Profiling the host defense responses against Candida auris in a reliable Drosophila melanogaster infection model
Jie Li, Guangsheng Chen, Xiangkang Zeng, Jiaxin Lin, Xiaoqing Chen, Wenqiang Wang, Yueru Tian, Xinhua Huang, Yun Zou, Ming Guan, Zhiyi He, Hailei Wang, Changbin Chen, Lei Pan
TL;DR
Researchers developed a fruit fly model to study how Candida auris, a dangerous fungus, interacts with the host's immune system, revealing new insights into infection mechanisms.
Contribution
The study introduces a reliable Drosophila melanogaster model to investigate Candida auris-host interactions and identifies novel immune factors.
Findings
Toll and JAK-STAT pathways mediate antifungal responses in Drosophila following C. auris infection.
Conserved novel factors involved in host-C. auris interactions were identified.
The Drosophila model enables high-throughput genetic screening for immune mechanisms and therapeutic strategies.
Abstract
The “superbug” Candida auris has been ranked as a priority fungal pathogen and is becoming a serious threat to public health. However, the underlying mechanisms of real‐world pathogen–host interactions remain elusive, in part due to the lack of powerful immunocompetent animal models. Here, we report that selected wild‐type strains of Drosophila melanogaster can be developed as a promising infection model to recapitulate C. auris systemic infection. The systemic and organ‐specific responses to C. auris infection in vivo were evaluated, as well as the corresponding transcriptional profiling. Our findings confirmed that Toll and JAK‐STAT signaling pathways mediate antifungal responses in the Drosophila model following C. auris infection. Moreover, we identified certain conserved novel factors required for host–C. auris interactions, highlighting the fly model's potential to reveal subtle…
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Taxonomy
TopicsInvertebrate Immune Response Mechanisms · Neurobiology and Insect Physiology Research · Fungal and yeast genetics research
INTRODUCTION
Candida auris is an emerging human fungal pathogen that was originally identified in Japan in 20091. Since then, it has become a significant public health threat due to its multidrug resistance, high mortality, lack of surveillance, and rapid environmental spread2, 3. Notably, both the US Center for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have classified the emerging superbug C. auris as an “urgent threat” and a “critical priority group,” as infections with this multidrug‐resistant fungal pathogen have rapidly spread across five continents, leading to severe outbreaks and high mortality, particularly in healthcare settings. This fungus is capable of causing severe disseminated or invasive infections by infiltrating the body, entering the bloodstream, and spreading throughout the body, posing a serious global health threat associated with mortality rates of about 30%–60%4, 5, 6. Given the importance of nosocomial fungemia caused by C. auris, this fungus has become a hot topic and subject of increasing interest in clinical and basic science research. Although extensive information has been gathered on the characteristics and physiological responses of C. auris under various culture conditions7, 8, the interactions between C. auris and the host, particularly the mechanisms underlying its virulence and the host immune responses, are still largely unknown. This gap in knowledge is primarily due to the challenges in identifying the pathogen, the scarcity of clinical data, and, importantly, the lack of robust animal models.
To overcome the limitations and weaknesses in the study of the diversity of host–C. auris interactions, several in vivo infection models have been developed. However, challenges remain. Based on its transmission routes, C. auris frequently colonizes the mucosa and can subsequently cause bloodstream infections. For the former, the C. auris skin infection model in mice is widely used9, 10, 11, 12. However, mechanical disruption of the skin barrier is often required in this model to make sure that bloodstream infection occurs, which may only partially recapitulate the process of natural infection. Moreover, establishing C. auris infection in immunocompetent mice is difficult because these mice display strong resilience to its colonization and few observable phenotypes13, 14, 15. Thus, immunocompromised mice are selected for studying C. auris infections16, 17. However, these systems may obscure the complex host immune responses due to their defective background. The zebrafish is a very useful vertebrate animal model; however, there is only one report describing the application of this model to study neutrophil function against C. auris 18, making it difficult to verify its efficacy. In addition to vertebrate animal models, invertebrates also offer advantages for studying C. auris infections. For instance, Galleria mellonella has been used to screen for C. auris virulence factors and drug resistance19, 20, but the lack of genetic manipulation in this system hinders in‐depth investigation of host factors involved in pathogenesis. Caenorhabditis elegans is another choice for studying genetic interactions and has been applied in several C. auris studies21, 22, 23. However, worms tend to avoid eating fungal conidia24 and are always difficult to separate from media containing C. auris 25.
Drosophila melanogaster has been confirmed as an ideal model organism for studying the genetic control of innate physiological and pathological responses to microbial pathogens, due to the high degree of conservation between the innate immune systems of flies and mammals26. Moreover, despite the fact that the circulatory system of D. melanogaster is open, its blood flow remains steady in a fixed direction27, 28, which enables the transportation of local pathogens to other parts of the body. This characteristic enables the partial mimicking of a bloodstream infection by this system. Two distinct and highly conserved signaling pathways have been identified in D. melanogaster that act against invading pathogens, including immune deficiency (Imd) signaling (TNF/NF‐κB pathway in mammals) against Gram‐negative bacteria and Toll signaling (TLR/lectin pathway in mammals) against fungi and Gram‐positive bacteria29, 30. Fungal challenges initiate a protease cascade in which the Toll receptor binds to a cleaved form of Spätzle, ultimately leading to the expression of potent fungicidal peptides regulated by the NF‐κB‐like transcription factors Dif and Dorsal31, 32. The D. melanogaster system has been successfully used to study other Candida species33, 34, 35. A recent study also developed a model using Toll‐deficient flies to assess the virulence of C. auris and the efficacy of antifungal treatment36. And, inevitably, this model has limitations, as its immunocompromised genetic background may mask the innate response to C. auris and hinder the study of true physiological and pathological changes in the host.
In this study, a robust model was established for the purpose of recapitulating C. auris systemic infection, which was achieved by optimizing infection conditions using immunocompetent D. melanogaster strains. The study characterized significant strain‐dependent lethality, sustained fungal proliferation in vivo, and organ‐specific transcriptional reprogramming. The present findings demonstrated an additional animal platform that enables high‐throughput genetic screening. This screening is for the discovery of novel mechanisms of host–C. auris interactions and the advancement of therapeutic development against this priority pathogen.
