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.

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.

Use of a human small airway epithelial cell line to study the interactions of Aspergillus fumigatus with pulmonary epithelial cells.

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5ß1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5ß1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

Novel Host Pathways Govern Epithelial Cell Invasion of Aspergillus fumigatus.

Invasive aspergillosis is initiated when Aspergillus fumigatus adheres to and invades the pulmonary epithelial cells that line the airways and alveoli. To gain deeper insight into how pulmonary epithelial cells respond to A. fumigatus invasion, we used transcriptome sequencing (RNA-seq) to determine the transcriptional response of the A549 type II alveolar epithelial cell line to infection with strains CEA10 and Af293, two clinical isolates of A. fumigatus. Upstream regulator analysis of the data indicated that while both strains activated virtually identical host cell signaling pathways after 16 h of infection, only strain CEA10 activated these pathways after 6 h of infection. Many of the pathways that were predicted to be activated by A. fumigatus, including the tumor necrosis factor (TNF), interleukin-1α (IL-1α), IL-1ß, IL-17A, Toll-like receptor 2 (TLR2), and TLR4 pathways, are known to be critical for the host defense against this fungus. We also found that the platelet-derived growth factor BB (PDGF BB) and progesterone receptor (PGR) pathways were activated by A. fumigatus. Using pharmacologic inhibitors, we determined that blocking the PDGF receptor or PGR inhibited the endocytosis of both strains of A. fumigatus in an additive manner. Both the PDGF BB and PGR pathways are also predicted to be activated by infection of A549 cells with other molds, such as Rhizopus delemar and Rhizopus oryzae. Thus, these pathways may represent a common response of pulmonary epithelial cells to mold infection. IMPORTANCE Invasive aspergillosis is a deadly invasive fungal infection that initiates when Aspergillus fumigatus spores are inhaled and come into contact with the epithelial cells that line the airways and alveoli. Understanding this fungus-host interaction is important for the development of novel therapeutics. To gain a deeper understanding of how these airway epithelial cells respond to A. fumigatus during infection, we used RNA-seq to determine the transcriptional response of alveolar epithelial cells to infection with two different clinical isolates of A. fumigatus. Our analysis identified new host response pathways that have not previously been tied to infection with A. fumigatus. Pharmacological inhibition of two of these pathways inhibited the ability of A. fumigatus to invade airway epithelial cells. These two pathways are also predicted to be activated by infection with other filamentous fungi. Thus, these pathways may represent a common response of alveolar epithelial cells to mold infection.

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.

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.

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.

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.

Identification of Host Receptors for Fungi Using Whole Cell Affinity Purification.

Receptors on endothelial and epithelial cells often recognize molecules that are expressed by fungi, and only a limited number of these receptors have been identified to date. Here, we describe a method for identifying novel host cell receptors for fungi that uses intact organisms to precipitate biotin-labelled host cell membrane proteins, which are then detected by immunoblotting with an anti-biotin antibody. Presented here is the method to use for identification of membrane proteins that bind to C. albicans.

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.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Plasma Membrane Phosphatidylinositol-4-Phosphate Is Not Necessary for Candida albicans Viability yet Is Key for Cell Wall Integrity and Systemic Infection.

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi apparatus is required for the budding-to-filamentous-growth transition in the human-pathogenic fungus Candida albicans; however, the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e., Efr3, Ypp1, and Stt4. The amounts of plasma membrane PI(4)P in the efr3Δ/Δ and ypp1Δ/Δ mutants were ∼60% and ∼40%, respectively, of that in the wild-type strain, whereas it was nearly undetectable in the stt4Δ/Δ mutant. All three mutants had reduced plas7ma membrane phosphatidylserine (PS). Although these mutants had normal yeast-phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity, and had an increased exposure of cell wall ß(1,3)-glucan, yet they induced a range of hyphal-specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3Δ/Δ mutant had wild-type virulence, the ypp1Δ/Δ mutant had attenuated virulence, and the stt4Δ/Δ mutant caused no lethality. In the mouse model of oropharyngeal candidiasis, only the ypp1Δ/Δ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity, and virulence in C. albicans. IMPORTANCE While the PI-4-kinases Pik1 and Stt4 both produce PI(4)P, the former generates PI(4)P at the Golgi apparatus and the latter at the plasma membrane, and these two pools are functionally distinct. To address the importance of plasma membrane PI(4)P in Candida albicans, we generated deletion mutants of the three putative plasma membrane PI-4-kinase complex components and quantified the levels of plasma membrane PI(4)P in each of these strains. Our work reveals that this phosphatidylinositol phosphate is specifically critical for the yeast-to-hyphal transition, cell wall integrity, and virulence in a mouse systemic infection model. The significance of this work is in identifying a plasma membrane phospholipid that has an infection-specific role, which is attributed to the loss of plasma membrane PI(4)P resulting in ß(1,3)-glucan unmasking.

Flotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections.

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.

Proteomic profiling of the monothiol glutaredoxin Grx3 reveals its global role in the regulation of iron dependent processes.

Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell.

Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus.

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity.IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis.

Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin ß1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin ß1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin ß1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Genetic variation of DNA methyltransferase-3A contributes to protection against persistent MRSA bacteremia in patients.

The role of the host in development of persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is not well understood. A cohort of prospectively enrolled patients with persistent methicillin-resistant S. aureus bacteremia (PB) and resolving methicillin-resistant S. aureus bacteremia (RB) matched by sex, age, race, hemodialysis status, diabetes mellitus, and presence of implantable medical device was studied to gain insights into this question. One heterozygous g.25498283A > C polymorphism located in the DNMT3A intronic region of chromosome 2p with no impact in messenger RNA (mRNA) expression was more common in RB (21 of 34, 61.8%) than PB (3 of 34, 8.8%) patients (P = 7.8 × 10-6). Patients with MRSA bacteremia and g.25498283A > C genotype exhibited significantly higher levels of methylation in gene-regulatory CpG island regions (Δmethylation = 4.1%, P < 0.0001) and significantly lower serum levels of interleukin-10 (IL-10) than patients with MRSA bacteremia without DNMT3A mutation (A/C: 9.7038 pg/mL vs. A/A: 52.9898 pg/mL; P = 0.0042). Expression of DNMT3A was significantly suppressed in patients with S. aureus bacteremia and in S. aureus-challenged primary human macrophages. Small interfering RNA (siRNA) silencing of DNMT3A expression in human macrophages caused increased IL-10 response upon S. aureus stimulation. Treating macrophages with methylation inhibitor 5-Aza-2'-deoxycytidine resulted in increased levels of IL-10 when challenged with S. aureus In the murine sepsis model, methylation inhibition increased susceptibility to S. aureus These findings indicate that g.25498283A > C genotype within DNMT3A contributes to increased capacity to resolve MRSA bacteremia, potentially through a mechanism involving increased methylation of gene-regulatory regions and reduced levels of antiinflammatory cytokine IL-10.

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

CARD9+ microglia promote antifungal immunity via IL-1ß- and CXCL1-mediated neutrophil recruitment.

The C-type lectin receptor-Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1ß served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1ß and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1ß, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1ß and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host-pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.