Recent experiments and simulations are starting to answer some fundamental questions about how life came to be.
Image credit: Shutterstock/Radoslaw Lecyk.
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Edited by Frederick M. Ausubel, Harvard Medical School, Boston, MA, and approved April 14, 2008 (received for review October 8, 2007)
Abstract
Zygomycosis is an emerging frequently fatal opportunistic mycosis whose immunopathogenesis is poorly understood. We developed a zygomycosis model by injecting Drosophila melanogaster flies with a standardized amount of fungal spores from clinical Zygomycetes isolates to study virulence and host defense mechanisms. We found that, as opposed to most other fungi, which are nonpathogenic in D. melanogaster (e.g., Aspergillus fumigatus), Zygomycetes rapidly infect and kill wild-type flies. Toll-deficient flies exhibited increased susceptibility to Zygomycetes, whereas constitutive overexpression of the antifungal peptide Drosomycin in transgenic flies partially restored resistance to zygomycosis. D. melanogaster Schneider 2 phagocytic cells displayed decreased phagocytosis and caused less hyphal damage to Zygomycetes compared with that to A. fumigatus. Furthermore, phagocytosis-defective eater mutant flies displayed increased susceptibility to Zygomycetes infection. Classic enhancers of Zygomycetes virulence in humans, such as corticosteroids, increased iron supply, and iron availability through treatment with deferoxamine dramatically increased Zygomycetes pathogenicity in our model. In contrast, iron starvation induced by treatment with the iron chelator deferasirox significantly protected flies infected with Zygomycetes. Whole-genome expression profiling in wild-type flies after infection with Zygomycetes vs. A. fumigatus identified genes selectively down-regulated by Zygomycetes, which act in pathogen recognition, immune defense, stress response, detoxification, steroid metabolism, or tissue repair or have unknown functions. Our results provide insights into the factors that mediate host–pathogen interactions in zygomycosis and establish D. melanogaster as a promising model to study this important mycosis.
Fungi of the class Zygomycetes, order Mucorales, are significant causes of life-threatening angioinvasive infections in patients with a wide range of immunosuppressive conditions and, occasionally, immunocompetent individuals (1, 2). Rhizopus species cause the majority of Zygomycetes infections, whereas Mucor, Rhizomucor, and Cunninghamella bertholletiae are less frequently encountered pathogens (1, 2). C. bertholletiae is considered the most pathogenic Zygomycetes species in humans (3).
Once thought to be an uncommon infection, zygomycosis has recently emerged as the second most common opportunistic invasive mold infection after aspergillosis in patients with hematological malignancies and transplant recipients (2, 4–6). Zygomycosis has a particularly poor prognosis in these patients, with mortality rates >90% in disseminated infection (3, 5, 6).
Quantitative and functional defects in immune effector cells associated with poorly controlled diabetes mellitus and receipt of corticosteroids or other immunosuppressive treatments are the principal predisposing factors for zygomycosis (1, 2). In addition, iron metabolism plays a central role in the pathobiology of zygomycosis. Thus, patients with elevated serum iron levels are at increased risk for zygomycosis (1–3), and treatment with deferoxamine, an iron-chelating agent that acts as a siderophore and supplies iron to Zygomycetes, promotes the development of severe disseminated infections in animal models and humans (7). However, unlike other medically important fungi (8), the epidemiology and immunopathogenesis of zygomycosis are poorly understood (2). Furthermore, mammalian models represent a bottleneck for large-scale genomic studies on microbial pathogenesis because of ethical considerations and logistic restraints associated with their use, the complexity of their immune systems, and difficulties in studying the dynamics of host–pathogen interaction in vivo (9).
We hypothesized that Drosophila melanogaster, a simple genetically amenable minihost with well characterized and evolutionarily conserved innate immunity, could serve as a suitable model for studying the immunopathogenesis of zygomycosis. D. melanogaster is capable of mounting efficient innate immune responses against a variety of fungal pathogens largely mediated by induction of the evolutionarily conserved Toll (Tl) pathway (9–12). Hence, upon challenge by fungi, Tl-pathway activation leads to rapid and selective induction of antifungal peptides, mainly Drosomycin (Drs) and Metchnikowin (Mtk), into the fly hemolymph and allows D. melanogaster to successfully combat infection by most fungal invaders (9–12). In addition, researchers have increasingly recognized that the D. melanogaster cellular immune response, comprising hemocytes circulating in the hemolymph, plays an instrumental role in early recognition and elimination of bacterial and fungal pathogens (9, 13, 14). Specifically, D. melanogaster Schneider 2 (S2) embryonic phagocytic cells share many characteristics with mammalian phagocytic cells, and S2 RNA interference libraries have been used to identify evolutionarily conserved genes involved in phagocytosis of bacteria (15) and Candida albicans (16).
