Publications by authors named "Scott G Filler"

179 Publications

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

PLoS Pathog 2021 Mar 29;17(3):e1009235. Epub 2021 Mar 29.

Division of Infectious Diseases, Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, United States of America.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.
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http://dx.doi.org/10.1371/journal.ppat.1009235DOI Listing
March 2021

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

PLoS Pathog 2021 Jan 20;17(1):e1009221. Epub 2021 Jan 20.

Division of Infectious Diseases, Harbor-UCLA Medical Center, Torrance, California, United States of America.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.
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http://dx.doi.org/10.1371/journal.ppat.1009221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850503PMC
January 2021

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

Nat Microbiol 2021 Mar 18;6(3):313-326. Epub 2021 Jan 18.

Division of Infectious Diseases, The Lundquist Institute for Biomedical Innovation, Harbor-University of California at Los Angeles (UCLA) Medical Center, Torrance, CA, USA.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.
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http://dx.doi.org/10.1038/s41564-020-00837-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914224PMC
March 2021

Aberrant type 1 immunity drives susceptibility to mucosal fungal infections.

Science 2021 01;371(6526)

Fungal Pathogenesis Section, Laboratory of Clinical Immunology and Microbiology (LCIM), National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA.

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans.
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http://dx.doi.org/10.1126/science.aay5731DOI Listing
January 2021

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

Methods Mol Biol 2021 ;2260:27-36

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.

Receptors on endothelial and epithelial cells often recognize molecules that are expressed by fungi, and only a limited number of these receptors have been identified to date. Here, we describe a method for identifying novel host cell receptors for fungi that uses intact organisms to precipitate biotin-labelled host cell membrane proteins, which are then detected by immunoblotting with an anti-biotin antibody. Presented here is the method to use for identification of membrane proteins that bind to C. albicans.
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http://dx.doi.org/10.1007/978-1-0716-1182-1_3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895530PMC
March 2021

Identification of Candida glabrata Transcriptional Regulators That Govern Stress Resistance and Virulence.

Infect Immun 2021 Feb 16;89(3). Epub 2021 Feb 16.

Division of Molecular Medicine, Harbor-UCLA Medical Center, Torrance, California, USA.

The mechanisms by which resists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable to withstand these classes of stressors, a library of 215 transcriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin. We identified eight mutants that had increased susceptibility to both host defense peptides and caspofungin. Of these mutants, six were deleted for genes that were predicted to specify proteins involved in histone modification. These genes were , , , , , and Deletion of , , and also increased susceptibility to mammalian host defense peptides. The Δ and Δ mutants had increased susceptibility to other stressors, such as HO and SDS. In the model of disseminated infection, the Δ and Δ mutants had attenuated virulence, whereas in neutropenic mice, the virulence of the Δ and Δ mutants was decreased. Thus, histone modification plays a central role in enabling to survive host defense peptides and caspofungin, and Ada2 and Rpd3 are essential for the maximal virulence of this organism during disseminated infection.
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http://dx.doi.org/10.1128/IAI.00146-20DOI Listing
February 2021

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

Cell Rep 2020 08;32(7):108017

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), 07745 Jena, Germany; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, 07745 Jena, Germany. Electronic address:

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca ions and that inhibition of Ca-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.
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http://dx.doi.org/10.1016/j.celrep.2020.108017DOI Listing
August 2020

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

PLoS Genet 2020 06 11;16(6):e1008881. Epub 2020 Jun 11.

Department of Biological Chemistry, University of California, Irvine, California, United States of America.

Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell.
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http://dx.doi.org/10.1371/journal.pgen.1008881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319344PMC
June 2020

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

mBio 2020 06 2;11(3). Epub 2020 Jun 2.

Division of Infectious Diseases, The Lundquist Institute for Biomedical Innovation, Harbor-University of California Los Angeles Medical Center, Torrance, California, USA

Mucormycosis, caused by species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, CotH7 recognizes integrin β1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin β1 antibodies inhibit invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands. Mucormycosis caused by species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin β1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.
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http://dx.doi.org/10.1128/mBio.01087-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267888PMC
June 2020

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

mBio 2020 06 2;11(3). Epub 2020 Jun 2.

