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Drosophila melanogaster as a Facile Model for Large-Scale Studies of Virulence Mechanisms and Antifungal Drug Efficacy in Candida Species

  1. Georgios Chamilos1,
  2. Michail S. Lionakis1,
  3. Russell E. Lewis1,3,
  4. Jose L. Lopez-Ribot4,
  5. Stephen P. Saville4,
  6. Nathaniel D. Albert1,
  7. Georg Halder2 and
  8. Dimitrios P. Kontoyiannis1,3
  1. Departments of
  2. 1Infectious Diseases, Infection Control, and Employee Health and
  3. 2Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, and
  4. 3College of Pharmacy, University of Houston, Houston, and
  5. 4Division of Infectious Diseases, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio
  1. Reprints or correspondence: Dr. Dimitrios P. Kontoyiannis, Dept. of Infectious Diseases, Infection Control, and Employee Health, Unit 402, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (dkontoyi{at}mdanderson.org)
 
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Abstract

Candida species are the predominant fungal pathogens in humans and an important cause of mortality in immunocompromised patients. We developed a model of candidiasis in Toll (Tl)–deficient Drosophila melanogaster. Similar to the situation in humans, C. parapsilosis was less virulent than C. albicans when injected into Tl mutant flies. In agreement with findings in the mouse model of invasive candidiasis, cph1/cph1 and efg1/efg1 C. albicans mutants had attenuated virulence, and the efg1/efg1 cph1/cph1 double mutant was almost avirulent in Tl mutant flies. Furthermore, the conditional tet-NRG1 C. albicans strain displayed significantly attenuated virulence in flies fed food without doxycycline; virulence was restored to wild-type levels when the strain was injected into Tl mutant flies fed doxycycline-containing food. Fluconazole (FLC) mixed into food significantly protected Tl mutant flies injected with FLC-susceptible C. albicans strains, but FLC had no activity in flies injected with FLC-resistant C. krusei strains. The D. melanogaster model is a promising minihost model for large-scale studies of virulence mechanisms and antifungal drug activity in candidiasis

Candida species, which are commensal fungi of mucosal surfaces, are the predominant fungal pathogens in humans [1]. The incidence of invasive candidiasis, in particular, is increasing because of an expanding population of immunocompromised and debilitated patients [2–4], and the mortality attributable to candidiasis remains high [4]. C. albicans is the most frequently infecting pathogen, but the broad use of azole antifungals during the past 2 decades has led to the emergence of less susceptible, non-albicans species, such as C. glabrata and C. krusei [5–7]. Although the comparative virulence among different Candida species has not been extensively studied, C. parapsilosis another increasingly important Candida species that is frequently associated with device-related infections, appears to be the least virulent in humans and in animal models of candidiasis [7]

Understanding the molecular mechanisms of Candida pathogenesis is important for the development of novel, targeted therapeutic strategies. Conventional genetic studies of Candida species have long been hampered by the diploid genome and the lack of a known sexual cycle. During the past decade, however, gene disruption in C. albicans became feasible through the use of integrative transformation systems adapted from the closely related, nonpathogenic yeast Saccharomyces cerevisiae [8, 9]. Moreover, a tetracycline-regulatable (TR) promoter gene expression system was recently developed in C. albicans; in contrast to conventional genetic tools, this system allows the functional analysis of genes that are essential for cell growth and virulence [10, 11]

With the application of these molecular methods, it became evident that the ability to switch from a budding (yeast) form to a filamentous (hyphal) form is a key virulence mechanism that allows Candida species to invade host tissues and escape from phagocytic destruction [9–15]. This morphogenetic program in Candida species is under the control of a complex network of cross-talking signal transduction pathways [14]. For example, in response to various environmental cues (such as serum, temperature, and pH) the cyclic AMP/protein kinase A and mitogen-activated protein kinase pathways are activated, leading to up-regulation of the transcription factors Efg1p and Cph1p, expression of a set of hyphal-specific genes in Candida species, and filamentation [9, 14, 15]. In contrast, other transcription factors, such as Tup1p, Nrg1p, and Rfg1p, negatively regulate Candida filamentation [14]

