The complex roles of NADPH oxidases in fungal infection

Authors


Summary

NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signalling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signalling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell.

Introduction

Chronic granulomatous disease (CGD) due to NADPH oxidase deficiency was one of the first Mendelian traits linked to a gene and assigned a physiological basis (Babior, 2004; Nauseef, 2008; Segal et al., 2012). For much of the past 50 years, Occam's razor has balanced on its edge a single role for the phagocyte NADPH oxidase (Phox): production of superoxide radicals and thus reactive oxygen and nitrogen species that damage and kill invading microbes. It is manifestly clear that ROS and RNS created through Phox activity are microbicidal, but recent work is expanding the role of NADPH oxidases beyond strict and direct microbicidal functions. New work in the past ten years has focused attention on other roles for reactive oxygen species in autophagy, extracellular trap formation, metabolic transformations and signalling (Steinberg and Grinstein, 2007; Dupre-Crochet et al., 2013; Nathan and Cunningham-Bussel, 2013). Moreover, NADPH oxidase may even change an organism's gut physiology by depleting oxygen and creating localized hypoxia at sites of inflammation (Campbell et al., 2014). In addition to our new appreciation for the creative capacity of ROS, there is also new insight into how ROS levels are modulated. Superoxide and hydrogen peroxide are produced by NADPH oxidases and other enzymatic activities, and intracellular control of redox has emerged as an important post-translational tool (Aguirre et al., 2005; Heller and Tudzynski, 2011).

Human fungal pathogens are clinically relevant in both developed economies (largely in iatrogenically immunocompromised hosts) and underdeveloped countries (chiefly in the context of HIV/AIDS) (Brown et al., 2012a,b; Brown et al., 2014). The emerging nature of fungal disease in humans has meant that there is a great unknown in terms of the mechanisms whereby these pathogens colonize and cause morbidity. In the United States and Europe, Candida and Aspergillus species are responsible for the greatest number of opportunistic infections (Hidron et al., 2008; Brown et al., 2012a). Among HIV/AIDS patients, Cryptococcus neoformans is the most deadly culprit (Brown et al., 2014). Incongruously, the phagocyte oxidase plays a largely protective role against both Candida and Aspergillus (Pollock et al., 1995; Aratani et al., 2002a,b), whereas it can play a detrimental role in immunity to C. neoformans (Snelgrove et al., 2006). It is likely that these differences stem from the relative contributions of direct effects (i.e. oxidative damage) and indirect effects (i.e. regulation of adaptive T-cell responses) of Phox activity in immunity. This dichotomy highlights the potential for NADPH oxidase to play multiple roles in response to fungal pathogens. In addition to the ROS to which fungi are exposed during immune attack, fungi also respond to endogenously produced ROS in several ways, including in the regulation of key differentiation events (Fig. 1). The ability of pathogenic microbes to respond to both endogenous and host-derived ROS adds a new and potentially important dimension to host–pathogen interaction.

Figure 1.

Roles of NADPH oxidases and ROS in the context of fungal infection.

A. Host cells at the site of infection, including phagocytes and epithelial cells, create reactive oxygen species (ROS) through activation of NADPH oxidase complexes. (Left) Within phagocytes, these ROS have intracellular effects on both phagocyte physiology [e.g. autophagy, inflammasome, neutrophil extracellular traps (NETs)] and microbial physiology (damaging effects). (Right) ROS directly cause extracellular damage and act as chemoattractants, and their indirect effects on phagocyte signalling and adaptive immune responses lead to chemoattraction of immune cells to the site of infection, as well as programming of the inflammatory and adaptive immune responses locally and systemically.

B. Within the phagocyte, ROS drive recruitment of LC3 to the phagosome and activate autophagy, in most cases are required for NETosis, and lead to inflammasome activation.

C. Within the infecting fungus, ROS derived from either host or fungus can trigger the oxidative stress response, can enhance filamentous growth, or can activate apoptosis of the fungi.

