Hyphal Als proteins act as CR3 ligands to promote immune responses against Candida albicans.

Patients with decreased levels of CD18 (ß2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMß2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the ß-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the "outside-in signaling" for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1ß release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of ERG251 resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and ZRT2, a Zinc transporter. Notably, we determined that overexpression of ZRT2 is sufficient to increase azole tolerance in C. albicans. Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of ERG251 on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of ERG251 resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ERG251 (ERG251-A and ERG251-B) with ERG251-A exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of ERG251 on drug susceptibility, fitness, filamentation and stress responses.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Candida albicans stimulates formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance: Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Candida albicans stimulates the formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans . Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 were required for C. albicans stimulation of c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorated OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans . Highlights: c-Met is an oral epithelial cell receptor for Candida albicans C. albicans infection causes c-Met and the epidermal growth factor receptor (EGFR) to form a complex with E-cadherin, which is required for c-Met and EGFR function C. albicans Hyr1 and Als3 interact with c-Met and EGFR, inducing oral epithelial cell endocytosis and virulence during oropharyngeal candidiasis Dual blockade of c-Met and EGFR ameliorates oropharyngeal candidiasis.

Functional Dichotomy for a Hyphal Repressor in Candida albicans.

Nrg1 is a repressor of hypha formation and hypha-associated gene expression in the fungal pathogen Candida albicans. It has been well studied in the genetic background of the type strain SC5314. Here, we tested Nrg1 function in four other diverse clinical isolates through an analysis of nrg1Δ/Δ mutants, with SC5314 included as a control. In three strains, nrg1Δ/Δ mutants unexpectedly produced aberrant hyphae under inducing conditions, as assayed by microscopic observation and endothelial cell damage. The nrg1Δ/Δ mutant of strain P57055 had the most severe defect. We examined gene expression features under hypha-inducing conditions by RNA-sequencing (RNA-Seq) for the SC5314 and P57055 backgrounds. The SC5314 nrg1Δ/Δ mutant expressed six hypha-associated genes at reduced levels compared with wild-type SC5314. The P57055 nrg1Δ/Δ mutant expressed 17 hypha-associated genes at reduced levels compared with wild-type P57055, including IRF1, RAS2, and ECE1. These findings indicate that Nrg1 has a positive role in hypha-associated gene expression and that this role is magnified in strain P57055. Remarkably, the same hypha-associated genes affected by the nrg1Δ/Δ mutation in strain P57055 were also naturally expressed at lower levels in wild-type P57055 than those in wild-type SC5314. Our results suggest that strain P57055 is defective in a pathway that acts in parallel with Nrg1 to upregulate the expression of several hypha-associated genes. IMPORTANCE Hypha formation is a central virulence trait of the fungal pathogen Candida albicans. Control of hypha formation has been studied in detail in the type strain but not in other diverse C. albicans clinical isolates. Here, we show that the hyphal repressor Nrg1 has an unexpected positive role in hypha formation and hypha-associated gene expression, as revealed by the sensitized P57055 strain background. Our findings indicate that reliance on a single type strain limits understanding of gene function and illustrate that strain diversity is a valuable resource for C. albicans molecular genetic analysis.

Candida albicans Oropharyngeal Infection Is an Exception to Iron-Based Nutritional Immunity.

Candida albicans is a commensal of the human gastrointestinal tract and a common cause of human fungal disease, including mucosal infections, such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Overall, 97 genes were differentially expressed between the 2 infection sites. Virulence-associated genes, such as hyphae-specific transcripts, were expressed similarly in the 2 sites. Genes expressed during growth in a poor carbon source (ACS1 and PCK1) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, strains lacking transcription factors that regulate iron responsive genes (SEF1, HAP5) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron, zinc, and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but not to iron nutritional immunity during oral infection. IMPORTANCE Nutritional immunity is a response by which infected host tissue sequesters nutrients, such as iron, to prevent the microbe from efficiently replicating. Microbial pathogens subjected to iron nutritional immunity express specific genes to compensate for low iron availability. By comparing the gene expression profiles of the common human fungal pathogen Candida albicans in 2 infection sites, we found that C. albicans infecting the kidney has the transcriptional profile of iron starvation. By contrast, the C. albicans expression profile during oropharyngeal infection indicates the fungus is not iron starved. Two transcription factors that activate the transcriptional response to iron starvation are not required for C. albicans virulence during oral infection but are required for disseminated infection of the kidney. Thus, our results indicate that C. albicans is subject to nutritional iron immunity during disseminated infection but not during oropharyngeal infection, and highlight niche specific differences in the host-Candida albicans interaction.