Department of Pathology & Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca-ATPases in the human-pathogenic mold We demonstrate that acute ER stress increases transcription of the gene, encoding a member of the sarco/endoplasmic reticulum Ca-ATPase (SERCA) family, as well as that of , encoding a secretory pathway Ca-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of and transcription during ER stress, defining these ER/Golgi Ca pumps as novel downstream targets of this pathway. While deletion of alone caused no major deficiencies, a Δ/Δ mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca pumps. The Δ mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca influx at the ER and Golgi membranes and that the coupling between the UPR and Ca import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca signaling.
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http://dx.doi.org/10.1128/mBio.01060-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267887PMC
June 2020

Fosmanogepix (APX001) Is Effective in the Treatment of Pulmonary Murine Mucormycosis Due to Rhizopus arrhizus.

Antimicrob Agents Chemother 2020 05 21;64(6). Epub 2020 May 21.

The Lundquist Institute for Biomedical Innovations at Harbor-University of California Los Angeles (UCLA) Medical Center, Torrance, California, USA

Mucormycosis is a life-threatening infection with high mortality that occurs predominantly in immunocompromised patients. Manogepix (MGX) is a novel antifungal that targets Gwt1, a protein involved in an early step in the conserved glycosylphosphotidyl inositol (GPI) posttranslational modification pathway of surface proteins in eukaryotic cells. Inhibition of fungal inositol acylation by MGX results in pleiotropic effects, including inhibition of maturation of GPI-anchored proteins necessary for growth and virulence. MGX has been previously shown to have activity against some strains of Mucorales. Here, we assessed the activity of the prodrug fosmanogepix, currently in clinical development for the treatment of invasive fungal infections, against two strains with high (4.0 μg/ml) and low (0.25 μg/ml) minimum effective concentration (MEC) values. In both invasive pulmonary infection models, treatment of mice with 78 mg/kg or 104 mg/kg fosmanogepix, along with 1-aminobenzotriazole to enhance the serum half-life of MGX in mice, significantly increased median survival time and prolonged overall survival by day 21 postinfection compared to placebo. In addition, administration of fosmanogepix resulted in a 1 to 2 log reduction in both lung and brain fungal burden. For the 104 mg/kg fosmanogepix dose, tissue clearance and survival were comparable to clinically relevant doses of isavuconazole (ISA), which is FDA approved for the treatment of mucormycosis. These results support continued development of fosmanogepix as a first-in-class treatment for invasive mucormycosis.
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http://dx.doi.org/10.1128/AAC.00178-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269494PMC
May 2020

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

PLoS Genet 2020 01 21;16(1):e1008582. Epub 2020 Jan 21.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.
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http://dx.doi.org/10.1371/journal.pgen.1008582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994163PMC
January 2020

Cost effective, experimentally robust differential-expression analysis for human/mammalian, pathogen and dual-species transcriptomics.

Microb Genom 2020 01;6(1)

Institute for Genome Sciences, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine 14 pathogen or host-pathogen differential gene expression data sets to assess whether using longer reads is warranted. A variety of data sets was used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons and intron length. No genome attribute was found to influence the data in principal components analysis, hierarchical clustering with bootstrap support, or regression analyses of pairwise comparisons that were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36, 54, 72 and 101 bp. Read pairing had the greatest effect when there was little variation in the samples from different conditions or in their replicates (e.g. little differential gene expression). But overall, 54 and 72 bp reads were typically most similar. Given differences in costs and mapping percentages, we recommend 54 bp reads for organisms with no or few introns and 72 bp reads for all others. In a third of the data sets, read pairing had absolutely no effect, despite paired reads having twice as much data. Therefore, single-end reads seem robust for differential-expression analyses, but in eukaryotes paired-end reads are likely desired to analyse splice variants and should be preferred for data sets that are acquired with the intent to be community resources that might be used in secondary data analyses.
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http://dx.doi.org/10.1099/mgen.0.000320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067034PMC
January 2020

Describing the dynamic translational science landscape through Core Voucher utilization.

J Clin Transl Sci 2019 Jun 14;3(2-3):105-112. Epub 2019 Jun 14.

Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, CA, USA.

