevisiae and Cryptococcus neoformans, bacteria and protozoa, so far it has not been studied in C. glabrata, C. krusei, C. parapsilosis, or C. tropicalis. So, it will be necessary to prove whether this enzyme has the same function in these species, enabling them to detoxify RNS and thus colonize different tissues in the human host.
Response Mechanisms to ROS by Candida Species ROS, unlike RNS, have been extensively studied in both pathogenic and non-pathogenic microorganisms. This interest is probably driven by the fact that ROS are not only produced within phagolysosomes, but also in normal aerobic metabolism. Specifically, the delicate equilibrium in the concentration of ROS is one of the differences which make Candida behave as a pathogen. Generally, ROS are mainly formed in the mitochondrial electron transport chain by the partial reduction of O2, by transfer of one, two, • or three electrons, generating O-• 2 , H2O2, and OH. ROS attack DNA, proteins, and lipids [36]. To mitigate and repair the damage caused by these free radicals, Candida species have developed antioxidant mechanisms like Sods, Cats, Trxs, Grxs, and Prxs [36]. Accordingly, superoxide
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dismutase is an antioxidant enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide [24]. The C. albicans genome includes a family of six SOD genes encoding superoxide dismutase [32]. Four out of these are copper–zinc (CuZn) dependent (Sod1p, Sod4p, Sod5p, and Sod6p) and two are manganese dependent (Sod2p and Sod3p) [5]. These enzymes are located in different organelles; accordingly, Sod1p and Sod3p are found in the cytoplasm, Sod2p in the mitochondria, and the rest on the cell surface. Among these, Sod1p and Sod5p have been shown to be involved in pathogenesis of C. albicans [24] and those cells lacking Sod1p are susceptible to menadione, a compound that generates O-• 2 , and are killed by macrophages in comparison with wild strains [24]. For SOD5, it was observed that the mutant sod5D/D decreases its expression in the presence of polymorphonuclear neutrophils compared with native C. albicans cells [16]. SOD5 is also upregulated under conditions of oxidative stress, as well as during the yeast to hyphae transition [32, 40]. Interestingly, the sod1D/D mutant is hypersensitive to killing by a macrophage cell line in vitro, which is not seen with the mutant sod5D/D [24, 32]. These results indicate that the contribution of Sod5p to virulence may be due to resistance to neutrophils but not to macrophages. Sod2p has been dismissed as a virulence factor. Yet, it protects the cell against superoxide produced intracellularly [23]. In one study where behavior of SODs mutants of C. albicans was analyzed in the presence of macrophages and dendritic cells in vitro, it was observed that viability of the sod4D/D mutant was lost in the presence of macrophages [17]. This indicates that Sod4p is involved in the virulence of C. albicans, as it is essential for its survival in macrophages. In contrast, the mutants sod2D/D, sod3D/D, and sod6D/D showed no change in ROS production [17], and apparently these superoxide dismutases are not involved in the protection of C. albicans against superoxide ions. Collectively, these data suggest that C. albicans has a large number of Sods because they help to neutralize the superoxide generated during the respiratory burst in several anatomical sites of the human body, thus allowing them to survive. Sequence analysis has shown that C. glabrata genome carries the genes SOD1 and SOD2, which encode for CgSod1p and CgSod2p superoxide dismutases, respectively. The Cgsod1D mutant is highly sensitive to superoxide, suggesting that CgSod1p is involved in its detoxification in C. glabrata [49]. The difference in the number of SOD genes present in C. albicans and C. glabrata suggests that each pathogen adapts to different environments to survive in the human body. Studies on superoxide dismutases involved in superoxide detoxification have not been conducted in C. krusei, C. parapsilosis, and C. tropicalis.
