Fungal Adaptation to the Mammalian Host: It’s a New World, After All

Summary

Adaptation to environmental conditions is key to fungal survival during infection of human hosts. Although the host immune system is often considered the primary obstacle to fungal colonization, invading fungi must also contend with extreme abiotic stresses. Recent work with human pathogenic fungi has uncovered systems for detecting and responding to changes in temperature, carbon source, metal ion availability, pH, and gas tension. These systems play a major role in adaptation to the host niche and are essential factors for persistence in a mammalian host. Future investigations into fungal responses to these and other abiotic components of the host environment have the potential to uncover novel targets for anti-fungal therapy.

Introduction

Fungi survive not in isolation but as inhabitants of environmental niches that are composed of both biotic and abiotic factors. Despite our tendency to think of a niche as a static, consistent environment, many of these factors are highly dynamic and in a constant state of flux. Therefore, for a fungus to survive in its niche, it must be able to adapt to these constantly changing parameters. To this end, fungi possess sensory and regulatory systems whereby a change in a specific environmental component can be detected to induce transcriptional and translational changes that promote survival under the newest environmental conditions.

For fungal pathogens of humans, the requirement for environmental adaptation is extreme, since the transition from a saprophytic lifestyle in the environment to a parasitic lifestyle within a mammalian host entails abrupt changes in a number of environmental parameters. When considering the interaction between host and pathogen, we commonly think of biotic host components like immune cells as the major antagonists to the survival of fungal pathogens. By considering the host as an environment with its own set of abiotic parameters, however, we can outline a different set of host factors with which pathogens must contend in order to successfully colonize their new niche. As saprophytes, fungi survive in an environment with a moderate ambient temperature and pH, a defined set of sources for essential nutrients like carbon and metal ions, and atmospheric concentrations of key gases like carbon dioxide and oxygen. Upon invasion of a human host, these environmental factors undergo an immediate and drastic change. Ambient temperature is suddenly replaced with the restrictively high temperature of the human body. Ambient pH is replaced with acidic mucosal surfaces or neutral blood and tissues. Familiar sources of carbon and metal ions are missing in an environment where essential nutrients are sequestered from microbes to support host survival. Carbon dioxide and oxygen concentrations are reversed in host tissues, leaving fungi to cope with hypoxia and high levels of carbon dioxide. Here we focus on recently identified systems for adaptation to the abiotic stresses that fungi encounter during colonization and infection of their human hosts and review the role of these systems in fungal survival and virulence.

Temperature Adaptation

Fungal survival at the elevated temperature of a human host is essential for virulence. Despite the universality of thermotolerance among human fungal pathogens, temperature adaptation within the host manifests itself in a variety of ways. The systemic dimorphic fungi Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Penicillium marneffei undergo a morphological change from hyphal mold to budding yeast. In contrast, Candida albicans converts from commensal yeast to invasive hyphae. The fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are simply better able to survive at 37°C than their non-pathogenic counterparts.

A Saccharomyces cerevisiae pathway controlling filamentous growth during nitrogen starvation has recently been implicated in the survival of pathogenic fungi at 37°C. In this pathway, a G protein-coupled receptor activates a Ras GTPase, triggering the guanine nucleotide exchange factor Cdc24p to activate the GTPase Cdc42p. Downstream signaling, initiated by the Ste20p kinase, triggers transcriptional changes that mediate filamentous growth (1). In C. neoformans, Ras1 and Cdc24 mutants show reduced growth at elevated temperatures and are attenuated in a mouse model of cryptococcosis (2). In P. brasiliensis, Ras1 expression responds to heat shock, and inhibition of farnesylation, an essential modification of Ras1, causes filamentous growth at 37°C, suggesting that Ras1 is essential for thermal dimorphism (3). An exciting study with P. marneffei revealed a temperature-dependent role for the Ste20p homologue PakA in conidial germination. PakA mutants showed minor defects in filamentous germination at ambient temperature, but were severely impaired during germination and subsequent growth as budding yeast at 37°C. This defect was also apparent during growth in host macrophages, suggesting that activation of PakA at 37°C could play a role in virulence (4).

