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1.  Role of Retrograde Trafficking in Stress Response, Host Cell Interactions, and Virulence of Candida albicans 
Eukaryotic Cell  2014;13(2):279-287.
In Saccharomyces cerevisiae, the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of Candida albicans. We found that C. albicans vps15Δ/Δ and vps51Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells in vitro and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the vps51Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members CHR11, UTR2, CRZ1, CNA1, and CNA2 were elevated in the vps15Δ/Δ and vps51Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of CHR11 and UTR2 further increased the stress susceptibility of these mutants. In contrast, overexpression of CRH11 and UTR2 partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in C. albicans is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables C. albicans to withstand environmental stress.
PMCID: PMC3910971  PMID: 24363364
2.  In vitro models of hematogenously disseminated candidiasis 
Virulence  2014;5(2):240-242.
PMCID: PMC3956498  PMID: 24384501
virulence; endothelial cells; epithelial cells; adherence; invasion; damage
3.  Investigation of the Function of Candida albicans Als3 by Heterologous Expression in Candida glabrata 
Infection and Immunity  2013;81(7):2528-2535.
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 als3Δ/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.
PMCID: PMC3697595  PMID: 23630968
4.  Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans 
mBio  2013;4(6):e00542-13.
Candida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism.
During hematogenously disseminated infection, Candida albicans invades the endothelial cell lining of the blood vessels to invade the deep tissues. C. albicans can invade endothelial cells by inducing its own endocytosis, which is triggered when the C. albicans Als3 and Ssa1 invasins bind to N-cadherin on the endothelial cell surface. How this binding induces endocytosis is incompletely understood. Septins are intracellular GTP-binding proteins that influence the function and localization of cell surface proteins. We found that C. albicans Als3 and Ssa1 bind to a complex containing N-cadherin and septin 7, which in turn interacts with endothelial cell microfilaments, thereby inducing endocytosis of the organism. The key role of septin 7 in governing receptor-mediated endocytosis is likely relevant to host cell invasion by other microbial pathogens, in addition to C. albicans.
PMCID: PMC3870263  PMID: 24345743
5.  Different tumor necrosis factor α antagonists have different effects on host susceptibility to disseminated and oropharyngeal candidiasis in mice 
Virulence  2014;5(5):625-629.
Tumor necrosis factor α is important for the host defense against intracellular pathogens. We tested the effect of mouse analogs of human TNF-α antagonists, the rat anti-mouse TNF-α monoclonal antibody (XT22) and the soluble mouse 75 kDa TNF-α receptor fused to the Fc portion of mouse IgG1 (p75-Fc), on the susceptibility of mice to hematogenously disseminated candidiasis (HDC) and oropharyngeal candidiasis (OPC). Both XT22 and p75-Fc significantly reduced mice survival, increased kidney fungal burden, and reduced leukocyte recruitment during HDC. However, only XT22 significantly increased the oral fungal burden and reduced leukocyte recruitment during OPC. This result suggests that XT22 and p75-Fc affect host susceptibility to different types of Candida albicans infections by different inhibitory mechanisms.
PMCID: PMC4105312  PMID: 25007095
Candida albicans; hematogenously disseminated candidiasis; tumor necrosis factor antagonists; host susceptibility; murine model; oropharyngeal candidiasis
7.  Bcr1 Functions Downstream of Ssd1 To Mediate Antimicrobial Peptide Resistance in Candida albicans 
Eukaryotic Cell  2013;12(3):411-419.
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 bcr1Δ/Δ mutant, like the ssd1Δ/Δ mutant, had increased susceptibility to the antimicrobial peptides, protamine, RP-1, and human β defensin-2. Homozygous deletion of BCR1 in the ssd1Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1Δ/Δ and ssd1Δ/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 ssd1Δ/Δ mutant, and the forced expression of BCR1 in the ssd1Δ/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 bcr1Δ/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.
PMCID: PMC3629773  PMID: 23314964
8.  Activation and Alliance of Regulatory Pathways in C. albicans during Mammalian Infection 
PLoS Biology  2015;13(2):e1002076.