Despite the acceleration of research into the molecular mechanisms of Candida virulence, in vivo testing of Candida mutants in animal models is essential to fulfill Koch’s postulates for pathogenicity. Mammalian models, however, represent a bottleneck for large-scale virulence studies, because they are laborious. In addition, these models usually allow for the testing of only 1 Candida mutant, by comparing its virulence to that of the isogenic wild-type (wt) parental Candida strain in a small number of animals. Minihost models with well-characterized genetics and simple immunity against pathogens have been effectively used to study both microbial virulence factors and host immune-defense mechanisms [16–19]. For example, Drosophila melanogaster lacks adaptive immune responses but has fully developed innate immunity [16, 17]. In D. melanogaster the evolutionarily conserved Toll (Tl) signaling pathway plays a critical role in the control of innate immune responses against fungi and gram-positive pathogens [16, 17]. On challenge with a fungal pathogen, activation of the Tl pathway leads to the rapid and selective induction of a large amount of antifungal peptides in the fly hemolymph, allowing D. melanogaster to successfully combat infection [16, 17, 19]. Tl-deficient flies are, therefore, extremely susceptible to fungi and have been successfully used to study the pathogenesis of infection with various fungal pathogens [16, 17, 20–22]

In the present study, we developed a model of systemic candidiasis in Tl mutant D. melanogaster that enabled us to study the virulence of different Candida species and mutants and the antifungal activity of the oral drug fluconazole (FLC). We found that the D. melanogaster model has considerable similarities with the much more complex mammalian models of candidiasis. This system holds promise as a tool for the evaluation of novel virulence genes, the comparative study of virulence attributes of different Candida species, and high-throughput screening of antifungal compounds against Candida species

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Materials and Methods

D. melanogaster stocksOregon R flies were used as wt flies. Tl-deficient transheterozygote mutants were generated by crossing flies carrying a thermosensitive allele of Tl (Tlr632) with flies carrying a null allele of Tl (Tll-RXA). All fly strains were a gift from T. Y. Ip (University of Massachusetts, Worchester). We used standard procedures for manipulation, feeding, and housing of flies in all experiments [16, 20]

Fungal strains and antifungal agentsThe Candida strains used in the present study are listed in table 1. We used Candida strains (2 strains of C. albicans 2 strains of C. parapsilosis and 1 strain of C. krusei) that were isolated from patients with cancer and invasive candidiasis at the University of Texas M. D. Anderson Cancer Center. We also used the cph1/cph1 and efg1/efg1 mutants and the efg1/efg1 cph1/cph1 double mutant; their isogenic wt C. albicans parental strain, CAF2-1 [8, 9, 20]; and the conditional tet-NRG1 C. albicans strain, SSY50-B [11]. In the latter strain, up-regulation of the TR promoter in the absence of DOX leads to NRG1 overexpression, blockade of filamentation, and attenuated virulence. Full virulence is restored to this strain when it is in the presence of DOX [11]

FLC (Pfizer) and DOX (Sigma) dilutions were prepared in distilled water and PBS (pH ∼7.2), respectively, and stored at −80°C until use. All Candida strains were grown and maintained on yeast-peptone-dextrose (YPD) medium. For the infection experiments, cultures of each strain were grown overnight at 30°C in YPD liquid medium. Yeast cells were collected by centrifugation and washed 3 times in sterile, pyrogen-free saline. The cells were then counted with a hemacytometer (Hausser Scientific), and appropriate dilutions were made to achieve the required concentrations

D. melanogaster infection modelsFor the injection assay, 2–4-day-old female flies (30/group) were injected in the thorax with a thin sterile needle that was 0.25 mm in diameter and that had been dipped into a concentrated solution of Candida yeast cells (1×107–1×1010 yeast cells/mL), as described elsewhere [16, 20]. Flies that died within 3 h of infection (<5%) were excluded from the survival analysis. After infection, flies were housed at 29°C (the optimum temperature for susceptibility to microbial challenge [16]) and transferred to fresh vials every 2 days. Survival was assessed daily until day 8 after infection. Each experiment was performed at least in triplicate on different days. Quantification of Candida yeast cells in the injection inoculum was performed by transferring cells from the tip of a needle that had been previously dipped into a concentrated C. albicans solution (1×107–1×1010 yeast cells/mL) into 1 mL of 0.85% normal saline. Serial dilutions (100 μL each) of that solution were plated onto YPD plates and incubated at 30°C. Colony-forming units were counted after 48 h

For the ingestion assay, groups of 30 flies were placed in special fly-food vials containing YPD agar medium, on which a lawn of 1×107 yeast cells had grown for 24 h. Flies were fed Candida-infected food for 48 h and then were housed in vials containing regular fly food for 8 days and maintained at 29°C [20]. Each experiment was performed at least in triplicate on different days