RESULTS
Assessment of the host defense against systemic infection of C. auris in different strains of D. melanogaster
D. melanogaster has been proven to be a productive genetically tractable model organism for modeling host–pathogen interactions and a powerful system for large‐scale in vivo screening and analyses. Considering the clinical significance of C. auris as one of the four critical priority fungal pathogens on the WHO list37, we sought to determine whether flies could serve as an effective research model to explore C. auris–host interactions. To test this, we used a clinical isolate of C. auris from China (yCB799)13, which belongs to clade I, to infect each of the four “wild‐type” fly strains that are often used as genetic background controls (Ore‐R, Canton‐S, w ^ dah ^, and w ^1118^). 50.6 nl of C. auris cell suspension (10,000 yeast cells) was injected into each of the indicated fly strains using a Drummond Nanoject II injector (n = 60; three biological replicates) (Figure 1A). As shown in Figure 1B, it is clear that all test flies were susceptible to C. auris infection, but each fly strain displayed distinct survival patterns. The Ore‐R strain showed the highest susceptibility to C. auris infection, with rapid mortality observed: half of the flies even died within the first day postinjection. In contrast, the w ^1118^ strain was found to be the least susceptible, with half of the flies surviving up to 15 days postinfection (dpi). To rule out the possibility that the dose of the pathogen may have an effect on the mortality readout in fly strains with differing genetic backgrounds, a low‐dose infection (1000 cells per fly) was also used. In a consistent manner, Ore‐R flies showed the greatest sensitivity to C. auris infection, with an LD_50_ at around 8 dpi, whereas the w ^1118^ strain showed the lowest susceptibility, with an LD_50_ at around 16 dpi (Figure S1A). Additionally, for the most susceptible strain, Ore‐R, the mortality rate in them infected with C. auris was well demonstrated in a dose‐dependent manner (Figure 1C). This pattern was further confirmed by infecting the flies with the C. auris genome reference strain B8441 (Figure S1B), which belongs to clade I and was isolated in Pakistan in 2008 (NCBI Genbank ID: PEKT00000000.2)38. Collectively, our data validate the utility of D. melanogaster as a model for studying C. auris–host interactions. The easily observable survival phenotype in D. melanogaster allows for the development of large‐scale screening for mutualistic factors between C. auris and the host. Moreover, our results indicate that establishment of an optimal infection model, whether susceptible or tolerant, depends on the genetic background of the chosen fly strain and the dosage of fungal cells injected.
*Establishment of a Candida auris infection model in Drosophila melanogaster. (A) Schematic diagram showing the experimental approach of C. auris inoculation in fly. (B) Survival curves of different fly strains (Ore‐R, Canton‐S, w
dah , and w 1118) after C. auris (yCB799, 10,000 cells/fly) infection. (C) Survival curves of Ore‐R flies injected with different doses of C. auris (yCB799). (D) Fungal burden in whole Ore‐R flies after infection with a high dose of C. auris (yCB799; 10,000 cells per fly). (E) Fungal burden in whole Ore‐R flies after infection with a low dose of C. auris (yCB799; 1000 cells per fly). (F) Survival curves of Ore‐R flies infected with different clades of C. auris. Error bars represent the mean ± SEM.*
As a susceptible strain, Ore‐R fly strain showed a high mortality rate when challenged with a higher dose of C. auris (10,000 cells/fly) for 1–2 days (Figure 1B). Consistently, this was accompanied by a rapid increase in fungal load within the fly body, peaking at 24 hours post infection (hpi) (Figure 1D). The notion that C. auris is able to multiply in the fly body reflects the fact that the rapid onset of lethality, observed within 1 to 2 days, is likely due to the swift expansion of C. auris, as the number of fungal cells increased approximately 10‐fold within 24 h. It is well known that the activation of the innate immune system contributes to the eradication of pathogens. Interestingly, after the initial infection, we observed a gradual reduction in tissue fungal loads, which stabilized at a relatively constant level (around 10^4^ colony‐forming units [CFU] per fly) (Figure 1D). This hints at the presence of an innate immune evasion mechanism by C. auris. This phenomenon was also observed when a lower inoculum size (1000 cells per fly) was used, as the fungal burden remained stable at a high level (approximately 10^4^ cells per fly) even 15 days postinoculation (Figure 1E). These results not only suggest that a long‐term interaction between C. auris and Ore‐R fly host can be established in vivo but also that C. auris may use an immune evasion strategy in flies, just like its role in mammalian hosts13. Notably, CFU analysis of other fly strains provided further support for our observations in the survival curves (Figures 1B and S1C). The initial robust increase of C. auris resulted in Ore‐R flies being the most susceptible strain during the early infectious stage, while the gradient expansion of C. auris in other strains delayed the mortality onset in them. Additionally, given the fact that the global C. auris population comprises five genetically distinct clades1, we investigated whether Ore‐R flies could be infected by strains from clades other than Clade I. Survival assays with strains from different clades confirmed similar susceptibility patterns (Figure 1F) and reinforced the feasibility and suitability of D. melanogaster as an ideal working model for C. auris infection.
Transcriptional profile in the whole body of wild‐type flies following C. auris infection
The successful establishment of a wild‐type D. melanogaster model for C. auris infection prompted us to examine the dynamic global gene expression profiles of the host using RNA‐seq technology. Transcriptomic analyses were conducted with three independent biological replicates from the whole body of wild‐type Ore‐R flies at 6 and 24 hpi with either 10,000 cells of the C. auris strain (yCB799) or PBS as a mock control. Illumina NovaSeq. 6000 sequencing generated millions of 150 bp sequence reads. The sequencing data are available on the GEO (Gene Expression Omnibus) database under accession number GSE279369. Sequencing reads were aligned to the reference genome of D. melanogaster, identifying 42,043,467 average total clean reads with approximately 95% genome coverage at both time points (Figure S2). Differentially expressed gene (DEG) analyses, restricted to genes with fold changes ≥ 2 and p < 0.05, revealed a progressive transcriptional response to infection: 60 upregulated genes and 25 downregulated genes at 6 hpi, while increased to 108 upregulated genes and 42 downregulated genes at 24 hpi in whole body (Figures 2A, S3A and Table S1). These findings suggest that C. auris inoculation triggers an increasingly complex host response, with the number of responsive genes nearly doubling from 6 to 24 hpi.
Tissue‐specific and temporal transcriptional responses to C. auris infection in D. melanogaster. (A) Number of differentially expressed genes (DEGs) identified between C. auris infected and PBS control groups in the whole body and fat body at 6 and 24 hours post infection (hpi). (B) Venn diagram showing overlap of up‐ and downregulated DEGs between 6 and 24 hpi time points in fat body. Red: upregulated at 6 hpi, yellow: upregulated at 24 hpi, blue: downregulated at 6 hpi, and green: downregulated at 24 hpi. (C) Scatter plot of log2(fold changes) in fat body at 6 hpi (x‐axis) versus 24 hpi (y‐axis), with selected genes highlighted. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs categorized by temporal expression patterns. See also Figure S4C for Gene Ontology (GO) enrichment analysis of temporal gene expression patterns.