Introduction: Core facilities play crucial roles in carrying out the academic research mission by making available to researchers advanced technologies, facilities, or expertise that are unfeasible for most investigators to obtain on their own. To facilitate translational science through support of core services, the University of California, Los Angeles Clinical and Translational Science Institute (UCLA CTSI) created a Core Voucher program. The underlying premise is that by actively promoting interplay between researchers and core facilities, a dynamic feedback loop could be established that could enhance both groups, the productivity of the former and the relevance of the latter. Our primary goal was to give translational investigators what they need to pursue their immediate projects at hand.

Methods: To implement this system across four noncontiguous campuses, open-source web-accessible software applications were created that were scalable and could efficiently administer investigator submissions and subsequent reviews in a multicampus fashion.

Results: In the past five years, we have processed over 1400 applications submitted by over 750 individual faculty members across both clinical and nonclinical departments. In total, 1926 core requests were made in conjunction with 1467 submitted proposals. The top 10 most popular cores accounted for 50% of all requests, and the top half of the most popular cores accounted for 90% of all requests.

Conclusion: Tracking investigator demand provides a unique window into what are the high- and low-priority core services that best support translational research.
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http://dx.doi.org/10.1017/cts.2019.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802412PMC
June 2019

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

Proc Natl Acad Sci U S A 2019 10 16;116(40):20087-20096. Epub 2019 Sep 16.

Division of Infectious Diseases, Department of Medicine, Duke University School of Medicine, Durham, NC 27710;

The role of the host in development of persistent methicillin-resistant (MRSA) bacteremia is not well understood. A cohort of prospectively enrolled patients with persistent methicillin-resistant bacteremia (PB) and resolving methicillin-resistant bacteremia (RB) matched by sex, age, race, hemodialysis status, diabetes mellitus, and presence of implantable medical device was studied to gain insights into this question. One heterozygous g.25498283A > C polymorphism located in the intronic region of chromosome 2p with no impact in messenger RNA (mRNA) expression was more common in RB (21 of 34, 61.8%) than PB (3 of 34, 8.8%) patients ( = 7.8 × 10). Patients with MRSA bacteremia and g.25498283A > C genotype exhibited significantly higher levels of methylation in gene-regulatory CpG island regions (Δmethylation = 4.1%, < 0.0001) and significantly lower serum levels of interleukin-10 (IL-10) than patients with MRSA bacteremia without mutation (A/C: 9.7038 pg/mL vs. A/A: 52.9898 pg/mL; = 0.0042). Expression of was significantly suppressed in patients with bacteremia and in -challenged primary human macrophages. Small interfering RNA (siRNA) silencing of expression in human macrophages caused increased IL-10 response upon stimulation. Treating macrophages with methylation inhibitor 5-Aza-2'-deoxycytidine resulted in increased levels of IL-10 when challenged with In the murine sepsis model, methylation inhibition increased susceptibility to These findings indicate that g.25498283A > C genotype within contributes to increased capacity to resolve MRSA bacteremia, potentially through a mechanism involving increased methylation of gene-regulatory regions and reduced levels of antiinflammatory cytokine IL-10.
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http://dx.doi.org/10.1073/pnas.1909849116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778225PMC
October 2019

Candidalysin Is Required for Neutrophil Recruitment and Virulence During Systemic Candida albicans Infection.

J Infect Dis 2019 09;220(9):1477-1488

Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, United Kingdom.

Background: Candidalysin is a cytolytic peptide toxin secreted by Candida albicans hyphae and has significantly advanced our understanding of fungal pathogenesis. Candidalysin is critical for mucosal C albicans infections and is known to activate epithelial cells to induce downstream innate immune responses that are associated with protection or immunopathology during oral or vaginal infections. Furthermore, candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. However, the role of candidalysin in driving systemic infections is unknown.

Methods: In this study, using candidalysin-producing and candidalysin-deficient C albicans strains, we show that candidalysin activates mitogen-activated protein kinase (MAPK) signaling and chemokine secretion in endothelial cells in vitro.

Results: Candidalysin induces immune activation and neutrophil recruitment in vivo, and it promotes mortality in zebrafish and murine models of systemic fungal infection.