Regulation of virulence in the tet-NRG1 strain by DOX We mixed 2 different DOX concentrations (10 mg/mL and 100 mg/mL) with regular fly food, as described elsewhere [20]. In pilot experiments, these concentrations were found to be nontoxic to the flies (data not shown). Because DOX oxidizes rapidly in light, food was stored in the dark for a maximum of 24 h. After 6–8 h of starvation, groups of 30 flies were housed in vials with DOX-containing food for 2 days before infection with the tet-NRG1 C. albicans strain. After infection, the flies were transferred daily for 8 days to fresh vials with DOX-containing food. Each experiment was performed at least in triplicate on different days

FLC protection experimentsFor assessment of FLC protection, flies were housed in empty vials for 6–8 h to starve and then were transferred into vials with FLC-containing food (1 mg/mL), as described elsewhere [20]. After 24 h, flies were infected with Candida strains by injection and then were transferred daily for 8 days into fresh vials with FLC-containing food and maintained at 29°C. As a control, flies were starved for 6–8 h, transferred to vials containing regular fly food (without FLC), infected, and then maintained in vials containing regular fly food. FLC protection was assessed daily until day 8 after infection. Each experiment was preformed at least in triplicate on different days

Assessment of tissue fungal burdenGroups of 20 flies infected with Candida strains were collected 0, 12, 24, and 36 h after infection; placed in plastic tubes; and ground in 1 mL of 0.85% NaCl solution. Serial dilutions were plated onto YPD agar medium, and the number of colony-forming units was counted after 48 h of incubation at 30°C. Results shown are the mean of 4 experiments

Histopathological analysisOn day 2 after infection with Candida strains in the injection or ingestion assay, flies were fixed with 10% (vol/vol) formaldehyde, processed, and embedded in paraffin wax. Matched tissue sections were stained with Grocott-Gomori methenamine-silver nitrate (GMS), and representative sections were examined for visible fungal burden by light microscopy

Statistical analysesSurvival curves were plotted using Kaplan-Meier analysis, and differences in survival rates between the groups were analyzed using the log-rank test. The Mann-Whitney U test was used to determine statistically significant differences in fungal burden (expressed as colony-forming units) between the treatment groups. Statistical analyses were performed with GraphPad Prism software (version 4.0; GraphPad Software). P<.05 was considered to be statistically significant

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Results

Establishment of a reproducible model of disseminated Candida infection in D. melanogaster. We initially infected wt and Tl mutant flies with 2 C. albicans clinical isolates (CA2328 and CA2330) by injection of different inocula into the fly hemolymph and then assessed survival and fungal burden. Injection of either of the C. albicans strains into Tl mutant flies resulted in acute infection and significantly lower survival rates, compared with those in wt flies (P<.0001) (figure 1A). The wt flies were resistant to infection after injection of any of the inocula tested. The fungal burden (as determined by counts of colony-forming units) was significantly lower in wt flies than in Tl mutant flies within 12 h after infection (P<.03) (figure 1B). The difference in fungal burden further increased over time, and there was an ∼10-fold increase in the number of colony-forming units in Tl mutant flies 36 h after infection (P<.003). Similarly, in histopathological sections stained with GMS 48 h after injection of flies with C. albicans a much higher fungal burden and much more extensive infection were observed in Tl mutant flies than in wt flies (figure 1D and 1E)

Figure 1
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Figure 1

A Survival of Toll (Tl) mutant and wild-type (wt) flies infected by injection with different inocula of Candida albicans (n=30 flies/group). P<0.0001 for all comparisons, except for the Tl mutant 3×103 yeast cells group vs. either the Tl mutant 5×102 yeast cells group or the Tl mutant 8×102 yeast cells group (not significant [NS]). B Differences in fungal burden (expressed as colony-forming units) between wt and Tl mutant flies assessed at different time points after injection with ∼8×102C. albicans yeast cells (n=20 flies/group). C Survival rates of Tl mutant and wt flies after infection through the gastrointestinal route (ingestion assay; n=30 flies/group). Data shown in panels A–C are the means of 4 independent experiments; error bars represent SDs. D and E Representative histopathological sections from the thoraces (stained with Grocott-Gomori methenamine-silver nitrate) of wt (D) and Tl mutant (E) flies infected by injection with ∼8 × 102C. albicans yeast cells. Candida hyphae have dark staining (arrow) and the fungal burden is much higher in Tl mutant flies; Drosophila melanogaster muscle cells have green staining. F Representative histopathological section from the gastrointestinal tract of a fly infected by the oral route. Candida yeast cells and budding forms are shown as dark areas; epithelial cells have green staining (arrow)