Given the relatively modest number of DEGs at both 6 and 24 hpi time points, we used Gene Set Enrichment Analysis (GSEA) for functional interpretation, which does not require predefined gene sets and can capture subtle but coordinated changes in gene expression. Gene Ontology (GO) analysis at 6 hpi identified 5 activated biological processes, predominantly defense‐related processes including antimicrobial humoral response and defense response to symbionts, alongside 9 suppressed metabolic processes, particularly carbohydrate and organic acid metabolism (Figure S3B). By 24 hpi, the immune response expanded with 17 activated processes, encompassing humoral immunity, innate immune response, and specific antifungal and antibacterial defenses, while 48 processes were suppressed, reflecting continuous metabolic reprogramming (the top 10 are shown in Figure S3C). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed temporal progression in host response patterns. At 6 hpi, only the immune pathways, Toll and Imd signaling (TLR/NF‐κB signaling pathway in mammals39), were activated, while five metabolic pathways including pentose metabolism, starch metabolism, and biosynthesis of unsaturated fatty acids were suppressed (Figure S3D). By 24 hpi, pathway suppression expanded to 27 pathways spanning multiple metabolic networks including glycolysis, oxidative phosphorylation, amino acid metabolism, and lipid metabolism, while the Toll and Imd signaling pathway remained activated (Figure S3E). This pattern suggests that immune response is initiated rapidly, and metabolic adjustment intensifies to support sustained immune activation.
Notably, KEGG analysis revealed that the majority of upregulated genes were classified within immune‐related pathways, in particular the Toll and Imd signaling pathways (Figure S3D–E). This highlights the central role of these pathways in the fly immune response to C. auris. For example, these significantly upregulated genes upon infection with C. auris at 24 hpi included genes encoding antimicrobial peptides (drs and mtk), immune‐induced peptides with antifungal properties (BomBc1, BomS1, BomS2, BomS3, BomS5, BomT2, and IMPPP), serine proteases (Sp212 and SPE), and the serine protease inhibitor (Spn88Eb) (Figure S3F). More importantly, these Toll pathway‐related genes have been previously reported to be induced during fungal infection and are known to contribute to host antifungal responses40, 41, 42, 43, 44, 45, 46. This supports the conclusion that, similar to infections with other fungal pathogens, systemic infection with C. auris is sufficient to trigger the innate immune activation in flies.
Transcriptional profile in the fat body of wild‐type flies following C. auris infection
While we have obtained transcriptional profiles at the whole‐body level, this approach may mask weak signals or signals from tissues or organs, making it difficult to disentangle the contributions of individual tissues. To address this issue, we used the same inoculation protocol as above and conducted RNA‐seq analysis on the fat body of Ore‐R flies postinfection with C. auris or PBS (mock). This is because the fat body, functionally analogous to the mammalian liver47, plays a crucial role in both immune and metabolic processes in flies.
We analyzed gene induction in the infected FB relative to uninfected controls. The sequence reads mapped to an average of 95.83% of the D. melanogaster genome (Figure S2). Using the same stringent filtering criteria (a cutoff of +/−2.0 fold and adjusted p value of < 0.05), we found that after C. auris infection, DEGs in the fat body of fruit flies were substantially more numerous than those in the whole body at both time points: 1199 upregulated versus 60 upregulated and 212 downregulated versus 25 downregulated at 6 hpi, and 552 upregulated versus 108 upregulated and 389 downregulated versus 42 downregulated at 24 hpi (Figure 2A, Table S1). The fat body showed a 16.6‐fold higher number of DEGs than whole body at 6 hpi, and a 6.3‐fold at 24 hpi. These findings highlight the central role of FB against fungal infection. Notably, the total number of DEGs in fat body decreased by 33.3% from 6 to 24 hpi, reflecting a temporal shift in transcriptional response.
To better understand the temporal dynamics of gene expression in fat body during C. auris infection, we compared DEG sets between the two time points (6 and 24 hpi) and performed comprehensive Over Representation Analysis (Figures 2B,C and S4A,B). Among the 1411 DEGs at 6 hpi, 1079 genes were exclusively upregulated at this early time point, suggesting an acute transcriptional response. Between the two time points, only 77 genes showed persistent upregulation and 61 genes showed persistent downregulation, indicating dynamic regulation of the transcriptional landscape. At 24 hpi, 451 genes showed late‐specific upregulation, representing a shift in cellular priorities during persistent infection. Each categorized gene set was subjected to KEGG pathway and GO enrichment analysis to identify functional themes (Figures 2D and S4C).
The temporal comparison of gene expression patterns revealed distinct functional priorities at each infection stage. Both KEGG pathway and GO biological process enrichment analyses provided complementary insights into the developmental progression of C. auris infection (Figures 2D and S4C). Early response (6 hpi) was characterized by broad metabolic activation, with significant enrichment in organic acid metabolism, protein metabolism, and energy metabolism pathways. GO analysis identified carbohydrate metabolic processes and monosaccharide metabolism as significantly enriched early responses. The 6 hpi time point showed remarkable specificity for cilium‐related processes, potentially reflecting rapid cellular remodeling during initial pathogen recognition. As infection progressed to 24 hpi, the response transitioned to specialized energy production pathways including glycolysis and oxidative phosphorylation. Functional analysis of different temporal expression categories revealed specialized defensive roles: genes persistently upregulated across both time points showed enrichment for humoral immune response and antimicrobial humoral response, suggesting sustained activation of core defense mechanisms. In contrast, genes consistently downregulated demonstrated significant enrichment for protein refolding and heat shock processes, potentially compromising cellular stress protection. Late‐specific (24 hpi only) changes maintained immune process enrichment, indicating continued refinement of defensive responses during persistent infection. By further analysis of fat body‐specific DEGs, several genes including FucTA, PPO1, PPO2, and Adgf‐A (Figure 2C), as well as genes related to lipid metabolism (Figure S4D) and heat shock proteins (Figure S4E), were highlighted, suggesting their potential role during infection.
In summary, the extensive differential expression of D. melanogaster genes upon C. auris infection demonstrates that the fly uses distinct immune and metabolic strategies to fight against this emerging fungal pathogen. These findings underscore the conservation of antifungal responses across species and highlight the potential of D. melanogaster as a model for studying fungal infections.
The role of D. melanogaster Toll signaling in response to C. auris infection
Our aforementioned RNA‐seq data, either from the whole body or fat body, revealed a significant induction of genes related to the Toll signaling pathway following systemic infection with C. auris, highlighting the importance of this highly conserved signaling cascade in host resistance. Indeed, genes encoding antimicrobial peptides such as Drosomycin (drs) and Metchnikowin (mtk), which are well‐established markers of Toll pathway activation47, were significantly upregulated. These results were further validated by qRT‐PCR analyses, confirming the robustness and accuracy of our RNA‐seq data (Figure 3A–D).
