Drosophila melanogaster as a model host to dissectthe immunopathogenesis of zygomycosisGeorgios Chamilos*, Russell E. Lewis*†, Jianhua Hu‡, Lianchun Xiao‡, Tomasz Zal§, Michel Gilliet§, Georg Halder¶,and Dimitrios P. Kontoyiannis*†�

Departments of *Infectious Diseases, Infection Control and Employee Health, ‡Bioinformatics, and Computational Biology, and §Immunology and¶Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; and †College of Pharmacy,University of Houston, Houston, TX 77030

Edited by Frederick M. Ausubel, Harvard Medical School, Boston, MA, and approved April 14, 2008 (received for review October 8, 2007)

Zygomycosis is an emerging frequently fatal opportunistic mycosiswhose immunopathogenesis is poorly understood. We developeda zygomycosis model by injecting Drosophila melanogaster flieswith a standardized amount of fungal spores from clinical Zygo-mycetes isolates to study virulence and host defense mechanisms.We found that, as opposed to most other fungi, which are non-pathogenic in D. melanogaster (e.g., Aspergillus fumigatus), Zygo-mycetes rapidly infect and kill wild-type flies. Toll-deficient fliesexhibited increased susceptibility to Zygomycetes, whereas con-stitutive overexpression of the antifungal peptide Drosomycin intransgenic flies partially restored resistance to zygomycosis. D.melanogaster Schneider 2 phagocytic cells displayed decreasedphagocytosis and caused less hyphal damage to Zygomycetescompared with that to A. fumigatus. Furthermore, phagocytosis-defective eater mutant flies displayed increased susceptibility toZygomycetes infection. Classic enhancers of Zygomycetes viru-lence in humans, such as corticosteroids, increased iron supply, andiron availability through treatment with deferoxamine dramati-cally increased Zygomycetes pathogenicity in our model. In con-trast, iron starvation induced by treatment with the iron chelatordeferasirox significantly protected flies infected with Zygomyce-tes. Whole-genome expression profiling in wild-type flies afterinfection with Zygomycetes vs. A. fumigatus identified genesselectively down-regulated by Zygomycetes, which act in patho-gen 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. melano-gaster as a promising model to study this important mycosis.

animal models � Rhizopus � Toll receptor � zygomycetes � innate immunity

Fungi of the class Zygomycetes, order Mucorales, are significantcauses of life-threatening angioinvasive infections in patients

with a wide range of immunosuppressive conditions and, occasion-ally, immunocompetent individuals (1, 2). Rhizopus species causethe majority of Zygomycetes infections, whereas Mucor, Rhizomu-cor, and Cunninghamella bertholletiae are less frequently encoun-tered pathogens (1, 2). C. bertholletiae is considered the mostpathogenic Zygomycetes species in humans (3).

Once thought to be an uncommon infection, zygomycosis hasrecently emerged as the second most common opportunistic inva-sive mold infection after aspergillosis in patients with hematologicalmalignancies and transplant recipients (2, 4–6). Zygomycosis has aparticularly poor prognosis in these patients, with mortality rates�90% in disseminated infection (3, 5, 6).

Quantitative and functional defects in immune effector cellsassociated with poorly controlled diabetes mellitus and receipt ofcorticosteroids or other immunosuppressive treatments are theprincipal predisposing factors for zygomycosis (1, 2). In addition,iron metabolism plays a central role in the pathobiology of zygo-mycosis. Thus, patients with elevated serum iron levels are atincreased risk for zygomycosis (1–3), and treatment with deferox-amine, an iron-chelating agent that acts as a siderophore and

supplies iron to Zygomycetes, promotes the development of severedisseminated infections in animal models and humans (7). How-ever, unlike other medically important fungi (8), the epidemiologyand immunopathogenesis of zygomycosis are poorly understood(2). Furthermore, mammalian models represent a bottleneck forlarge-scale genomic studies on microbial pathogenesis because ofethical considerations and logistic restraints associated with theiruse, the complexity of their immune systems, and difficulties instudying the dynamics of host–pathogen interaction in vivo (9).