In the present study, we developed a zygomycosis model by injecting D. melanogaster flies with a standardized amount of Zygomycetes spores. We found that, as opposed to other fungi, Zygomycetes rapidly infect and kill D. melanogaster WT flies despite early activation of the Tl pathway. In addition, comparative studies using the D. melanogaster S2 phagocytic cell line, the phagocytosis-defective eater-null fly mutant and transcriptome analysis in flies infected with Zygomycetes vs. A. fumigatus demonstrated that the pathogenicity of Zygomycetes is linked with impaired phagocytic cell activity and suppression of induction of genes involved in host defense, stress responses, and tissue repair. These results provide insight into the factors that mediate host–Zygomycetes interactions and imply that D. melanogaster is an attractive model for studying immunopathogenesis of zygomycosis.
Results
Zygomycetes Rapidly Infect and Kill WT D. melanogaster.
Previous studies in D. melanogaster demonstrated that injection of various fungi into the hemolymph of WT flies did not induce mortality (10–12). In contrast with these studies and our previous experience with A. fumigatus (17) and Candida species (18), injection of a standardized amount of spores of Zygomycetes clinical isolates into WT flies resulted in acute infection and high mortality rates (Figs. 1 A and B), whereas injection of A. fumigatus into WT flies did not significantly affect survival regardless of the inoculum size tested (Fig. 1 B). As evidenced by histopathological studies, Zygomycetes spores injected into the fly hemolymph germinated rapidly to form hyphae that subsequently disseminated and invaded multiple structures of the fly body, leading to death (Fig. 1 C). Fungal burden, determined by real-time PCR (RT-PCR), increased significantly over time, exceeding 4 logs 24 h after infection of WT flies with Rhizopus (Fig. 1 D).
Fig. 1.
Infection of WT, Toll mutant (Tl), and Drs overexpressing (U-Drs) flies with clinical isolates of Zygomycetes. (A) Survival rates of Tl and WT flies infected by injection (108 spores per ml) of R. oryzae or M. circinelloides; P = 0.004 for WT vs. Tl flies infected with R. oryzae or M. circinelloides; P = nonsignificant for other comparisons. (B) Survival rates of Tl-mutant and WT flies infected with C. bertholletiae (108 spores per ml); infection of WT flies with A. fumigatus clinical isolate Af293 (range of inoculum, 108 to 109 spores per ml) served as control; P < 0.0001 for WT flies infected with A. fumigatus vs. C. bertholletiae; P = nonsignificant for WT vs. Tl flies infected with C. bertholletiae. (C) Representative histopathological sections (stained with crystal violet) from the thorax and abdomen of a WT fly 24 h after infection with R. oryzae (D) Measurement of fungal burden at different time points after infection of WT and Tl-mutant flies with R. oryzae (108 spores per ml), assessed by qPCR; fungal burden is expressed as conidial equivalents of DNA (E) Survival rates of Drs overexpressing (da-Gal4/U-Drs) and control (da-Gal4/+) flies after infection with R. oryzae (108 spores per ml); P < 0.0001 for da-Gal4/U-Drs vs. control (da-Gal4/+) flies. (F) Survival rates of Tl flies after infection with different inocula of R. oryzae; P < 0.0001 for heat-killed (HK) R. oryzae spores vs. 106 R. oryzae spores per ml and 106 R. oryzae spores per ml vs. 107 R. oryzae spores per ml; P = 0.05 for 107 vs. 108 R. oryzae spores per ml.