The degree of fungal burden is typically reflective of the amount of cells in the inoculum and has prognostic significance in patients and in mammalian models of candidiasis [23]. We quantified the cells in the infection inocula that we used and found that Tl mutant flies injected with a needle dipped in a solution containing 1×1010, 1×109, 1×108, or 1×107 yeast cells/mL were inoculated with ∼2×104, ∼5×103, ∼8×102, and ∼5×102 yeast cells, respectively. The survival of Tl mutant flies was inoculum dependent over a wide range of inocula (figure 1A). For example, Tl mutant flies injected with ∼2×104, ∼8×102, and ∼5×102 yeast cells had a median survival time of 2, 3, and 4 days, respectively (figure 1A)

We next attempted to develop an alternative route of infection by use of an ingestion assay in which Tl mutant flies were fed food that was infected with Candida yeast cells, as has been previously done using other yeasts (e.g., Cryptococcus species) and molds (e.g., Aspergillus species) with D. melanogaster [20, 21], because the gastrointestinal tract is considered to be an important route of entry for Candida species in human candidiasis [1]. We housed wt and Tl mutant flies for 48 h in vials containing a fresh lawn of Candida yeast cells. Both groups were resistant to infection by this method (figure 1C and 1F). After establishing a reproducible model of Candida infection via injection and characterizing the effects of inoculum size, we used this optimized model to examine Candida virulence and protection by antifungal drugs

Differences in virulence of Candida species in Tlmutant fliesWe injected Tl mutant flies with 2 strains of C. parapsilosis (ATCC 22019 and CA2537), 3 strains of C. albicans (CA2338, CA2330, and CAF2-1), and 1 strain of C. krusei (CA2355). Tl mutant flies infected with either C. parapsilosis isolate had significantly better survival rates than did flies infected with the other 2 Candida species on day 3 (65% vs. <20%; P<.009) and day 8 (30% vs. <5%; P<.01) after infection (figure 2)

Figure 2
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Figure 2

Survival rates of Toll mutant flies after injection with ∼8×102 yeast cells of representative isolates of different Candida species (C. albicans CA2328, C. krusei CA2355, and C. parapsilosis ATCC 22019). Data shown are the means of 4 independent experiments (n=30 flies/group)

Attenuated virulence in C. albicans mutants with defective filamentation in Tlmutant fliesThe C. albicans efg1/efg1 cph1/cph1 double mutant is locked in the yeast phase and is avirulent in the mouse model of candidiasis; the cph1/cph1 and efg1/efg1 mutants also show defects in filamentation and attenuated virulence [9]. In agreement with the findings in the mouse model of candidiasis, Tl mutant flies infected by injection with the efg1/efg1 and cph1/cph1 mutants had a significantly better survival rate (>60%) 8 days after infection than did Tl mutant flies infected with the isogenic wt strain CAF2-1 (0%) (P<.0001). The efg1/efg1 cph1/cph1 double mutant was almost avirulent in Tl mutant flies, which had a survival rate of >90% 8 days after infection (figure 3)

Figure 3
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Figure 3

Survival rates of Toll mutant flies after injection with ∼8×102 yeast cells of the efg1/efg1 or cph1/cph1 mutants, the efg1/efg1 cph1/cph1 double mutant, or the isogenic wild-type CAF2-1 strain of Candida albicans. Data shown are the means of 4 independent experiments (n=30 flies/group)

Modulation of virulence of tet-NRG1 C. albicans by DOX in Tlmutant fliesWe also used our D. melanogaster model to test the virulence of the genetically engineered tet-NRG1 C. albicans strain, in which filamentation is exogenously modulated (in vivo and in vitro) through a TR promoter [11]. As was previously shown, up-regulation of the TR promoter leads to NRG1 overexpression, blocks filamentation, and renders the tet-NRG1 strain avirulent in mice in the absence of DOX [11]. Being in the presence of DOX completely restores the virulence of tet-NRG1 to a level comparable with that of the isogenic wt strain CAF2-1 [11]. We injected the tet-NRG1 strain into different groups of Tl mutant flies that had been fed for 48 h with food containing various concentrations of DOX. We then compared the survival rates of those flies with the survival rates of flies infected with the CAF2-1 strain. Tl mutant flies injected with the engineered strain and fed regular fly food had a survival rate of >95% 8 days after infection, which is in agreement with the findings in the mouse model [11]. In contrast, Tl mutant flies fed food containing a high concentration of DOX (100 mg/mL) and infected with the same inoculum of the tet-NRG1 strain had a low survival rate (<5%), comparable with that after infection with the isogenic wt CAF2-1 strain, 8 days after infection. The tet-NRG1 strain displayed attenuated virulence in Tl mutant flies fed food containing a lower concentration of DOX (10 mg/mL) (figure 4). Thus, the D. melanogaster model seems to be a promising one for the study of Candida virulence genes regulated by inducible promoters