We hypothesized that Drosophila melanogaster, a simple genet-ically amenable minihost with well characterized and evolutionarilyconserved innate immunity, could serve as a suitable model forstudying the immunopathogenesis of zygomycosis. D. melanogasteris capable of mounting efficient innate immune responses against avariety of fungal pathogens largely mediated by induction of theevolutionarily conserved Toll (Tl) pathway (9–12). Hence, uponchallenge by fungi, Tl-pathway activation leads to rapid and selec-tive induction of antifungal peptides, mainly Drosomycin (Drs) andMetchnikowin (Mtk), into the fly hemolymph and allows D. mela-nogaster to successfully combat infection by most fungal invaders(9–12). In addition, researchers have increasingly recognized thatthe D. melanogaster cellular immune response, comprising hemo-cytes circulating in the hemolymph, plays an instrumental role inearly recognition and elimination of bacterial and fungal pathogens(9, 13, 14). Specifically, D. melanogaster Schneider 2 (S2) embryonicphagocytic cells share many characteristics with mammalian phago-cytic cells, and S2 RNA interference libraries have been used toidentify evolutionarily conserved genes involved in phagocytosis ofbacteria (15) and Candida albicans (16).

In the present study, we developed a zygomycosis model byinjecting D. melanogaster flies with a standardized amount ofZygomycetes spores. We found that, as opposed to other fungi,Zygomycetes rapidly infect and kill D. melanogaster WT fliesdespite early activation of the Tl pathway. In addition, comparativestudies using the D. melanogaster S2 phagocytic cell line, thephagocytosis-defective eater-null fly mutant and transcriptomeanalysis in flies infected with Zygomycetes vs. A. fumigatus dem-onstrated that the pathogenicity of Zygomycetes is linked withimpaired phagocytic cell activity and suppression of induction ofgenes 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 anattractive model for studying immunopathogenesis of zygomycosis.

Author contributions: G.C., G.H., and D.P.K. designed research; G.C. and R.E.L. performedresearch; 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.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

�To whom correspondence should be addressed. E-mail: dkontoyi@mdanderson.org.

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

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ResultsZygomycetes Rapidly Infect and Kill WT D. melanogaster. Previousstudies in D. melanogaster demonstrated that injection of variousfungi into the hemolymph of WT flies did not induce mortality(10–12). In contrast with these studies and our previous experiencewith A. fumigatus (17) and Candida species (18), injection of astandardized amount of spores of Zygomycetes clinical isolates intoWT flies resulted in acute infection and high mortality rates (Figs.1 A and B), whereas injection of A. fumigatus into WT flies did notsignificantly affect survival regardless of the inoculum size tested(Fig. 1B). As evidenced by histopathological studies, Zygomycetesspores injected into the fly hemolymph germinated rapidly to formhyphae that subsequently disseminated and invaded multiple struc-tures of the fly body, leading to death (Fig. 1C). Fungal burden,determined by real-time PCR (RT-PCR), increased significantlyover time, exceeding 4 logs 24 h after infection of WT flies withRhizopus (Fig. 1D).

Injection of Tl-mutant fruit flies, with either Rhizopus or Mucorspp. resulted in a higher mortality rate at 48 h (95%) whencompared with injection of WT fruit flies (65%; P � 0.004) [Fig.1A and supporting information (SI) Fig. S1A]. As a furtherindication of the role of the Toll pathway in fly defense againstZygomycetes, transgenic flies constitutively expressing the antifun-gal peptide Drosomycin (Drs) were significantly less susceptible toZygomycetes infection when compared with control flies (survivalrate at 48 h 63% vs. 0%; P � 0.0001) (Fig. 1E). Interestingly, C.bertholletiae infection caused hyperacute mortality in both WT andTl-mutant flies, with all flies dying of the infection within 48 h (Fig.1B). Finally, survival of both WT and Tl-mutant flies was inoculum-dependent (Fig. 1F). These experiments thus established an easyreproducible model of zygomycosis and demonstrated that Zygo-

mycetes are among the few reported fungi that are pathogenic inWT D. melanogaster.

