Human Fungal Pathogens a Model for The

Immune-Deficient Drosophila melanogaster: A Model for the Innate Immune Response to Human Fungal Pathogens

This information is current as Anne-Marie Alarco, Anne Marcil, Jian Chen, Beat Suter, of September 28, 2021. David Thomas and Malcolm Whiteway J Immunol 2004; 172:5622-5628; ; doi: 10.4049/jimmunol.172.9.5622 http://www.jimmunol.org/content/172/9/5622 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immune-Deﬁcient Drosophila melanogaster: A Model for the Innate Immune Response to Human Fungal Pathogens1,2

Anne-Marie Alarco,3* Anne Marcil,* Jian Chen,† Beat Suter,† David Thomas,‡ and Malcolm Whiteway*†

We explored the host-pathogen interactions of the human opportunistic fungus Candida albicans using Drosophila melanogaster. We established that a Drosophila strain devoid of functional Toll receptor is highly susceptible to the human pathogen C. albicans. Using this sensitive strain, we have been able to show that a set of specific C. albicans mutants of different virulence in mammalian infection models are also impaired in virulence in Drosophila and remarkably display the same rank order of virulence. This immunodeficient insect model also revealed virulence properties undetected in an immunocompetent murine model of infection. The genetic systems available in both host and pathogen will enable the identification of host-specific components and C. albicans genes involved in the host-fungal interplay. The Journal of Immunology, 2004, 172: 5622Ð5628. Downloaded from

andida albicans is an opportunistic human pathogen that tion of antimicrobial peptides (AMPs)4 in the ﬂy fat body (14, 15). asymptomatically colonizes a wide variety of body loca- Interestingly, infection of Drosophila with different pathogens C tions. However, upon alteration of immune system func- leads to the preferential induction of the appropriate group of AMP tions, C. albicans can proliferate and cause infections termed can- (8). Fungal infection results in the induction of an antifungal pep-

didiasis. The significance of our understanding of these fungal tide, drosomycin, but not of the antibacterial peptide diptericin (7, http://www.jimmunol.org/ infections has been heightened by the increased incidence of can- 16). The expression of drosomycin is controlled by a signaling didiasis, primarily due to the expansion of the immunocompro- pathway orchestrated by the Toll receptor (7, 16). Identification of mised population associated mostly with the AIDS pandemia and Toll-like receptors (TLRs) in mammals revealed that these pro- the wider use of medical therapies altering the immune system teins regulate signaling pathways similar to that of the Drosophila such as anticancer treatments and organ transplantation. Toll-dependent pathway (17, 18). Although both innate and acquired immunity play important Our work establishes that Drosophila is an appropriate host for roles in the resistance of mammals to C. albicans infections (1, 2), C. albicans and provides a powerful model to study the interplay the innate immune response is the first line of defense against C. between pathogen virulence and the host innate immune system. albicans in the systemic circulation (3, 4). Model organisms have by guest on September 28, 2021 proven to be powerful tools for the elucidation of many biological processes. Mammalian models have been developed to explore the Materials and Methods very complex relationship between C. albicans and its hosts (5, 6). Drosophila stocks Recently, genetically well-defined model organisms such as the OregonR flies were used as wild-type standard. Toll transheterozygotes fruit fly Drosophila melanogaster and the nematode Caenorhab- were generated through crossing of flies carrying a loss of function allele 1-RXA ditis elegans have been used to decipher diverse host-pathogen of Toll (Tl ; generous gift of D. Ferrandon, Strasbourg, France) and of flies carrying a thermosensitive allele of Toll, with a strong phenotype at interactions (7Ð11). Drosophila, although devoid of an adaptive 29¡C (Tlr632; obtained from the Bloomington Stock Center). Transgenic fly immune system, harbors an innate immune response with striking line expressing the fusion protein drosomycin-GFP was generated by D. similarities with plant and mammalian defense mechanisms (12). Ferrandon (Ref. 19; a generous gift). All stocks were maintained on stan- The Drosophila innate immune response uses pattern recognition dard fly medium at 25¡C, except during infection experiments. receptors, which, depending on the receptor, activate phagocytosis by plasmatocytes (the equivalent of mammalian macrophages Infection experiments (13)), proteolytic clotting cascades in the hemolymph, and produc- Injection of flies (2- to 4-day-old adult females; 30 per experimental group) was performed as described (8) with a thin needle dipped in a concentrated cell pellet containing 200 OD of the yeast cells used in our study. The Ϸ 3 *Genetics Group, Biotechnology Research Institute/National Research Council, and inoculum size was evaluated at 10 cells per fly. Following infection, Departments of †Biology and ‡Biochemistry, McGill University, Montreal, Quebec, flies were maintained at 30¡C on regular fly medium. Infection experiments Canada were performed at least three independent times, and SDs were calculated. Received for publication March 19, 2003. Accepted for publication July 15, 2003. The costs of publication of this article were defrayed in part by the payment of page Microscopy charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. For microscopic observations, flies were either ether anesthetized for direct observation or crushed with a mini-tissue homogenizer (Fisher Scientific, 1 A.-M.A. was supported by a visiting fellowship from the National Science and Ottawa, Ontario, Canada) in Drosophila Ringer solution. Microscopy was Engineering Research Council of Canada. This project was funded by the Genome performed using an upright Leitz (Oberkochen, Germany) Aristoplan mi- Health Initiative of the National Research Council of Canada and a Canadian Insti- ϫ ϫ ϫ tutes of Health Research grant to M.W. croscope with a 2or 40 objective and a 10 projection lens. 2 This is National Research Council Publication No. 46201. 3 Address correspondence and reprint requests to Dr. Anne-Marie Alarco, Genetics Group, Biotechnology Research Institute, 6100 Royalmount, Montreal, Quebec, H4P 4 Abbreviations used in this paper: AMP, antimicrobial peptide; TLR, Toll-like re- 2R2, Canada. E-mail address: [email protected] ceptor; GFP, green fluorescent protein; YEPD, yeast extract peptone dextrose.