Figure 4
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Figure 4

Survival rates of Toll (Tl) mutant flies after injection with ∼8×102 yeast cells of the engineered tet-NRG1 Candida albicans strain SSY50-B or the isogenic wild-type CAF2-1 strain. Flies were exposed to different doxycycline (DOX) concentrations in their food. Different groups of Tl mutant flies (n=30 flies/group) were fed in vials containing either regular fly food (-DOX) or fly food containing different concentrations of DOX (+DOX, 10 mg/mL; ++DOX, 100 mg/mL). Data shown are the means of 4 independent experiments

FLC protection of Tlmutant flies injected with FLC-susceptible C. albicans and FLC-resistant C. krusei strainsThe increased incidence of azole-resistant non-albicans infections mandates acceleration of the discovery of new antifungal agents with novel cellular targets in Candida species [24]. Because the D. melanogaster model is simpler and less laborious to use than are conventional animal models, it might be suitable for mass screening of candidate compounds with anti-Candida activity. As proof of principle, we evaluated whether FLC, an effective oral antifungal in human candidiasis [5], can protect Tl mutant flies infected with a C. albicans strain that is susceptible to FLC in vitro (CA2330; MIC, 0.25 μg/mL, determined by CLSI M 27-A2 methods [25]). We housed Tl mutant flies in vials containing food with 1 mg/mL FLC. Flies fed FLC-containing food had a significantly higher survival rate 8 days after infection (>70%) than did control flies (<5%) (P<.0001) (figure 5A). Furthermore, flies fed FLC-containing food had significantly lower fungal burdens than did control flies after 12 h of infection, as determined by counts of colony-forming units (P<.03) (figure 5C) and histopathological analysis (figure 5E and 5F)

Figure 5
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Figure 5

Fluconazole (FLC) protection of Toll (Tl) mutant flies infected by injection with ∼8×102 yeast cells of FLC-susceptible Candida albicans or FLC-resistant C. krusei. A and B Survival rates of FLC-treated and untreated flies infected with C. albicans (A) or C. krusei (B). C and D Fungal burden (expressed as colony-forming units), at different time points, of FLC-treated and untreated Tl mutant flies infected as described above with FLC-susceptible C. albicans (C) or FLC-resistant C. krusei (D). Data shown are the means of 4 independent experiments (n=30 flies/group); error bars represent SDs. E and F Histopathological sections from the thoraces of Tl mutant flies infected by injection with FLC-susceptible C. albicans and fed regular fly food (without FLC) (E) or FLC-containing food (F). Candida hyphae have dark staining (arrow) and the fungal burden is higher in untreated flies; Drosophila melanogaster muscle cells have green staining. NS, not significant

Table 1
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Table 1

Candida strains

The in vitro resistance of Candida species to FLC may be associated with a lack of response to FLC in both human candidiasis and animal models of this infection [24]. We evaluated whether our D. melanogaster model is capable of distinguishing the protection offered by FLC after infection with Candida species with different in vitro FLC susceptibilities. A group of Tl mutant flies was fed FLC-containing food and infected 24 h later with C. krusei a species intrinsically resistant to FLC (CA2355; MIC, 64 μg/mL). Tl mutant flies preexposed to FLC-containing food and subsequently infected with the FLC-resistant C. krusei strain had survival rates and fungal burdens that were comparable with those of control Tl mutant flies fed regular fly food and infected with the same C. krusei strain (figure 5C and figure 5D)

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Discussion

We have developed a reproducible model of acute systemic candidiasis induced through injection of a standardized amount of Candida yeast cells into the hemolymph of Tl mutant flies. Although the small size of invertebrate organisms is considered to be a limitation in establishing standardized infection models, the use of an injection assay allowed us to quantify the inocula and enabled us to study their effects reliably in D. melanogaster

We found that infection of D. melanogaster with different Candida species resulted in differences in mortality, with C. parapsilosis being the least virulent species. These findings imply that this simple host might be a suitable model for comparative studies of candidiasis caused by different Candida species