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When C. albicans is exposed to hydrogen peroxide, the activities of catalase and glutathione reductase are increased [19]. C. albicans has a catalase (Cta1p) that confers resistance to H2O2 and disrupting of the CTA1 gene generates a mutant strain that is sensitive to this compound and exhibits an attenuated virulence [62]. These results show that Cta1p is important for virulence of C. albicans [39]. C. albicans, like C. glabrata, has a single catalase. The analysis of the cta1D mutant of C. glabrata in both the logarithmic and stationary phases showed that the strain is sensitive to H2O2 in both phases, suggesting that Cta1p participates in the hydrolysis of this oxidizing agent [9]. Interestingly, it was found that Cta1p is not required for virulence of C. glabrata in a mouse model test, suggesting the operation in this organism of an alternative mechanism to compensate for the absence of the Cta1p in vivo, such as glutathione reductase that neutralizes H2O2 [9]. Additionally, when analyzing whether the Cta1p is involved in the detoxification of H2O2 generated from other oxidizing agents such as menadione and cumene hydroperoxide, Cta1p was found to be indispensable in the presence of menadione in the logarithmic phase, but not in the stationary phase [10]. Yet, Cta1p is not required in the presence of cumene hydroperoxide [10]. These observations indicate that C. glabrata responds differentially with respect to C. albicans in OSR and that this difference may confer advantages to each species in colonizing different organs of the human body. So far, catalases of C. krusei, C. parapsilosis, and C. tropicalis have not been described in OSR. However, there are two reports that describe the catalase sequence in C. tropicalis, and this might be useful for studying OSR in this species [38, 43]. Other enzymes with a high affinity for hydrogen peroxide and organic hydroperoxides are Pxs. Some Pxs use glutathione as reducing agent while others function coupled to the thioredoxin system. C. albicans has a peroxidase (Px1p) and two putative peroxidases (encoded by orf19.87 and orf19.85) with high homology for Px1p. However, in the presence of H2O2, only Px1p is expressed [28]. Recently, a peroxidase with the ability to reduce H2O2 and tert-butyl hydroperoxide (t-BOOH) was characterized in C. albicans [59]. Deletion of the corresponding gene generates a mutant susceptible to t-BOOH but less sensitive to H2O2, diamide, or menadione [59], suggesting that each peroxidase plays a different role in detoxification of ROS induced by different chemical compounds. Unlike C. albicans, C. glabrata has neither Px1p nor Px3p. However, sequence analysis revealed that it has CgPx2p which is 74 % identical to one of peroxidases of S. cerevisiae (ScPx2p). In addition to catalases and peroxidases, the expression of glutaredoxins has been shown to increase in response to H2O2 [12]. Grxs are cytosolic enzymes that protect and repair the thiol groups of proteins, and are responsible for
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reducing the disulfide glutathione linkages [7]. Depending on their structure and catalytic activity, these enzymes are classified as monothiolic and dithiolic, which contain one and two cysteine residues in their catalytic domain. They also differ in their mechanism of reduction. Thus, whereas monothiolic species act on an intermediate formed between glutathione and the disulfide bridge of oxidized protein [47], dithiolic enzymes catalyze the reduction in two steps: first, the cysteine located toward the N-terminal end reduces a cysteine of the dithiol protein disulfide bridge forming an intermediate between this and glutaredoxin. Next, the second cysteine attacks the intermediate releasing the oxidized Grx and the reduced protein [47]. According to the Candida Genome Database (candidagenome.org), C. albicans has four genes, namely GRX1, GRX2, GRX3, and GRX5, encoding Grxs. Chaves et al. (2007) showed that a mutant lacking the GRX2 gene presented a deficiency in the formation of hyphae and was more susceptible to killing by polymorphonuclear macrophages [6]. Also, the virulence of the grx2D strain was attenuated after systemic infection in a murine model and was susceptible to menadione and resistant to diamide [6, 7]. These observations indicate that in C. albicans, Grx2p participates in the detoxification of ROS. On the other hand, C. glabrata has three monothiol Grxs, two of which exhibit a Trx–Grx structure. In contrast, C. albicans has only one nuclear monothiol Grx with the hybrid Trx–Grx structure [20]. However, the role of the Grxs remains to be defined in C. glabrata, C. krusei, C. tropicalis and C. parapsilosis. Other enzymes involved in protection against oxidative stress are thioredoxins (Trx) which are specific NADPHdependent oxidoreductases involved in reduction of disulfide bridges [53]. In C. albicans, the location of the thioredoxin-dependent peroxidase CaTsa1p depends on growth conditions. Accordingly, an immunocytochemical study showed that this enzyme is located in the cytoplasm in the yeast morphotype while it is translocated into the nucleolus during transition to hyphae [55]. Interestingly, in mutants lacking TSA1, the concentration of H2O2 increases by an order of magnitude, suggesting that the function of CaTsa1p is not redundant with other enzymes that neutralize this peroxide. However, CaTsa1p is indispensable in the yeast to hyphae transition under stress [55]. Also, using mass spectrometry analysis, it was found that Trxs are overexpressed in other cells that form biofilms [54]. In C. glabrata, CgTsa1p is overexpressed in cells that change phenotypically from white to dark brown in the presence of CuSO4 [58]. This change may be considered as a virulence factor and that CgTsa1p may be involved in non-obvious function of colonization and virulence [50]. Although Candida species have developed efficient enzymatic mechanisms to counteract the ROS, other proteins have recently been identified that also protect these