Thus, NADPH oxidases and the ROS they produce can drive changes in both the host and the pathogenic fungi causing infection. In this Review, we will focus on recent work to highlight the roles of NADPH oxidases and ROS in fungal virulence and antifungal immunity of human fungal pathogens. First we will look at links between NADPH oxidases and immune migration, then cover unexpected physiological roles of NADPH oxidases in antifungal immunity, and finally examine the roles of ROS in the physiology and virulence of fungal pathogens (Fig. 1).

Alternative roles of NADPH oxidases in tissue homeostasis and immunity to fungi

There has been a recent expansion in our perspective on the ways in which NADPH oxidases can impact immunity. These alternative roles have been comprehensively reviewed recently (Deken et al., 2013; Paiva and Bozza, 2014; van der Vliet and Janssen-Heininger, 2014) so here we will focus on their impacts on fungal infection. NADPH oxidases have important roles in tissue homeostasis and can direct and participate in chemotaxis. Recent work in the zebrafish model suggests that these activities may be important in promoting early fungal containment to limit invasive growth. In the context of anti-fungal immunity, Phox activity has also been connected with activation of three potentially effective immune weapons: neutrophil extracellular traps (NETs), autophagy and the inflammasome. We will cover each of these immune mechanisms in turn, with particular attention to recent developments in the field.

Immune migration and tissue homeostasis

Work in the last ten years has established that NADPH oxidase-derived reactive oxygen species play an important role in attracting immune cells to the sites of damage and inflammation (van der Vliet and Janssen-Heininger, 2014). Although the phagocyte NADPH oxidase (Phox) is the best-studied vertebrate member of this enzyme class, it is the activity of alternate NADPH oxidase enzyme complexes that has been most closely linked to immune migration in vivo. Influx of macrophages to damaged tissue has been linked to Nox1 and Nox4 in endothelial tissue, as well as Duox in epithelial tissue (Kvietys and Granger, 2012). Similar pathways are active in Drosophila development and C. elegans gut immunity, suggesting that regulated ROS production is not only a ‘damage’ signal, but is part and parcel of creating and maintaining a multicellular organism (Hurd et al., 2012).

The dual-specific oxidase Duox is a predominantly epithelial enzyme that has been implicated in leucocyte chemotaxis to wounds, cancerous cells and infected tissue (Feng et al., 2010; 2012; Deken et al., 2013). Groundbreaking work in the zebrafish wounding model has since proven to be applicable in other models of wounding and inflammation (van der Vliet and Janssen-Heininger, 2014). Current molecular models suggest a post-transcriptional signalling pathway that includes activity of intracellular calcium transients, purine receptors and hydrogen peroxide generation to propagate signals (van der Vliet and Janssen-Heininger, 2014). The proximal biochemical mechanisms by which hydrogen peroxide drive leucocyte attraction are still unknown, although elegant work in the zebrafish model led to the discovery that a key thiol in the Lyn tyrosine kinase cell autonomously regulates neutrophil chemotaxis in zebrafish and their human counterparts (Yoo et al., 2011).

In addition to recruiting to wound sites, NADPH oxidases also work in multiple cell types in the lung to drive macrophages, neutrophils and eosinophils into allergic airways (van der Vliet, 2011). Contributions from Duox1/2, Nox1, Nox2, Nox3 and Nox4 combine to upregulate ROS production in lung tissue. It is notable that these different NADPH oxidase complexes function in a number of cell types, including epithelial, endothelial, muscle and immune cells. Thus, NADPH oxidase-produced ROS can enhance immune migration combinatorially.

Initial work in the Drosophila, roundworm and zebrafish models has also uncovered a role for the evolutionarily well-conserved Duox enzyme in producing microbicidal hydrogen peroxide and directing gut immunity (Bae et al., 2010; Flores et al., 2010; Hoeven et al., 2011; Deken et al., 2013; Jain et al., 2013; Lee et al., 2013; Strengert et al., 2013). Notably, ablation of Duox makes flies, worms and fish more sensitive to gut pathogens. Follow-up studies in mice have shown increased susceptibility to Helicobacter felis and influenza A virus in the absence of Duox activity (Grasberger et al., 2013; Strengert et al., 2013). This suggests that Duox-mediated mucosal immunity is conserved from worms to mammals.