*Toll signaling is essential for the host defense against C. auris infection. (A–D) Expression levels of antimicrobial peptide (AMP) genes (Drs and Mtk) in whole body and fat body of flies at 24 hpi with C. auris detected by qRT‐PCR, which were normalized to the ribosomal protein gene rp49. (E) Survival curves of the indicated flies (Dif
1 , Myd88
KG03447 and Tl
r3) with Toll signaling deficiency following C. auris infection. w 1118 flies were used as controls. (F) Survival curves of the indicated flies (Myd88‐RNAi, spz‐RNAi‐1and spz‐RNAi‐2) with knockdown of Toll signaling in the whole body after C. auris infection. (G, H) Fungal burden measured in indicated flies at 1 day postinfection (dpi) and 2 dpi. (I) Survival curves of the indicated flies with knockdown of Toll signaling in the fat body after C. auris infection. (J, K) Fungal burden measured in indicated flies at 1 dpi (J) and 8 dpi (K). The values represent mean ± SEM. Data were analyzed using a t‐test for panels (A–D, G, H, J, K) and Kaplan–Meier analysis for survival curves (E, F, I). *p < 0.05; **p < 0.01; ***p < 0.001; and ***p < 0.0001.
To further elucidate the contribution of Toll signaling in mounting an effective innate immune response, we tested the susceptibility of several D. melanogaster mutants with targeted gene mutations to systemic infection with C. auris. We focused on three key players of the Toll pathway48, 49, including the transmembrane receptor‐encoding gene Toll (Tl), the adaptor protein‐encoding gene Myd88 that operates downstream of the Toll receptor, and the transcription factor Dorsal‐related immunity factor (Dif). Since Ore‐R flies are too sensitive to C. auris infection to test the required function of mutant genes, we used the w ^1118^ strain as the genetic control, as it shares the same genetic background as our mutant strains. Survival analysis revealed that flies lacking any of these components were significantly more susceptible to C. auris infection compared to control flies (Figure 3E). This finding suggests that activation of the Toll signaling pathway is crucial for D. melanogaster resistance to fungal infection, notably to the fungus C. auris. This phenotype was further corroborated by using the ubiquitous driver daughterless(da)‐Gal4 to generate RNAi‐mediated knockdown of Myd88 and spätzle (spz) RNAi flies, the latter being an inducer of the Toll pathway. We observed that whole‐body knockdown of either Myd88 or Spatzle led to a striking increase in mortality rates upon C. auris challenge compared to the genetic controls (Figure 3F, the knockdown efficiency is shown in Figure S5). Correspondingly, fungal loads in the RNAi flies were significantly higher (Figure 3G,H). Taken together, our results strongly suggest that the fly, like the mammalian host, requires the Toll signaling pathway to respond to and combat the invasion of fungal pathogens like C. auris.
In addition to the whole‐body response, our data have indicated that during C. auris infection, genes related to the Toll signaling were upregulated in the fat body, the major organ responsible for humoral immunity in flies. To specifically assess the contribution of the fat body to host resistance, we used the binary ppl‐Gal4 system to generate fat body‐specific knockdowns of Myd88 (ppl‐GAL4 > Myd88‐RNAi) and spz (ppl‐GAL4 > spz‐RNAi). The results recapitulated those observed in the whole‐body knockdowns, with significantly reduced survival rates (Figure 3I) and increased fungal loads (Figure 3J,K) following C. auris infection in the conditional knockdown lines. These findings underscore the vital role of the fat body, and by extension, the Toll signaling pathway, in defending against C. auris invasion.
Immune protection of the JAK‐STAT signaling pathway from C. auris infection
To explore the signaling pathways involved in the host response to C. auris infection, we utilized PathON (https://www.flyrnai.org/tools/pathon/web), a web‐based tool developed for analyzing the major signaling pathways and expression patterns of their signature target genes in D. melanogaster 50. Applying this bioinformatics tool to our RNA‐seq data, we sought to identify other signaling pathways presumably involved in the interaction between C. auris and the host. Our analyses revealed significant enrichment of signature genes from the Imd, Insulin, JAK/STAT, JNK, and Toll pathways among the DEGs in both the whole body and fat body of flies at 24 hpi after C. auris infection (Figure 4A). Notably, our qRT‐PCR analyses, as shown in Figure 4B–H, confirmed the induced expression of key genes operating in the JAK/STAT signaling pathway, including unpaired2 (upd2) and upd3, the ligands of the JAK/STAT signaling pathway, which are homologs to mammalian IL6 51, and Socs36E, known as the suppressor of cytokine signaling at 36E, a homolog of mammalian SOCS5 52. These transcriptional changes suggest that JAK/STAT signaling is involved in C. auris–host interactions.
*The JAK/STAT signaling is required for the protection from C. auris infection. (A) Pathway analysis tool for Drosophila (PathON) of differentially expressed genes in the whole body and fat body at 24 hpi with C. auris. (B–G) Expression levels of upd2, upd3, and Socs36E in the whole body and fat body at 24 hpi detected by qRT‐PCR. (H) Heatmap showing the expression of core JAK/STAT signaling genes after C. auris infection. (I) Survival curve of hop mutant flies postinfection. Relative survivals were calculated by normalizing the survival rates of C. auris‐infected flies (hop 25) to the PBS injection flies. (J) Survival curves of the indicated flies (Stat92E‐RNAi and hop‐RNAi) with knockdown of JAK/STAT signaling in the whole body after infection. (K) Survival curves of the indicated flies (Stat92E‐RNAi, hop‐RNAi, and dome
DN ) with knockdown of JAK/STAT signaling in the fat body postinfection. (L) Fungal burden in indicated flies. (M) Relative expression levels of JAK/STAT signaling reporter genes (totB and totM) in the fat body of GS106‐GAL4 > UAS‐hop
Tuml flies with or without RU486 treatment, in which the JAK/STAT signaling is activated or not, respectively. (N) Fungal burden in GS106‐GAL4 > UAS‐hop
Tuml flies with or without RU486 treatment after C. auris infection. (O) Survival curves of the different flies infected with C. auris. The values represent mean ± SEM. Data were analyzed using a t‐test (B–G, L–O) and the log‐rank test for survival curves (I, J, and K). *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001; and ns, not significant.
As stated previously53, the D. melanogaster JAK/STAT cascade is initiated by the binding of Upd ligands to the receptor Domeless (Dome), which, upon dimerization, induces Hopscotch (hop) phosphorylation and provides a docking site for the recruitment of a STAT dimer, which undergoes phosphorylation and translocation into the nucleus, subsequently altering the expression of target genes. To assess the direct involvement of JAK/STAT signaling in the host response to C. auris infection, we compared the survival rates of hop mutant flies to those of wild‐type flies. As anticipated, C. auris‐infected flies harboring the hop mutation (hop ^25^) showed increased mortality compared to their genetic controls (Figure 4I). Similar reductions in survival were also observed when hop or Stat92E expression was knocked down via RNAi, either throughout the whole body using da‐GAL4 or especially in the fat body using ppl‐Gal4 (Figure 4J,K, the knockdown efficiency is shown in Figure S5). Measurement of fungal abundance in infected flies further confirmed the protective role of JAK/STAT signaling against C. auris infection (Figure 4L). In addition, flies overexpressing the dominant‐negative form of Domeless (dome ^ DN ^) were more susceptible to C. auris infection, as evidenced by decreased survival rates and increased fungal loads (ppl‐GAL4 > dome ^ DN ^ versus ppl‐GAL4 > +) (Figure 4J–L). Interestingly, when the JAK/STAT signaling was activated by using a RU486‐induced (RU486+), fat body‐specific GAL4 (GS106‐GAL) to overexpress hop (reporters of signaling were measured in Figure 4M)54, we observed significantly lower fungal loads in flies overexpressing hop (hop ^ Tumi ^) compared to wild‐type controls (Figure 4N). However, the overall resistance curves in response to C. auris infection in these mutant flies showed only a mild improvement (Figure 4O).