Construction of a GFP-CAI4 strain An ADH1 promoter driving green fluorescent protein (GFP) expression plasmid was constructed from a Renilla luciferase expression plasmid as follows. A 1.5-kb NotI-EcoRV fragment from pYPB1-ADHpL (20) was blunt ended and subcloned in SmaI-digested pCRW3 (21). The resulting plasmid, pAM1.3, was digested with NcoI-SacI, generating a 2.8-kb fragment containing the ADH1 promoter, luciferase gene, and WH11 termina- tor sequence. This fragment was subcloned in the SmaI-digested pJA39 (a kind gift from J. Ash, Montreal, Canada) to generate pAM3. A 723-bp fragment, containing the GFP gene, was obtained by HindIII-PstI digestion of pYeGFP3 (22), blunt ended, and subcloned in XhoI-BglII-digested and blunt ended pAM3 to remove the luciferase gene. The resulting plasmid, pAM5.6, also contained the CARS, the TRP locus, and the URA3 selectable marker. It was transformed in CAI4 (URA3Ϫ) strain using the rapid lithium acetate method (23). Transformants were streaked on ϪURA plates and replica plated on yeast extract peptone dextrose (YEPD). After two rounds on ϪURA followed by replica plating to YEPD, clones that stably maintained the selectable marker were identified. Yeast manipulations The C. albicans and Saccharomyces cerevisiae strains used in this study are listed in Table I. Yeasts were grown in YEPD medium at 30¡C. Downloaded from Northern blot analysis Total RNA was extracted from 20 adult flies using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s specification. RNA samples (10 ␮g) were electrophoresed on a 7.5% formaldehyde-1% agarose gel and transferred by capillarity to a ␨-Probe nylon membrane (Bio-Rad Labora- tories, Mississauga, Ontario, Canada). Detection of specific RNAs was http://www.jimmunol.org/ performed by hybridization with 32P-labeled DNA probes as previously described (24). PCR-generated probes were used for the detection of FIGURE 1. Infection of D. melanogaster with CAI4-GFP. GFP ex- metchnikowin (forward, 5Ј-GCCACCGAGCTAAGATG; reverse, 5Ј- pressing C. albicans was injected in Drosophila. A, Flies were observed by AACGACATCAGCAGTGTG) and RP49 (forward, 5Ј-CGTGAA epifluorescence microscopy 1 and 48 h postinfection. B, Infected flies 48 h GAAGCGCACCAA; reverse, 5Ј-GAATCTTAAGCTTACTCG). A probe postinfection were crushed in Drosophila Ringer’s solution and observed ,ءء ;Yeast-like cell ,ء .for drosomycin was generated by digesting plasmid pMC804 (19) with under light microscopy or epifluorescence EcoRI and HindIII. Densitometry analyses (data not shown) were per- Pseudohyphae cells. formed using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at