Immune recruitment to fungal infection in the zebrafish

Hydrogen peroxide-driven phagocyte influx to inflammatory sites suggests the possibility that it may also play a role in recruitment of immune cells to sites of infection. Accordingly, recent work in the zebrafish has examined the contributions of NADPH oxidases to infection-driven leucocyte migration. Independent work from two leading laboratories suggests that NADPH oxidases play minimal or redundant roles in phagocyte recruitment to sites of bacterial infection. There is no apparent contribution of NADPH oxidase-produced hydrogen peroxide to neutrophil recruitment in response to Gram-positive or Gram-negative pathogens in the otic vesicle (Deng et al., 2011). Nor is there any apparent role for p22phox in leucocyte recruitment to mycobacterial granulomas (Yang et al., 2012). The NADPH oxidase-independent early chemoattraction of neutrophils to sites of bacterial infection indicates that, in contrast to the wound response, other mechanisms such as lipid chemoattractants and chemokines play predominant roles in early chemoattraction to these bacterial infections.

In contrast to the inconsequential or redundant roles played by NADPH oxidases in early response to bacteria, both Phox and Duox were shown to play positive roles in containment of fungi by phagocytes in the zebrafish hindbrain ventricle infection model of candidemia (Brothers et al., 2013). The reduced phagocyte recruitment and lack of early containment strongly increased susceptibility to infection, as the unengulfed fungi germinated hyphae that caused extensive tissue damage and morbidity. The mechanism(s) whereby Phox and Duox promote early recruitment of phagocytes to C. albicans are still unknown. Notably, although Phox and Duox are more highly expressed in phagocytes or the epithelium, respectively, the compartment(s) within which they function in these circumstances are still not known. While both Phox and Duox are required for early responses in the hindbrain ventricle, early recruitment of neutrophils to a mucosal C. albicans infection in the swimbladder is insensitive to NADPH oxidase inhibition by diphenyleneiodonium. Thus, the requirement for NADPH oxidase activity appears to be tissue-specific and limited to only some infections.

The unusual requirements of both Phox and Duox for phagocyte recruitment to C. albicans infection in the hindbrain ventricle suggests that this tissue and/or the fungus provide an unusual stimulus that is sensitive to ROS signalling. Further work with a C. albicans mutant that does not efficiently make the switch from yeast to hypha suggests that fungi may actively inhibit other modes of chemoattraction (Brothers et al., 2013). Specifically, it was found that recruitment to and containment of this ‘yeast-locked’ knockout of the EDT1 gene was unaffected by DPI inhibition. Either this mutant promoted chemoattraction in a novel way or it failed to limit NADPH oxidase-independent chemoattraction. Consistent with the latter possibility, inactivated wild type yeast drive phagocyte recruitment even with DPI inhibition, suggesting that an active process linked to the morphogenetic switch is responsible for limiting Nox-independent chemoattraction (S.E. Barker and R.T. Wheeler, unpublished).

Support for the idea that phagocyte NADPH oxidase can also play an important role in directing chemotaxis in mammals comes from work with purified chemoattractants in vitro and in vivo. Work in vitro with mouse and human neutrophils and in vivo with mouse neutrophils clearly implicates the Phox complex in chemotaxis towards fMLP and TNFα (Hattori et al., 2010a,b). This was the first demonstration of a signalling role for Phox within migrating phagocytes, revealing that activation of Phox promotes directional movement. Independently, it was also found that macrophage chemotaxis to purified M-CSF requires Phox in the macrophages (Chaubey et al., 2013). Taken together, this work suggests that Phox is important in mammalian neutrophils and macrophages for active chemotaxis in vivo.