The pivotal role played by dimorphism in Candida pathogenicity has been widely recognized [14]. We studied the efg1/efg1 and cph1/cph1 mutants and the efg1/efg1 cph1/cph1 double mutant, which have well-characterized defects in filamentous growth and attenuated virulence in a mouse model of systemic candidiasis [9]. In the D. melanogaster model—in contrast to other invertebrate models, such as the Galleria mellonela moth model [26]—the efg1/efg1 and cph1/cph1 mutants were hypovirulent, whereas the efg1/efg1 cph1/cph1 double mutant was almost completely avirulent. Our finding is similar to what has been shown in the mouse model of candidiasis [9]; however, the cph1/cph1 mutant was less virulent in the D. melanogaster model, compared with its virulence in the mouse model [9]. In agreement with our results, Alarco et al. recently tested another group of C. albicans mutants (cdc35, cla4, and sap4–6) in Tl mutant flies and showed that all of them displayed attenuated virulence that was similar to that seen in mammalian models of candidiasis [22]. Importantly, the complete genomic sequence of C. albicans is available [27], and modern genetic tools are being applied to the development of large libraries of mutants with single gene deletions [28, 29]. As minihost organisms are increasingly used to screen collections of mutants from a range of pathogenic microorganisms [30, 31], Tl mutant flies might emerge as an important system for high-throughput studies of potential virulence genes in Candida species

We also examined an alternative strategy for studying Candida virulence by use of a conditional mutant whose virulence is under the control of a TR promoter system. Unlike conventional gene disruption, this system allows the exogenous modulation of gene expression both in vivo and in vitro [10, 11]. We studied the virulence of the tet-NRG1 strain and showed that exogenous modulation of NRG1 expression is achievable in our D. melanogaster model by feeding flies food containing various concentrations of DOX. Importantly, the virulence of the tet-NRG1 strain in the presence of different DOX concentrations in Tl mutant flies was similar to that seen in the mouse model of candidiasis

Finally, we assessed the efficacy of antifungal agents in our D. melanogaster model. We selected FLC for these studies, because it is the preferred oral antifungal for treatment of human candidiasis [5] and is also one of the few antifungal agents for which susceptibility breakpoints have been established in Candida species [24]. We found that FLC had a significant protective effect in flies infected with an FLC-susceptible C. albicans strain, whereas FLC had no effect on mortality in flies infected with the FLC-resistant species C. krusei. These results were verified by assessment of fungal burden. We believe that the D. melanogaster model might represent a reliable alternative for studying the effects of orally absorbed antifungal compounds on Candida strains with different in vitro susceptibilities. Because modern genetic tools now allow the disruption of essential Candida genes that might be targets for new drugs, an array of novel and selective anti-Candida compounds will soon become available [27]. The D. melanogaster model provides a fast and economical in vivo assay that shows promise for mass screening of these candidate compounds

Like all in vivo models, this D. melanogaster one has limitations. For example, the amounts of orally absorbed antifungal agents cannot be precisely quantified; as a result, pharmacodynamic (dose response) and pharmacokinetic studies are difficult to perform using this model. Furthermore, the administration of parenteral antifungal agents to D. melanogaster is technically challenging [32]. In addition, because D. melanogaster infection experiments are performed at 29°C and not at human body temperature (37°C), there may be differences in the expression of some virulence factors in Candida species. Finally, even though innate immune mechanisms are evolutionarily conserved, invertebrate models are not directly comparable to the situation in mammals [33]. For example, invertebrates lack adaptive immunity, and significant differences exist between the innate immune responses of invertebrates and those of mammals [33]. Overall, the considerable similarities of the D. melanogaster model with other mammalian models of systemic candidiasis, as well as its simplicity and economical nature, suggest that it is a useful minihost model for the elucidation of Candida virulence mechanisms

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Footnotes

  • Presented in part: 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 30 October–2 November 2004 (abstract M-240)

    Financial support: University of Texas M. D. Anderson Cancer Center (institutional research grant and M. D. Anderson Faculty E. N. Cobb Scholar Award Research Endowment to D.P.K.)

    Potential conflicts of interest: D.P.K. has received research support and honoraria from Merck, Fujisawa, Enzon, Pfizer, and Schering Plough. R.E.L. has received research support from Merck, Fujisawa, Pfizer, and Schering Plough. All other authors: no conflicts

  • Received July 16, 2005.
  • Accepted November 1, 2005.
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  1. J Infect Dis. (2006) 193 (7): 1014-1022. doi: 10.1086/500950
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