http://rsb.info.nih.gov/nih-image). by guest on September 28, 2021 C. albicans is a dimorphic fungus. It has been suggested that the Results yeast-to-hyphae transition plays a pivotal role in virulence, allow- The human pathogen C. albicans can infect the fruit fly D. ing C. albicans to penetrate mammalian host tissues (26). It was melanogaster thus of interest to examine the morphology of the CAI4-GFP Several studies have shown that infection of D. melanogaster with strain, injected as blastospores, in our insect model. Flies infected various pathogens can be achieved through pricking of the dorso- for 48 h with C. albicans and showing strong fluorescence under lateral cuticle of the fly with a needle dipped in a concentrated microscopy were selected. Whole fly bodies were then crushed and pathogen pellet (7, 9). This bypasses the natural defense mecha- examined microscopically. C. albicans remained primarily with nisms that are found on the cuticle of the adult fly and has been the crushed tissues (presumably muscles), while very few fungal used to study the behavior of nonentomopathogen organisms in cells were found in the hemolymph (data not shown). C. albicans Drosophila (9, 25). We used a C. albicans strain carrying an in- proliferated extensively as pseudohyphae, with some yeast forms tegrated version of the GFP gene (CAI4-GFP), which allowed us visible (Fig. 1B, top). Thus, in a manner similar to that which to follow the progression of the infection through epifluorescence occurs in a mammalian host model, proliferation within tissues of anesthetized flies. One hour after Drosophila infection with seems to be linked with C. albicans morphological switching. CAI4-GFP, fluorescence can detected surrounding the site of We further characterized the infection by determining the sur- pricking (Fig. 1A). In some flies, the infection progresses from the vival of flies infected with the virulent C. albicans strain SC5314. initial infection site to neighboring tissues (Fig. 1A; 48 h), leading Wild-type Drosophila is highly resistant to infection with C. al- eventually to multiplication of the fungus in the whole Drosophila bicans, with an average survival of 85%, 48 h postinfection (Fig. body cavity. 2). Thus, even though we were able to bypass the physical barrier

Table I. C. albicans and S. cerevisiae strains used in this study

Strains Relevant Genotype Reference

SC5314 URA3/URA3 56 CAI4-GFP URA3-GFP This study cla4 ⌬CaCla4::hisG/⌬CaCla4::hisG-URA3-hisG23 cdc35 ⌬cdc35::hisG/⌬cdc35::hisG-URA3-hisG22 sap4,5,6 ⌬sap6::hisG/⌬sap6::hisG ⌬sap4::hisG/⌬sap4::hisG ⌬sap5::hisG/⌬sap5::hisG-URA3-hisG24 D665Ð1A (S. cerevisiae) URA3/URA3 Mat a CSH stock 5624 ROLE OF Drosophila TOLL SIGNALING PATHWAY IN RESISTANCE TO Candida albicans

