Oxidative stress defense: survival mechanism in fungi

Organisms produce reactive oxygen species (ROS) as by-products of aerobic respiration and other metabolic functions due to oxygen excitation, partial reduction and radical and peroxide

formation (Aguirre et al., 2005). ROS are used as intracellular signaling molecules as well as for inter-species communication, for example in symbioses and in pathogenic processes (Marschall and Tudzynski, 2016; Nath et al., 2016; Zhang et al., 2016). The production of ROS, and thus oxidative stress during development can actively be regulated in fungi (Pöggeler et al., 2006). ROS are produced as defense mechanism by host immune systems of animals and their counterparts in plants (Camejo et al., 2016; Moye-Rowley, 2003). ROS can damage all kinds of biomolecules like nucleotides, proteins and lipids (Breitenbach et al., 2015; Sato et al., 2009). Therefore, fast and potent mechanisms to counteract ROS stress are crucial for fungal fitness and success.

ROS are detoxified by enzymatic mechanisms and redox systems, which provide reducing power (Aguirre et al., 2005; Matsuzawa, 2017). Several enzymes, such as superoxide dismutases and catalases are involved in the oxidative stress response (OSR). At least five catalases exist in A. nidulans: catalases A-D and the uncharacterized AN8553 gene product (Bayram et al., 2016; Kawasaki et al., 1997; Kawasaki and Aguirre, 2001; Navarro et al., 1996). Deletion of catA, catB and catC, as well as double and triple deletions did not have developmental influences in A. nidulans (Kawasaki et al., 1997; Kawasaki and Aguirre, 2001). CatA is preferentially found in conidiospores, whereas CatB is a hyphal catalase and both protect against external as well as internal H₂O₂ (Kawasaki et al., 1997; Navarro et al., 1996). CatC is proposed to act on very specific stress situations since expression of catC is not induced during oxidative or osmotic stress and only slightly upregulated by heat shock stress (Kawasaki and Aguirre, 2001). The catalase-peroxidase CatD functions as a H₂O₂ scavenger and during heat stress only in old mycelia (24 h and thereafter) (Kawasaki and Aguirre, 2001).

1.7.2 Thioredoxin and glutathione system

Besides the enzymatic OSR proteins, several oxidative stress defense systems have evolved.

Key mechanisms in the OSR are the thioredoxin and the glutathione system (Aguirre et al., 2005; Bakti et al., 2017; Carmel-Harel and Storz, 2000; Sato et al., 2009; Thön et al., 2007).

The main cellular oxidative stress defense system is the glutathione system, a redox-buffer system (Bakti et al., 2017; Breitenbach et al., 2015). Glutathione peroxidase, for which glutathione functions as electron donor, specifically reduces H2O2 to H2O (Breitenbach et al., 2015; Meister and Anderson, 1983; Sato et al., 2009). The glutathione system exhibits interplay with a second redox system, the thioredoxin system (Sato et al., 2009; Thön et al., 2007). Thioredoxins are small, omnipresent proteins of 12 to 13 kDa, which function as

oxidoreductases. They act as electron donors for thioredoxin peroxidases, similarly to glutathione (Sato et al., 2009; Thön et al., 2007). Glutathione and thioredoxin are reduced by their specific reductases, which use NADPH as electron donor, after the oxidation processes (Breitenbach et al., 2015; Sato et al., 2009; Thön et al., 2007)

1.7.3 Transcription factors involved in the oxidative stress response

Rapid transcriptional regulation events are important for the fungal defense against ROS stress. The OSR is mainly regulated by nuclear localization control of specific transcription factors and their protein phosphorylation (Moye-Rowley, 2003). Several examples of fungal transcription factors are known, where nuclear localization is regulated by oxidative stress (da Silva Dantas et al., 2015; Glover-Cutter et al., 2014; Jin et al., 2015; Morano et al., 2012;

Moye-Rowley, 2003). Yap1 from Saccharomyces cerevisiae, which corresponds to NapA of A. nidulans, is required for expression of thioredoxin TRX2 and involved in the regulation of the glutathione biosynthesis (Asano et al., 2007; Kuge and Jones, 1994; Moye-Rowley, 2003;

Wu and Moye-Rowley, 1994). Transcriptional regulation by Yap1 in the OSR is regulated via an exportin: Yap1 enters the nucleus in unstressed situation but is rapidly shuttled out again, whereas oxidative stress leads to a nuclear Yap1 accumulation and subsequent transcriptional regulation of target genes (Isoyama et al., 2001). Localization of its homologs from other yeasts is controlled in an oxidant-responsive manner as well (Moye-Rowley, 2003). Skn7 is, together with Yap1, required for oxidative stress tolerance (Moye-Rowley, 2003). Yap1 and Skn7 are interdependent and likely function in the same OSR pathway via activation of TRX2 (thioredoxin) expression (Krems et al., 1996; Morgan et al., 1997). NapA (A. nidulans AP-1 homolog A) is the Yap1 ortholog in A. nidulans. It is important for the stress-mediated activation of several genes of the OSR, such as catB, trxR and trxA in A. nidulans. Strains, which lost napA are not able to grow on medium supplemented with oxidative stress inducers (Asano et al., 2007). Several transcription factors are activated by mitogen-activated protein kinase (MAPK) phosphorylation cascades upon oxidative stress, such as Atf1 of Schizosaccharomyces pombe or its homolog in A. nidulans, AtfA (Hagiwara et al., 2008;

Lara-Rojas et al., 2011; Shiozaki and Russelp, 1996). Deletion of the gene encoding the MAPK SakA, which interacts with AtfA, leads to increased sensitivity of conidiospores to oxidative stress and decreased spore viability in A. nidulans (Kawasaki et al., 2002; Lara-Rojas et al., 2011).

Another fungal mechanism in response to stresses is the adjustment of the cellular protein composition. Target proteins are labeled for degradation by multi-subunit SCF Cullin RING

ligases, which employ F-box proteins as substrate specific adaptors (Jöhnk et al., 2016; Yu, 2010). The F-box protein Fbx15 in A. fumigatus is necessary to shuttle SsnF into the nucleus in response to oxidative stress (Jöhnk et al., 2016). SsnF is a subunit of the transcriptional co-repressor complex RcoA-SsnF and mislocalization of SsnF in Δfbx15 correlates with an upregulation of catB (Jöhnk et al., 2016). In S. cerevisiae, the corresponding Ssn6-Tup1 co-repressor complex coordinates the expression of three to five percent of the whole genome and is involved in mating, nutrient sensing, DNA-damage repair and stress response (Derisi et al., 1997; Parnell and Stillman, 2011).