Aspergillus fumigatus is currently the major air-borne fungal pathogen. It is able to cause several forms of disease in humans of which invasive aspergillosis is the most severe. The high mortality rate of this disease prompts increased efforts to disclose the basic principles of A. fumigatus pathogenicity. According to our current knowledge, A. fumigatus lacks sophisticated virulence traits; it is nevertheless able to establish infection due to its robustness and ability to adapt to a wide range of environmental conditions. This review focuses on two crucial aspects of invasive aspergillosis: (i) properties of A. fumigatus that are relevant during infection and may distinguish it from non-pathogenic Aspergillus species and (ii) interactions of the pathogen with the innate and adaptive immune systems.
Aspergilli are saprophytes that commonly grow on decaying plant material. They are able to utilize a wide range of organic substrates and adapt well to a broad range of environmental conditions. In contact with air the mycelium forms specialized structures, so-called conidiophores. These produce large numbers of conidia (asexual spores) that are efficiently dispersed through the air and inhaled by humans.
Aspergillus fumigatus is currently the most important air-borne fungal pathogen causing different kinds of disease depending on the immune status of the host (e.g. invasive and non-invasive pulmonary infections or allergic bronchopulmonary aspergillosis). Most cases of invasive aspergillosis are associated with haematological malignancies, particularly haematopoietic stem cell transplantation, leukaemia or lymphoma. The risk of invasive Aspergillus infection is particularly high for patients with persistent neutropenia, graft-versus-host disease (especially with concomitant steroid therapy) and certain types of allogeneic transplantation (Segal et al., 2002; Camps, 2008). In all cases, recovery of granulocytes is pivotal for the survival of these patients.
What makes the difference?
Fungal conidia of many species are inhaled by humans in substantial numbers, but invasive aspergillosis is caused predominantly by A. fumigatus and only to a much lesser extent by Aspergillus terreus, A. flavus and others (Marr et al., 2002; Morgan et al., 2005), while the other approximately 650 Aspergillus species are unable to provoke severe infections. This indicates a selective process that operates even in immunocompromised patients and eliminates most fungal invaders before or when they reach the lower respiratory tract (Fig. 1). Regrettably, our knowledge about this protective mechanism and the stage at which innocuous fungi are eliminated is still in its infancy.
Fungi are important pathogens for insects, amphibians and plants, and since most fungi grow best at ambient temperatures, it was speculated that vertebrate endothermy evolved primarily for protection against fungal infections (Casadevall, 2005). Invasive Aspergillus infections usually start in the non-inflamed lung, hence at normal body temperature. Under this condition many Aspergillus species are able to germinate and grow. A. fumigatus is a particularly thermotolerant organism: its temperature optimum ranges from 37°C to 42°C, but it can grow at up to 55°C and thereby approaches the upper temperature limit of eukaryotic organisms. This suggests that A. fumigatus evolved distinct mechanisms of stress resistance that might provide the basis of its virulence. Several mutants obtained by chemical mutagenesis were identified that grow at 42°C, but not at 48°C. Interestingly, none of them was attenuated in virulence (Chang et al.,2004). In contrast, disruption of the cgrA gene, which reduced growth at 37°C, but not at 25°C, led to an attenuation in virulence in a murine (37°C), but not in a Drosophila melanogaster (25°C) model of infection (Bhabhra et al., 2004). Further support for a correlation between thermotolerance and pathogenicity came from studies that compared different A. fumigatus isolates (Paisley et al., 2005) or Aspergillus species (Araujo and Rodrigues, 2004). Thus, certain genes that are required for thermotolerance seem to be also relevant for virulence. Thermotolerance may reflect a general hardiness that helps A. fumigatus to cope with different stress conditions. Although some traits have been implicated, it is still ambiguous whether they are distinct for A. fumigatus. It will be a challenge in the future to prove that human body temperature is the critical parameter that obviates infections by the numerous non-pathogenic Aspergillus species.