Infection of D. melanogaster with C. albicans induces drosomycin expression Drosophila possesses pathogen recognition receptors that will induce, upon activation, signaling pathways leading to the production of AMPs (15). Infection of adult Drosophila with a fungal entomopathogen or Gram-positive bacteria induces most notably the production of the antifungal peptide drosomycin in the Dro- sophila fat body, the equivalent of the mammalian liver (7, 31). We tested the production of drosomycin in response to C. albicans infection with a transgenic Drosophila line expressing a GFP- drosomycin fusion protein (19). Injected wild-type C. albicans SC5314 gave strong drosomycin-GFP expression after 48 h when compared with control flies pricked with a clean needle (Fig. 3). FIGURE 2. Pathogenicity of C. albicans in D. melanogaster. Two- to Similar drosomycin-GFP expression levels were also observed 4-day-old OregonR females were infected with wild-type C. albicans upon challenge of the transgenic flies with the C. albicans mutants (SC5314) or with C. albicans carrying different gene deletions (cdc35, sap4Ð6, cla4, and cdc35 (Fig. 3). Thus, C. albicans is recognized sap4Ð6, and cla4). A prototroph diploid S. cerevisiae strain was also used. by Drosophila as a pathogen, leading to the activation of signaling Control flies (CTL) were pricked with a clean needle in the thorax. Survival pathways causing drosomycin levels to increase. Downloaded from rates did not change significantly after 48 h. Results are presented as the The production of AMPs by the fat body and their secretion in mean of three independent experiments with SDs. the hemolymph is a component of the Drosophila innate immune response (32). Among these peptides, drosomycin and metchnikowin have been shown to display antifungal activity (31, 33) and of the cuticle of Drosophila, the fly has significant defenses against are transcriptionally up-regulated upon fungal challenge (7, 34). We monitored drosomycin and metchnickowin transcript levels

infection with C. albicans. This is consistent with the fact that http://www.jimmunol.org/ Candida species have not often been reported as entomopathogens upon challenge with C. albicans using Northern blot analysis (Fig. (27). Deletion mutants of C. albicans affecting the virulence in 4). The results confirmed those obtained with the drosomycin-GFP rodent models have been characterized. Among those, the cdc35, and showed that drosomycin and metchnikowin transcript levels which carries a deletion for the gene coding for adenylyl cyclase, increase over time after infection with wild-type and mutant C. and cla4, carrying a deletion in a p21-activated kinase homolog, albicans of OregonR Drosophila (Fig. 4, compare lane 2 with had been shown to be avirulent in mouse models of hematog- lanes 4, 5, 6, and 7). Conversely, there was no effect on the expres- enously disseminated candidiasis (28, 29), whereas in a similar sion levels of diptericin, which encodes an antibacterial peptide model, the sap4Ð6 strain, deleted of three aspartyl-proteases, was (data not shown; Ref. 7). Drosomycin and metchnikowin tran- strongly attenuated in virulence (30). In wild-type Drosophila, all scripts are also elevated after challenge of Drosophila with heat- by guest on September 28, 2021 of these mutants were also avirulent (Fig. 2), thus indicating that killed C. albicans (Fig. 4, lane 8), suggesting that recognition of the fruit fly is a useful host model for the study of C. albicans. this pathogen by the flies is similar to mammalian recognition

FIGURE 3. Expression of drosomycin-GFP in response to C. albicans. Two- to 4-day-old transgenic female Drosophila carrying a drosomycin-GFP transgene were infected with various strains of C. albicans and observed through epiﬂuorescence 48 h postinfection. HK, Heat killed; CTL, ﬂies were pricked with a clean needle. The Journal of Immunology 5625

type and Toll mutant flies following infection with C. albicans (Fig. 6). These data show that C. albicans is able to stably maintain itself in the OregonR flies over 12 h postinfection, with little change in CFU over that time period (Fig. 6). In contrast, CFU obtained for infected Toll mutants increased readily 6 h postinfection, and active proliferation of the pathogen was maintained up to 12 h postinfection (Fig. 6). The increase in drosomycin and metchnikowin transcript levels FIGURE 4. Northern blot analysis of drosomycin and metchnikowin. observed in wild-type flies following infection with C. albicans is Total RNA, extracted 6 h postinfection, was used to monitor the expression strongly reduced in the Toll mutants (Fig. 4, compare lanes 2, 3, of drosomycin (DRS) and metchnikowin (MTK) using radiolabelled cDNA and 4). It is thus tempting to attribute the susceptibility of the Toll encoding these antifungal peptides. A control cDNA was used to monitor mutants to challenge with C. albicans to insufficient levels of the Drosophila constitutive gene RP49. Lanes are as follows: Lane 1, Ore- drosomycin. Indeed, this hypothesis is supported by the recent R R gon , no pricking; 2, Oregon , pricked with a clean needle; 3, Toll mu- demonstration that reinjection of drosomycin in Drosophila im- R tants, pricked with C. albicans SC5314; 4, Oregon pricked with C. albi- paired in multiple AMP production was sufficient to restore wild- cans SC5314; 5, OregonR pricked with C. albicans cdc35; 6, OregonR type susceptibility to the fungi Neurospora crassa and Fusarium pricked with C. albicans cla4; 7, OregonR pricked with C. albicans sap4Ð6; and 8, OregonR pricked with heat killed C. albicans. oxysporum (38) We examined the susceptibility of the Toll mutants to our panel