An immunomodulatory role to limit tissue damage in fungal infection

In humans suffering from CGD due to loss of phagocyte oxidase activity, patients suffer from granulomatous lesions and inflammatory bowel disease, the consequences of an overly robust immune response (Schappi et al., 2008; Segal et al., 2012). Sterile inflammation models have shown that Phox −/− knockout mice have a stronger initial recruitment of neutrophils to chemoattractants (Segal et al., 2012). Intravital imaging in the zebrafish has also elucidated a dampening effect of phagocyte NADPH oxidase on neutrophil influx to inflammatory lesions through myeloperoxidase-mediated signal inactivation (Pase et al., 2012; Robertson et al., 2014).

Recent work has sought to determine how this hyperactive response relates to immunity to fungal infection, focusing on A. fumigatus, which causes the most frequent and debilitating invasive fungal infections in CGD patients. It has been found that, in addition to roles in mediating leukocyte attraction to fungi, NADPH oxidase also limits lung-damaging immune infiltrates in pulmonary fungal infection. Segal and co-workers found that phagocyte oxidase plays an important role in dampening neutrophil activity and recruitment through, in part, activation of the Nrf2 transcriptional repressor (Segal et al., 2010; Grimm et al., 2011). This provides important mechanistic insight into how phagocyte oxidase can have a dampening role in detrimental neutrophil infiltration. The Phox −/− mice are also more susceptible to infection, despite the exaggerated neutrophilic infiltrate, and this increased susceptibility may be due to both increased fungal proliferation and increased toxicity from neutrophilic activity. It remains an open but important question whether this ability of Phox to dampen neutrophil responses requires its activity in the myeloid compartment and/or in the stroma. Although the expression of Phox components is greatest in phagocytes, this enzyme complex clearly plays important roles in other cell types (Bedard and Krause, 2007; Bedard et al., 2007; Kvietys and Granger, 2012).

The double-edged sword of phagocyte NADPH oxidase activity in fungal infection is also brought out in surprising work that implicates Phox-produced ROS in exacerbation of Cryptococcus neoformans lung infection. C. neoformans is a primary fungal pathogen that typically causes life-threatening disease in immunocompromised hosts. This predilection has made it a devastating infection among HIV/AIDS patients in Africa, where it competes with tuberculosis for the most important AIDS-associated lethal infection. Using first a Phox −/− mouse and then treating mice intranasally with an antioxidant, it was found that mice were protected against intranasal cryptococcal infection by abrogating NADPH oxidase activity or reducing ROS (Snelgrove et al., 2006). The loss of Phox-derived ROS was associated with a more protective Th1-type response and containment in pulmonary granulomatous lesions. Thus, counter intuitively, loss of this key phagocyte weapon rendered the mice more resistant to fungal infection. This work identifies important indirect consequences of Phox activity on downstream events, pointing to its influence on T-helper cell activity and adaptive immunity.

It is notable that Phox plays anti-protective roles in two fungal lung infections by promoting detrimental neutrophil-mediated inflammation in Aspergillus infection and limiting protective Th1 responses to cryptococcosis. Taken together, these studies highlight the important role of Phox and ROS in dampening immune responses, especially in the lung. Interestingly, there appears to be an important lung-specific activity for Phox, as proinflammatory response to TNFα is affected more in Phox knockout animals in the lung than other tissues (Zhang et al., 2011). The mechanistic basis for tissue-specific activity of Phox against infection is still unknown but represents an important area in the context of therapy.

Neutrophil extracellular traps

Granulocytes from vertebrates from fish to man can undergo a process called NET formation, or NETosis (Yipp and Kubes, 2013). NETs can play an important role in limiting the spread of infection and/or directly damaging microbes (Ermert et al., 2009). Both in vitro and in vivo experiments have implicated Phox as a key player in production of NETs, although results have differed somewhat depending on experimental setup (Yipp and Kubes, 2013). Human neutrophil NETs are stimulated by C. albicans recognition and limit C. albicans proliferation in vitro (Urban et al., 2006). These NETs contain calprotectin, which is important in their ability to damage and kill fungi (Urban et al., 2009). Intriguingly, calprotectin (S100A8/A9) is a chemoattractant that is also associated with symptomatic Candida vaginitis (Peters et al., 2014). Although these studies implicate NETs in protection against C. albicans, it is currently not known if C. albicans stimulates NET formation that protects against candidiasis in vivo.