Clinically Important Potentiators of Zygomycetes Virulence in Humansalso Increase Zygomycetes Pathogenicity in Fruit Flies. We nextexamined ‘‘classic’’ enhancers of Zygomycetes virulence in humansto determine how they affect survival of WT fruit flies infected witha representative Rhizopus oryzae clinical isolate. Importantly, ironacquisition from the host plays a pivotal role in successful infectionby Zygomycetes in humans and animal models (1–3). Because ironmetabolism in D. melanogaster has many similarities with that inmammals (19, 20), we hypothesized that increased iron availabilityalso modulates pathogenesis of zygomycosis in fruit flies.

Indeed, we found that WT flies fed with deferoxamine andinfected with R. oryzae (108 spores/ml) developed rapidly dissem-inated infections with higher fungal burdens upon histopathology(Fig. 2A) and a significantly higher mortality rate when comparedwith infected control flies (no deferoxamine) (100% vs. 55% at 24 h;P � 0.0001) (Fig. 2B). Also, WT flies fed with iron (FeCl3) hadincreased susceptibility to zygomycosis when compared with con-trol flies (no iron) (mortality rate at 48 h: 100% vs. 65%; P � 0.04)(Fig. 2B).

Importantly, deferasirox is a iron chelator that, in contrast todeferoxamine, induces iron starvation to Zygomycetes and im-proves survival in a murine model of zygomycosis (21). We foundthat 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. 2B). Thus, D. melanogaster couldserve as a valuable model to study the role of iron metabolismduring host–Zygomycetes interactions.

Corticosteroids have a wide range of complex immunosuppres-sive effects, mainly by affecting cellular immunity, and increase host

Fig. 1. Infection of WT, Toll mutant (Tl), and Drsoverexpressing (U-Drs) flies with clinical isolates of Zy-gomycetes. (A) Survival rates of Tl and WT flies infectedby injection (108 spores per ml) of R. oryzae or M.circinelloides; P � 0.004 for WT vs. Tl flies infected withR. oryzae or M. circinelloides; P � nonsignificant forother comparisons. (B) Survival rates of Tl-mutant andWT flies infected with C. bertholletiae (108 spores perml); infection of WT flies with A. fumigatus clinicalisolate Af293 (range of inoculum, 108 to 109 spores perml) served as control; P � 0.0001 for WT flies infectedwith A. fumigatus vs. C. bertholletiae; P � nonsignifi-cant for WT vs. Tl flies infected with C. bertholletiae. (C)Representative histopathological sections (stainedwith crystal violet) from the thorax and abdomen of aWT fly 24 h after infection with R. oryzae (D) Measure-ment of fungal burden at different time points afterinfection of WT and Tl-mutant flies with R. oryzae (108

spores per ml), assessed by qPCR; fungal burden isexpressed as conidial equivalents of DNA (E) Survivalrates 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 in-fection with different inocula of R. oryzae; P � 0.0001for heat-killed (HK) R. oryzae spores vs. 106 R. oryzaespores per ml and 106 R. oryzae spores per ml vs. 107 R.oryzae spores per ml; P � 0.05 for 107 vs. 108 R. oryzaespores per ml.

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susceptibility to invasive fungal infections in humans (8, 22). Sim-ilarly, treatment with corticosteroids in invertebrates results inimpaired cellular immune responses and increased susceptibility toboth bacteria and entomopathogenic fungi (23, 24). We found thatWT flies fed with dexamethasone and infected with R. oryzae hadsignificantly higher mortality rates than did infected control flies(no dexamethasone) (P � 0.02; Fig. S1B). Therefore, receipt ofcorticosteroids, a major risk factor for zygomycosis in humans, alsoincreased the susceptibility of flies to this infection.

