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Candida albicans contains four ORFs (GIT1,2,3,4) predicted to encode proteins involved in the transport of glycerophosphodiester metabolites. Previously, we reported that Git1, encoded by ORF 19.34, is responsible for the transport of intact glycerophosphoinositol but not glycerophosphocholine (GroPCho). Here, we report that a strain lacking both GIT3 (ORF 19.1979) and GIT4 (ORF 19.1980) is unable to transport [3H]GroPCho into the cell. In the absence of a GroPCho transporter, C. albicans can utilize GroPCho via a mechanism involving extracellular hydrolysis. Upon reintegration of either GIT3 or GIT4 into the genome, measurable uptake of [3H]GroPCho is observed. Transport assays and kinetic analyses indicate that Git3 has the greater transport velocity. We present evidence that GDE1 (ORF 19.3936) codes for an enzyme with glycerophosphodiesterase activity against GroPCho. Homozygous deletion of GDE1 results in a buildup of internal GroPCho that is restored to wild type levels by reintegration of GDE1 into the genome. The transcriptional regulator, Pho4, is shown to regulate the expression of GIT3, GIT4, and GDE1. Finally, Git3 is shown to be required for full virulence in a mouse model of disseminated candidiasis, and Git3 sequence orthologs are present in other pathogenic Candida species. In summary, we have characterized multiple aspects of GroPCho utilization by C. albicans and have demonstrated that GroPCho transport plays a key role in the growth of the organism in the host.

During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro , a C. albicans als3 Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata , a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3 -expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3 -expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro , demonstrating a potential mechanism for ALS3 -mediated neurotropism. In addition, upon initiation of infection, the ALS3 -expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3 -expressing strain had increased resistance to neutrophil killing in vitro . These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

NanoString nCounter is a recently developed platform that can make direct multiplexed measurement of gene expression using color-coded probe pairs (Geiss et al., Nat Biotechnol 26(3):317-325, 2008; Malkov et al., BMC Res Notes 2:80, 2009). We have found that this platform is uniquely suitable for quantification of pathogen gene expression during infection, where pathogen RNA comprises a tiny portion of total RNA isolated from the infected tissue. Here, we describe a protocol that we have successfully applied to a number of pathogens across multiple infection models, including both invasive and mucosal infection by Candida albicans, and lung infection by Aspergillus fumigatus and Cryptococcus neoformans.

Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1 Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.

Oropharyngeal candidiasis is a frequent cause of morbidity in patients with defects in cell-mediated immunity or saliva production. Animal models of this infection are important for studying disease pathogenesis and evaluating vaccines and antifungal therapies. Here we describe a simple murine model of oropharyngeal candidiasis. Mice are rendered susceptible to oral infection by injection with cortisone acetate and then inoculated by placing a swab saturated with Candida albicans sublingually. This process results in a reproducible level of infection, the histopathology of which mimics that of pseudomembranous oropharyngeal candidiasis in patients. Using this model, data are obtained after 5–9 days of work.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress., Author summary Candida albicans is the species most often isolated from patients with invasive fungal disease, and is also a common colonizer of healthy people. It is well equipped to compete for nutrients with bacteria co-inhabiting human gastrointestinal mucous membranes, since it possesses multiple transporters to internalize important nutrients like sugars, nitrogen sources, and phosphate. During infection, the fungus needs to withstand human defense cells that attack it with noxious chemicals, among which reactive oxygen species (ROS) are critical. We found that a high-affinity phosphate transporter, Pho84, is required for C. albicans’ ability to successfully invade animal hosts and to eliminate ROS. Levels of a fungal enzyme that breaks down ROS, Sod3, were decreased in cells lacking Pho84. A connection between this phosphate transporter and the ROS-detoxifying enzyme was identified in the Target of Rapamycin (TOR) pathway, to which Pho84 is known to provide activating signals when phosphate is abundant. Small molecules that block Pho84 activity impair the ability of C. albicans to detoxify ROS. Since humans manage phosphate differently than fungi and have no Pho84 homolog, a drug that inhibits Pho84 could disable the defense of the fungus against the host.