Conclusions: The data demonstrate a key role for candidalysin in neutrophil recruitment and fungal virulence during disseminated systemic C albicans infections.
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http://dx.doi.org/10.1093/infdis/jiz322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761979PMC
September 2019

EphA2 Is a Neutrophil Receptor for Candida albicans that Stimulates Antifungal Activity during Oropharyngeal Infection.

Cell Rep 2019 07;28(2):423-433.e5

Division of Infectious Diseases, Harbor-UCLA Medical Center, Torrance, CA 90502, USA; Institute for Infection and Immunity, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA; David Geffen School of Medicine at UCLA, Los Angeles, CA 90024, USA. Electronic address:

During oropharyngeal candidiasis (OPC), Candida albicans proliferates and invades the superficial oral epithelium. Ephrin type-A receptor 2 (EphA2) functions as an oral epithelial cell β-glucan receptor that triggers the production of proinflammatory mediators in response to fungal infection. Because EphA2 is also expressed by neutrophils, we investigated its role in neutrophil candidacidal activity during OPC. We found that EphA2 on stromal cells is required for the accumulation of phagocytes in the oral mucosa of mice with OPC. EphA2 on neutrophils is also central to host defense against OPC. The interaction of neutrophil EphA2 with serum-opsonized C. albicans yeast activates the MEK-ERK signaling pathway, leading to NADPH subunit p47 site-specific phospho-priming. This priming increases intracellular reactive oxygen species production and enhances fungal killing. Thus, in neutrophils, EphA2 serves as a receptor for β-glucans that augments Fcγ receptor-mediated antifungal activity and controls early fungal proliferation during OPC.
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http://dx.doi.org/10.1016/j.celrep.2019.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638578PMC
July 2019

Genome Sequence for Candida albicans Clinical Oral Isolate 529L.

Microbiol Resour Announc 2019 Jun 20;8(25). Epub 2019 Jun 20.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

The diploid heterozygous yeast is the most common cause of fungal infection. Here, we report the genome sequence assembly of the clinical oral isolate 529L. As this isolate grows as a commensal, this genome will serve as a reference for experimental and genetic studies of mucosal colonization.
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http://dx.doi.org/10.1128/MRA.00554-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588375PMC
June 2019

Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis.

Sci Adv 2019 06 12;5(6):eaaw1327. Epub 2019 Jun 12.

Los Angeles Biomedical Research Institute at Harbor-University of California at Los Angeles (UCLA) Medical Center, Torrance, CA, USA.

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated that , the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuates virulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing of . Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused by and other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice from infection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.
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http://dx.doi.org/10.1126/sciadv.aaw1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561750PMC
June 2019

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

PLoS Genet 2019 05 15;15(5):e1008137. Epub 2019 May 15.

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, United States of America.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.
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http://dx.doi.org/10.1371/journal.pgen.1008137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538192PMC
May 2019

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

Nat Immunol 2019 05 17;20(5):559-570. Epub 2019 Apr 17.

Fungal Pathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy & Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

The C-type lectin receptor-Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1β served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1β and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1β, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1β and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host-pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.
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http://dx.doi.org/10.1038/s41590-019-0377-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494474PMC
May 2019

AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus.

mBio 2019 03 12;10(2). Epub 2019 Mar 12.

Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

Aspergillosis associated with azole-resistant has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of as well as that of an important membrane transporter protein gene called In this work, we characterize two different mutant alleles of , either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the and promoters. The ATRE in was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis. is the major filamentous fungal pathogen in humans. Infections associated with are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene as an important contributor to resistance in Here, we demonstrate that the transcription factor AtrR binds to a region in the promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of .
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http://dx.doi.org/10.1128/mBio.02563-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414702PMC
March 2019

endocarditis diagnosed by fungemia plus serum antigen testing.

Med Mycol Case Rep 2019 Mar 25;23:1-3. Epub 2018 Oct 25.

Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509, USA.

Fungal endocarditis remains an uncommon clinical diagnosis, though is likely to become more frequent due to the global increase in transplantations and cardiac valvular surgery. A case of prosthetic valve endocarditis due to Aspergillus fumigatus is described that was diagnosed with serologic fungal markers and confirmed with positive blood cultures, an uncommon finding.
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http://dx.doi.org/10.1016/j.mmcr.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216080PMC
March 2019

Protective immunity in recurrent infection reflects localized immune signatures and macrophage-conferred memory.