A. fumigatus also stimulates NET formation, and a series of studies from several laboratories has established the requirement of Phox for NET formation both in vivo in the mouse lung and in vitro in the Petri dish (Bruns et al., 2010; Rohm et al., 2014). NETs were also shown to inhibit growth of A. fumigatus in vitro (McCormick et al., 2010). Remarkably, gene therapy in an X-CGD patient was monitored with respect to NET formation and reconstitution of Phox function was found to correlate with restored NET formation (Bianchi et al., 2009). Neutrophils from this patient were also used to implicate calprotectin in Phox-dependent human NET formation (Bianchi et al., 2011). The recent work from Urban and colleagues further demonstrated that Phox is required for NET induction in a pulmonary aspergillosis model (Rohm et al., 2014). Remarkably, in this hyphal infection model CGD neutrophils failed to efficiently undergo apoptosis, which could potentially contribute to hyper-inflammation. These elegant studies combine to implicate Phox-dependent NET formation in control of pulmonary A. fumigatus infection in human disease.

Autophagy

Autophagy is an important cellular mechanism both for cellular recycling and pathogen containment. ROS have been linked to activation of the autophagy pathway in both cellular recycling and in response to intracellular microbes (Huang et al., 2009; Scherz-Shouval and Elazar, 2011). Key early experiments showing that NADPH oxidase activity is required for efficient recruitment of autophagic proteins to microbe-containing phagosomes used the fungal particle zymosan (Sanjuan et al., 2007; Huang et al., 2009). This work was followed up by several groups who have shown that LC3 accumulation on fungi-containing phagosomes is dependent on ROS production through NADPH oxidase.

In 2012, two groups independently showed that the autophagy reporter protein LC3 is recruited to phagosomes containing live C. albicans and C. neoformans. In one case, it was shown that the recognition of C. albicans by Dectin-1 in RAW264.7 mouse macrophages leads to LC3 recruitment to phagosomes, which facilitates presentation of fungal antigens to CD4+ T cells (Ma et al., 2012). LC3 recruitment was shown to require the Dectin-1 receptor, the Syk tyrosine kinase, NADPH oxidase-derived ROS, and the activity of Atg5. Intriguingly, LC3 recruitment was not linked to intracellular fungal containment or killing. In the other work, myeloid expression of Atg5 was shown to mediate resistance to intravenous C. albicans infection but not intraperitoneal or intratracheal C. neoformans infection (Nicola et al., 2012). Intriguingly, knockdown of Atg5 enhanced C. neoformans intramacrophage survival but mice lacking Atg5 in myeloid cells exhibited decreased pathology compared to littermate controls. An altered macrophage polarization profile in the Atg5 myeloid-specific knockout mice may help to explain the decreased pathology. The disconnect between in vitro activity and in vivo activity suggests that autophagy may be playing other important roles in immune responses – beyond containment of intracellular pathogens.

Phagosomes of ingested Aspergillus fumigatus spores have also been shown to recruit LC3 in macrophages (Kyrmizi et al., 2013). Similar to the case for C. albicans, this localization requires the β-glucan receptor Dectin-1 and NADPH oxidase. However, autophagy does contribute to preventing germination and proliferation of intracellular fungi. Remarkably, treatment of monocytes with hydrocortisone ex vivo, or isolation of monocytes from patients treated with hydrocortisone, results in reduced containment of A. fumigatus. This finding uncovers a surprising ability of hydrocortisone to block signalling through the Dectin-1 pathway and forges a mechanistic connection between autophagy and corticosteroid-mediated immunosuppression in susceptibility to fungal disease.

The zebrafish model has also been used to examine links among NADPH oxidase activity, autophagy and C. albicans infection. A transgenic line expressing the GFP-LC3 fusion protein has been found to report robustly on autophagic flux, and was used to demonstrate recruitment of LC3 to bacteria-containing phagosomes in vivo for the first time (He et al., 2009; Meijer et al., 2014). Remarkably, C. albicans-containing phagosomes only rarely recruit significant levels of GFP-LC3 in vivo, and this low level recruitment is not abolished by either the antioxidant vitamin E or the NADPH oxidase inhibitor diphenyleneiodonium (Brothers et al., 2013). This suggests that phagosomal recruitment of LC3 may be less important in the context of vertebrate C. albicans infection.