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.
PMCID: PMC4333574  PMID: 25693184
9.  Using Bayesian modelling to investigate factors governing antibiotic-induced Candida albicans colonization of the GI tract 
Scientific Reports  2015;5:8131.
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.
PMCID: PMC4314636  PMID: 25644850
10.  A systematic evaluation of high-dimensional, ensemble-based regression for exploring large model spaces in microbiome analyses 
BMC Bioinformatics  2015;16(1):31.
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.
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.
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.
PMCID: PMC4339743  PMID: 25638274
Microbiome analysis; Sparse ensemble-based regression; Simulation design; Stability selection; Bayesian model averaging; Evaluation
11.  Pharmacokinetics of Posaconazole Within Epithelial Cells and Fungi: Insights Into Potential Mechanisms of Action During Treatment and Prophylaxis 
The Journal of Infectious Diseases  2013;208(10):1717-1728.
Background. The antifungal posaconazole concentrates within host cells and protects against Aspergillus fumigatus. The specific subcellular location of posaconazole and the mechanism by which cell-associated posaconazole inhibits fungal growth remain uncharacterized.
Methods. Posaconazole was conjugated with the fluorophore boron-dipyrromethene (BDP-PCZ). A549 pulmonary epithelial cells and A. fumigatus were exposed to BDP-PCZ individually and in coculture. BDP-PCZ subcellular localization and trafficking were observed using confocal microscopy and flow cytometry.
Results. BDP-PCZ concentrated within A549 cell membranes, and in particular within the endoplasmic reticulum. Epithelial cell-associated BDP-PCZ rapidly transferred to and concentrated within A. fumigatus cell membranes on contact. BDP-PCZ transfer to conidia did not require phagocytosis and was markedly enhanced by the conidial hydrophobin RodA. Within AF, BDP-PCZ also concentrated in membranes including the endoplasmic reticulum and colocalized with the azole target enzyme CYP51a. Concentration of BDP-PCZ within host and fungal cell membranes persisted for >48 hours and could be competitively inhibited by posaconazole but not voriconazole.
Conclusions. Posaconazole concentrates within host cell membranes and rapidly transfers to A. fumigatus, where it accumulates to high concentrations and persists at the site of its target enzyme. These intracellular and intercellular pharmacokinetic properties probably contribute to the efficacy of PCZ.
PMCID: PMC3805237  PMID: 23908482
posaconazole; localization; pharmacokinetics; endoplasmic reticulum; epithelial cells; Aspergillus; postantifungal effect; hydrophobin rodA
12.  Synergistic regulation of hyphal elongation by hypoxia, CO2, and nutrient conditions controls the virulence of Candida albicans 
Cell host & microbe  2013;14(5):499-509.
Candida albicans reversibly switches between yeast and hyphal morphologies, with hyphae being associated with virulence. Hyphal initiation and maintenance depends 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 CO2, 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 CO2. 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.
PMCID: PMC4049569  PMID: 24237696
14.  The pH-Responsive PacC Transcription Factor of Aspergillus fumigatus Governs Epithelial Entry and Tissue Invasion during Pulmonary Aspergillosis 
PLoS Pathogens  2014;10(10):e1004413.
Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.
Author Summary
Inhaled spores of the pathogenic mould Aspergillus fumigatus cause fungal lung infections in humans having immune defects. A. fumigatus spores germinate within the immunocompromised lung, producing invasively growing, elongated cells called hyphae. Hyphae degrade the surrounding pulmonary tissue, a process thought to be caused by secreted fungal enzymes; however, A. fumigatus mutants lacking one or more protease activities retain fully invasive phenotypes in mouse models of disease. Here we report the first discovery of a non-invasive A. fumigatus mutant, which lacks a pH-responsive transcription factor PacC. Using global transcriptional profiling of wild type and mutant isolates, and in vitro pulmonary invasion assays, we established that loss of PacC leads to a compound non-invasive phenotype characterised by deficits in both contact-mediated epithelial entry and protease expression. Consistent with an important role for epithelial entry in promoting invasive disease in mammalian tissues, PacC mutants remain surface-localised on mammalian epithelia, both in vitro and in vivo. Our study sets a new precedent for involvement of both host and pathogen activities in promoting epithelial invasion by A. fumigatus and supports a model wherein fungal protease activity acting subsequently to, or in parallel with, host-mediated epithelial entry provides the mechanistic basis for tissue invasion.