Early Induction of the Tl Pathway After Infection with Zygomycetes IsNot Sufficient to Protect Against Zygomycetes-Induced Mortality inWT Fruit Flies. Importantly, some bacterial pathogens suppress theimmune response during the early stages of infection in D. mela-nogaster by limiting antimicrobial peptide gene expression (25). Totest whether this was also the case during host-Zygomycetes inter-action, we studied the comparative kinetics of Drs and Mtk geneexpression in WT flies after injection with Zygomycetes sporescompared with injection with A. fumigatus, which is not pathogenicin WT fruit flies (10). We found that expression of Drs and MtkmRNA was increasingly induced at 1, 6, 12, and 24 h after injectionof R. oryzae in WT flies and to a similar degree after injection ofA. fumigatus (Fig. 3A). In addition, Drs and Mtk mRNA weresignificantly induced 12 h after infection of WT flies with differentZygomycetes species (Fig. 3B). These findings indicate that Zygo-mycetes do not suppress the induction of major antifungal peptidesin the early stages of infection in D. melanogaster.

D. melanogaster S2 Phagocytic Cells Exhibit Decreasing Rates ofPhagocytosis and Hyphal Damage of Zygomycetes Compared with A.fumigatus. Phagocytic cell responses have an increasingly recog-nized role in D. melanogaster defense against invading pathogens(9). Furthermore, D. melanogaster S2 phagocytic cells have consid-

erable similarities with human phagocytic cells, and S2 cells are thusused to study phagocytosis of C. albicans (16). We used S2 cells todetermine whether defective cellular immune responses to Zygo-mycetes partially account for the higher pathogenicity of these fungiwhen compared with A. fumigatus in fruit flies. Light and confocalmicroscopy imaging studies showed that S2 cells engulf Zygomy-cetes spores [Fig. 4 A and B and 3D animation (Movie S1)] andattach avidly to hyphae within 15 min of exposure (Fig. 4C andFig. S2A). However, the rate of phagocytosis of Zygomycetes sporeswas significantly lower than that of A. fumigatus spores (medianrates of phagocytosis at 2 h, 40% vs. 67%; P � 0.0001) (Fig. 4B). Thefact that Zygomycetes spores are much larger than A. fumigatusspores (1) may provide a mechanistic explanation for the differ-ences in the rates of phagocytosis and virulence of these fungi in D.melanogaster (26).

Switching from the conidial (spore) to the hyphal stage of growthis a key virulence mechanism in molds that facilitates invasivefungal growth into the host tissues and dissemination of infection(8). Professional phagocytes rapidly recognize and eliminate hy-phae, preventing infection in immunocompetent hosts. Therefore,we next sought to determine whether the ability of S2 cells to causehyphal damage to Zygomycetes as compared with A. fumigatuscorrelates 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 cellsthan were hyphae of a clinical isolate of A. fumigatus (Af293)according to an assay based on the colorimetric reduction of XTTto formazan derivatives by metabolically active hyphal cells (P �0.0001) (Fig. 4D and Fig. S2C) and by immunofluorescence studiesusing the cellular morbidity dye DiBAC (Fig. S2B). In agreementwith these findings, a recent study showed that S2 cells are capableof efficient killing of C. albicans, which is similar to A. fumigatusnonpathogenic in D. melanogaster (27). These ex vivo studies implythat less efficient phagocytic cell responses partially account for

Fig. 2. Effects of increased iron supply and availability through treatmentwith deferoxamine and iron starvation through treatment with deferasirox inWT flies infected with R. oryzae. (A) Representative histopathological sectionsof deferoxamine-treated and untreated control WT flies 24 h after infectionwith 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. untreatedcontrol flies; P � 0.04 for iron-treated vs. untreated control flies.

Fig. 3. Induction of Drosomycin (Drs) and Metchnikowin (Mtk) mRNA in WTflies infected with Zygomycetes vs. A. fumigatus assessed by RT-PCR. (A) Normal-ized expression of Drs and Mtk mRNA in WT flies after infection with R. oryzaeand A. fumigatus (Af293) at different time points (1, 6, and 12 h). (B) Normalizedexpression of Drs and Mtk mRNA in WT flies 12 h after infection with C. berthol-letiae, M. circinelloides, and A. fumigatus (Af293). Relative mRNA expression isreported compared with that induced by aseptic injury in control WT flies.