Injection of Tl-mutant fruit flies, with either Rhizopus or Mucor spp. resulted in a higher mortality rate at 48 h (95%) when compared with injection of WT fruit flies (65%; P = 0.004) [Fig. 1 A and supporting information (SI) Fig. S1A ]. As a further indication of the role of the Toll pathway in fly defense against Zygomycetes, transgenic flies constitutively expressing the antifungal peptide Drosomycin (Drs) were significantly less susceptible to Zygomycetes infection when compared with control flies (survival rate at 48 h 63% vs. 0%; P < 0.0001) (Fig. 1 E). Interestingly, C. bertholletiae infection caused hyperacute mortality in both WT and Tl-mutant flies, with all flies dying of the infection within 48 h (Fig. 1 B). Finally, survival of both WT and Tl-mutant flies was inoculum-dependent (Fig. 1 F). These experiments thus established an easy reproducible model of zygomycosis and demonstrated that Zygomycetes are among the few reported fungi that are pathogenic in WT D. melanogaster.
Clinically Important Potentiators of Zygomycetes Virulence in Humans also Increase Zygomycetes Pathogenicity in Fruit Flies.
We next examined “classic” enhancers of Zygomycetes virulence in humans to determine how they affect survival of WT fruit flies infected with a representative Rhizopus oryzae clinical isolate. Importantly, iron acquisition from the host plays a pivotal role in successful infection by Zygomycetes in humans and animal models (1–3). Because iron metabolism in D. melanogaster has many similarities with that in mammals (19, 20), we hypothesized that increased iron availability also modulates pathogenesis of zygomycosis in fruit flies.
Indeed, we found that WT flies fed with deferoxamine and infected with R. oryzae (108 spores/ml) developed rapidly disseminated infections with higher fungal burdens upon histopathology (Fig. 2 A) and a significantly higher mortality rate when compared with infected control flies (no deferoxamine) (100% vs. 55% at 24 h; P < 0.0001) (Fig. 2 B). Also, WT flies fed with iron (FeCl3) had increased susceptibility to zygomycosis when compared with control flies (no iron) (mortality rate at 48 h: 100% vs. 65%; P = 0.04) (Fig. 2 B).
Fig. 2.
Effects of increased iron supply and availability through treatment with deferoxamine and iron starvation through treatment with deferasirox in WT flies infected with R. oryzae. (A) Representative histopathological sections of deferoxamine-treated and untreated control WT flies 24 h after infection with R. oryzae. (B) Survival rates of WT flies fed deferoxamine or iron (FeCl3) or deferasirox and subsequently infected with R. oryzae (108 spores per ml) and untreated control WT flies; P < 0.0001 for deferoxamine-treated vs. untreated control flies; P < 0.0001 for deferasirox-treated vs. untreated control flies; P = 0.04 for iron-treated vs. untreated control flies.
Importantly, deferasirox is a iron chelator that, in contrast to deferoxamine, induces iron starvation to Zygomycetes and improves survival in a murine model of zygomycosis (21). We found that deferasirox significantly protected WT flies infected with R. oryzae when compared with control flies (mortality rate at 48 h: 29% vs. 65%; P < 0.0001; Fig. 2 B). Thus, D. melanogaster could serve as a valuable model to study the role of iron metabolism during host–Zygomycetes interactions.
Corticosteroids have a wide range of complex immunosuppressive effects, mainly by affecting cellular immunity, and increase host susceptibility to invasive fungal infections in humans (8, 22). Similarly, treatment with corticosteroids in invertebrates results in impaired cellular immune responses and increased susceptibility to both bacteria and entomopathogenic fungi (23, 24). We found that WT flies fed with dexamethasone and infected with R. oryzae had significantly higher mortality rates than did infected control flies (no dexamethasone) (P = 0.02; Fig. S1B ). Therefore, receipt of corticosteroids, a major risk factor for zygomycosis in humans, also increased the susceptibility of flies to this infection.
Early Induction of the Tl Pathway After Infection with Zygomycetes Is Not Sufficient to Protect Against Zygomycetes-Induced Mortality in WT Fruit Flies.
Importantly, some bacterial pathogens suppress the immune response during the early stages of infection in D. melanogaster by limiting antimicrobial peptide gene expression (25). To test whether this was also the case during host-Zygomycetes interaction, we studied the comparative kinetics of Drs and Mtk gene expression in WT flies after injection with Zygomycetes spores compared with injection with A. fumigatus, which is not pathogenic in WT fruit flies (10). We found that expression of Drs and Mtk mRNA was increasingly induced at 1, 6, 12, and 24 h after injection of R. oryzae in WT flies and to a similar degree after injection of A. fumigatus (Fig. 3 A). In addition, Drs and Mtk mRNA were significantly induced 12 h after infection of WT flies with different Zygomycetes species (Fig. 3 B). These findings indicate that Zygomycetes do not suppress the induction of major antifungal peptides in the early stages of infection in D. melanogaster.