Proc Natl Acad Sci U S A 2018 11 8;115(47):E11111-E11119. Epub 2018 Oct 8.

Los Angeles Biomedical Research Institute, Harbor-University of California, Los Angeles Medical Center, Torrance, CA 90502;

is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Our prior studies discovered a role for protective innate memory against recurrent methicillin-resistant (MRSA) SSSI. In the present study, the dynamics and mechanisms of this response were explored in recurrent SSSI in WT mice. Priming by prior infection reduced skin lesion severity and MRSA burden, and protected against dissemination at day 7 but not day 2. Cytokine and cellular signatures in SSSI differed at day 2 versus 7, and were distinct in skin versus blood or spleen. Cytokines associated with protection in skin included increased IL-17, IL-6, monokine inducible by IFN-γ (MIG), and RANTES, while increased IP-10 correlated with protection from dissemination. Cellular signatures of protection included increased Th17, M1 macrophage, and dendritic cell populations in abscesses, and total macrophages in lymph nodes. Priming potentiated -specific phagocytic killing by bone marrow-derived macrophages in vitro, and their adoptive transfer into naïve skin afforded protective efficacy in vivo. Present findings indicate that protective immunity in recurrent infection is locally targeted, and involves specific memory conferred by macrophages. These insights provide targets for vaccine and immunotherapeutic development against MRSA.
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http://dx.doi.org/10.1073/pnas.1808353115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255181PMC
November 2018

Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus .

mBio 2018 10 2;9(5). Epub 2018 Oct 2.

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany

is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)-pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated onidial ell wall rotein (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of Δ resting conidia appeared normal. However, trypsin shaving of Δ conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen Δ conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with Δ conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition. The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of , the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.
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http://dx.doi.org/10.1128/mBio.01557-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168859PMC
October 2018

A possible role for fumagillin in cellular damage during host infection by Aspergillus fumigatus.

Virulence 2018 ;9(1):1548-1561

a Fungal and Bacterial Biomics Research Group, Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology , University of the Basque Country (UPV/EHU) , Leioa , Spain.

Virulence mechanisms of the pathogenic fungus Aspergillus fumigatus are multifactorial and depend on the immune state of the host, but little is known about the fungal mechanism that develops during the process of lung invasion. In this study, microarray technology was combined with a histopathology evaluation of infected lungs so that the invasion strategy followed by the fungus could be described. To achieve this, an intranasal mice infection was performed to extract daily fungal samples from the infected lungs over four days post-infection. The pathological study revealed a heavy fungal progression throughout the lung, reaching the blood vessels on the third day after exposure and causing tissue necrosis. One percent of the fungal genome followed a differential expression pattern during this process. Strikingly, most of the genes of the intertwined fumagillin/pseurotin biosynthetic gene cluster were upregulated as were genes encoding lytic enzymes such as lipases, proteases (DppIV, DppV, Asp f 1 or Asp f 5) and chitinase (chiB1) as well as three genes related with pyomelanin biosynthesis process. Furthermore, we demonstrate that fumagillin is produced in an in vitro pneumocyte cell line infection model and that loss of fumagillin synthesis reduces epithelial cell damage. These results suggest that fumagillin contributes to tissue damage during invasive aspergillosis. Therefore, it is probable that A. fumigatus progresses through the lungs via the production of the mycotoxin fumagillin combined with the secretion of lytic enzymes that allow fungal growth, angioinvasion and the disruption of the lung parenchymal structure.
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http://dx.doi.org/10.1080/21505594.2018.1526528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177242PMC
April 2019

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Sci Rep 2018 09 6;8(1):13377. Epub 2018 Sep 6.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.
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http://dx.doi.org/10.1038/s41598-018-31420-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127098PMC
September 2018

Inhibition of EGFR Signaling Protects from Mucormycosis.

mBio 2018 08 14;9(4). Epub 2018 Aug 14.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of var. () infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by , and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug. Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following var. infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.
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http://dx.doi.org/10.1128/mBio.01384-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094478PMC
August 2018

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

PLoS Pathog 2018 07 30;14(7):e1007076. Epub 2018 Jul 30.

Division of Infectious Diseases, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085062PMC
July 2018