Hgc1 Independence of Biofilm Hyphae in Candida albicans.

Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

Candida albicans oropharyngeal infection is an exception to iron-based nutritional immunity.

Candida albicans is a commensal of the human gastrointestinal tract and one of the most causes of human fungal disease, including mucosal infections such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Although the expression of a set of environmentally responsive genes were correlated in the two infection sites (Pearson R 2 , 0.6), XXX genes were differentially expressed. Virulence associated genes such as hyphae-specific transcripts were expressed similarly in the two sites. Genes expressed during growth in a poor carbon source ( ACS1 and PCK1 ) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron-replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, deletion of two transcription factors that activate iron uptake genes ( SEF1 , HAP5 ) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but is exempted from iron nutritional immunity during oral infection.

IL-23 signaling prevents ferroptosis-driven renal immunopathology during candidiasis.

During infection the host relies on pattern-recognition receptors to sense invading fungal pathogens to launch immune defense mechanisms. While fungal recognition and immune effector responses are organ and cell type specific, during disseminated candidiasis myeloid cells exacerbate collateral tissue damage. The ß-glucan receptor ephrin type-A 2 receptor (EphA2) is required to initiate mucosal inflammatory responses during oral Candida infection. Here we report that EphA2 promotes renal immunopathology during disseminated candidiasis. EphA2 deficiency leads to reduced renal inflammation and injury. Comprehensive analyses reveal that EphA2 restrains IL-23 secretion from and migration of dendritic cells. IL-23 signaling prevents ferroptotic host cell death during infection to limit inflammation and immunopathology. Further, host cell ferroptosis limits antifungal effector functions via releasing the lipid peroxidation product 4-hydroxynonenal to induce various forms of cell death. Thus, we identify ferroptotic cell death as a critical pathway of Candida-mediated renal immunopathology that opens a new avenue to tackle Candida infection and inflammation.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells.

During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.

Use of the Iron-Responsive RBT5 Promoter for Regulated Expression in Candida albicans.

Engineered conditional gene expression is used in appraisal of gene function and pathway relationships. For pathogens like the fungus Candida albicans, conditional expression systems are most useful if they are active in the infection environment and if they can be utilized in multiple clinical isolates. Here, we describe such a system. It employs the RBT5 promoter and can be implemented with a few PCRs. We validated the system with RBT5 promoter fusions to two genes that promote filamentation and polarized growth, UME6 and HGC1, and with efg1Δ/Δ mutants, which are defective in an activator of filamentous growth. An RBT5 promoter fusion to either gene enabled filamentous growth of an efg1Δ/Δ mutant of strain SC5314 in iron-limited media, including RPMI with serum and yeast extract-peptone-dextrose with bathophenanthrolinedisulfonic acid. The RBT5-UME6 fusion promoted filamentation of efg1Δ/Δ mutants in RPMI with serum of four other clinical C. albicans isolates as well. In a mouse model of disseminated candidiasis, the RBT5-UME6 fusion promoted filamentation of the SC5314 efg1Δ/Δ mutant in kidney tissue, an indication that the RBT5 promoter is active in the iron-limited host environment. The RBT5 promoter expands the conditional expression toolkit for C. albicans genetics. IMPORTANCE Genetic strategies have been vital for mechanistic analysis of biological processes. Here, we describe a genetic tool for the fungal pathogen Candida albicans.

Systematic Genetic Interaction Analysis Identifies a Transcription Factor Circuit Required for Oropharyngeal Candidiasis.

Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions and can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans biofilm formation on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine if the in vitro biofilm transcriptional regulatory network also plays a role in OPC, we carried out a systematic genetic interaction analysis in a mouse model of C. albicans OPC. Whereas each of the six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1ΔΔ, bcr1ΔΔ, and rob1ΔΔ) and one heterozygous mutant (tec1Δ/TEC1) have reduced infectivity in the mouse model of OPC. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors during OPC. Using epistasis analysis, we have formulated a genetic circuit, [EFG1+BRG1]→TEC1→BCR1, that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation, such as efg1ΔΔ, rob1ΔΔ, and brg1ΔΔ filament, during oral infection and that reduced filamentation does not correlate with infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections during infection are distinct from those observed in vivo. IMPORTANCE The pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that the network of transcription factors that regulates in vitro biofilm formation has similarities and differences during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

Control of ß-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence.

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes ß-glucan in the fungal cell wall. C. albicans ß-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how ß-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates ß-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated ß-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated ß-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.

The Globular C1q Receptor Is Required for Epidermal Growth Factor Receptor Signaling during Candida albicans Infection.

During oropharyngeal candidiasis, Candida albicans activates the epidermal growth factor receptor (EGFR), which induces oral epithelial cells to endocytose the fungus and synthesize proinflammatory mediators. To elucidate EGFR signaling pathways that are stimulated by C. albicans, we used proteomics to identify 1,214 proteins that were associated with EGFR in C. albicans-infected cells. Seven of these proteins were selected for additional study. Among these proteins, WW domain-binding protein 2, Toll-interacting protein, interferon-induced transmembrane protein 3 (IFITM3), and the globular C1q receptor (gC1qR) were found to associate with EGFR in viable oral epithelial cells. Each of these proteins was required for maximal endocytosis of C. albicans, and all regulated fungus-induced production of interleukin-1ß (IL-1ß) and/or IL-8, either positively or negatively. gC1qR was found to function as a key coreceptor with EGFR. Interacting with the C. albicans Als3 invasin, gC1qR was required for the fungus to induce autophosphorylation of both EGFR and the ephrin type A receptor 2. The combination of gC1qR and EGFR was necessary for maximal endocytosis of C. albicans and secretion of IL-1ß, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human oral epithelial cells. In mouse oral epithelial cells, inhibition of gC1qR failed to block C. albicans-induced phosphorylation, and knockdown of IFITM3 did not inhibit C. albicans endocytosis, indicating that gC1qR and IFITM3 function differently in mouse versus human oral epithelial cells. Thus, this work provides an atlas of proteins that associate with EGFR and identifies several that play a central role in the response of human oral epithelial cells to C. albicans infection. IMPORTANCE Oral epithelial cells play a key role in the pathogenesis of oropharyngeal candidiasis. In addition to being target host cells for C. albicans adherence and invasion, they secrete proinflammatory cytokines and chemokines that recruit T cells and activated phagocytes to foci of infection. It is known that C. albicans activates EGFR on oral epithelial cells, which induces these cells to endocytose the organism and stimulates them to secrete proinflammatory mediators. To elucidate the EGFR signaling pathways that govern these responses, we analyzed the epithelial cell proteins that associate with EGFR in C. albicans-infected epithelial cells. We identified four proteins that physically associate with EGFR and that regulate different aspects of the epithelial response to C. albicans. One of these is gC1qR, which is required for C. albicans to activate EGFR, induce endocytosis, and stimulate the secretion of proinflammatory mediators, indicating that gC1qR functions as a key coreceptor with EGFR.

Response to Comments on "Aberrant type 1 immunity drives susceptibility to mucosal fungal infections".

Puel and Casanova and Kisand et al. challenge our conclusions that interferonopathy and not IL-17/IL-22 autoantibodies promote candidiasis in autoimmune polyendocrinopathy­candidiasis­ectodermal dystrophy. We acknowledge that conclusive evidence for causation is difficult to obtain in complex human diseases. However, our studies clearly document interferonopathy driving mucosal candidiasis with intact IL-17/IL-22 responses in Aire-deficient mice, with strong corroborative evidence in patients.

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.

Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects ß-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.