During infection Aspergillus must procure nutrition from the host. The finding that methylcitrate synthase is required for invasive Aspergillus infections (Ibrahim-Granet et al., 2008) indicates that the fungus feeds mainly on amino acids. This implies that the degradation of proteins is crucial during infection and A. fumigatus is well equipped with numerous proteases to make amino acids available (Monod et al., 2009). However, attempts to define relevant proteases by single mutations failed, as have recent studies in which PrtT, a regulator controlling a subset of extracellular proteases, was deleted (Bergmann et al., 2009; Sharon et al., 2009).
According to our current knowledge A. fumigatus lacks sophisticated virulence factors that are solely dedicated to permit a pathogenic lifestyle. This distinguishes A. fumigatus from many bacterial pathogens and reflects its evolutionary background as a saprophytic soil dweller. Further research will have to uncover the secrets of its flexibility and robustness that distinguish this important and life-threatening opportunistic pathogen from its numerous non-pathogenic colleagues.
First encounter: host–pathogen interactions in the alveoli
Conidia of A. fumigatus are inhaled by humans and, due to their small size, travel deep into the respiratory system. Alveoli are the principal origin of systemic Aspergillus infections, although infection may also start from other anatomical sites, like the sinus. In the alveolus the fungus germinates in a highly specialized anatomical niche that consists of type I and type II epithelial cells, alveolar macrophages, interstitial fibroblasts and endothelial cells (Herzog et al., 2008) (Fig. 2A). The thin and flat alveolar type I cells cover 95% of the alveolar surface and mediate the gas exchange in collaboration with underlying endothelial cells. Fibroblasts produce extracellular matrix proteins and thereby build up the scaffold for the alveolus. Type II cells cover only 5% of the alveolar surface, but play an important role by keeping the alveolar space free of fluid. Type II cells are additionally involved in the innate immune response. They release opsonins, such as complement and surfactant proteins, to the alveolar space and are able to respond to microbial infections with the production of cytokines (Herzog et al., 2008).
Conidia of different Aspergillus species were shown to activate the alternative complement cascade and asexual spores from clinical isolates induce a stronger response than non-pathogenic environmental isolates (Dumestre-Pérard et al., 2008). Thus, complement produced by alveolar cells might be an important player at this stage of infection.
In vitro, A. fumigatus conidia bind efficiently to the surface of A549 cells. These type II-like cells represent the standard model for studying interactions of Aspergillus with the alveolar epithelium. Conidia also bind to several matrix proteins, e.g. fibrinogen, laminin, and type I and type IV collagen (Bromley and Donaldson, 1996), and pre-incubation with fibrinogen or laminin impaired conidial binding to A549 cells (Bromley and Donaldson, 1996; Bouchara et al., 1997). Hence, receptors for matrix proteins may reside in the surface layer of resting conidia and mediate the primary adhesion to host tissue in the lung. However, the relevance of these findings for the virulence of A. fumigatus is still unclear.
Conidia are also internalized by A549 cells and travel to an acidic compartment comprising lysosomal markers. It has been reported that only 3% of these asexual spores survive, suggesting that A549 cells have the ability to kill conidia in a phagosomal compartment. The few conidia that survived in this hostile environment formed germ tubes, breached host membranes and escaped from the infected cell (Wasylnka and Moore, 2003). A549 cells infected with viable A. fumigatus conidia release IL-6 and IL-8 (Zhang et al., 2002), which underlines the role of type II cells in the innate immune response to fungi and in particular in the recruitment of neutrophils to the site of infection.