Fig. 3.
Induction of Drosomycin (Drs) and Metchnikowin (Mtk) mRNA in WT flies infected with Zygomycetes vs. A. fumigatus assessed by RT-PCR. (A) Normalized expression of Drs and Mtk mRNA in WT flies after infection with R. oryzae and A. fumigatus (Af293) at different time points (1, 6, and 12 h). (B) Normalized expression of Drs and Mtk mRNA in WT flies 12 h after infection with C. bertholletiae, M. circinelloides, and A. fumigatus (Af293). Relative mRNA expression is reported compared with that induced by aseptic injury in control WT flies.
D. melanogaster S2 Phagocytic Cells Exhibit Decreasing Rates of Phagocytosis and Hyphal Damage of Zygomycetes Compared with A. fumigatus.
Phagocytic cell responses have an increasingly recognized role in D. melanogaster defense against invading pathogens (9). Furthermore, D. melanogaster S2 phagocytic cells have considerable similarities with human phagocytic cells, and S2 cells are thus used to study phagocytosis of C. albicans (16). We used S2 cells to determine whether defective cellular immune responses to Zygomycetes partially account for the higher pathogenicity of these fungi when compared with A. fumigatus in fruit flies. Light and confocal microscopy imaging studies showed that S2 cells engulf Zygomycetes spores [Fig. 4 A and B and 3D animation (Movie S1)] and attach avidly to hyphae within 15 min of exposure (Fig. 4 C and Fig. S2A ). However, the rate of phagocytosis of Zygomycetes spores was significantly lower than that of A. fumigatus spores (median rates of phagocytosis at 2 h, 40% vs. 67%; P < 0.0001) (Fig. 4 B). The fact that Zygomycetes spores are much larger than A. fumigatus spores (1) may provide a mechanistic explanation for the differences in the rates of phagocytosis and virulence of these fungi in D. melanogaster (26).
Fig. 4.
S2 D. melanogaster phagocytic cell ex vivo assays. (A) The D. melanogaster S2 embryonic phagocytic cell line engulfs GFP expressing R. oryzae spores. (B) Percentage of phagocytosis of A. fumigatus (Af293) and R. oryzae spores by S2 cells; P = 0.01 for phagocytosis rates of A. fumigatus vs. R. oryzae spores at 0.5 h (*) and 1 h (**); P < 0.0001 for phagocytosis rates of A. fumigatus vs. R. oryzae spores at 2 h (***). (C) S2 cell attachment to GFP expressing R. oryzae hyphae 30 min after exposure. (D) Percentage of damage induced by S2 cells in hyphae of A. fumigatus (Af293) and R. oryzae with or without preexposure to dexamethasone (Dexa; 100 μM) assessed by the XTT assay; (*) P = 0.0022; (**) P < 0.0001. (E) Effect of dexamethasone (Dexa;100 μM) on the rate of phagocytosis of A. fumigatus and R. oryzae FITC-labeled spores by S2 cells, assessed by flow cytometry. The results of one representative experiment of three performed are shown.
Switching from the conidial (spore) to the hyphal stage of growth is a key virulence mechanism in molds that facilitates invasive fungal growth into the host tissues and dissemination of infection (8). Professional phagocytes rapidly recognize and eliminate hyphae, preventing infection in immunocompetent hosts. Therefore, we next sought to determine whether the ability of S2 cells to cause hyphal damage to Zygomycetes as compared with A. fumigatus correlates with the differences in virulence of these fungi in D. melanogaster. We found that hyphae of both R. oryzae and C. bertholletiae were significantly more resistant to damage by S2 cells than were hyphae of a clinical isolate of A. fumigatus (Af293) according to an assay based on the colorimetric reduction of XTT to formazan derivatives by metabolically active hyphal cells (P < 0.0001) (Fig. 4 D and Fig. S2C ) and by immunofluorescence studies using the cellular morbidity dye DiBAC (Fig. S2B ). In agreement with these findings, a recent study showed that S2 cells are capable of efficient killing of C. albicans, which is similar to A. fumigatus nonpathogenic in D. melanogaster (27). These ex vivo studies imply that less efficient phagocytic cell responses partially account for increased virulence of Zygomycetes compared with that of nonpathogenic fungi such as A. fumigatus in D. melanogaster.