of C. albicans mutant strains. Infection with the cdc35, cla4, and Downloaded from of fungal heat-resistant ␣-linked mannans and ␤-linked sap4Ð6 mutants shows that all these strains display, to different glucans (35Ð37). extents, a reduced virulence toward the Toll mutants when compared with SC5314 (Fig. 5). This is evident through both the Resistance of Drosophila to infection with C. albicans is slower effect on viability and the higher survival rates of the Toll dependent on the Toll receptor ﬂies infected with our C. albicans mutants. Comparing the survival

Infection of Drosophila by the entomopathogenic fungus Beau- of Toll mutants 48 h postinfection with the different mutants, http://www.jimmunol.org/ Ͼ varia bassiana leads to an up-regulation of genes encoding AMPs, 90% of the ﬂies infected survived infection with the cdc35 strain, via the activation to the Toll pathway (7, 8). Furthermore, it was compared with an average of 60% survival in the infections with shown that Drosophila Toll mutants, in which Toll expression is the cla4 and sap4Ð6 mutants (sap4Ð6 mutants killing faster than abolished, are very sensitive to fungal infection and have concom- cla4 mutants) and 15% with the wild-type C. albicans SC5314 itantly a marked reduction of Toll-dependent AMPs (7, 8). We over the same period. These results are in agreement with the body examined the role of the Toll pathway in C. albicans infection of work that explored the effects of cla4 and sap4Ð6 deletions on using Toll transheterozygotes that carried a loss-of-function allele virulence. Indeed, compared with the cdc35 strain, the cla4 and sap4Ð6 mutants do not display any in vitro growth rate difference

of Toll and a thermosensitive allele of Toll. The susceptibility of by guest on September 28, 2021 the Toll mutant flies to C. albicans was determined as for the under our growth conditions (29, 30). In addition, the increased wild-type flies. The Toll mutants proved to be highly susceptible to ability of the sap4Ð6 and cla4 mutants to kill the Toll mutants the wild-type C. albicans SC5314, with an average survival rate of compared with cdc35 may also be associated with their ability to ϳ15% after 48 h (Fig. 5) compared with the OregonR wild-type undergo pseudohyphal differentiation (29, 39Ð41). Of all the C. flies (Fig. 2). This susceptibility is dose dependent (data not albicans strains used in our study, the cdc35 knockout strain dis- shown). Furthermore, the death of Toll mutant flies correlates with plays the most severely reduced virulence. These results are cor- massive proliferation of C. albicans throughout the body of the related with the fact that the cdc35 strain is unable to show any insect with no external signs visible (data not shown). The susceptibility of the Toll mutants to infection with C. albicans was also assessed by comparing the total fungal load of wild-