Oral epithelial cells discriminate between pathogenic and non-pathogenic stimuli, and only induce an inflammatory response when they are exposed to high levels of a potentially harmful microorganism. The pattern recognition receptors (PRRs) in epithelial cells that mediate this differential response are poorly understood. Here, we demonstrate that the ephrin type-A receptor 2 (EphA2) is an oral epithelial cell PRR that binds to exposed β-glucans on the surface of the fungal pathogen Candida albicans. Binding of C. albicans to EphA2 on oral epithelial cells activates signal transducer and activator of transcription 3 and mitogen-activated protein kinase signalling in an inoculum-dependent manner, and is required for induction of a proinflammatory and antifungal response. EphA2

Aspergillus fumigatus causes serious and frequently fatal infections in immunocompromised patients. To investigate the regulation of virulence of this fungus, we constructed and analysed an A. fumigatus mutant that lacked the transcription factor Ace2, which influences virulence in other fungi. The Deltaace2 mutant had dysmorphic conidiophores, reduced conidia production and abnormal conidial cell wall architecture. This mutant produced an orange pigment when grown on solid media, although its conidia had normal pigmentation. Conidia of the Deltaace2 mutant were larger and had accelerated germination. The resulting germlings were resistant to hydrogen peroxide, but not other stressors. Non-neutropenic mice that were immunosuppressed with cortisone acetate and infected with the Deltaace2 mutant had accelerated mortality, greater pulmonary fungal burden, and increased pulmonary inflammatory responses compared with mice infected with the wild-type or Deltaace2::ace2-complemented strains. The Deltaace2 mutant had reduced ppoC, ecm33 and ags3 mRNA expression. It is known that A. fumigatus mutants with absent or reduced expression of these genes have increased virulence in mice, as well as other phenotypic similarities to the Deltaace2 mutant. Therefore, reduced expression of these genes likely contributes to the increased virulence of the Deltaace2 mutant.

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41 , Orf19.5412, Orf19.1277, MSB2 , Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41 Δ/ sun41 Δ deletion mutants and sun41 Δ/ sun41 Δ+p SUN41 -complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41 Δ/ sun41 Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41 Δ/ sun41 Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs., Author Summary The ubiquitous mold A. fumigatus is isolated in over 80% of all patients with invasive aspergillosis (IA). A. nidulans is a relatively non-pathogenic species that rarely causes IA except in patients with chronic granulomatous disease (CGD), a hereditary disease characterized by impaired neutrophil function due to mutations in the NADPH oxidase complex. Here, we demonstrate that one factor underlying the differences in the intrinsic virulence between A. fumigatus and A. nidulans is the amount of the exopolysaccharide galactosaminogalactan that is associated with the cell wall of these species. A. fumigatus produces higher amounts of cell wall-associated galactosaminogalactan and is more resistant than A. nidulans to neutrophil killing by NADPH-oxidase dependent extracellular traps (NETs). Increasing cell wall-associated galactosaminogalactan in A. nidulans enhanced resistance to NETs and increased the virulence of this species to the same level as A. fumigatus in mice with intact NET formation. Collectively, these data suggest that A. nidulans is more sensitive than A. fumigatus to NADPH-oxidase dependent NETosis due to lower levels of cell wall-associated GAG.

Background Microbiome studies incorporate next-generation sequencing to obtain profiles of microbial communities. Data generated from these experiments are high-dimensional with a rich correlation structure but modest sample sizes. A statistical model that utilizes these microbiome profiles to explain a clinical or biological endpoint needs to tackle high-dimensionality resulting from the very large space of variable configurations. Ensemble models are a class of approaches that can address high-dimensionality by aggregating information across large model spaces. Although such models are popular in fields as diverse as economics and genetics, their performance on microbiome data has been largely unexplored. Results We developed a simulation framework that accurately captures the constraints of experimental microbiome data. Using this setup, we systematically evaluated a selection of both frequentist and Bayesian regression modeling ensembles. These are represented by variants of stability selection in conjunction with elastic net and spike-and-slab Bayesian model averaging (BMA), respectively. BMA ensembles that explore a larger space of models relative to stability selection variants performed better and had lower variability across simulations. However, stability selection ensembles were able to match the performance of BMA in scenarios of low sparsity where several variables had large regression coefficients. Conclusions Given a microbiome dataset of interest, we present a methodology to generate simulated data that closely mimics its characteristics in a manner that enables meaningful evaluation of analytical strategies. Our evaluation demonstrates that the largest ensembles yield the strongest performance on microbiome data with modest sample sizes and high-dimensional measurements. We also demonstrate the ability of these ensembles to identify microbiome signatures that are associated with opportunistic Candida albicans colonization during antibiotic exposure. As the focus of microbiome research evolves from pilot to translational studies, we anticipate that our strategy will aid investigators in making evaluation-based decisions for selecting appropriate analytical methods. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0467-6) contains supplementary material, which is available to authorized users.

Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive Candida albicans infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either ZAP1 or the Zap1-dependent zinc transporter gene ZRT2 restores pathogenicity to a sut1 mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host–pathogen interaction occurs., A study of the invasive infection of a mammalian host by the pathogenic fungus Candida albicans reveals characteristic gene regulation patterns in response to the host environment, distinct from those seen when growing in vitro., Author Summary We have a limited understanding of how the expression of pathogens’ genes changes during infection of humans or other animal hosts, in contrast to in vitro models of infection. Here we profile the alteration in gene expression over time as a predictor of functional consequences during invasive growth of Candida in the kidney; a situation in which the limited number of pathogen cells makes gene expression challenging to assay. Our findings reveal that there are distinct early and late phases of infection, and identify new genes that govern the early zinc acquisition response necessary for proliferation in vivo—and thus required for infection. We also find that the response to drug treatment that manifests during infection can be distinct from that detected in vitro. We show that a well-known gene expression response to the antifungal drug caspofungin is naturally down-regulated in infecting cells, suggesting that the efficacy of the drug may be enhanced by a susceptible state of the pathogen during invasive proliferation.

Candida albicans is part of the normal commensal microbiota of mucosal surfaces in a large percentage of the human population. However, perturbations of the host's immune response or bacterial microbiota have been shown to predispose individuals to the development of opportunistic Candida infections. It was recently discovered that a defect in the chemokine receptor CX 3 CR1 increases susceptibility of mice and humans to systemic candidiasis. However, whether CX 3 CR1 confers protection against mucosal C. albicans infection has not been investigated. Using two different mouse models, we found that Cx3cr1 is dispensable for the induction of interleukin 17A (IL-17A), IL-22, and IL-23 in the tongue after infection, as well as for the clearance of mucosal candidiasis from the tongue or lower gastrointestinal (GI) tract colonization. Furthermore, the dysfunctional human CX 3 CR1 allele CX 3 CR1-M280 was not associated with development of recurrent vulvovaginal candidiasis (RVVC) in women. Taken together, these data indicate that CX 3 CR1 is not essential for protection of the host against mucosal candidiasis, underscoring the dependence on different mammalian immune factors for control of mucosal versus systemic Candida infections.

The ability to switch from yeast to hyphal growth is essential for virulence in Candida albicans. The cell surface is the initial point of contact between the fungus and the host. In this work, a free-gel proteomic strategy based on tryptic digestion of live yeast and hyphae cells and protein identification using LC-MS/MS methodology was used to identify cell surface proteins. Using this strategy, a total of 943 proteins were identified, of which 438 were in yeast and 928 were in hyphae. Of these proteins, 79 were closely related to the organization and biogenesis of the cell wall, including 28 GPI-anchored proteins, such as Hyr1 and Sod5 which were detected exclusively in hyphae, and Als2 and Sap10which were detected only in yeast. A group of 17 proteins of unknown function were subsequently studied by analysis of the corresponding deletion mutants. We found that four new proteins, Pst3, Tos1, Orf19.3060 and Orf19.5352 are involved in cell wall integrity and in C. albicans' engulfment by macrophages. Moreover, the putative NADH-ubiquinone-related proteins, Ali1, Mci4, Orf19.287 and Orf19.7590, are also involved in osmotic and oxidative resistance, yeast to hypha transition and the ability to damage and invade oral epithelial cells. This article is part of a Special Issue entitled: HUPO 2014.

Receipt of broad-spectrum antibiotics enhances Candida albicans colonization of the GI tract, a risk factor for haematogenously-disseminated candidiasis. To understand how antibiotics influence C. albicans colonization, we treated mice orally with vancomycin or a combination of penicillin, streptomycin and gentamicin (PSG) and then inoculated them with C. albicans by gavage. Only PSG treatment resulted in sustained, high-level GI colonization with C. albicans. Furthermore, PSG reduced bacterial diversity in the colon much more than vancomycin. Both antibiotic regimens significantly reduced IL-17A, IL-21, IL-22 and IFN-γ mRNA levels in the terminal ileum but had limited effect on the GI fungal microbiome. Through a series of models that employed Bayesian model averaging, we investigated the associations between antibiotic treatment, GI microbiota and host immune response and their collective impact on C. albicans colonization. Our analysis revealed that bacterial genera were typically associated with either C. albicans colonization or altered cytokine expression but not with both. The only exception was Veillonella, which was associated with both increased C. albicans colonization and reduced IL-21 expression. Overall, antibiotic-induced changes in the bacterial microbiome were much more consistent determinants of C. albicans colonization than either the GI fungal microbiota or the GI immune response.