Other fungal proteins have also been implicated in adaptation to elevated temperature. A recent review in the pages of this journal highlighted the identification of Drk1, a hybrid histidine kinase that is essential for growth of B. dermatitidis as a yeast at 37°C. This kinase controls expression of the virulence factors Bad1 and α-(1,3)-glucan and is essential for virulence (5,6). More recently, work in H. capsulatum identified Ryp1, a homologue of the C. albicans transcriptional regulator Wor1, as essential for thermal dimorphism in response to elevated temperature. Ryp1 binds its own promoter and likely acts as an autoregulatory transcriptional regulator in H. capsulatum as it does in C. albicans. Ryp1 mutants grow as hyphae and are unable to induce expression of the vast majority of yeast phase-specific genes at 37°C (7). Because yeast phase-specific genes include most known virulence factors for this fungus, Ryp1 is likely to be essential for initiation of histoplasmosis.

Carbon Metabolism

Fungal survival in any environment requires assimilation of available carbon via metabolic enzymes. It was shown in 2001 that C. albicans requires isocitrate lyase (ICL), a key component of the glyoxylate cycle, for full virulence (8). This suggested that in the host, rich carbon sources like glucose, which repress ICL expression, are limited and C. albicans activates the glyoxylate cycle for utilization of poor carbon sources like acetate and fatty acids. This hypothesis, however, was contradictory to the fact that glucose levels in the blood are high enough to prevent ICL expression. The use of a C. albicans strain expressing GFP behind the ICL promoter demonstrated that ICL expression in the host is confined to specific micro-environments. ICL expression is repressed in the blood, but induced in the nutrient-limited phagosome of immune cells (9). The role of alternative carbon metabolism in C. albicans virulence was further underscored by the elucidation of a network of carbon utilization genes, including those that mediate the glyoxylate cycle, fatty acid oxidation, and gluconeogenesis, which are all required for persistence in the host (10).

Other fungal pathogens have also been shown to upregulate glyoxylate cycle genes during host-simulative conditions. Yeast phase cells of P. brasiliensis have increased transcript levels of both glyoxylate cycle enzymes after exposure to host macrophages (11). This suggests that in P. brasiliensis, as in C. albicans, alternative carbon metabolism plays a role in survival within the host. Work in P. marneffei demonstrated a unique temperature-dependent regulation of ICL transcription. In known models of ICL regulation, transcription is induced by acetate and fatty acids through the FacB transcriptional activator (12). In P. marneffei, both FacB-dependent acetate induction and AbaA-dependent temperature induction of ICL were observed (13). This finding represents a novel mechanism for regulation of this important metabolic gene in a thermally dimorphic intracellular pathogen.

Increased expression of ICL was also observed in A. fumigatus after exposure to host macrophages, leading to the hypothesis that ICL would be required for virulence (14). Surprisingly, subsequent work demonstrated that ICL is dispensible for virulence (15). Further investigation revealed that A. fumigatus ICL mutants were still able to grow in vitro with lipids as the sole carbon source (16). This suggested that Aspergillus might possess additional mechanisms for fatty acid utilization that compensate for loss of ICL during infection. A recent microarray study exploring transcriptional changes in A. fumigatus spores upon exposure to host neutrophils demonstrated upregulation of dozens of genes involved in carbon metabolism (17). This pool of genes is likely to contain proteins required for fatty acid utilization in the host and for the development of invasive aspergillosis.

Iron Acquisition

Within the host, iron is sequestered from microbes by iron carrier proteins, creating an iron-limited environment in which fungal pathogens must encode mechanisms for iron acquisition in order to survive (18). Many fungi employ siderophores, high affinity iron chelators, to efficiently bind host iron in the extracellular space and safely store it within the fungal cytoplasm. Recent work in H. capsulatum identified the siderophore synthesis gene SID1 as an essential factor for growth in low iron environments. This growth defect was extended to include defects in both intracellular replication and virulence within mice (19). Additionally, the iron-dependent transcriptional repressor Sre1 was shown to repress transcription of a suite of siderophore synthesis genes in H. capsulatum (20). Recent work in A. fumigatus demonstrated for the first time that different types of siderophores are employed for distinct purposes in different developmental stages and niches, with all identified siderophores being essential for virulence (21).