FIGURE 6. Fungal load of infected Drosophila. Wild-type OregonR FIGURE 5. Infection of Drosophila Toll mutants with C. albicans. flies and Toll transheterozygotes were infected with C. albicans SC5314. Two- to 4-day-old female Toll transheterozygotes were infected with wild- Groups of 10 flies were collected immediately after infection (0) or at 6, 9, type C. albicans (SC5314) or with C. albicans carrying different gene and 12 h postinfection. Collected flies were crushed in Drosophila Ringer’s deletions (cdc35, sap4Ð6, and cla4). A prototroph diploid S. cerevisiae solution using an Eppendorf and an adapted grinder. Serial dilutions were strain was also used. Results presented are the means of four independent plated on YEPD agar and incubated for 2 days at 30¡C, and CFU were experiments with SD. counted. Results are the mean of two experiments. 5626 ROLE OF Drosophila TOLL SIGNALING PATHWAY IN RESISTANCE TO Candida albicans sign of morphological differentiation under a multitude of condi- istic of this strain (29). Proliferating cells of the cdc35 strain in the tions and displays a reduced growth rate (28). Interestingly, our Toll mutants are, as for SC5314, mainly associated with tissue. model also indicates that the cdc35 mutant is not totally avirulent, However, cdc35 cells are only found as yeast-like cells, forming because the survival associated with infection with the cdc35 strain grape-like shapes (Fig. 7). decreases significantly 48 h postinfection, reaching ϳ45% after 96 h (Fig. 5). The inability of S. cerevisiae to have any effect on the Discussion survival of the Toll mutants further supports the idea that the cdc35 In common with most insects, the fruit fly D. melanogaster is very mutant still contains some virulence potency (Fig. 5). Taken to- resistant to microbial infection. This is due to the presence of the gether, our results establish that the ranking of virulence of these physical barriers, formed by the external cuticle as well as the C. albicans strains in Toll mutants, i.e., SC5314 Ͼ sap4Ð6 Ͼ chitinous membranes of the gut and the trachea. In addition, Dro- cla4 Ͼ cdc35, is similar to that which is observed in mammals. sophila maintains a low internal pH hostile to many pathogens. We examined the morphology displayed by our panel of C. al- Finally, a plethora of inducible immune reactions in response to bicans strains proliferating in infected Toll mutant flies. As was invasion of a pathogen will lead to the secretion to high concen- observed for infection in wild-type flies, SC5314 in Toll mutants trations of AMPs in the hemolymph (38). However, a growing was a mixture of yeast-like cells together with pseudohyphae at- body of work indicates that D. melanogaster can be infected by tached to the host tissue (Fig. 7). Observation of Toll mutants various pathogens through natural routes or by injections, and thus infected with the sap4Ð6 and cla4 mutants shows that most of the this genetically tractable organism can serve as a model to dissect cells of both strains display germ tubes and are mainly associated host-pathogen interactions, without the overlying complexities of with Drosophila tissue, as was seen for infection with the wild- complement and Ab responses (38, 42). Downloaded from type SC5314 strain (Fig. 7). The differentiated cla4 mutant cells We used thoracic injection of adult wild-type Drosophila to observed inside the fly showed aberrant morphologies character- evaluate the use of the fruit fly as a host model for the human opportunistic fungal pathogen C. albicans. This strategy allowed us to successfully infect Drosophila. The mode of infection used (i.e., pricking through the cuticule) overcomes limitations encoun-