PMCID: PMC4199764  PMID: 25329394
15.  Cryptococcus gattii VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal 
PLoS Pathogens  2014;10(8):e1004285.
Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.
Author Summary
The environmentally-acquired human pathogen C. gattii is responsible for ongoing and expanding outbreaks in the Western United States and Canada. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV. Molecular types VGI, VGII, and VGIII have been isolated from patients and animals throughout the Western US. The Pacific Northwest and Canadian outbreak is primarily caused by C. gattii VGII. VGIII is responsible for ongoing infections in HIV/AIDS patients in Southern California. However, only two environmental C. gattii isolates have ever been identified from the Californian environment: CBS7750 (VGII) and WM161 (VGIII). We sought to collect environmental samples from areas that had confirmed reports of clinical or veterinary infections. Here we report the isolation of C. gattii VGI and VGIII from environmental soil and tree samples. C. gattii isolates were obtained from three novel tree species: Canary Island pine, American sweetgum, and a Pohutukawa tree. Genetic analysis provides robust evidence that these environmental isolates are the source of human infections.
PMCID: PMC4140843  PMID: 25144534
16.  Candida albicans transcription factor Rim101 mediates pathogenic interactions through cell wall functions 
Cellular microbiology  2008;10(11):2180-2196.
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 (OPC) model. Rim101 affects the yeast-hyphal morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the OPC model is independent of the yeast-hyphal transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Δ/Δ 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 rim101Δ/Δ 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.
PMCID: PMC2701370  PMID: 18627379
17.  Can host receptors for fungi be targeted for treatment of fungal infections? 
Trends in microbiology  2013;21(8):389-396.
The invasion and stimulation of normally non-phagocytic host cells, such as epithelial and endothelial cells, is a key step in the pathogenesis of many fungal infections. In most cases, host cell invasion and/or stimulation of a pro-inflammatory response is induced when proteins or carbohydrates on the fungal cell surface bind to receptors on the host cell. While many of these fungal–host cell interactions have only been investigated in vitro, the therapeutic efficacy of blocking the host cell receptors for Candida albicans and Rhizopus oryzae has been demonstrated in experimental animal models of infection. Here we summarize recent studies of the fungal receptors on normally non-phagocytic host cells and the therapeutic implications of blocking these receptors.
PMCID: PMC3735786  PMID: 23796589
epithelial cells; endothelial cells; fungi; receptor; pathogenesis
18.  Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism 
Cellular microbiology  2009;11(8):1179-1189.
Candida albicans is a major cause of oropharyngeal, vulvovaginal and hematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes. InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non classical stimulation of the clathrin-dependent endocytosis machinery was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans. Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans, we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans, like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.
PMCID: PMC4098847  PMID: 19416270
19.  Genome Mining of a Prenylated and Immunosuppressive Polyketide from Pathogenic Fungi 
Organic letters  2013;15(4):780-783.
Activation of the polycyclic polyketide prenyltransferases (pcPTase)-containing silent clusters in Aspergillus fumigatus and Neosartorya fischeri led to isolation of a new metabolite neosartoricin (3). The structure of 3 was solved by X-ray crystallography and NMR to be a prenylated anthracenone. 3 Exhibits T-cell antiproliferative activity with an IC50 of 3 μM, suggestive of a physiological role as an immunosuppressive agent.
PMCID: PMC3576815  PMID: 23368997
20.  Efficacy of Liposomal Amphotericin B and Posaconazole in Intratracheal Models of Murine Mucormycosis 
Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections.