In agreement with a more nuanced role for autophagy in vivo, recent work argues that autophagy is not crucially important for human control of C. albicans infection (Smeekens et al., 2013). These authors found that myeloid-specific knockout of Atg7 in mice did not affect infection outcome. In addition, no associations were found between fungal infection and single-nucleotide polymorphisms in autophagy pathway genes in humans. Similar to what was found for RAW264.7 mouse macrophages, there was no significant contribution of autophagy to phagocytosis or killing of C. albicans by human monocytes.

Taken together, these studies support a complex view of autophagy and suggest that some components of the autophagy machinery may be more important than others when considered in the whole animal. Further, these combined findings are consistent with other work that has defined roles for autophagy in immune signalling as well as phagosome maturation.

Inflammasome

Autophagy is closely linked to inflammasome activity, which regulates production of mature IL-1 and IL-18, activates pyroptosis, and plays an important role in resistance to fungal infection (Gross et al., 2011; Skeldon and Saleh, 2011; Rodgers et al., 2014). Inflammasomes can be activated by NADPH oxidase activation, but it may be a negative role played by Phox that is a key event in controlling immune response to fungi.

Several fungi trigger NLRP3 inflammasome activation, including dermatophytes and invasive opportunistic pathogens (Hise et al., 2009; Joly and Sutterwala, 2010; Gross et al., 2011; Li et al., 2013; Pietrella et al., 2013; Mao et al., 2014). NLRP3 activation by A. fumigatus in THP-1 monocytes requires activation of the β-glucan receptor Dectin-1 and NADPH oxidase activation, emphasizing the role of Phox in this process (Said-Sadier et al., 2010). In the context of both mucosal and disseminated candidiasis, NLRP3 seems to play an important protective role (Hise et al., 2009; Tomalka et al., 2011). This includes both regulating innate immunity and adaptive immunity (Hise et al., 2009; Tomalka et al., 2011; van de Veerdonk et al., 2011). In addition to NLRP3, the NLRC4 inflammasome also activates in response to C. albicans and plays a protective role against oropharyngeal candidiasis, primarily in the stroma (Tomalka et al., 2011).

New work highlights another role for inflammasome activity in C. albicans interaction with macrophages in vitro. Namely, the NLRP3 inflammasome has been implicated in activating pyroptosis of macrophages in response to C. albicans that have germinated within them (Wellington et al., 2012; 2014; Uwamahoro et al., 2014). It had been previously thought that fungal germination itself destroyed macrophages, but this new work from two laboratories shows that changes to the fungal cell surface upon this morphotypic switching event trigger NLRP3 activation and death of the macrophage. These studies are also in agreement with previous work that first identified the hyphal-specific activation of inflammasome activation (Cheng et al., 2011). Although the role for NADPH oxidase activity in NLRP3 inflammasome-mediated pyroptosis is unclear, its requirement for NLRP3 activation downstream of A. fumigatus recognition suggests it might play a similar role.

The inflammasome is also implicated in damaging hyper-inflammatory states, such as the gastrointestinal granulomas and inflammatory bowel disease (IBD) seen in some human CGD patients. Exciting new work shows that IL-1 production through the inflammasome is a key mediator of the hyper-inflammatory state, both in CGD-associated IBD and in pulmonary A. fumigatus infection (de Luca et al., 2014). It appears that the phagocyte NADPH oxidase normally limits a positive feedback loop between inflammasome IL-1 production and autophagy both in vivo and in vitro. The mechanism whereby NADPH oxidase downregulates autophagy and upregulates IL-1 production is not yet known. Importantly, blockade of IL-1R activation through a clinically approved drug (anakinra) can short-circuit this loop in vitro and in vivo in both mice and in CGD patients to ameliorate gut inflammation. Furthermore, anakinra also reduced gut inflammation in experimental IBD that did not involve loss of phagocyte NADPH oxidase, suggesting that this drug may be effective in treating the majority of IBD patients that do not have defective Phox. Consistent with previous work that suggests Phox exacerbates pulmonary fungal infection, use of anakinra in Phox −/− mice reduced fungal load in A. fumigatus infection. While the target tissue for anakinra action in the context of IBD or A. fumigatus infection has not been positively identified in vivo, previous work suggests that CGD monocytes play an important role in exacerbated IL-1 production (Meissner et al., 2010). These studies highlight the power of IL-1 and provoke a number of new and interesting questions about the relationships among NADPH oxidase activity, IL-1 and IL-17 production in the context of fungal infection (Mills et al., 2013).