The innate immune response
Aspergillus conidia are able to withstand harsh conditions. This is due to a reduced water content, the accumulation of protective molecules in the cytoplasm, and a protective surface layer comprising two hydrophobin proteins (Thau et al., 1994; Paris et al., 2003) and a melanin layer (Langfelder et al., 1998; Tsai et al., 1998). Resting conidia shrouded in this hydrophobic mantle are immunologically inert particles (Aimanianda et al., 2009). Activation of resting conidia leads to an isotrophic growth that bursts open the rigid surface layer and thereby exposes the carbohydrates of the cell wall. Evidence is mounting to support the importance of fungal-specific glycostructures as target molecules for invariant, germ line-encoded pattern recognition receptors (PRRs) that are crucial for the innate immune response. Currently three main PRRs are believed to participate in the response to A. fumigatus: Dectin-1 and the Toll-like receptors TLR2 and TLR4. At least dectin-1 is of general importance for the recognition of fungal pathogens (Herre et al., 2004). Recognition of its ligand, β-1,3-glucan, by the innate immune system is evolutionarily old and can be traced back to ancient invertebrates, like the horse-shoe crab Limulus polyphemus. Aspergillusβ-1,3-glucan triggers a strong inflammatory response and enhances phagocytosis by macrophages (Steele et al., 2005; Luther et al., 2007). Immunocompetent mice are prone to Aspergillus infections, if they lack dectin-1 (Werner et al., 2009), whereas TLR2 or TLR4 are only required after immunosuppression (Dubourdeau et al., 2006). The ligand of dectin-1, β-1,3-glucan, is hardly detectable on resting conidia, but prominent on swollen conidia and germ tubes. Interestingly, it is not traceable on hyphae (Hohl et al., 2005), a fact that has been discussed as a fungal stealth strategy. If this in vitro observation holds true during infection, the essential role of dectin-1 in the defence of Aspergillus is solely based on its importance in the combat of swollen conidia and germ tubes and therefore restricted to a very early stage of infection (Fig. 2B).
The Aspergillus molecules that are recognized by other PRRs are still under debate. Given their surface exposure and specificity for fungi, certain carbohydrates are excellent candidates for pathogen-associated molecular patterns (PAMPs) and the exemplary fungal pathogen Candida albicans was recently shown to be recognized by the concerted action of three PRRs that detect β-1,3-glucan (dectin-1), O-linked mannan (TLR4) and N-linked mannan (mannose receptor) (Netea et al., 2006).
In contrast to Aspergillus, C. albicans is a yeast and has a long record as a human pathogen. Thus, lessons learned from Candida may not necessarily apply to Aspergillus. Since purified carbohydrate ligands are usually not available, unambiguous proof for the relevance of certain Aspergillus glycostructures as PAMPs depends on appropriate mutants. Mutants in key enzymes of protein O-glycosylation and glycolipid synthesis have been analysed, but revealed no phenotype with respect to cytokine release in murine macrophages (Wagener et al., 2008; Kotz et al., 2010). This might be the consequence of a fundamental difference between yeasts and filamentous fungi: C. albicans produces highly mannosylated proteins and glycolipids, whereas comparatively smaller glycoconjugates are characteristic of A. fumigatus.
Resident alveolar macrophages engulf conidia and respond to this encounter by producing cytokines and chemokines. This triggers a massive recruitment of neutrophils, which is the hallmark that distinguishes a substantial inflammation from a daily skirmish. Neutrophils patrol through the bloodstream and have to be attracted to the site of infection. They are the executors of the acute inflammatory response and the particular susceptibility of granulopenic patients to severe Aspergillus infections underlines their relevance. Depletion experiments also assigned a critical importance to neutrophils, but not to alveolar macrophages (Mircescu et al., 2009). The ability of neutrophils to attack and kill A. fumigatus depends on TLR2, TLR4 and dectin-1 (Bellocchio et al., 2004; Werner et al., 2009). Elimination of conidia and small germ tubes is accomplished by phagocytosis, while the release of microcidal molecules enables neutrophils to attack larger hyphal cells. Recently, the formation of neutrophil extracellular traps (NETs) triggered by A. fumigatus was demonstrated in the infected lung (Bruns et al., 2010). NETs represent an anti-microbial effector mechanism that mediates killing of a diverse range of bacterial pathogens as well as C. albicans (Papayannopoulos and Zychlinsky, 2009). NETs are unable to eliminate A. fumigatus, but reduce hyphal growth by depleting zinc ions (McCormick et al., 2010), a mechanism that might be valuable to confine infection.