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increased virulence of Zygomycetes compared with that of non-pathogenic fungi such as A. fumigatus in D. melanogaster.

Dexamethasone Attenuates Hyphal Damage and Blocks Phagocytosisof Fungi by D. melanogaster S2 Cells in Vitro. Corticosteroids causemultiple immunosuppressive effects in human phagocytic cell func-tion in vivo and in vitro (28). We studied the effect of corticosteroidsin effector activity of S2 cells against both Zygomycetes and A.fumigatus. Exposure of S2 cells to dexamethasone resulted in asignificant decrease in their ability to induce damage in hyphae ofR. oryzae and A. fumigatus (Fig. 4D). In addition, dexamethasone(100 �M) completely inhibited phagocytosis of both Rhizopus andAspergillus FITC-labeled spores by S2 cells (Fig. 4E). These resultsfurther validate S2 cells as a tool to study cellular immune responsesagainst fungi in vitro.

Eater Null Flies Exhibit Increased Susceptibility to Zygomycetes In-fection. Eater is a recently identified scavenger receptor in Dro-sophila macrophages capable of recognizing a broad spectrum ofbacterial pathogens and yeasts (29). Flies lacking the eater genedisplayed normal humoral immune responses but showed de-creased survival to bacterial infection (29). We found that eater nullflies exhibited increased susceptibility to R. oryzae as compared withcontrol WT flies (median survival rates 2 days vs. 4 days, respec-tively, P � 0.01; Fig. S3). Overall, these in vivo studies further

validate the important role of cellular immune responses in flydefense to Zygomycetes infection.

Whole-Genome Profiling in Fruit Flies Infected with Zygomycetes vs.A. fumigatus Reveals Candidate Genes with Important Roles in Zygo-mycetes Pathogenicity. To gain insights into the global host immuneresponse of D. melanogaster after infection with Zygomycetes, weperformed a whole-genome expression analysis of WT fruit flies12 h after infection with R. oryzae or A. fumigatus; flies with sterileinjury (mock-inoculated) and untreated (naıve) flies served ascontrols. We selected the 12-h time point after infection to analyzemolecular events of fly immune response during the initial stages ofinvasive fungal growth. Analysis of genomic data were performedbased on a recent study of Pseudomonas pathogenesis (25), focusingon genes whose induction was selectively up-regulated (�1.5-fold)or down-regulated (�1.5-fold) after infection with R. oryzae (patho-genic) or A. fumigatus (nonpathogenic). The complete list of genes(n � 98) induced by Rhizopus vs. aseptic injury control is providedin Table S1. We found that 54 genes were differentially induced inflies 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 smallimmune-inducible peptides under the transcriptional control of theTl pathway (25, 30), and CG18594 accounts for an immune-inducedprotein homologous to a mammalian serpin with a regulatory rolein the mitogen-activated protein kinase and nuclear factor �Bsignaling pathways (32). Of the three down-regulated genes relatedto pathogen recognition, CG13422 is predicted to encode a GNBP-soluble receptor (30). The down-regulated genes also includedHsp70-Ab, which has pleiotropic functions in stress responses; theodorant receptor 47a (Or47a); and MtnD (metallothionein D) andCG9897, which are involved in detoxification.

Notably, we found that a striking fraction of down-regulatedgenes (n � 13) function in skeletal muscle repair and tissuereconstruction (Table S2); researchers recently reported down-regulation of some of these genes (CG18255, CG7216, CG10297,CG5494, CG1919, and CG10287) after infection with a highlypathogenic Pseudomonas aeruginosa species (25). In support to ourgenomic data, a recent study in Drosophila showed that genesinvolved in skeletal muscle repair are regulated by the cJun-N-terminal Kinase pathway and play an important role in increasedsusceptibility to infection by Pseudomonas both in flies and mam-mals (33).