PMCID: PMC3697351  PMID: 23650163
21.  Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus 
Recent perspectives forecast a new paradigm for future “third generation” vaccines based on commonalities found in diverse pathogens or convergent immune defenses to such pathogens. For Staphylococcus aureus, recurring infections and a limited success of vaccines containing S. aureus antigens imply that native antigens induce immune responses insufficient for optimal efficacy. These perspectives exemplify the need to apply novel vaccine strategies to high-priority pathogens. One such approach can be termed convergent immunity, where antigens from non-target organisms that contain epitope homologs found in the target organism are applied in vaccines. This approach aims to evoke atypical immune defenses via synergistic processes that (1) afford protective efficacy; (2) target an epitope from one organism that contributes to protective immunity against another; (3) cross-protect against multiple pathogens occupying a common anatomic or immunological niche; and/or (4) overcome immune subversion or avoidance strategies of target pathogens. Thus, convergent immunity has a potential to promote protective efficacy not usually elicited by native antigens from a target pathogen. Variations of this concept have been mainstays in the history of viral and bacterial vaccine development. A more far-reaching example is the pre-clinical evidence that specific fungal antigens can induce cross-kingdom protection against bacterial pathogens. This trans-kingdom protection has been demonstrated in pre-clinical studies of the recombinant Candida albicans agglutinin-like sequence 3 protein (rAls3) where it was shown that a vaccine containing rAls3 provides homologous protection against C. albicans, heterologous protection against several other Candida species, and convergent protection against several strains of S. aureus. Convergent immunity reflects an intriguing new approach to designing and developing vaccine antigens and is considered here in the context of vaccines to target S. aureus.
PMCID: PMC4176462  PMID: 25309545
Staphylococcus aureus; vaccines; NDV-3; Als3; convergent immunity; convergent antigen
22.  CotH3 mediates fungal invasion of host cells during mucormycosis 
Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.
PMCID: PMC3871245  PMID: 24355926
23.  NDV-3, a Recombinant Alum-Adjuvanted Vaccine for Candida and Staphylococcus aureus is Safe and Immunogenic in Healthy Adults 
Vaccine  2012;30(52):7594-7600.
The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300 μg of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-γ and IL-17A, which peaked at day 7 for subjects receiving the 300 μg dose and at day 28 for those receiving the 30 μg dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30 μg dose. A memory T-cell response was also noted for IFN-γ in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections.
PMCID: PMC3513491  PMID: 23099329
vaccine; phase 1; first-in-human; Als3p; Candida; Staphylococcus aureus
24.  SR-Like RNA-Binding Protein Slr1 Affects Candida albicans Filamentation and Virulence 
Infection and Immunity  2013;81(4):1267-1276.
Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.
PMCID: PMC3639594  PMID: 23381995
25.  Aspergillus Galactosaminogalactan Mediates Adherence to Host Constituents and Conceals Hyphal β-Glucan from the Immune System 
PLoS Pathogens  2013;9(8):e1003575.
Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.
Author Summary
Invasive aspergillosis is the most common mold infection in humans, predominately affecting immunocompromised patients. The mechanisms by which the mold Aspergillus fumigatus adheres to host tissues and causes disease are poorly understood. In this report, we compared mutants of Aspergillus with different adhesive properties to identify fungal factors involved in adherence to host cells. This approach identified a cell wall associated polysaccharide, galactosaminogalactan, which is required for adherence to a wide variety of substrates. Galactosaminogalactan was also observed to suppress inflammation by concealing β-glucans, key pattern associated microbial pattern molecules in Aspergillus hyphae, from recognition by the innate immune system. Mutants that were deficient in galactosaminogalactan were less virulent in mouse models of invasive aspergillosis. These data identify a bifunctional role for galactosaminogalactan in the pathogenesis of invasive aspergillosis, and suggest that it may serve as a useful target for antifungal therapy.
PMCID: PMC3749958  PMID: 23990787

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