A rapid influx of neutrophils into the lung can be observed in mice that inhaled larger numbers of conidia. After 2–3 h, samples obtained by bronchoalveolar lavage contained large aggregates of neutrophils and conidia, and germination was shown to be inhibited over a period of 24 h (Bonnett et al., 2006). In contrast, gp91phox−/− mice, which are deficient in phagocyte NADPH oxidation and therefore production of reactive oxygen species (ROS), are already susceptible to low doses of conidia (Bonnett et al., 2006). Alveolar macrophages from p47phox−/− mice, which are also deficient in ROS production, are impaired in killing of A. fumigatus (Philippe et al., 2003). These findings are in line with the fact that patients with Chronic Granulomatous Disease (CGD) who are deficient in ROS production are also more susceptible to Aspergillus infections. However, the concept that ROS are pivotal for killing of Aspergillus is still under debate. A yap1 mutant, although highly sensitive to ROS, behaved as wild type in confrontation experiments with human neutrophils and in a murine model of infection (Lessing et al., 2007), whereas a triple mutant lacking all three superoxide dismutase genes was more efficiently killed by macrophages, but not attenuated in virulence (Lambou et al., 2010). Mutations in the tmpL and the conidial catalase A gene are sensitive to oxidative stress in vitro and attenuated in virulence (Kim et al., 2009; Ben-Ami et al., 2010); however, killing assays with murine alveolar macrophages revealed no difference between the catA mutant and the wild type (Paris et al., 2003).
Recent data demonstrate that CGD patients have an impaired ability to form NETs. Restoration of ROS production by gene therapy was shown to reconstitute NET formation and to protect a CGD patient from a severe Aspergillus nidulans infection (Bianchi et al., 2009). The recent findings that NADPH oxidase restrains the innate immune response and limits inflammation provides another important tool to better understand the particular sensitivity of CGD patients to recurrent infections (Segal et al., 2010). Thus, apart from a potential direct action on microbes, ROS seem to play an important role in directing the innate immune response.
Natural killer (NK) cells represent a further facet of innate immunity. They are recruited early during Aspergillus infection and participate in the anti-fungal response (Morrison et al., 2003). At this stage, NK cells are the major source of IFN-γ (Park et al., 2009), a cytokine that is known to increase the microbicidal activity of phagocytes. Further studies are clearly required to define the role of NK cells in anti-Aspergillus immunity.
The large pentraxin PTX3 belongs to a family of acute phase proteins that represent the major humoral arm of innate immunity. PTX3 is produced by macrophages and epithelial cells in response to infection. Moreover, it is stored in larger quantities in the granules of neutrophils that release PTX3 during NETosis (Jaillon et al., 2007). PTX3 is an opsonin that mutually binds to the complement protein C1q and ficolin-2, a recognition molecule of the lectin complement pathway (Ma et al., 2009). Thus, PTX3, C1q and ficolin-2 might form complexes on the conidial surface and thereby amplify the innate immune response. Remarkably, PTX3 deficiency renders immunocompetent mice highly susceptible to A. fumigatus infection (Garlanda et al., 2002). Early administration of PTX3 enhances the conidiocidal activity of neutrophils and limits the inflammatory pathology (D'Angelo et al., 2009). The latter effect can be attributed to a faster elimination of PTX3-opsonized conidia (Garlanda et al., 2002) and a reduced neutrophil recruitment due to the binding of PTX3 to P-selectin (Deban et al., 2010). A fast elimination of PTX3-opsonized conidia and a concomitantly restrained inflammation provide a rationale for the fact that NADPH oxidase-deficient mice can be protected by the exogenous administration of PTX3 (D'Angelo et al., 2009).