Collectively, our results strongly indicate that activation of the JAK/STAT signaling pathway enhances host protection, enabling the elimination of the invasion of C. auris.
Identification of novel factors required for C. auris–host interactions
C. auris infection in immunocompetent mammalian hosts often results in minimal physiological and pathological changes13, 14, making it challenging to study host–pathogen interactions and identify potential therapeutic targets. To address this, we leveraged our highly sensitive D. melanogaster model to explore novel host factors involved in C. auris infection. We conducted an in‐depth analysis of our fly RNA‐seq data, comparing it with two other RNA‐seq data sets (Figure 5A), C. auris‐infected mouse bone marrow–derived macrophages (mBMDMs) (GEO accession number GSE203508)13, and C. auris‐infected human peripheral blood mononuclear cells (hPBMCs) (GEO accession number GSE154911)44. We focused on D. melanogaster DEGs that have orthologs in mammals. In addition to the genes with similar expression patterns across D. melanogaster, mouse, and human species, certain genes stood out due to their significant differential expression in flies while remaining unperceived in mammals at corresponding time points (Figure S6). In particular, tissue‐specific DEGs were worthy of further consideration.
*Search for novel host factors responding to C. auris infection. (A) Schematic of identifying novel host factors involved in C. auris infection. Venn diagram represents the upregulated DEGs of mBMDMs, hPBMCs, and flies after infection. Orthologous genes were identified using the DRSC Integrative Ortholog Prediction Tool (DIOPT). (B, C) Survival curves (B) and fungal burden (C) of the indicated mutant flies (srl −/−, CrebA −/−, Cdc7 −/−, and CG8303 −/−) after C. auris infection. w 1118 flies were used as controls. (D, E) Survival curves (D) and fungal burden (E) of the indicated flies (srl‐RNAi and CrebA‐RNAi) with fat body‐specific gene knockdown after C. auris infection. The values represent mean ± SEM. The log‐rank test was used to analyze statistical differences (B, D). Data were analyzed using a t‐test (C, E). *p < 0.05; ***p < 0.001; and ***p < 0.0001. hPBMCs, human peripheral blood mononuclear cells; mBMDMs, mouse bone marrow‐derived macrophages.
For instance, four outstanding genes were tested in our study: Spargel (srl, dPGC1), which shares significant functional similarity to vertebrate PGC‐1 53; Cyclic‐AMP response element binding protein A (CrebA), with the closest human orthologs, Creb3L1 and Creb3L2 55; CG8303, which has a mammalian homolog, FAR1; and Cdc7, a highly conserved serine‐threonine kinase56. We observed that loss of each of these genes in the whole fly exhibited accelerated mortality following C. auris infection (Figure 5B), with increased fungal burden compared to controls (Figure 5C). Interestingly, certain genes were significantly upregulated specifically in the D. melanogaster fat body compared to whole‐body RNA‐seq postinfection, suggesting tissue‐specific roles for these factors. Consistent with the mutant results, D. melanogaster with fat body‐specific knockdown of srl and CrebA displayed similar susceptibility to systemic C. auris infection (Figure 5D,E, the knockdown efficiency is shown in Figure S7), highlighting their protective roles in the fat body in response to C. auris challenge.
To further support the advantage of our fly system, we sought to validate the functions of these four genes in mammalian cell lines. We individually silenced the expression of these genes using siRNA in the murine macrophage cell line, RAW264.7 (Figure S8A–D). Cell counting kit‐8 (CCK‐8) assays revealed that only knockdown of Far1 had a slight effect on cell viability (Figure S8E). Meanwhile, lactate dehydrogenase (LDH) release assays demonstrated that none of these four genes compromised cell membrane integrity (Figure S8F). However, flow cytometry analysis revealed that knockdown of each gene resulted in a variable but consistent reduction in the phagocytic capacity of macrophages toward C. auris compared to the siRNA‐control (Figure 6A,B), suggesting that these genes may contribute to the host defense against C. auris in mammalian hosts. In parallel, we assessed the intracellular survival of C. auris post‐siRNA transfection. Knockdown of Creb3l2 or Cdc7 enhanced intracellular C. auris proliferation (Figure 6C). These results further support the requirement of these candidate genes in the control of C. auris infection.
*Functions of genes in macrophage cell lines against C. auris. (A) Flow cytometry analysis of RAW264.7 macrophages (F4/80‐APC and CFW‐PB450). siRNA‐transfected RAW 264.7 cells were co‐incubated with C. auris (MOI = 3) stained with Calcofluor White (CFW), and the percentage of CFW positive cells within the F4/80 population (Q2 quadrant) was quantified by flow cytometry to assess phagocytosis. Creb3l2, cAMP responsive element binding protein 3‐like 2; Cdc7, cell division cycle 7; Far1, fatty acyl CoA reductase 1; Pprc1, peroxisome proliferative activated receptor, gamma, coactivator‐related 1. (B) Phagocytosis efficiency in macrophages determined by flow cytometry (n = 3). (C) Intracellular survival of C. auris in RAW264.7 cells following siRNA transfection. The values represent mean ± SEM. Data were analyzed using a t‐test (B, C). *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001; and ns, not significant.
In summary, our findings suggest that the D. melanogaster model can help identify factors that may be overlooked in C. auris infection models of mammals, revealing subtle novel mechanisms. This provides new avenues for studying C. auris–host interactions and offers a deeper understanding of the pathology of C. auris infections.
DISCUSSION
C. auris has been recognized as an urgent public health concern, particularly among critically ill patients57. To address this challenge, there is a pressing need for effective in vivo animal models that can mimic the dynamic interactions between C. auris and its host, as well as facilitate large‐scale screening for antifungal drugs. Despite various attempts to establish such models, many existing systems have notable limitations21, 58, 59, 60.
