Beside its saprophytic life style A. fumigatus also acts as an opportunistic pathogen, which can infect humans and animals. Among the more than 200 Aspergillus species only a small subset of approximately 10% have been associated with human pathogenicity, with A. fumigatus as the most important cause for life-threatening mycoses (Horn et al., 2012; Lamoth et al., 2016). The infective agents are the conidia, which are inhaled and because of their small size easily reach the lung alveoli. On average every human inhales several hundreds of A. fumigatus conidia per day (Kwon-Chung and Sugui, 2013). In immunocompetent hosts this rarely causes problems since the conidia are efficiently eliminated by the innate immune response (Denning, 1998; Gazendam et al., 2016).
However, especially in patients with structural lung diseases like tuberculosis or patients suffering from cystic fibrosis or asthma, conidia are able to cause allergic reactions and aspergilloma, which refers to growing mycelia in the lung cavities, called chronic pulmonary aspergillosis (CPA) with the potential to disrupt surrounding blood vessels (Agarwal et al., 2013; Hedayati et al., 2015; Latgé, 1999). The most severe disease caused by A. fumigatus is the life threatening invasive pulmonary aspergillosis (IPA), which affects predominantly patients with a disturbed immune system. IPA is typified by growing mycelia, which penetrates the surrounding tissues with the potential to spread to other tissues with the blood stream, leading to mortality rates of 40% to 90% (Dagenais and Keller, 2009; Lamoth et al., 2016). Particularly patients with the auto-immune deficiencies like chronic granulomatous disease (CGD), characterized by dysfunctional neutrophils, patients suffering from leukemia or patients with heart and lung transplantation carry high risks for IPA, with an incidence of 25-40%, 48% and 19-26%
respectively (Denning, 1998; Georgiadou and Kontoyiannis, 2012). The number of high-risk patients for IPA has increased during recent decades for reasons like intensified usage of chemotherapy during cancer treatment, an increasing number of patients with solid organ transplantations or the appearance and rise of AIDS (Cramer, 2016;
Steinbach, 2013). Treatment of IPA patients is complicated by poor diagnosis methods and the limited availability of antifungal agents. Commonly used antifungals like
8 Introduction
voriconazole or amphotericin B may have toxic side-effects for the host, whereas second line agents like echinocardins exhibit fungi-static properties instead of being fungicidal (Lamoth et al., 2016; Smith and Kauffman, 2012). Therefore, severity and increasing incidence of IPA yield in a growing interest into this medical important fungus.
1.2.1 Virulence determinants
The identification of specific virulence factors, which set A. fumigatus apart from other Aspergilli, thus making it the most prevalent cause for aspergillosis in humans, has gained an increasing interest during recent decades. Consequently more than 400 A. fumigatus mutant strains have been constructed and assessed for their virulence (Horn et al., 2012). Especially the availability of genome sequences for A. oryzae, A. nidulans and A. fumigatus, which allowed comparative studies among these Aspergilli and the addition of new genetic tools like extended genetic marker availability, new mutagenesis systems and akuA/akuB-mutant strains that allow a more efficient transformation procedure led to a substantial progress in the determination of virulence traits for A. fumigatus (da Silva Ferreira et al., 2006; Galagan et al., 2005; Krappmann, 2006;
Krappmann et al., 2006b; Machida et al., 2005; Nierman et al., 2005; Zhang et al., 2016).
Although specific virulence determinants could be identified (see Table 1), the largely accepted hypothesis claims that virulence of A. fumigatus is based on a multifactorial basis. The ability of A. fumigatus to grow and adapt to the harsh conditions of compost piles, its nutritional versatility, a sophisticated system to prevent damage from oxidative stress and the ability to produce a variety of secondary metabolites including mycotoxins are the main reasons for its success as a human pathogen (Brandon et al., 2015; Frisvad and Larsen, 2016; Hillmann et al., 2015; Miao et al., 2015; Rhodes, 2006).
Table 1: Putative virulence determinants, involved genes and their impact on
rodA, rodB Rodlet layer, oxidative stress resistance, dispersion of spores, prevents immune-recognition
Yes (rodA) (Aimanian-da et al., 2009; Paris et al., 2003a)
pksP PKS, key enzyme for DHN-melanin biosynthesis, ROS protection, prevents phagocytical killing of conidia
Yes (Heinekamp et al., 2012)
Thermotolerance thtA, pmt1 Required for growth above 37 °C (pmt1) or 42 °C (thtA)
No (Chang et al., 2004;
Zhou et al., 2007) mnt1, cgrA Required for growth above 25 °C (cgrA)
and 30 °C (mnt1), essential for conidial germination at 48 °C
cpcC, cpcA Sensor kinase and transcriptional activa-tor of cross-pathway control of amino
pkaC, pkaR Cyclic AMP-dependent protein kinase, regulation of carbon catabolite
Catalases for scavenging H2O2 No (cat1/cat2 double mutants exhibit slower yap1, skn7 Transcriptional regulators for oxidative
stress response
Siderophor biogenesis, iron uptake Yes (Schrettl et al., 2007)
gliZ, gliP Transcription factor and core NRPS enzyme of the gliotoxin biosynthesis
veA, laeA global regulators for morphology and secondary metabolism
Yes (Bok et al., 2005;
Dhingra et al., 2012)
10 Introduction
1.2.2 Development and virulence in Aspergilli
The rapid responses to environmental changes are subject to distinct mechanisms that seem to be evolutionary conserved in A. fumigatus due to its natural habitat (Cramer, 2016; Latgé, 1999; Rhodes, 2006; Tekaia and Latgé, 2005). Several of these stress response mechanisms are linked to development regulating processes, which were thoroughly described for the related model organism A. nidulans (Cramer, 2016; Dhingra et al., 2013; Smith and Calvo, 2014). A key element for the regulation of asexual/sexual development in A. nidulans is the velvet protein VeA, which is known to promote sexual development and secondary metabolism while it delays the formation of asexual conidiospores (see also Discussion section 2) (Bayram et al., 2016; Gerke and Braus, 2014; Sarikaya Bayram et al., 2014; Terfrüchte et al., 2014). In A. fumigatus, however, the deletion of veA just merely affects asexual sporulation on nitrate containing medium, but has significant effects on the production of secondary metabolites like gliotoxin, fumagillin and many more (Alkhayyat et al., 2015; Dhingra et al., 2012; 2013; Dolan et al., 2015; Krappmann et al., 2005).
This example emphasizes possible connections between essential developmental regu-lators of A. nidulans, which play only minor roles for development in A. fumigatus, but instead have evolved as prominent virulence contributing factors.
2 Targeted protein degradation