Dexamethasone Attenuates Hyphal Damage and Blocks Phagocytosis of Fungi by D. melanogaster S2 Cells in Vitro.
Corticosteroids cause multiple immunosuppressive effects in human phagocytic cell function in vivo and in vitro (28). We studied the effect of corticosteroids in effector activity of S2 cells against both Zygomycetes and A. fumigatus. Exposure of S2 cells to dexamethasone resulted in a significant decrease in their ability to induce damage in hyphae of R. oryzae and A. fumigatus (Fig. 4 D). In addition, dexamethasone (100 μM) completely inhibited phagocytosis of both Rhizopus and Aspergillus FITC-labeled spores by S2 cells (Fig. 4 E). These results further validate S2 cells as a tool to study cellular immune responses against fungi in vitro.
Eater Null Flies Exhibit Increased Susceptibility to Zygomycetes Infection.
Eater is a recently identified scavenger receptor in Drosophila macrophages capable of recognizing a broad spectrum of bacterial pathogens and yeasts (29). Flies lacking the eater gene displayed normal humoral immune responses but showed decreased survival to bacterial infection (29). We found that eater null flies exhibited increased susceptibility to R. oryzae as compared with control WT flies (median survival rates 2 days vs. 4 days, respectively, P = 0.01; Fig. S3). Overall, these in vivo studies further validate the important role of cellular immune responses in fly defense to Zygomycetes infection.
Whole-Genome Profiling in Fruit Flies Infected with Zygomycetes vs. A. fumigatus Reveals Candidate Genes with Important Roles in Zygomycetes Pathogenicity.
To gain insights into the global host immune response of D. melanogaster after infection with Zygomycetes, we performed a whole-genome expression analysis of WT fruit flies 12 h after infection with R. oryzae or A. fumigatus; flies with sterile injury (mock-inoculated) and untreated (naïve) flies served as controls. We selected the 12-h time point after infection to analyze molecular events of fly immune response during the initial stages of invasive fungal growth. Analysis of genomic data were performed based on a recent study of Pseudomonas pathogenesis (25), focusing on genes whose induction was selectively up-regulated (≥1.5-fold) or down-regulated (≥1.5-fold) after infection with R. oryzae (pathogenic) or A. fumigatus (nonpathogenic). The complete list of genes (n = 98) induced by Rhizopus vs. aseptic injury control is provided in Table S1. We found that 54 genes were differentially induced in flies injected with R. oryzae vs. those injected with A. fumigatus (Table S2, Fig. S4).
Of the down-regulated genes pertaining to humoral immunity, immune molecule 23 and immune molecule 10 encode for small immune-inducible peptides under the transcriptional control of the Tl pathway (25, 30), and CG18594 accounts for an immune-induced protein homologous to a mammalian serpin with a regulatory role in the mitogen-activated protein kinase and nuclear factor κB signaling pathways (32). Of the three down-regulated genes related to pathogen recognition, CG13422 is predicted to encode a GNBP-soluble receptor (30). The down-regulated genes also included Hsp70-Ab, which has pleiotropic functions in stress responses; the odorant receptor 47a (Or47a); and MtnD (metallothionein D) and CG9897, which are involved in detoxification.
Notably, we found that a striking fraction of down-regulated genes (n = 13) function in skeletal muscle repair and tissue reconstruction (Table S2); researchers recently reported down-regulation of some of these genes (CG18255, CG7216, CG10297, CG5494, CG1919, and CG10287) after infection with a highly pathogenic Pseudomonas aeruginosa species (25). In support to our genomic data, a recent study in Drosophila showed that genes involved in skeletal muscle repair are regulated by the cJun-N-terminal Kinase pathway and play an important role in increased susceptibility to infection by Pseudomonas both in flies and mammals (33).