Recent studies have also shown that, unlike C. albicans, C. auris natural infection barely kills the wild‐type mice and yields noticeable severe phenotypes13, 61, creating hurdles in the study of host–pathogen interactions with this system. Our work using the “wild‐type” D. melanogaster has successfully addressed these limitations. Herein, we evaluated various fly strains usually served as genetic controls for their response to C. auris invasion and found significant differences based on the genetic background. OreR flies were the most susceptible, whereas w ^1118^ flies were the least susceptible, and Canton‐S and w ^ dah ^ showed intermediate susceptibility. These results actually provide a basis for selecting optimal fly strains tailored to specific research needs, establishing a reliable and flexible D. melanogaster model for C. auris studies. Additionally, the pathogen can consistently proliferate within the fly body, leading to a long‐term in vivo interaction with each other. Notably, similar to human hosts, flies were unable to completely eliminate C. auris during infection; instead, the immune system was able to reduce but not fully eradicate the fungal burden. This observation suggests that C. auris uses immune evasion strategies that allow it to persist and replicate within the host, highlighting the importance of further research into these mechanisms to better understand host–pathogen coevolution and the development of drug resistance.
Interestingly, transcriptomic studies in mice have shown that systemic infection with C. auris resulted in relative minor changes in gene expression13. However, our findings in D. melanogaster revealed significant alterations in the transcriptional profile following C. auris infection, particularly in the fat body, the key immune‐metabolic organ in flies. C. auris elicits a more pronounced and common response in the fly model, highlighting its potential as an alternative system for investigating the physiological and pathological changes induced by C. auris. Notably, fat body showed much more sensitivity to infection, showing a significantly greater number of DEGs compared to the whole body, suggesting its pivotal role in coordinating immune response and metabolic reprograming during C. auris infection. The dramatic decrease of DEGs in fat body from the initial infectious stage to a later stage may reflect an transition of adjustment from immediate defensive responses to sustained metabolic adaptation required for prolonged host survival during persistent fungal infection.
Among these DEGs, several genes stood out beyond the above‐mentioned immune‐related genes. FucTA, encoding a Golgi apparatus fucosyltransferase, was upregulated in the fat body of C. auris‐infected flies (Figure 2C). Fucosylation is known to play a role in the mammalian immune system, including M1 macrophage polarization62, B cell development62, IgG antibody activity63, and host resistance to pathogens such as Staphylococcus aureus and C. albicans 64, 65. Our results hint that, similar to its role in mammals, fucosylation might be important for survival in flies following C. auris infection. On the other hand, we also found that the downregulation of certain genes in the fat body of C. auris‐infected flies may be linked to the host immune response to combat C. auris infections. For example, melanization, a key immune mechanism in arthropods, involves the enzyme phenoloxidase (PO), which synthesizes melanin at infection sites66, 67, 68, 69. In line with a previous study showing that PPO (the gene encoding PO enzymes) mutants are more susceptible to C. albicans than wild‐type flies70, we observed in the fat body that systemic infection with C. auris significantly downregulates the expression of PPO1 and PPO2 (Figure 2C), a pattern that may potentially facilitate C. auris colonization in the host. In other words, melanization involving the expression of PPO1 and PPO2 may significantly enhance host defense against C. auris. Additionally, C. auris infection led to the significant downregulation of ADGF‐A (Figure 2C), a gene encoding adenosine deaminase‐related growth factor A. This may result in a compromised immune response and disrupted metabolism, as adenosine deaminase deficiency is known to cause immunodeficiency and increased susceptibility to pathogens in patients71. We also observed downregulation of genes related to lipid metabolism (including Acbp2, Acbp3, Acbp5, Acat1, and Acox57D‐p), suggesting that reduced lipid metabolism during C. auris infection may hinder the fly's ability to generate sufficient energy to eliminate invading pathogens (Figures 2C and S4D). Interestingly, ten genes encoding heat shock proteins (HSPs) were clustered and downregulated in the fat body following C. auris infection (Figures 2C and S4E), hinting at a potential role for HSPs in protecting flies from C. auris invasion. HSPs are inducible by factors like growth factors, infection, and inflammation, and play a critical role in cellular protection72. This is supported by studies showing that infections, such as Helicobacter pylori and Mycobacterium bovis, can alter HSP expression and affect pathogen proliferation73, 74. These findings highlight the systemic response mediated by the fat body, orchestrating immune and metabolic functions against fungal infection.
The involvement of the classical antifungal Toll signaling pathway was confirmed in this study, with increased fungal loads correlating with higher mortality, reinforcing the model's validity for studying C. auris–host interactions. Furthermore, digging into our RNA sequencing data, an important signaling pathway, the JAK/STAT signaling pathway, was enriched in our analysis. It is known for its roles in inflammation, antibacterial, and antiviral activity75, 76, 77, as well as in development and metabolism78. The JAK/STAT pathway was activated upon C. auris infection, as confirmed by both transcriptional analysis and real‐time PCR. Inhibition of this pathway markedly increased susceptibility of flies to C. auris, with increased fungal load, whereas activation of the JAK/STAT pathway provided slight protection with reduced fungal burden. However, prolonged or ectopic activation of the JAK/STAT pathway usually leads to developmental defects and even lethality79, so short‐term activation may not be sufficient to clear C. auris and extend lifespan. These results also indicate that achieving a balance between its inflammatory and antimicrobial functions is crucial for effective host defense. For a comprehensive understanding of the JAK/STAT pathway in the interplay between host and C. auris infection, further investigation is needed.
Additionally, and importantly, the D. melanogaster model offers a significant additional advantage in identifying novel conserved factors that are often overlooked in mammalian models, thanks to its heightened sensitivity. Also, the ability to perform precise genetic manipulations in D. melanogaster allows us to explore the tissue‐specific roles of these genes in response to C. auris infection. For example, in our fly system, we identified four highly conserved genes that confer resistance to C. auris in flies but were not prominent in RNA‐seq analyses of mammalian BMDMs13 and PBMCs44. Among these, Spargel (the D. melanogaster ortholog of human PGC1) is known to coordinate mitochondrial function with nutrient availability and cellular metabolism80, 81, while CrebA (homologous to human CREB3L family transcription factors) regulates processes such as the acute‐phase response, lipid metabolism, cell development, organelle homeostasis, and protein secretion82. Both genes may influence intracellular C. auris infection through metabolic regulation. Additionally, CG8303 (human FAR1), encoding a highly conserved fatty acyl‐CoA reductase, converts fatty acyl‐CoA into fatty alcohol and has been implicated in saturated fatty acid (SFA)‐mediated ferroptosis83, suggesting a potential link between ferroptosis and host defense against C. auris. Cdc7, a conserved serine‐threonine kinase essential for DNA replication initiation56, also emerged as critical for resistance to C. auris invasion. Furthermore, we confirmed their function in mammalian cell lines. More impressively, when these genes were knocked down in mammalian cell lines, the resulting infectious severity closely mirrored those observed in the fly system, reinforcing their conserved functional roles. These findings underscore the power of the D. melanogaster model in uncovering novel host–C. auris interactions that may be missed in conventional mammalian studies. Thus, our work not only highlights previously unrecognized players in C. auris defense but also demonstrates the utility of D. melanogaster as a discovery platform for identifying key mechanisms in fungal–host interactions.