Invasive pulmonary aspergillosis: tissue invasion and inflammation
After penetration of the epithelial layer of the alveoli, the fungus immediately comes in direct contact with the underlying blood vessels (Fig. 2C). Here, A. fumigatus requires no sophisticated adhesion and invasion mechanisms to breach epithelial or endothelial barriers. Instead it can rely on the robust architecture of its cell wall and the enormous driving force of the polarized hyphal growth. Aspergillus is a so-called angiotrophic fungus and infection of vessels is a characteristic histopathological feature of invasive Aspergillus infections (Kradin and Mark, 2008). As an organism that is used to growing in complex organic matter, A. fumigatus has a well-developed ability to follow gradients; during infection this will guide hyphae to blood vessels that transport oxygen and carbohydrates. Angioinvasion often results in infarction and consequently in reduced oxygen supply (Fig. 2C). Recruitment of neutrophils will furthermore disturb the integrity of the endothelial and epithelial barriers. Local obstruction of the airways may induce oedema, alveolar flooding and completely shut down the oxygen supply. Consequently, the fungus has to adapt to a hypoxic environment. According to our current knowledge, A. fumigatus relies on its oxidative energy metabolism to do so. The putative transcription factor SrbA is essential for hypoxic adaptation and virulence (Willger et al., 2008). This important finding demonstrates that the adaptation to hypoxia is a prerequisite for the survival of A. fumigatus in the inflamed tissue and its ability to spread to different organs.
It has become evident only recently that hypoxia is also a strong signal to immune cells. Effector cells that are recruited from the bloodstream, like neutrophils and monocytes, travel along an oxygen gradient when entering inflamed tissue. Hypoxia is deciphered by these cells as an activating signal and HIF-1, the central transcriptional activator of hypoxic adaptation in mammalian cells, activates the anti-microbial activities of phagocytes and has been discussed as a master regulator of the innate immune response (Nizet and Johnson, 2009). It will be a challenge, in future analyses of the innate immune response to A. fumigatus, to consider the hypoxic adaptation of both the pathogen and the host.
The adaptive immune response to A. fumigatus
The innate and the adaptive immune responses generally collaborate to defeat infections. T and B lymphocytes represent the two parts of the adaptive immune system. In contrast to the combat-ready innate defence, the adaptive response follows afterwards and reacts to signals originating from the innate immune response. The daily housekeeping work of eliminating inhaled fungal conidia relies solely on the innate immune system, whereas a concerted action of the innate and adaptive immune systems is required to fight established and potentially life-threatening infections.
Aspergillus-specific antibodies have been detected in immunocompromised patients suffering from invasive aspergillosis, but their functional importance is often considered minor. However, administration of β-1,3-glucan-specific antibodies can be protective (Torosantucci et al., 2005) and the importance of antibodies in protection against aspergillosis clearly deserves more attention.
While the role of B cells is still under debate, it is generally accepted that T cells play an important role in the defeat of aspergillosis and fungal infections in general. The T cell response is in many ways linked to the innate immune response. Dendritic cells (DCs) infiltrate the infected region, differentiate in response to the pathogen and, when loaded with antigen, migrate to the draining lymph nodes to instruct T cells. T cells have the ability to either activate phagocytes or limit the immune response. The collateral tissue damage caused by an exaggerated inflammatory response contributes substantially to the morbidity of Aspergillus infections and the control of immune effector cells is therefore of prime importance. DCs are located at the cross-roads and direct the immune system either towards a balanced and protective Th1 or towards an excessive, inflammatory Th17 response. Production of IFN-γ by Th1 cells is fundamental to optimize the microbicidal activity of phagocytes. In contrast, stimulation of Th17 cells and the production of IL-23 by DCs promote a destructive inflammatory response and impair anti-fungal resistance (Zelante et al., 2007). Regulatory T cells (Tregs) limit the inflammatory response steered by Th1 cells and act in an antagonistic fashion to Th17 cells.
In conclusion, an efficient anti-Aspergillus immune response requires the coordinated actions of innate and adaptive immunity. Both arms are part of a highly interconnected and interdependent network that must be finely tuned in order to find balance between protection and immunopathology. The adaptive immune system represents the regulatory part and is crucial to activate, direct and finally limit the innate immune response, especially neutrophils which act as the major executors of aggressive anti-fungal measures.