Role of reactive oxygen species in fungal development

As discussed above, ROS play important roles in mammalian signalling. Similarly, ROS from various sources, including internally generated and host-generated, regulate important processes in fungal cells that have been implicated in pathogenesis. Some fungi have one or more NADPH oxidases that play various roles in development, as recently reviewed (Takemoto et al., 2007; Scott and Eaton, 2008). Even those fungi that don't encode NADPH oxidases encounter ROS generated from metabolism or non-respiratory enzymatic activity, or from a mammalian host or neighbouring bacterial or fungal species. The potential sources of ROS encountered during the life of the fungus can be demonstrated using C. albicans as an example. C. albicans is thought to generally reside in the presence of mammalian hosts either as a commensal or a pathogen. In the presence of host-associated microbiota in the mouth, intestinal or female reproductive tract, C. albicans encounters hydrogen peroxide-producing microbes such as Lactobacillus species (Collins and Aramaki, 1980; Fitzsimmons and Berry, 1994). Normal fungal metabolism generates endogenous ROS, and Miramon et al. (2012) found, using GFP-reporters, that both phagocytosed and non-phagocytosed C. albicans induces genes indicative of ROS exposure in the presence of immune cells. In addition, a small molecule signal produced by C. albicans can also impact intracellular ROS levels. Antifungal therapy can also promote intracellular ROS levels (Delattin et al., 2014). In this section, we will highlight regulated genes in intracellular ROS in C. albicans, and the consequences of ROS in the control of developmental processes including morphogenesis, biofilm formation, and apoptosis. We will also briefly mention ROS in the regulation of fungal processes in other fungi.

Candida albicans produces a quorum-sensing molecule called farnesol. This small molecule accumulates in culture supernatants to concentrations that can repress hyphal growth despite the presence of hypha-inducing cues (Hornby et al., 2001). Farnesol acts, at least in part, through direct inhibition of adenylate cyclase activity (Davis-Hanna et al., 2008; Hall et al., 2011). The consequences of decreased cAMP signalling, due to inhibition of adenylate cyclase, include the induction of stress response genes such as those that are protective against reactive oxygen species (e.g. catalase) (Deveau et al., 2010). This induced protection to ROS upon exposure to farnesol may be advantageous because farnesol itself can induce ROS in C. albicans (Westwater et al., 2005) and farnesol-induced ROS may contribute to apoptosis (Shirtliff et al., 2009). The ROS generated by farnesol may be a consequence of altered metabolic activity, perhaps caused by interaction with the electron transport chain, as has been demonstrated in S. cerevisiae (Machida et al., 1998; Machida and Tanaka, 1999). Farnesol, which is also produced when cells grow as biofilms (Martins et al., 2007), may inhibit the further accumulation of cells in the community (Ramage et al., 2002). Though it is not yet known if ROS generated by farnesol also modulate the activity of the Ras1-controlled signalling pathway in C. albicans, low concentrations of hydrogen peroxide appear to directly impact Ras activity in Paracoccidioides brasiliensis Pb18, potentially explaining the observed stimulation of hyphal growth by ROS in this fungus (Haniu et al., 2013).