In comparison, we found that induction of a smaller number ofgenes (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 Turan-dot C are immune-inducible peptides under the transcriptionalcontrol of the JAK/STAT pathway, which plays a pivotal rolein stress responses and immunity (34). In addition, Mthl12(methuselah-like 12) and Fst (Frost), which act in aging and coldresponses 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 incarbohydrate metabolism.

Overall, infection of D. melanogaster with R. oryzae suppressedthe induction of several genes involved in host defense and an arrayof other important cellular repair functions. Notably, our genomicstudies confirmed that Drs and Mtk were significantly induced afterinfection with Zygomycetes (Table S1). Finally, our transcriptomedata are in line with similar studies of the D. melanogaster tran-scriptome during host–pathogen interactions (25).

DiscussionIn this study, we developed a simple, inexpensive, and robust modelof zygomycosis using injection of Zygomycetes spores into the flyhemolymph. Remarkably, our experiments indicated that, as op-

Fig. 4. S2 D. melanogaster phagocytic cell ex vivo assays. (A) The D. melano-gaster S2 embryonic phagocytic cell line engulfs GFP expressing R. oryzae spores.(B) Percentage of phagocytosis of A. fumigatus (Af293) and R. oryzae spores byS2cells;P�0.01forphagocytosis ratesofA. fumigatusvs.R.oryzae sporesat0.5h(*) and 1 h (**); P � 0.0001 for phagocytosis rates of A. fumigatus vs. R. oryzaespores at 2 h (***). (C) S2 cell attachment to GFP expressing R. oryzae hyphae 30min 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 �M) assessed by the XTT assay; (*) P � 0.0022; (**) P � 0.0001. (E) Effectof dexamethasone (Dexa;100 �M) on the rate of phagocytosis of A. fumigatusand R. oryzae FITC-labeled spores by S2 cells, assessed by flow cytometry. Theresults of one representative experiment of three performed are shown.

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posed to other medically important fungi, Zygomycetes are able tocause fulminant infections not only in Tl-mutant but also in WT D.melanogaster.

Notably, our Drosophila model simulates important pathophys-iological aspects of zygomycosis in humans. We found considerablesimilarities in the patterns of infection and factors enhancing theseverity of zygomycosis in both D. melanogaster and humans. First,C. bertholletiae, the most pathogenic Zygomycetes species in hu-mans (3), exhibited the highest degree of pathogenicity in ourmodel. Second, classic enhancers of Zygomycetes virulence inhumans, such as administration of corticosteroids, increased ironsupply, and increased iron availability resulting from treatment withthe iron chelator deferoxamine, enhanced the lethality of zygomy-cosis in our model. Third, treatment with another iron chelator(deferasirox) that induces iron starvation for the fungus andprotects mice from zygomycosis significantly improved survival ofZygomycetes-infected flies. Overall, these results indicate thatZygomycetes use common virulence strategies for invading evolu-tionarily disparate organisms such as D. melanogaster and humansand establish D. melanogaster as a relevant model for studying thepathobiology of zygomycosis.

In an effort to understand key molecular aspects of the immu-nopathogenesis of zygomycosis in D. melanogaster, we initiallyexamined the role of Tl signaling in fly defense against Zygomy-cetes. We found that Tl-mutant flies exhibited increased suscepti-bility to Zygomycetes infection, which was partially restored byconstitutive overexpression of the Tl-dependent antifungal peptideDrs in transgenic flies. These studies reveal the important role of Tlpathway 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 inductionof the two major antifungal peptides, Drs and Mtk, which are underthe control of the Tl pathway, we found that the Tl pathway wasrapidly and efficiently activated by Zygomycetes and A. fumigatus toa comparable degree. Nonetheless, we cannot exclude selectivedegradation of antifungal peptides in D. melanogaster by Zygomy-cetes toxins (40), or that Zygomycetes spores might be less suscep-tible to the effects of D. melanogaster antifungal peptides than A.fumigatus spores (41).