In comparison, we found that induction of a smaller number of genes (n = 18 [33%]) were selectively up-regulated by R. oryzae vs. A. fumigatus. Most of these genes (n = 12 [67%]) are immune-induced (27, 30, 31, 34–36). For example, Turandot M and Turandot C are immune-inducible peptides under the transcriptional control of the JAK/STAT pathway, which plays a pivotal role in stress responses and immunity (34). In addition, Mthl12 (methuselah-like 12) and Fst (Frost), which act in aging and cold responses respectively, are strongly linked with immunity in D. melanogaster (25, 30, 37). Finally, two other up-regulated genes, Uro (urate oxidase) and CG11669 (alpha-galactosidase) have a role in carbohydrate metabolism.
Overall, infection of D. melanogaster with R. oryzae suppressed the induction of several genes involved in host defense and an array of other important cellular repair functions. Notably, our genomic studies confirmed that Drs and Mtk were significantly induced after infection with Zygomycetes (Table S1). Finally, our transcriptome data are in line with similar studies of the D. melanogaster transcriptome during host–pathogen interactions (25).
Discussion
In this study, we developed a simple, inexpensive, and robust model of zygomycosis using injection of Zygomycetes spores into the fly hemolymph. Remarkably, our experiments indicated that, as opposed to other medically important fungi, Zygomycetes are able to cause fulminant infections not only in Tl-mutant but also in WT D. melanogaster.
Notably, our Drosophila model simulates important pathophysiological aspects of zygomycosis in humans. We found considerable similarities in the patterns of infection and factors enhancing the severity of zygomycosis in both D. melanogaster and humans. First, C. bertholletiae, the most pathogenic Zygomycetes species in humans (3), exhibited the highest degree of pathogenicity in our model. Second, classic enhancers of Zygomycetes virulence in humans, such as administration of corticosteroids, increased iron supply, and increased iron availability resulting from treatment with the iron chelator deferoxamine, enhanced the lethality of zygomycosis in our model. Third, treatment with another iron chelator (deferasirox) that induces iron starvation for the fungus and protects mice from zygomycosis significantly improved survival of Zygomycetes-infected flies. Overall, these results indicate that Zygomycetes use common virulence strategies for invading evolutionarily disparate organisms such as D. melanogaster and humans and establish D. melanogaster as a relevant model for studying the pathobiology of zygomycosis.
In an effort to understand key molecular aspects of the immunopathogenesis of zygomycosis in D. melanogaster, we initially examined the role of Tl signaling in fly defense against Zygomycetes. We found that Tl-mutant flies exhibited increased susceptibility to Zygomycetes infection, which was partially restored by constitutive overexpression of the Tl-dependent antifungal peptide Drs in transgenic flies. These studies reveal the important role of Tl pathway activation in fly immune response to Zygomycetes.
We then tested whether, similar to other microbial pathogens, early induction of the Tl pathway is suppressed by Zygomycetes (25, 38, 39). By studying the comparative kinetics of mRNA induction of the two major antifungal peptides, Drs and Mtk, which are under the control of the Tl pathway, we found that the Tl pathway was rapidly and efficiently activated by Zygomycetes and A. fumigatus to a comparable degree. Nonetheless, we cannot exclude selective degradation of antifungal peptides in D. melanogaster by Zygomycetes toxins (40), or that Zygomycetes spores might be less susceptible to the effects of D. melanogaster antifungal peptides than A. fumigatus spores (41).
Next, we examined whether defects in cellular immune responses in D. melanogaster are associated with the increased pathogenicity of Zygomycetes. Importantly, the increased virulence of some entomopathogenic fungi in insects is linked to impaired phagocytosis of fungal spores (26). Similarly, we found that D. melanogaster S2 phagocytic cells exhibited impaired activity against Zygomycetes as evidenced by decreased rates of conidial phagocytosis and hyphal damage when compared with that against A. fumigatus. Furthermore, in agreement with in vitro studies in human phagocytic cells (28), dexamethasone inhibited phagocytosis of Rhizopus and Aspergillus spores by S2 cells and attenuated their ability to induce hyphal damage. Little is known about the molecular mechanisms of action of dexamethasone in insects. Previous studies demonstrated that the immunosuppressive effect of dexamethasone in cellular immunity of insects depended on inhibition of phospholipase A2 (PLA2) and was reversed by administration of arachidonic acid (23, 24, 43). Nonetheless, similar to mammals, it is plausible that corticosteroids act by inhibiting multiple pathways of insect innate immunity. Overall, our studies validate the use of S2 cells to study cellular immune responses against fungi in vitro. Importantly, the availability of high-throughput genetic screens in D. melanogaster S2 cells using RNA interference technology makes it possible to identify genes involved in Zygomycetes recognition, phagocytosis, and hyphal damage feasible (16).