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pathogens from a stress such as heat-shock proteins and enolases [11]. It has been shown that enolase and the heatshock proteins Hsp90 and Hsp70 in C. albicans are the major immunodominant proteins detected in patients with IC [25, 29]. Recently, it was determined that proteins induced by OSR in C. albicans are heat-shock proteins, while in C. glabrata and C. krusei these are enolases. These observations suggest that these immunomodulatory proteins are seemingly the primary ROS-protecting factors in these species [11]. In addition, other mechanisms that allow C. albicans to rapidly adapt to ROS inside the phagosome have been identified. These include changes in the expression of proteins involved in different metabolic pathways such as the downregulation of the carbon metabolism, and the upregulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, which indicates that yeast shifts to a starvation mode [14]. The differences in OSR in Candida species probably help each of these microorganisms to avoid the immediate, early immune response allowing them to colonize different tissues and survive in the human host. It is thus important to elucidate the mechanisms by which each of the Candida species are capable of withstanding the reactive species generated during the respiratory burst.
Transcription Factors Regulating the Response to Oxidative Stress Nitrosative stress response regulation in Candida species has not been extensively studied. The transcriptional response to RNS in C. albicans is dependent on the transcription factor Cta4p [8] that activates the nitrosative YHB1 stress gene induced in the presence of NO• [26]. Another transcription factor recently identified in NSR is Cwt1p. This factor was first characterized as a regulator of cell wall damage and morphogenesis in C. albicans [37]. By the combination of expression analysis and genomewide location, it was shown that Cwt1p is essential for the proper suppression of Yhb1p [52]. Several transcription factors have been described in Candida that regulate the expression of genes encoding enzymes involved in OSR. For instance, the transcription factor Yap1p controls gene activation in OSR. In C. albicans, the ortholog gene of YAP1 is CAP1 and deletion of this gene results in hypersensitivity to hydrogen peroxide, indicating that Cap1p is involved in OSR in C. albicans [1]. In C. glabrata, the ortholog gene of YAP1 responds to oxidative stress and compounds such as benomyl, diamide, and menadione. In oxidative stress tests, the mutant strain yap1D was susceptible to H2O2, indicating that Yap1p is required in C. glabrata for activating genes in OSR [9].
M. Cue´llar-Cruz et al.: Differential Response of Candida albicans
Skn7p is another transcription factor involved in OSR [27] and is part of a two-component system. This two-component signal transduction system consists of a histidine kinase located in the cell membrane which senses the signal and phosphorylates a histidine residue in the second component which is the regulating response. In C. albicans, Skn7 participates in OSR since the skn7D mutant is susceptible to H2O2 [56]. As in C. albicans, the mutant skn7D of C. glabrata is sensitive to H2O2 in both the logarithmic and stationary phases [9]. Collectively, these data show that in both pathogenic species, Skn7p is required to activate transcription factor genes in OSR. Msn2p and Msn4p are transcription factors that were identified in S. cerevisiae as involved in general stress response [33]. Msn2p/Msn4p respond to many types of stress including heat stress, lack of nitrogen and glucose and oxidative, ethanol, and osmotic stresses [13, 51]. C. albicans mutants, msn4D/D and mnl1D/D (for Msn2- and Msn4-like), were constructed to evaluate the role of these transcription factors, and it was observed that their deletion had no effect on the resistance to different types of stress [42]. This suggests that Mnl1p and CaMsn4p do not participate in the stress response in C. albicans [42]. Unlike C. albicans, an evaluation of the simple mutants msn2D and msn4D of C. glabrata in the logarithmic and stationary phases demonstrated that adaptation to H2O2 in the logarithmic phase partly depends on Msn4p and Msn2p. On the other hand, in the stationary phase, simple mutants showed no susceptibility to H2O2. Yet, in the stationary phase, the double msn2Dmsn4D mutant was more susceptible to an oxidizing agent in comparison with the control strain. These data indicate that Msn2p and Msn4p are partially required in conjunction with OSR in the stationary phase [9]. Unfortunately, OSR for C. krusei, C. tropicalis, and C. parapsilosis has not been analyzed and consequently the transcription factors that regulate their response to this stress have not been identified.