Fungal pathogens must also possess mechanisms for controlling and coordinating the utilization of acquired iron. The recent identification of the transcriptional regulator HapX in A. nidulans has provided insight into a mechanism by which fungi can adapt to iron limitation by reducing iron utilization. HapX acts primarily during iron-depleted conditions as a transcriptional repressor for genes that utilize iron, effectively reducing the cell’s iron requirement (22). A related protein in C. albicans called Hap43, however, was found to act as a transcriptional activator for the ferric reductases, which are crucial for the removal and utilization of iron from chelators including both siderophores and host carrier proteins (23). A novel mechanism by which C. albicans scavenges iron from host hemoglobin was also recently described. Receptor-mediated endocytosis of hemoglobin facilitates extraction of iron, likely by a heme oxygenase in the vacuole (24). This hemoglobin utilization system represents an additional iron acquisition system that will likely be linked to persistence of C. albicans within the host.

pH Adaptation

Within human hosts fungal pathogens must adapt to changing pH levels, which range from acidic mucosal surfaces to neutral or slightly alkaline blood and tissues. One pathway implicated in fungal responses to changing pH involves activation of the transcription factor PacC/Rim101 (25). PacC is essential for A. nidulans virulence in a model of pulmonary aspergillosis (26). Rim101 is essential for C. albicans virulence in models of mucosal invasion and systemic candidiasis (27). PacC/Rim101 is activated downstream of a signaling cascade involving a plasma membrane complex, an endosomal membrane complex, and the proteasome.

The proteins of the plasma membrane signaling complex undergo pH-dependent activation. PalI/Rim9 is responsible for localization of the pH-sensor PalH/Rim21 to the plasma membrane (28). This facilitates PalH-dependent phosphorylation and ubiquitylation of PalF/Rim8. PalF is an arrestin-like protein thought to trigger endocytosis of the plasma membrane complex upon PalH-dependent modification, mediating transduction of the pH signal from the plasma membrane to the endosomal membrane (29). PalH is also required for localization of the endosomal complex protein PalC to the endosomal membrane (30). This occurs through interaction of PalC with the endosomal sorting complex protein Vps32, which is essential for both the pH response and the virulence of C. albicans (31). Vps32 also interacts with endosomal membrane complex proteins PalA/Rim20 and PalB/Rim13. PalA recruits the inactive transcription factor PacC to the endosomal membrane complex at neutral or alkaline pH, facilitating cleavage of PacC to a smaller precursor by the protease PalB (25). In the final processing step, PacC is cleaved from inactive precursor to active transcription factor by the proteasome (32). Active PacC translocates to the nucleus and mediates expression changes of pH-responsive genes.

Several effector proteins downstream of PacC/Rim101 are essential for adaptation to the host. In A. nidulans, activation of PacC induces upregulation of iron acquisition system genes including siderophore biosynthesis genes (33). Similarly, expression of ferric reductase genes occurs downstream of Rim101 activation in C. albicans (23). This links the pH response to known virulence factors and is likely one reason why PacC/Rim101 mutants show reduced virulence. Mucosal invasion by C. albicans requires degradation of epithelial cell junctions by the protease Sap5p, which is upregulated downstream of Rim101. Expression of Sap5p in a Rim101 mutant strain restored the ability of C. albicans to invade an epithelial barrier in an in vitro model (34). Changes in cell wall composition also occur downstream of Rim101 activation in C. albicans, with over-expression of several cell wall-modulating proteins partially restoring virulence to a Rim101 mutant strain (35). These results indicate the importance of pH sensing in fungal adaptation to mammalian hosts and highlight components of the pH signaling cascade as potential drug targets.