tered with natural infection routes (e.g., size of the organisms with http://www.jimmunol.org/ C. elegans (11)) and thus broadens the spectra of fungi that might be studied in Drosophila. In Drosophila, C. albicans undergoes morphological differentiation in a manner similar to what occurs during infection of mammalian hosts (43). This morphological transition from yeast to pseudohyphae occurs in vitro and in vivo in response to a range of environmental cues such as temperature above 35¡C, high pH, or the presence of serum. Because our infection experiments were performed at 30¡C (the nonpermissive temperature of the Toll- by guest on September 28, 2021 thermosensitive allele), it is not possible to attribute the pseudohyphal differentiation of C. albicans in Drosophila to elevated temperature but rather to factors associated with the host environ- ment such as pH, ion distribution, or intercellular contacts. C. albicans survives in a wide variety of mucosa and can cause diseases in different body locations, displaying a versatility far greater than most other commensal microorganisms. These abili- ties can be categorized as virulence traits. Genetic approaches have been use to try to unravel the virulence traits contributing to C. albicans pathogenesis (6). To do so, C. albicans carrying deletion in specific genes are most frequently tested in acute-lethality mouse models. We have used three such C. albicans mutant strains to infect Drosophila. Our work shows that C. albicans genes essential for infection in mammalian models are also critical to successfully infect wild-type Drosophila, validating the use of this insect model. In addition, Drosophila Toll mutants revealed that the rank order of virulence of these C. albicans mutants, consid- ering both the lethality and the rate of killing, is similar in the Toll mutants and in mouse models. Interestingly, the rank order of virulence of the mutant C. albicans strains followed the ability of these strains to form hyphae. Hence, these data support the common view of the importance of morphological differentiation for virulence. However, decreased virulence has also been associated with gene deletions not affecting hyphal formation (44, 45). In addition, Odds et al. (46) established that injection of very high numbers of a given C. albicans strain can lead to pathologically FIGURE 7. Morphology of C. albicans in Toll transheterozygotes. Fe- significant tissue burdens independent of hyphal formation. Our males Toll mutant flies infected with C. albicans SC5314 were crushed experiments showed that tissue invasion is not solely dependent on 12Ð24 h postinfection in Drosophila Ringer’s solution and observed under morphological differentiation, because the cdc35 mutant is able to light microscopy. invade tissues in the Drosophila Toll mutants, even though this The Journal of Immunology 5627 strain does not undergo yeast-to-hyphae transition. This is a par- innate immune response. We showed that C. albicans genes im- ticularly interesting observation, because infection in the Drosoph- portant for virulence in mammalian models are also important for ila Toll mutants allows for the uncoupling of hyphal differentiation infecting Drosophila and that the Toll pathway, conserved in and tissue proliferation. The observed virulence of the cdc35 mu- higher eukaryotes, is essential for the resistance of Drosophila to tant strain in Toll mutant flies indicates that deletion of the CDC35 C. albicans. Our pathogenesis model, in which both the pathogen gene strongly hampers, but may not totally block the virulence of and the host are genetically tractable, will undoubtedly provide a C. albicans, which underscores the fact that infection with C. al- critical tool to decipher the function of C. albicans virulence fac- bicans of Drosophila Toll mutants—due to their higher suscepti- tors as well as host response against them, issues at the core of bility—revealed more subtle effects of specific mutations in C. host-pathogen interactions. albicans compared with infection of wild-type flies and, by exten- sion, to models of immunocompetent mice. This may not be sur- Acknowledgments prising in view of the facts that virulence studies with this strain We are very grateful to members of the Suter laboratory for their invalu- were performed in an immunocompetent mouse model (28), that able help in introducing us to the world of Drosophila. We are also grateful the cdc35 strain is able to colonize the murine vaginal mucosa for to Dr. D. Ferrandon for providing the transgenic flies and to Dr. D. up to 10 days (28), and that this strain is able to induce macrophage Sanglard for providing the C. albicans sap4Ð6 strain. death, albeit at a slower rate than a wild-type C. albicans strain (47). C. albicans is a commensal, hence, its virulence potency References although present in immunocompetent hosts, is silent and does not 1. Ashman, R. B., and J. M. Papadimitriou. 1995. Production and function of cy- tokines in natural and acquired immunity to Candida albicans infection. Micro- Downloaded from induce infection. Consequently, our immunosuppressed fly model biol. Rev. 59:646. is all the more relevant and should prove useful for the study of C. 2. Romani, L., and S. H. Kaufmann. 1998. Immunity to fungi: editorial overview. albicans virulence determinants. Res. Immunol. 149:277. 3. Fidel, P. L., Jr. 2002. Immunity to Candida. Oral Dis. 8(Suppl. 2):69. Infection of Toll mutants with the C. albicans mutant strains 4. Calderone, R. 2002. Candida and Candidiasis. Am. Soc. Microbiol., tested resulted in a similar threshold of ϳ40% survival, signifi- Washington, DC. cantly higher than the survival rate obtained with the SC5314 5. Samaranayake, Y. H., and L. P. Samaranayake. 2001. Experimental oral candidiasis in animal models. Clin. Microbiol. Rev. 14:398. strain. This may result from defects inherent to the mutant strains, 6. Navarro-Garcia, F., M. Sanchez, C. Nombela, and J. Pla. 2001. 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