Conclusions The differential response to nitrosative and oxidative stress observed for each Candida species is probably due to the fact that each pathogen confronts distinct environmental conditions characteristic of the specific tissue colonized. These responses enable them to adapt to adverse conditions and survive in the human host. Thus, understanding how each of these microorganisms responds to different types of stress should pave the way for the development of new therapeutic strategies to confront these mycoses. Acknowledgments The authors are thankful for the financial support provided by Grants: Proyecto-Institucional-UGTO-id202/2013
737 from the University of Guanajuato, Me´xico and PROMEP-UGTOPTC-328 (M Cue´llar-Cruz, respectively). Conflict of interest The authors declare that there were no conflicts of interest with any organization or entity with a financial interest or financial conflict with the material discussed in this review.
References 1. Alarco AM, Raymond M (1999) The bZip transcription factor Cap1p is involved in multidrug resistance and oxidative stress response in Candida albicans. J Bacteriol 181:700–708 2. Bhattacharjee A, Majumdar U, Maity D, Sarkar TS, Goswami AM, Sahoo R, Ghosh S (2010) Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae. Arch Biochem Biophys 496:109–116 3. Brown JP, Haynes K, Quinn J (2009) Nitrosative and oxidative stress responses in fungal pathogenicity. Curr Opin Microbiol 12:384–391 4. Calderone RA (2002) Candida and candidiasis. ASM Press, Washington, DC 5. Chauhan N, Latge JP, Calderone R (2006) Signalling and oxidant adaptation in Candida albicans and Aspergillus fumigatus. Nat Rev Microbiol 4:435–444 6. Chaves GM, Bates S, MacCallum DM, Odds FC (2007) Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model. Genet Mol Res 6:1051–1063 7. Chaves GM, da Silva WP (2012) Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide. Mem Inst Oswaldo Cruz 107:998–1005 8. Chiranand W, McLeod I, Zhou H, Lynn JJ, Vega LA, Myers H, Yates JR 3rd, Lorenz MC, Gustin MC (2008) CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans. Eukaryot Cell 7:268–278 9. Cue´llar-Cruz M, Briones-Martin-del-Campo M, Can˜as-Villamar I, Montalvo-Arredondo J, Riego-Ruiz L, Castan˜o I, De Las Pen˜as A (2008) High resistance to oxidative stress in the fungal pathogen Candida glabrata is mediated by a single catalase, Cta1p, and is controlled by the transcription factors Yap1p, Skn7p, Msn2p, and Msn4p. Eukaryot Cell 7:814–825 10. Cue´llar-Cruz M, Castan˜o I, Arroyo-Helguera O, De Las Pen˜as A (2009) Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata. Mem Inst Oswaldo Cruz 104:649–654 11. Cue´llar-Cruz M, Gutie´rrez-Sa´nchez G, Lo´pez-Romero E, RuizBaca E, Villago´mez-Castro JC, Rodrı´guez-Sifuentes L (2013) Identification of heat shock proteins in Candida albicans and enolases in Candida glabrata and Candida krusei involved in the response to oxidative stress. Cent Eur J Biol 8:337–345 12. Enjalbert B, Nantel A, Whiteway M (2003) Stress-induced gene expression in Candida albicans: absence of a general stress response. Mol Biol Cell 14:1460–1467 13. Estruch F, Carlson M (1993) Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13:3872–3881 14. Ferna´ndez-Arenas E, Cabezo´n V, Bermejo C, Arroyo J, Nombela C, Diez-Orejas R, Gil C (2007) Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction. Mol Cell Proteomics 6:460–478 15. Ferrari CK, Souto PC, Franc¸a EL, Honorio-Franc¸a AC (2011) Oxidative and nitrosative stress on phagocytes’ function: from
123
M. Cue´llar-Cruz et al.: Differential Response of Candida albicans
738
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