Gas Tension

Carbon Dioxide

During human infection, fungi are exposed to carbon dioxide (CO₂) concentrations from low atmospheric CO₂ on epithelial surfaces to higher physiological CO₂ within host tissues. Work in C. albicans and C. neoformans identified carbonic anhydrase (CA) and adenyly cyclase (AC) as CO₂ sensors. Conversion of atmospheric CO₂ to bicarbonate by CA is essential for fatty acid biosynthesis and growth. Physiological CO₂ levels, however, spontaneously generate sufficient bicarbonate for both growth and stimulation of AC. This activates the cAMP pathway and initiates filamentation and expression of virulence traits (36). Accordingly, CA is required for virulence in models with ambient CO₂ levels, such as C. albicans epithelial invasion (37). In contrast, CA is dispensible and AC is essential in models with elevated CO₂, such as systemic infection by both C. albicans and C. neoformans (37,38,39). Recent work implicates differential regulation of the cAMP-repressor NRG1 in response to host-specific factors in the differential virulence of Candida species. Host-specific cues like elevated CO₂ induce C. albicans downregulation of NRG1 and activation of the cAMP-dependent filamentation pathway. Non-pathogenic C. dubliniensis fails to downregulate NRG1 and maintains cAMP repression. Deletion of C. dubliniensis NRG1 increases virulence of this species in several infection models, indicating that activation of cAMP signaling in response to abiotic components of the host environment is a major determinant of virulence in Candida species (40).

Oxygen

In order to maintain metabolic and biosynthetic functions in the host, fungal pathogens must also be able to adapt to hypoxia within host tissues. Oxygen levels in mammalian tissues are below atmospheric levels. Moreover, inflammation, thrombosis, and necrosis associated with infection are thought to increase degrees of hypoxia. In C. albicans, the response to hypoxia depends on the coordination of specific transcriptional regulators. In hypoxic conditions, the transcription factor Ace2 represses oxidative metabolic processes and promotes filamentation (41). The transcriptional regulator Egf1p, however, antagonizes Ace2 by repressing filamentation during hypoxia (42). The hypoxic environment of the vaginal mucosa has been shown to induce iron uptake protein expression, likely linking the hypoxic response to both iron acquisition and virulence in C. albicans (43). In C. neoformans, two additional players in hypoxic adaptation have recently been described. The hybrid histidine kinase Tco1 is required for hypoxic growth and appears to play a post-transcriptional role in the hypoxia response (44). Sre1, a conserved regulator of sterol biosynthesis, is required for transcriptional changes that promote growth under hypoxic conditions. Sre1 is activated by proteolytic cleavage that likely occurs in response to reduced activity of oxygen-requiring sterol synthesis enzymes during hypoxia. Two independent laboratories have demonstrated an indispensible role for Sre1 in both hypoxic adaptation and virulence in a mouse model for cryptococcosis (44,45).

Conclusions

All fungal pathogens must possess systems for sensing and responding to changes in abiotic factors during colonization of human hosts. Where tested, these systems have proved essential for normal colonization and full virulence. It is thus clear that components of the fungal response to abiotic factors within the host are promising targets for anti-fungal drugs. Identification of factors divergent from or absent in mammalian cells will maximize the potential for successful human therapies. The advantages of thinking of the host as a set of environmental conditions become even more apparent if we consider other abiotic stimuli that have not been discussed in this review. In addition to the specific sensory and regulatory systems reviewed above, fungal pathogens must also adapt to changes in nitrogen, calcium, magnesium, and copper sources, pressure, and fluid flow rates. Viewing the host as a new environment for an invading pathogen will foster a more complete understanding of fungal pathogenesis and the advent of novel targets for anti-fungal drugs (Box 1).

BOX

Therapeutic Applications

One goal of studying fungal adaptation to mammalian hosts is the development of potential anti-fungal therapies. This goal came into the spotlight in 1994 when the anti-malarial drug chloroquine was shown to inhibit the growth of H. capsulatum within host macrophages. Upon supplementation with iron, intracellular growth was restored, indicating that chloroquine’s iron-chelating ability was responsible for the growth restriction. Acquisition of iron within mice proved essential for virulence, as chloroquine treatment led to a resolution of infection (46). Subsequently, chloroquine proved effective against C. neoformans and P. marneffei, but this time through its impact on a different aspect of the host environment: hydrogen ion concentration. In each of these cases, chloroquine’s alkalinizing activity was essential for restriction of fungal growth (47,48). Most recently, the story has come full circle, as chloroquine restricts intracellular growth of P. brasiliensis in an iron-dependent manner and is therapeutic in a mouse model for paracoccidoidomycosis (49,50). Chloroquine has thus emerged as a drug with broad anti-fungal capacity that acts by altering abiotic parameters of the host environment. These discoveries highlight the clinical importance of understanding the impact of abiotic stresses on fungi during persistence within mammalian hosts.