However, the fly system still has several limitations. Primarily, D. melanogaster has an open circulatory system, while in mammals, it is a closed vascular system. Thus, all the organs of the fly that are in the hemolymph are directly in contact with pathogens that reach the body cavity. Despite the fact that the blood flow in the fly remains steady in a fixed direction27, 28, local pathogens can be transmitted to distal organs following circulatory flow. However, this is not the real case for a bloodstream infection in mammals. Second, although the biological signalings and most genes (over 75%) in D. melanogaster have homologs to mammals, some defense mechanisms are specific to insect, such as encapsulation and melanization. In particular, several immune effectors are unique to insects, just like antimicrobial peptides (AMPs), the peptide sequences of which are distinct among species. However, the three‐dimensional structure and conserved physicochemical properties are instrumental in predicting immune effectors in higher organisms including humans84. Additionally, and importantly, the absence of adaptive immunity in D. melanogaster facilitates the study of innate immunity, which is essential for understanding the regulation of innate immunity without the hindrance of adaptive immunity. Nevertheless, this limitation restricts the comprehensive understanding of complex immune and inflammatory mechanisms in mammals85. Consequently, the utilization of the D. melanogaster model is more appropriate for the study of innate immune responses and drug screening, whereas the investigation of comprehensive immune responses in humans necessitates the use of an additional system59.
In summary, the present study provides an additional reliable animal system for studying the mechanisms underlying C. auris–host interactions. The fly model not only enables detailed investigation of innate immune responses but also serves as a powerful platform for large‐scale screening of novel host and pathogen interactors, as well as antifungal agents. Furthermore, the potential for sophisticated genetic manipulation facilitates comprehensive investigation of multiorgan systemic responses, encompassing numerous pathological and physiological singling points, including but not limited to neuron behavior, metabolism, and other relevant parameters, subsequent to C. auris infection. These findings can advance our understanding of C. auris pathogenicity and aid in the development of targeted antifungal strategies in the future.
MATERIALS AND METHODS
D. melanogaster stocks
All flies were maintained on a standard cornmeal diet, consisting of 77.7 g of cornmeal, 63.2 g of glucose, 32.19 g of yeast, 31.62 g of sucrose, 10.6 g of agar, 2 g of potassium sorbate, 0.726 g of CaCl_2_, and 15 ml of 5% Tegosept per liter of food. The environment was maintained at 50%–60% humidity with a 12‐h light/dark cycle. Unless otherwise noted, the fly stocks used in these experiments are detailed in Flybase (http://flybase.org/).
The wild‐type lines included Ore‐R (Bloomington Stock Center, 5), Canton‐S (a gift from Dr. Yi Rao, Capital Medical University), and w ^ dah ^ (a gift from Dr. Jiongming Lu, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences). The mutant lines were Dif ^ 1 ^ (Bloomington Stock Center, 36559), Myd88 ^ KG03447 ^ (Bloomington Stock Center, 14091), Tl ^ r3^ (Bloomington Stock Center, 3238), hop ^ 25 ^ (Bloomington Stock Center, 8494), srl ^ EY05931 ^ (Bloomington Stock Center, 20009), CrebA ^ 03576 ^ (Bloomington Stock Center, 10183), Cdc7 ^ EY07177 ^ (Bloomington Stock Center, 16796), and CG8303 ^ EY07698 ^ (Bloomington Stock Center, 17390). The control line was w ^1118^ (Bloomington Stock Center, 5905).
For RNAi, the following lines were used: UAS‐Myd88‐RNAi (Vienna Drosophila Resource Center, 25399), UAS‐spz‐RNAi 1 (Bloomington Stock Center, 28538), UAS‐spz‐RNAi 2 (Bloomington Stock Center, 34699), UAS‐Stat92E‐RNAi (Tsinghua Fly Center, 1915), UAS‐hop‐RNAi (Tsinghua Fly Center, 5763), UAS‐srl RNAi (Bloomington Stock Center, 33914), and UAS‐CrebA RNAi (Bloomington Stock Center, 31900). The UAS lines included UAS‐dome ^ DN ^ (CEMCS, Chinese Academy of Sciences) and UAS‐hop ^ Tuml ^ (CEMCS, Chinese Academy of Sciences).
The Gal4 driver lines used were ppl‐GAL4 (a gift from Dr. Xun Huang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), da‐GAL4 (a gift from Dr. Renjie Jiao, Guangzhou Medical University), and GS106‐GAL4 (Bloomington Stock Center, 8151). All flies used in this study were adult males.
Fungal preparations
All fungal strains used in this study are listed in Table S2, with yCB799 serving as the primary infecting strain unless otherwise noted. Strains were retrieved from glycerol stocks stored at −80°C, streaked onto YPD agar plates (2% Bacto peptone, 1% yeast extract, 2% glucose, and 2% agar), and incubated at 30°C. Before infection, yeast cells were transferred to YPD liquid medium and incubated at 30°C until they reached the exponential growth phase (OD_600_ = 0.6–0.8). The cells were then washed three times with PBS, counted using a hemocytometer, and diluted in PBS to the target concentration for infection.
Flies were anesthetized with CO_2_ and placed on a fly pad. A 50.6 nl volume of the yeast suspension was injected into the hemocoel at the anterior thorax using a Nanoject II microinjection system (Drummond) to induce systemic immune response. For control, 50.6 nl of sterile PBS was injected to assess the procedural impact on survival. Flies that died within 30 min postinjection were considered to have succumbed due to handling and were excluded from survival analysis.
Infected flies were kept at 29°C, and survivors were counted and transferred to fresh food daily. To quantify fungal burden, flies were euthanized at specific time points postinfection, homogenized, serially diluted, and plated onto YPD agar plates. The plates were incubated at 30°C for 48 h, and CFUs were counted to assess the fungal load.
qRT‐PCR
To assess mRNA levels, whole flies or dissected tissues were collected at specific time points postinfection and immediately homogenized in TRIZOL (Invitrogen) in 1.5 ml tubes. The samples were then frozen at −80°C until RNA extraction. Total RNA was isolated using TRIzol following the manufacturer's protocol. Reverse transcription was performed using the PrimerScript RT Kit (Takara) and PCR amplification was conducted using the SYBR Green system (Takara, TB Green Premix Ex Taq II Kit) on the ABI 7900HT Fast Real‐Time PCR System. Gene expression levels were normalized to the housekeeping gene rp49. Primer sequences are provided in Table S3.
Sample collection and RNA‐seq profiling
Ore‐R flies were infected with either PBS or live C. auris. Live flies (whole body or fat body) were collected 24 hpi, immediately frozen in liquid nitrogen, and stored at −80°C. Three biological replicates were performed for each infection condition. Total RNA was extracted following the previously described method. RNA quality was assessed before sequencing, which was conducted on the Illumina NovaSeq. 6000 platform at Beijing Novogene Bioinformatics Technology Co., Ltd.