We further assessed the role of cellular immunity in Drosophila defense to zygomycosis by infecting eater null flies with Zygomycetes. Importantly, because eater flies possess normal humoral immune responses (29), their use enabled us to address the specific role of phagocytosis in vivo. We found that eater flies displayed increased susceptibility to zygomycosis, further validating the important role of cellular immune responses in fly defense to Zygomycetes infection.
Finally, we took advantage of the powerful genomics of D. melanogaster to gain insights into the molecular aspects of global host immune response during the early stages of host–Zygomycetes interaction. Because the innate immune defense genes in D. melanogaster are well characterized for an array of pathogens (27, 30, 31, 34), we focused our analysis on genes with specific roles in Zygomycetes pathogenicity by comparing the transcriptomes of flies infected with R. oryzae with those of flies infected with A. fumigatus. We identified 54 genes with potential roles in Zygomycetes pathogenicity. The induction of two-thirds of those genes was down-regulated in response to R. oryzae infection and included genes acting in distinct aspects of innate immunity but also other important cellular functions, such as global stress response, detoxification, steroid and iron metabolism, cytoskeleton reconstruction, and tissue repair. Our genomic findings complement those of a previous study by Apidianakis et al. (25) of flies infected with virulent and nonvirulent Pseudomonas strains. Of particular interest is that most of these genes have homologues in humans and encode functions relevant to the pathophysiology of zygomycosis (Table S1). For example, a clinical observation regarding zygomycosis that deserves explanation is the propensity of Zygomycetes for angioinvasion and tissue necrosis (1, 2). The fact that a vast proportion (37%) of genes whose induction is down-regulated by R. oryzae encode for tissue repair may provide the molecular framework for unraveling the virulence attributes that enable this fungus to invade host tissues.
To our knowledge, our study represents a previously undescribed whole-genome and whole-organism expression analysis on Zygomycetes pathogenesis. Although we cannot preclude that important genes induced during earlier time points of Zygomycosis infection in Drosophila might have been missed, our genomic data identify a dynamic network of host-defense functions and reveal D. melanogaster genes that respond to fungal challenge. In addition, our genomic studies demonstrate that host factors involved in the infection process are not restricted to Tl-pathway-related immunity genes. Given the high degree of molecular and mechanistic conservation between the D. melanogaster and human innate immune systems (9), our results also provide insights into molecular aspects of the pathobiology of Zygomycetes in humans and should help in designing targeted therapeutic strategies for zygomycosis.
Methods
D. melanogaster Stocks.
OregonR flies were used as WT flies. Tl-deficient Tl r632/TlI−RXA transheterozygote mutants (Tl-mutant flies) were generated as described in ref. 17. For Drs constitutive expression, flies overexpressing Gal4 under the ubiquitous promoter daughterless were crossed to transgenic strains carrying Drs coding sequences under the control of upstream activating sequence enhancer elements (da-Gal4/U-Drs). eater-null transheterozygote mutants were generated as described in ref. 29. Standard procedures for manipulation, feeding, and housing of the flies were used in all experiments (10, 17, 18).
Zygomycetes Strains and Infection Model.
Three clinical Zygomycetes isolates (Mucor circinelloides 424760, R. oryzae 557969, and C. bertholletiae 506313) and the clinical A. fumigatus isolate Af293, were used in virulence studies (17). GFP-R. oryzae has been described (21).
WT and Tl-mutant flies were initially infected by injecting them with a thin sterile needle previously dipped in a concentrated solution of Zygomycetes (or A. fumigatus) spores (106-108 spores/ml; the optimized inoculum of 108 spores/ml, corresponding to ≈800 per fly, was then used in all further studies) (10, 15, 16). After injection, the flies were housed at 29°C on standard fly medium and transferred to fresh vials every 2 days.
Virulence studies were performed as described in SI Text .
Histopathological Analysis.
On day 1 after infection with Zygomycetes, fruit flies were fixed with 10% (vol/vol) formaldehyde, processed, and embedded in paraffin wax. Matched tissue sections were stained with crystal violet (in pilot experiments, we observed that crystal violet stained better hyphae of Zygomycetes as compared with other classic fungal stains, including Gomori methenamine-silver stain and H&E), and representative sections were examined for visible fungal burden under a light microscope.