As a tightly controlled innate immune response is pivotal to eliminate the pathogen, resolve inflammation and initiate tissue repair, attempts have been undertaken to develop new therapeutic concepts aimed at modulation of the adaptive immune response. The adoptive transfer of Th1 cells has already been successfully applied to treat human patients (Perruccio et al., 2005) and effective DC vaccination has been described in a murine model of infection (Bozza et al., 2003). More recently, an siRNA approach has been successfully applied in a similar infection model to optimize the host response by dampening PI3K/Akt/mTOR inflammatory pathways (Bonifazi et al., 2010).
Systemic spread of infection
During invasive Aspergillus infection, hyphae commonly target blood vessels, as mentioned above. This often results in thrombosed vessels and the appearance of targetoid lesions (Kradin and Mark, 2008). Hyphae and conidia activate platelets in vitro and this host–pathogen interaction probably promotes thrombosis and contributes to inflammation in vivo (Rødland et al., 2010). The propensity to invade blood vessels is also a means for dissemination via the bloodstream. Viable fungal cells are rarely found in the peripheral blood, a fact that severely hampers diagnosis of disseminated Aspergillus infections. This is a consequence of the hyphal architecture that establishes tight cellular cohesion by a common cell wall and prevents the release of single cells or fragments. However, a detachment of short hyphal segments may occasionally occur and drive the systemic spread of infection. Alternatively, phagocytes may ingest small fungal elements and displace them (Fig. 2C). Secondary blood vessel-borne infectious foci often grow with a characteristic sunburst appearance (Kradin and Mark, 2008). Thrombosis is a common feature of these lesions and instrumental to generate hypoxic conditions. Aspergillus furthermore inhibits angiogenesis through production of secondary metabolites, like gliotoxin, and thus enforces the formation of hypoxic conditions (Ben-Ami et al., 2009). The resulting disseminated abscesses are the hallmark of this late stage of infection and appear in different organs.
One of the major complications during Aspergillus infection is its dissemination into the central nervous system (CNS), surmounting the blood–brain barrier. CNS aspergillosis has been diagnosed with increasing frequency over the past decade; parenchymal abscesses represent the majority of these with true meningitis being rare. Although the blood–brain barrier consists of tight junctions between all endothelial cells in capillaries supplying brain cells, A. fumigatus is able to overcome this barrier and to penetrate into the cerebrospinal fluid (CSF). Infiltration of A. fumigatus into the CNS is often fatal because of reduced penetration capacities of most anti-fungal agents and impaired numbers of immune cells present in the CSF (Schwartz and Thiel, 2009).
Systemic infections by A. fumigatus are only found in patients with severely impaired immune defences. Clearly, such infections are rather a consequence of modern medicine and cannot have influenced the evolution of this opportunistic pathogen. So far, there is no evidence that Aspergillus acquired pathogenic traits in host–pathogen interactions with, for example, predatory protozoa. Also sequencing of the genome did not provide any hints for the presence of classical virulence factors. But in order to survive in the soil, A. fumigatus acquired a high level of stress tolerance and flexibility that could provide a basis for its pathogenicity. However, to distinguish itself from non-pathogenic moulds A. fumigatus appears to keep additional secrets that have yet to be disclosed.
Infections by filamentous fungi are a severe medical problem characterized by an increasing number of cases and limited therapeutic options. Hence, the identification of new therapeutic targets is an urgent need. Filamentous fungi rely on polarized hyphal growth to invade tissues and cross barriers. Therefore, studies on the hyphal organization and cell biology may uncover new Achilles heels of these pathogens.
During infection Aspergillus has to deal with changing conditions at different anatomical sites. For a deeper understanding of the interactions between the pathogen and the host, the experimental conditions have to be adapted to the reality of the infected tissue. Environmental parameters, such as the oxygen concentration, have to be considered and infection models have to mirror the complexity of the immune response. Travelling along this road will enable us to further shape the contours of this opportunistic pathogen.
We are grateful to Kirsten Niebuhr for critical reading of the manuscript. This work was supported by a grant of the Wilhelm-Sander-Stiftung (to J.L. and F.E.).