Summary Candida albicans reversibly switches between yeast and hyphal morphologies, with hyphae being associated with virulence. Hyphal initiation and maintenance depend on host environment sensing. Hyphal maintenance in vitro requires chromatin remodeling of hypha-specific gene promoters, although disrupting chromatin-remodeling does not disrupt C. albicans hyphal elongation and virulence during invasive infection. We find that the combination of hypoxia and high CO 2 , but neither condition alone, maintains hyphal elongation, even in mutants lacking the nutrient-responsive chromatin-remodeling pathway. Ume6, the transcriptional activator of hypha-specific genes, is stabilized via regulation by Ofd1, a prolyl hydroxylase family member inhibited by hypoxia, and by an uncharacterized pathway that senses high CO 2 . Virulence and hyphal elongation in vivo are attenuated only when the parallelly acting Ume6 stabilization and chromatin-remodeling pathways are both blocked. The evolution of redundant signaling pathways allowing C. albicans to adapt to varied host environments may explain this commensal's success as a pathogen.

In order to colonize the host and cause disease, Candida albicans must avoid being killed by host defense peptides. Previously, we determined that the regulatory protein Ssd1 governs antimicrobial peptide resistance in C. albicans . Here, we sought to identify additional genes whose products govern susceptibility to antimicrobial peptides. We discovered that a bcr1 Δ/Δ mutant, like the ssd1 Δ/Δ mutant, had increased susceptibility to the antimicrobial peptides, protamine, RP-1, and human β defensin-2. Homozygous deletion of BCR1 in the ssd1 Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1 Δ/Δ and ssd1 Δ/Δ mutants to RP-1 induced greater loss of mitochondrial membrane potential and increased plasma membrane permeability than with the control strains. Therefore, Bcr1 and Ssd1 govern antimicrobial peptide susceptibility and likely function in the same pathway. Furthermore, BCR1 mRNA expression was downregulated in the ssd1 Δ/Δ mutant, and the forced expression of BCR1 in the ssd1 Δ/Δ mutant partially restored antimicrobial peptide resistance. These results suggest that Bcr1 functions downstream of Ssd1. Interestingly, overexpression of 11 known Bcr1 target genes in the bcr1 Δ/Δ mutant failed to restore antimicrobial peptide resistance, suggesting that other Bcr1 target genes are likely responsible for antimicrobial peptide resistance. Collectively, these results demonstrate that Bcr1 functions downstream of Ssd1 to govern antimicrobial peptide resistance by maintaining mitochondrial energetics and reducing membrane permeabilization.

The fungus Candida albicans is the major cause of oropharyngeal candidiasis (OPC). A key feature of this disease is fungal invasion of oral epithelial cells, a process that can occur by active penetration and fungal-induced endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C. albicans , in part by binding to E-cadherin. However, inhibition of E-cadherin function only partially reduces C. albicans endocytosis, suggesting that there are additional epithelial cell receptors for this organism. Here, we show that the EGF receptor (EGFR) and HER2 function cooperatively to induce the endocytosis of C. albicans hyphae. EGFR and HER2 interact with C. albicans in an Als3- and Ssa1-dependent manner, and this interaction induces receptor autophosphorylation. Signaling through both EGFR and HER2 is required for maximal epithelial cell endocytosis of C. albicans in vitro. Importantly, oral infection with C. albicans stimulates the phosphorylation of EGFR and HER2 in the oral mucosa of mice, and treatment with a dual EGFR and HER2 kinase inhibitor significantly decreases this phosphorylation and reduces the severity of OPC. These results show the importance of EGFR and HER2 signaling in the pathogenesis of OPC and indicate the feasibility of treating candidal infections by targeting the host cell receptors with which the fungus interacts.

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.

Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells., Author Summary During hematogenously disseminated infection, the fungus Candida albicans is carried by the bloodstream to virtually all organs in the body, including the brain. C. albicans infection of the brain is a significant problem in premature infants with disseminated candidiasis. To infect the brain, C. albicans must adhere to and invade the endothelial cells that line cerebral blood vessels. These endothelial cells express unique proteins on their surface that are not expressed by endothelial cells of other vascular beds. Here, we show that C. albicans infects the brain by binding to gp96, a heat shock protein that is uniquely expressed on the surface of brain endothelial cells. Gp96 is bound by the C. albicans Als3 invasin, which induces the uptake of this organism by brain endothelial cells. The C. albicans Ssa1 invasin also mediates fungal uptake by brain endothelial cells, but does so by binding to a receptor other than gp96. Thus, during hematogenously disseminated infection, C. albicans traffics to and infects the brain by binding to gp96, a receptor that is expressed specifically on the surface of brain endothelial cells.

Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans , which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components ( C1q , C1r , and C1s ) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans , Cryptococcus neoformans , and Aspergillus fumigatus . In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans , in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis . We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis . Similar to C. albicans , C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease., Author Summary The fungus Candida albicans can proliferate in the mouth, causing oropharyngeal candidiasis. In other patients, it can enter the bloodstream and spread throughout the body, resulting in hematogenously disseminated candidiasis. Fungal invasion of host cells is a key feature of both types of infection. One mechanism by which C. albicans invades both the epithelial cell lining of the oropharynx and the endothelial cell lining of the blood vessels is by inducing its own uptake. This uptake is induced in part by the binding of the C. albicans invasin Als3 to host cell proteins, which include N- and E-cadherin. Here we show that C. albicans Ssa1, a member of the 70 kDa heat shock protein family, is expressed on the surface of C. albicans where it functions as an invasin. The key role of Ssa1 in host cell invasion is illustrated by the reduced capacity of an ssa1Δ/Δ null mutant to induce its own uptake by epithelial and endothelial cells in vitro, and by the significantly attenuated virulence of this mutant in mouse models of oropharyngeal candidiasis and disseminated candidiasis. Thus, Ssa1 is the second identified invasin of C. albicans.

The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a Δ dvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro . However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a Δ dvrA :: dvrA -complemented strain. This increase in damage required direct contact between the mutant and host cells. The Δ dvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the Δ dvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and Δ dvrA :: dvrA -complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.

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Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species., Author Summary Generation of cellular polarity – asymmetry in shape, protein distribution, and/or sub-cellular function – is an essential feature of most eukaryotic cells and underlies such diverse processes as differentiation, mating, nutrient acquisition, and growth. Localization of specific mRNAs is one mechanism through which cells achieve polarity. We describe an RNA transport system in Candida albicans, a fungal pathogen of humans, that grows in both single cell (budding yeast) and filamentous (hyphal and pseudohyphal) forms. Hyphae are chains of elongated cells that remain attached after cell division and exhibit highly polarized growth at their tips. We show that the C. albicans She3-dependent RNA transport system binds to 40 mRNAs and transports these mRNAs to yeast buds and to the tips of hyphae. Both the transport system itself and many of the genes encoded by transported mRNAs are required for normal growth and function of hyphae. Although the basic transport mechanism appears conserved with that of the model yeast, Saccharomyces cerevisiae, the cargo mRNAs are largely distinct. The apparently rapid evolution of the transported mRNAs probably reflects distinct selective pressures acting on the two organisms.

Candida albicans interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify C. albicans genes that govern interactions with these host cells in vitro. The transcriptional response of C. albicans to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that YCK2 , VPS51 , and UEC1 are required for C. albicans to cause normal damage to epithelial cells and resist antimicrobial peptides. YCK2 is also necessary for maintenance of cell polarity. VPS51 is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage. UEC1 encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some C. albicans genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream.

pH-responsive transcription factors of the Rim101/PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Delta/Delta mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101Delta/Delta mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.

Immunologic limitations make it difficult to study the pharmacokinetic effects of human tumor necrosis factor (TNF) blockers in murine models. To counter this, we have studied the pharmacokinetics in mice of two murine analogs of human TNF blockers, a murine p75-FC fusion protein (analogous to etanercept), and the rat MP6-XT22 anti-murine TNF mAb (analogous to infliximab). We analyzed the pharmacokinetics of the murine p75-Fc protein and MP6-XT22 antibody in mice that were uninfected and in mice with disseminated candidiasis in order to confirm dosing strategies and interpret future studies evaluating the efficacy and tolerability of these agents in mice. We propose that, while conducting safety or efficacy studies in murine disease models, it is reasonable to administer the murine p75-Fc protein to mice at10 mg/kg every 4-5 days, and the MP6-XT22 antibody at 10-20 mg/kg every 4-5 days.