Phagocytosis Assays Using a D. melanogaster S2 Embryonic Phagocytic Cell Line.
A D. melanogaster S2 embryonic phagocytic cell line was used for in vitro phagocytosis and hyphal killing assays (16). D. melanogaster S2 cells were grown in Schneider's medium (Invitrogen) supplemented with 10% heat-inactivated FBS, penicillin, and streptomycin.
The rate of phagocytosis of R. oryzae and A. fumigatus spores by S2 cells was visually assessed under a light microscope at different time points (0.5, 1, and 2 h) as described in ref. 42. Phagocytosis was recorded by counting the number of S2 cells containing fungal spores out of 100 cells in triplicate for each sample.
The effect of dexamethasone on phagocytosis of R. oryzae and A. fumigatus spores by S2 cells was evaluated by using flow cytometry. R. oryzae and A. fumigatus spores were first incubated with FITC (3 μg/ml; Sigma) overnight at 4°C and then washed extensively with PBS. S2 cells (105) were preexposed to dexamethasone (100 μM) for 1 h and subsequently challenged with FITC-labeled spores (ratio, 1:1) for 30 min at 29°C. S2 cells unexposed to dexamethasone served as controls. After a wash, rate of phagocytosis was measured by flow cytometric analysis. Flow cytometry was performed on a FACSCalibur (BD Biosciences).
Hyphal damage assays using D. melanogaster S2 cell line were performed as described in SI Text .
Imaging by Confocal Microscopy.
S2 cells were coincubated with GFP expressing R. oryzae spores or hyphae for 30 min. Imaging was performed on fixed S2 cells stained with Alexa 647-conjugated phalloidin (red; Molecular Probes) and DAPI (blue; Molecular Probes) using a Leica SP2 RS laser-scanning confocal microscope with an oil-immersion objective (Leica ×63/1.4 numerical aperture).
RNA extraction and RT-PCR analysis of Drs and Mtk mRNA and fungal burden analysis by RT-PCR were performed as described in SI Text .
Whole-Genome Expression Profiling Studies.
Drosophila 2.0 Affymetrix microarrays were used for whole-genome expression analysis. Briefly, the dChip software (www.dchip.org) was applied in processing the raw data contained in CEL files. The Li-Wong model using only the perfect-match probes was used to obtain the gene expression indexes. Probe sets with low expression in the background noise region or little variation across all samples (mean expression ≤79 or standard error ≤9) were eliminated. To assess the variance structure of the data, an unsupervised hierarchical clustering analysis based on Pearson correlation was performed to cluster samples by using these probe sets. To identify differentially expressed genes in fruit flies infected with A. fumigatus or R. oryzae, we used the following criteria, as proposed elsewhere (27): (i) the fold change of mean expression value of each gene between either A. fumigatus and injury or R. oryzae and injury ≥1.5 and (ii) the fold change of mean expression value of each gene between A. fumigatus and R. oryzae ≥ 1.5. Fifty-four probe sets were determined to be differentially expressed by meeting both of these criteria. Hierarchical clustering analysis based on Pearson correlation was performed on the 54 probe sets.
Acknowledgments
We thank Y. Apidianakis (Harvard Medical School, Boston) for useful comments and the U-Drs transgenic flies; T. Y. Ip (University of Massachusetts Medical School, Worcester, MA) for Tl fly mutants; C. Kocks (Harvard Medical School, Boston) for eater mutants; A. S. Ibrahim for the GFP Rhizopus strain; and N. D. Albert for excellent technical assistance. This work was supported by grants from the University of Texas M. D. Anderson Cancer Center [institutional research grant and M. D. Anderson Faculty E. N. Cobb Scholar Award Research Endowment (D.P.K.)].
Footnotes
- ‖To whom correspondence should be addressed. E-mail: dkontoyi{at}mdanderson.org
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Author contributions: G.C., G.H., and D.P.K. designed research; G.C. and R.E.L. performed research; T.Z., M.G., and G.H. contributed new reagents/analytic tools; G.C., R.E.L., J.H., L.X., T.Z., and M.G. analyzed data; and G.C. and D.P.K. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0709578105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
References
Drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis
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