Next, we examined whether defects in cellular immune responsesin D. melanogaster are associated with the increased pathogenicityof Zygomycetes. Importantly, the increased virulence of someentomopathogenic fungi in insects is linked to impaired phagocy-tosis of fungal spores (26). Similarly, we found that D. melanogasterS2 phagocytic cells exhibited impaired activity against Zygomycetesas evidenced by decreased rates of conidial phagocytosis and hyphaldamage when compared with that against A. fumigatus. Further-more, in agreement with in vitro studies in human phagocytic cells(28), dexamethasone inhibited phagocytosis of Rhizopus and As-pergillus spores by S2 cells and attenuated their ability to inducehyphal damage. Little is known about the molecular mechanisms ofaction of dexamethasone in insects. Previous studies demonstratedthat the immunosuppressive effect of dexamethasone in cellularimmunity 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 thatcorticosteroids act by inhibiting multiple pathways of insect innateimmunity. Overall, our studies validate the use of S2 cells to studycellular immune responses against fungi in vitro. Importantly, theavailability of high-throughput genetic screens in D. melanogaster S2cells using RNA interference technology makes it possible toidentify genes involved in Zygomycetes recognition, phagocytosis,and hyphal damage feasible (16).

We further assessed the role of cellular immunity in Drosophiladefense to zygomycosis by infecting eater null flies with Zygomy-cetes. Importantly, because eater flies possess normal humoralimmune responses (29), their use enabled us to address the specific

role of phagocytosis in vivo. We found that eater flies displayedincreased susceptibility to zygomycosis, further validating the im-portant role of cellular immune responses in fly defense to Zygo-mycetes infection.

Finally, we took advantage of the powerful genomics of D.melanogaster to gain insights into the molecular aspects of globalhost immune response during the early stages of host–Zygomycetesinteraction. Because the innate immune defense genes in D. mela-nogaster are well characterized for an array of pathogens (27, 30, 31,34), we focused our analysis on genes with specific roles in Zygo-mycetes pathogenicity by comparing the transcriptomes of fliesinfected with R. oryzae with those of flies infected with A. fumigatus.We identified 54 genes with potential roles in Zygomycetes patho-genicity. The induction of two-thirds of those genes was down-regulated in response to R. oryzae infection and included genesacting in distinct aspects of innate immunity but also other impor-tant cellular functions, such as global stress response, detoxification,steroid and iron metabolism, cytoskeleton reconstruction, andtissue repair. Our genomic findings complement those of a previousstudy by Apidianakis et al. (25) of flies infected with virulent andnonvirulent Pseudomonas strains. Of particular interest is that mostof these genes have homologues in humans and encode functionsrelevant to the pathophysiology of zygomycosis (Table S1). Forexample, a clinical observation regarding zygomycosis that deservesexplanation is the propensity of Zygomycetes for angioinvasion andtissue necrosis (1, 2). The fact that a vast proportion (37%) of geneswhose induction is down-regulated by R. oryzae encode for tissuerepair may provide the molecular framework for unraveling thevirulence attributes that enable this fungus to invade host tissues.

To our knowledge, our study represents a previously undescribedwhole-genome and whole-organism expression analysis on Zygo-mycetes pathogenesis. Although we cannot preclude that importantgenes induced during earlier time points of Zygomycosis infectionin Drosophila might have been missed, our genomic data identify adynamic network of host-defense functions and reveal D. melano-gaster genes that respond to fungal challenge. In addition, ourgenomic studies demonstrate that host factors involved in theinfection process are not restricted to Tl-pathway-related immunitygenes. Given the high degree of molecular and mechanistic con-servation between the D. melanogaster and human innate immunesystems (9), our results also provide insights into molecular aspectsof the pathobiology of Zygomycetes in humans and should help indesigning targeted therapeutic strategies for zygomycosis.

MethodsD. melanogaster Stocks. OregonR flies were used as WT flies. Tl-deficient Tlr632/TlI�RXA transheterozygote mutants (Tl-mutant flies) were generated as describedin ref. 17. For Drs constitutive expression, flies overexpressing Gal4 under theubiquitous promoter daughterless were crossed to transgenic strains carrying Drscoding sequences under the control of upstream activating sequence enhancerelements (da-Gal4/U-Drs). eater-null transheterozygote mutants were generatedas described in ref. 29. Standard procedures for manipulation, feeding, andhousing 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) andthe 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 thinsterile 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 andtransferred to fresh vials every 2 days.