Adapter sequences and low‐quality reads were trimmed from the raw sequencing data using Trim Galore and then aligned to the D. melanogaster genome assembly dm6 (Ensembl version 109) using STAR86. DESeq2 was utilized to perform differential expression analysis on gene counts generated by featureCounts87. Genes with an adjusted p‐value of < 0.05 and an absolute fold change > 2 were considered differentially expressed. GO enrichment analysis was carried out with clusterProfiler88. Orthologs of Drosophila genes in humans and mice were identified using the D. melanogaster RNAi Screening Center's Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt)89.
To investigate the response of orthologs to C. auris infection across species, two published RNA‐seq datasets were reanalyzed: C. auris‐infected mBMDMs (GEO: GSE203508) and C. auris‐infected hPBMCs (GEO: GSE154911). Based on these data, a set of genes was identified according to the following criteria: (1) significantly upregulated in the D. melanogaster fat body compared to whole‐body RNA‐seq at both 6 and 24 hpi; (2) conserved orthology across D. melanogaster, mice, and humans; and (3) significant differential expression in D. melanogaster but not in their mammalian orthologs, with no significant changes in mice (3 and 6 hpi) or humans (4 and 24 hpi).
RU486 treatment
RU486 (Mifepristone; Sigma) was first dissolved in DMSO to prepare a 10 mg/ml stock solution. This stock was further diluted with 100% ethanol and added to melted fly food, which was thoroughly mixed to obtain a final concentration of 25 µg/g of food. The mixture was poured into 2 cm‐diameter vials and allowed to dry at room temperature for half a day or overnight at 4°C. Flies were then transferred to the RU486‐treated food and maintained at 25°C, with the food replaced every 2 days.
siRNA transfection
RAW264.7 macrophage cells were maintained in RPMI‐1640 growth medium containing 10% fetal bovine serum (FBS). Cells were seeded in 24‐well plates at 30%–50% confluency and incubated overnight. siRNA transfection was performed using RFect^SP^ V2 transfection reagent (Biodai, 11050) according to the manufacturer's protocol. Cells were transfected with 20 nM siRNA and harvested 24 h later for RNA extraction and gene expression analysis. The siRNA sequences used were as follows: Creb3l2 (sense: 5′‐CAGAGAAGAGCGAGUCAAUTT‐3′, antisense: 5′‐AUUGACUCGCUCUUCUCUGTT‐3′); Far1 (sense: 5′‐GACCUUUAAUAUUGAUGUATT‐3′, antisense: 5′‐UACAUCAAUAUUAAAGGUCTT‐3′); Cdc7 (sense: 5′‐CGACAAGCUUCUGGACCUATT‐3′, antisense: 5′‐UAGGUCCAGAAGCUUGUCGTT‐3′); Pprc1 (sense: 5′‐GAAGGAGCGUGCAAUAGAATT‐3′, antisense: 5′‐UUCUAUUGCACGCUCCUUCTT‐3′); and negative control siRNA (sense: 5′‐UUCUCCGAACGUGUCACGUTT‐3′, antisense: 5′‐ACGUGACACGUUCGGAGAATT‐3′).
C. auris–RAW264.7 interaction assay
Phagocytosis assay
C. auris cells were harvested and stained with Calcofluor White (Sigma‐Aldrich, 18909‐100ML‐F) for 1 min, followed by three washes with PBS. The labeled fungal cells were co‐incubated with RAW264.7 macrophages at a multiplicity of infection (MOI) of 1:3 for 2 h at 37°C in 5% CO₂. After incubation, cells were washed twice with HBSS and detached by gentle pipetting. Cells were pelleted by centrifugation (1000 rpm, 3 min) in 1.5 ml microcentrifuge tubes. Following supernatant removal, the cell pellet was resuspended in HBSS. Cells were incubated on ice and protected from light with APC‐conjugated anti‐mouse F4/80 antibody for 20 min. Flow cytometric analysis was performed using a BECKMAN COULTER DxFLEX cytometer, detecting PB450 (450/45) and APC (660/10) fluorescence. At least 20,000 macrophages were analyzed per sample. C. auris cells were identified in the PB450 (blue) channel, while macrophages were gated using the APC (red) channel. Gating strategies were consistent across all analyses.
Fungal survival assay
RAW264.7 cells were co‐incubated with C. auris at an MOI of 1:1 for 6 h at 37°C in 5% CO₂. Following incubation, cells were washed twice with PBS to remove unbound fungi and non‐adherent cells. Cells were then lysed using lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X‐100, 1 mM EDTA). Lysates were serially diluted and plated onto YPD agar. Plates were incubated at 30°C for 48 h, after which CFUs were counted to assess fungal viability.
LDH assay
LDH activity in the cell culture supernatant was measured using the LDH Cytotoxicity Assay Kit (Beyotime, C0017) according to the manufacturer's instructions. Optical density (OD) was recorded at 490 nm using a microplate reader. LDH levels in each sample were expressed as fold change relative to the maximal LDH activity control.
CCK‐8 assay
Cells were transfected as described previously and seeded in 96‐well plates at 100 μl per well according to the manufacturer's protocol (DOJINDO, CK04). After incubation at 37°C in 5% CO₂ for 12 h, 10 μl of CCK‐8 solution was added to each well. Absorbance readings at 450 nm were taken on a microplate reader subsequent to the final incubation step.
Statistical analyses
Statistical significance was assessed using t‐test for pairwise comparisons, while survival curve comparisons were performed using the log‐rank test. Data from replicates are presented as the mean ± SEM. All analyses were conducted using GraphPad Prism version 8.0.2.
AUTHOR CONTRIBUTIONS
Jie Li: Formal analysis; investigation; methodology; writing—original draft; writing—review and editing. Guangsheng Chen: Formal analysis; software. Xiangkang Zeng: Data curation; investigation. Jiaxin Lin: Data curation; formal analysis; investigation; methodology. Xiaoqing Chen: Data curation; formal analysis; software. Wenqiang Wang: Data curation; validation. Yueru Tian: Validation. Xinhua Huang: Investigation; methodology. Yun Zou: Investigation; validation. Ming Guan: Investigation. Zhiyi He: Supervision. Hailei Wang: Supervision. Changbin Chen: Formal analysis; funding acquisition; resources; writing—review and editing. Lei Pan: Conceptualization; formal analysis; funding acquisition; methodology; project administration; resources; supervision; validation; writing—original draft; writing—review and editing.
ETHICS STATEMENT
An ethics statement was not required for this study.
CONFLICT OF INTERESTS
The authors declare no conflict of interests.
Supporting information
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Figure S5.
Figure S6.
Figure S7.
Figure S8.
Supplement figure legends.
Table S1.
Table S2.
Table S3.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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