effective defense to immunity evasion mechanisms. Arch Immunol Ther Exp 59:441–448 Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B (2005) Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56:397–415 Frohner IE, Bourgeois C, Yatsyk K, Majer O, Kuchler K (2009) Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 71:240–252 Gardner PR, Gardner AM, Martin LA, Salzman AL (1998) Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci USA 95:10378–10383 ´ , Arguelles JC (2003) Role of Gonzalez-Parraga P, Hernandez JA antioxidant enzymatic defences against oxidative stress H(2)O(2) and the acquisition of oxidative tolerance in Candida albicans. Yeast 20:1161–1169 Herrero E (2005) Evolutionary relationships between Saccharomyces cerevisiae and other fungal species as determined from genome comparisons. Rev Iberoam Micol 22:217–222 Horan S, Bourges I, Meunier B (2006) Transcriptional response to nitrosative stress in Saccharomyces cerevisiae. Yeast 23:519–535 Hromatka BS, Noble SM, Johnson AD (2005) Transcriptional response of Candida albicans to nitric oxide and the role of the YHB1 gene in nitrosative stress and virulence. Mol Biol Cell 16:4814–4826 Hwang CS, Baek YU, Yim HS, Kang SO (2003) Protective roles of mitochondrial manganese-containing superoxide dismutase against various stresses in Candida albicans. Yeast 20:929–941 Hwang CS, Rhie GE, Oh JH, Huh WK, Yim HS, Kang SO (2002) Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence. Microbiology 148:3705–3713 Jong AY, Chen SH, Stins MF, Kim KS, Tuan TL, Huang SH (2003) Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells. J Med Microbiol 52:615–622 Kaloriti D, Tillmann A, Cook E, Jacobsen M, You T, Lenardon M, Ames L, Barahona M, Chandrasekaran K, Coghill G, Goodman D, Gow NA, Grebogi C, Ho HL, Ingram P, McDonagh A, de Moura AP, Pang W, Puttnam M, Radmaneshfar E, Romano MC, Silk D, Stark J, Stumpf M, Thiel M, Thorne T, Usher J, Yin Z, Haynes K, Brown AJ (2012) Combinatorial stresses kill pathogenic Candida species. Med Mycol 50:699–709 Krems B, Charizanis C, Entian KD (1995) Mutants of Saccharomyces cerevisiae sensitive to oxidative and osmotic stress. Curr Genet 27:427–434 Kusch H, Engelmann S, Albrecht D, Morschha¨user J, Hecker M (2007) Proteomic analysis of the oxidative stress response in Candida albicans. Proteomics 7:686–697 Laı´n A, Elguezabal N, Amutio E, Ferna´ndez de Larrinoa I, Moragues MD, Ponto´n J (2008) Use of recombinant antigens for the diagnosis of invasive candidiasis. Clin Dev Immunol. doi:10. 1155/2008/721950 Lushchak OV, Inoue Y, Lushchak VI (2010) Regulatory protein Yap1 is involved in response of yeast Saccharomyces cerevisiae to nitrosative stress. Biochemistry (Mosc) 75:629–664 Lushchak OV, Lushchak VI (2008) Catalase modifies yeast Saccharomyces cerevisiae response towards S-nitrosoglutathione-induced stress. Redox Rep 13:283–291 Martchenko M, Alarco AM, Harcus D, Whiteway M (2004) Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal-induced SOD5 gene. Mol Biol Cell 15:456–467
123
33. Martı´nez-Pastor MT, Marchler G, Schu¨ller C, Marchler-Bauer A, Ruis H, Estruch F (1996) The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15:2227–2235 34. Miceli MH, Diaz JA, Lee SA (2011) Emerging opportunistic yeast infections. Lancet Infect Dis 11:142–151 35. Miramo´n P, Kasper L, Hube B (2013) Thriving within the host: Candida spp. interactions with phagocytic cells. Med Microbiol Immunol. doi:10.1007/s00430-013-0288-z 36. Moradas-Ferreira P, Costa V (2000) Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death. Redox Rep 5:277–285 37. Moreno I, Martinez-Esparza M, Laforet LC, Sentandreu R, Ernst JF, Valentin E (2010) Dosage-dependent roles of the Cwt1 transcription factor for cell wall architecture, morphogenesis, drug sensitivity and virulence in Candida albicans. Yeast 27:77–87 38. Murray WW, Rachubinski RA (1989) Nucleotide sequence of peroxisomal catalase from the yeast Candida tropicalis pK233: identification of an upstream BamHlI site polymorphism. Nucleic Acids Res 17:3600 39. Nakagawa Y, Kanbe T, Mizuguchi I (2003) Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents. Microbiol Immunol 47:395–403 40. Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, Bouin AP, Sensen CW, Hogues H, van het Hoog M, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M (2002) Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13:3452–3465 41. Nathan C, Shiloh MU (2000) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA 97:8841–8848 42. Nicholls S, Straffon M, Enjalbert B, Nantel A, Macaskill S, Whiteway M, Brown AJ (2004) Msn2- and Msn4-like transcription factors play no obvious roles in the stress responses of the fungal pathogen Candida albicans. Eukaryot Cell 3:1111–1123 43. Okada H, Ueda M, Sugaya T, Atomi H, Mozaffar S, Hishida T, Teranishi Y, Okazaki K, Takechi T, Kamiryo T, Tanaka A (1987) Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources. Eur J Biochem 170:105–110 44. Palmerini CA, Palombari R, Perito S, Arienti G (2003) NO synthesis in human saliva. Free Radic Res 37:29–31 45. Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163 46. Poole RK, Hughes MN (2000) New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol Microbiol 36:775–783 47. Rodrı´guez-Manzaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E (1999) Grx5 Glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Mol Cell Biol 19:8180–8190 48. Roetzer A, Gregori C, Jennings AM, Quintin J, Ferrandon D, Butler G, Kuchler K, Ammerer G, Schu¨ller C (2008) Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol Microbiol 69:603–620 49. Roetzer A, Klopf E, Gratz N, Marcet-Houben M, Hiller E, Rupp S, Gabaldo´n T, Kovarik P, Schu¨ller C (2011) Regulation of Candida glabrata oxidative stress resistance is adapted to host environment. FEBS Lett 585:319–327
M. Cue´llar-Cruz et al.: Differential Response of Candida albicans 50. Rogers PD, Vermitsky JP, Edlind TD, Hilliard GM (2006) Proteomic analysis of experimentally induced azole resistance in Candida glabrata. J Antimicrob Chemother 58:434–438 51. Schu¨ller C, Brewster JL, Alexander MR, Gustin MC, Ruis H (1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene. EMBO J 13:4382–4389 52. Sellam A, Tebbji F, Whiteway M, Nantel A (2012) A novel role for the transcription factor Cwt1p as a negative regulator of nitrosative stress in Candida albicans. PLoS ONE 7:e43956 53. Sen CK (1998) Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem Pharmacol 55:1747–1758 54. Seneviratne CJ, Wang Y, Jin L, Abiko Y, Samaranayake LP (2008) Candida albicans biofilm formation is associated with increased anti-oxidative capacities. Proteomics 8:2936–2947 55. Shin DH, Jung S, Park SJ, Kim YJ, Ahn JM, Kim W, Choi W (2005) Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans. Yeast 22:907–918 56. Singh P, Chauhan N, Ghosh A, Dixon F, Calderone R (2004) SKN7 of Candida albicans: mutant construction and phenotype analysis. Infect Immun 72:2390–2394 57. Sobko T, Reinders CI, Jansson E, Norin E, Midtvedt T, Lundberg JO (2005) Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide 13:272–278
739 58. Srikantha T, Zhao R, Daniels K, Radke J, Soll DR (2005) Phenotypic switching in Candida glabrata accompanied by changes in expression of genes with deduced functions in copper detoxification and stress. Eukaryot Cell 4:1434–1445 59. Srinivasa K, Kim NR, Kim J, Kim M, Bae JY, Jeong W, Kim W, Choi W (2012) Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans. Mol Cells 33:301–307 60. Tillmann A, Gow NAR, Brown AJP (2011) Nitric oxide and nitrosative stress tolerance in yeast. Biochem Soc Trans 39:219–223 61. Ullmann BD, Myers H, Chiranand W, Lazzell AL, Zhao Q, Vega LA, Lopez-Ribot JL, Gardner PR, Gustin MC (2004) Inducible defense mechanism against nitric oxide in Candida albicans. Eukaryot Cell 3:715–723 62. Wysong DR, Christin L, Sugar AM, Robbins PW, Diamond RD (1998) Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene. Infect Immun 66:1953–1961 63. Zhang X, De Micheli M, Coleman ST, Sanglard D, MoyeRowley WS (2000) Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p. Mol Microbiol 36:618–629 64. Znaidi S, Barker KS, Weber S, Alarco AM, Liu TT, Boucher G, Rogers PD, Raymond M (2009) Identification of the Candida albicans Cap1p regulon. Eukaryot Cell 8:806–820
123