Virulence studies were performed as described in SI Text.

Histopathological Analysis. On day 1 after infection with Zygomycetes, fruitflies were fixed with 10% (vol/vol) formaldehyde, processed, and embeddedin paraffin wax. Matched tissue sections were stained with crystal violet (inpilot experiments, we observed that crystal violet stained better hyphae of

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Zygomycetes as compared with other classic fungal stains, including Gomorimethenamine-silver stain and H&E), and representative sections were exam-ined 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 vitrophagocytosis and hyphal killing assays (16). D. melanogaster S2 cells were grownin Schneider’s medium (Invitrogen) supplemented with 10% heat-inactivatedFBS, penicillin, and streptomycin.

The rate of phagocytosis of R. oryzae and A. fumigatus spores by S2 cells wasvisually 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 S2cells containing fungal spores out of 100 cells in triplicate for each sample.

The effect of dexamethasone on phagocytosis of R. oryzae and A. fumigatusspores by S2 cells was evaluated by using flow cytometry. R. oryzae and A.fumigatus spores were first incubated with FITC (3 �g/ml; Sigma) overnight at 4°Cand then washed extensively with PBS. S2 cells (105) were preexposed to dexa-methasone (100 �M) for 1 h and subsequently challenged with FITC-labeledspores (ratio, 1:1) for 30 min at 29°C. S2 cells unexposed to dexamethasone servedas controls. After a wash, rate of phagocytosis was measured by flow cytometricanalysis. Flow cytometry was performed on a FACSCalibur (BD Biosciences).

Hyphal damage assays using D. melanogaster S2 cell line were performed asdescribed in SI Text.

Imaging by Confocal Microscopy. S2 cells were coincubated with GFP expressingR. oryzae spores or hyphae for 30 min. Imaging was performed on fixed S2 cellsstained with Alexa 647-conjugated phalloidin (red; Molecular Probes) and DAPI(blue; Molecular Probes) using a Leica SP2 RS laser-scanning confocal microscopewith an oil-immersion objective (Leica �63/1.4 numerical aperture).

RNA extraction and RT-PCR analysis of Drs and Mtk mRNA and fungalburden analysis by RT-PCR were performed as described in SI Text.

Whole-Genome Expression Profiling Studies. Drosophila 2.0 Affymetrix microar-rayswereusedforwhole-genomeexpressionanalysis.Briefly, thedChipsoftware(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 thegene expression indexes. Probe sets with low expression in the background noiseregion or little variation across all samples (mean expression �79 or standarderror �9) were eliminated. To assess the variance structure of the data, anunsupervised hierarchical clustering analysis based on Pearson correlation wasperformed to cluster samples by using these probe sets. To identify differentiallyexpressed genes in fruit flies infected with A. fumigatus or R. oryzae, we used thefollowing criteria, as proposed elsewhere (27): (i) the fold change of meanexpression 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 eachgene between A. fumigatus and R. oryzae � 1.5. Fifty-four probe sets weredetermined to be differentially expressed by meeting both of these criteria.Hierarchical clustering analysis based on Pearson correlation was performed onthe 54 probe sets.

ACKNOWLEDGMENTS. We thank Y. Apidianakis (Harvard Medical School,Boston) for useful comments and the U-Drs transgenic flies; T. Y. Ip (Universityof Massachusetts Medical School, Worcester, MA) for Tl fly mutants; C. Kocks(Harvard Medical School, Boston) for eater mutants; A. S. Ibrahim for the GFPRhizopus strain; and N. D. Albert for excellent technical assistance. This workwas supported by grants from the University of Texas M. D. Anderson CancerCenter [institutional research grant and M. D. Anderson Faculty E. N. CobbScholar Award Research Endowment (D.P.K.)].

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