Hydrogen peroxide can also modulate the morphology of C. albicans. Nasution et al. (2008) found that both exogenous hydrogen peroxide and endogenously-produced ROS induced hyphal growth in cells within colonies. It is not yet known if ROS signalling pathways play a role in morphogenesis, or if the change in morphology is an indirect effect of oxidizing molecules, but it is interesting to note that the authors also found increased levels of ROS in cells grown with serum, a potent inducer of hypha formation. Increased ROS levels in hyphae may be due to the repression of ROS scavenging enzymes that are repressed upon increased cAMP signalling (Harcus et al., 2004; Davis-Hanna et al., 2008). Alternatively, increases in respiration that are often concomitant with filamentation may lead to increased levels of ROS (Morales et al., 2013). Work by Srinivasa et al. (2012) showed that filamentation induced by H2O2 leads specifically to growth in the pseudohyphal morphology, and this morphogenic change involves multiple pathways including the Cek1-Cph1-dependent MAP kinase pathway. ROS are also predicted to impact the formation of chlamydospores, thick walled structures that may contribute to stress resistance in C. albicans, in part through the activity of the Hog1 MAP kinase (Alonso-Monge et al., 2003).

The role of reactive oxygen species in biofilm differentiation in S. cerevisiae and C. albicans was nicely reviewed in by Cap et al. (2012). The authors first discuss the potential benefits of hormesis, a process by which low concentrations of a stressor or toxin enhance survival upon exposure to higher concentrations of this stress and even other stresses. In addition, ROS impact biofilm development in other ways. Heterogeneity in ROS exposure in biofilms can create variability of signalling pathways within the population, and can contribute to localized cell death or changes in metabolism. C. albicans cells undergo an apoptosis-like process in response to ROS (Phillips et al., 2006).

While we focus on C. albicans in this review, A. fumigatus and C. neoformans are also influenced by ROS. As examples, in C. neoformans, Rac1 and Rac2 play roles in the localization of ROS (Ballou et al., 2013), either through the localization of Nox proteins, as has been reported for Cdc42 in A. nidulans (Rolke and Tudzynski, 2008) or through the regulation of Nox activity, as has been reported in Claviceps purpurea (Semighini and Harris, 2008) or Epichloë festucae (Takemoto et al., 2011). In these fungi, the proper localization of intracellular ROS is critical for establishing and maintaining polarized growth. Future studies will determine how NADPH-generated ROS, endogenous ROS produced by other pathways, and extracellular ROS work together to regulate key processes in these and other fungi.

Conclusion and perspectives

As detailed here, study of NADPH oxidase activity and the mechanistic consequences of spatiotemporally controlled ROS production have led to a greater understanding of fungal infection. NADPH oxidases play both effector and signalling roles in infection, which can lead to protective responses and dampened tissue damage. Phox-associated immunomodulation can promote protection (as in the case of resistance to C. albicans or A. fumigatus) or damage and exacerbated disease (as in pulmonary C. neoformans infection). Similarly, study of fungal infection can lead to insight into novel signalling roles of NADPH oxidases in immunity. This is illustrated by discussion of recent work that provokes new questions about autophagy-inflammasome connectivity and chemotaxis mechanisms.

Overall, the last few years have seen a shift in the view of ROS from microbicidal molecules to short-lived and targeted second messengers that regulate crucial signalling pathways in the immunocompetent host. In addition, it is also clear that ROS are produced and used as signals by eukaryotic pathogens. Thus, pathogenic fungi may respond to host-derived ROS by activating morphogenetic switching and thereby increased virulence.

The development of improved techniques for visualizing the presence of diverse reactive oxygen species in real time and for discerning the structural and biochemical effects of ROS on proteins, lipids, sugars and DNA will likely clarify how ROS work (Finkel, 2011; van der Vliet, 2011; Enyedi et al., 2013). This, in turn, will advance our understanding of the complex homeostatic and immune roles of NADPH oxidases, which may lead to more nuanced approaches to treatment of inflammatory dysregulation and fungal infections.

Acknowledgements

The authors gratefully acknowledge their funding from the National Institutes of Health (Grant No. R15AI094406 to R.T.W. and No. R01GM108492 to D.A.H.) and USDA (Project No. ME0-H-1-00517-13 to R.T.W.).

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