Publications by authors named "Olaf Kniemeyer"

100 Publications

The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in .

Int J Mol Sci 2021 Jul 20;22(14). Epub 2021 Jul 20.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The airborne fungus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine, sumoylation of lysine and phosphorylation of threonine. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in . .
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http://dx.doi.org/10.3390/ijms22147739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307855PMC
July 2021

Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.

Cell Microbiol 2021 Jul 10:e13378. Epub 2021 Jul 10.

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute (HKI), Jena, Germany.

The human pathogenic fungus Candida albicans is a frequent cause of mucosal infections. Although the ability to transition from the yeast to the hypha morphology is essential for virulence, hypha formation and host cell invasion per se are not sufficient for the induction of epithelial damage. Rather, the hypha-associated peptide toxin, candidalysin, a product of the Ece1 polyprotein, is the critical damaging factor. While synthetic, exogenously added candidalysin is sufficient to damage epithelial cells, the level of damage does not reach the same level as invading C. albicans hyphae. Therefore, we hypothesized that a combination of fungal attributes is required to deliver candidalysin to the invasion pocket to enable the full damaging potential of C. albicans during infection. Utilising a panel of C. albicans mutants with known virulence defects, we demonstrate that the full damage potential of C. albicans requires the coordinated delivery of candidalysin to the invasion pocket. This process requires appropriate epithelial adhesion, hyphal extension and invasion, high levels of ECE1 transcription, proper Ece1 processing and secretion of candidalysin. To confirm candidalysin delivery, we generated camelid V Hs (nanobodies) specific for candidalysin and demonstrate localization and accumulation of the toxin only in C. albicans-induced invasion pockets. In summary, a defined combination of virulence attributes and cellular processes is critical for delivering candidalysin to the invasion pocket to enable the full damage potential of C. albicans during mucosal infection. TAKE AWAYS: Candidalysin is a peptide toxin secreted by C. albicans causing epithelial damage. Candidalysin delivery to host cell membranes requires specific fungal attributes. Candidalysin accumulates in invasion pockets created by invasive hyphae. Camelid nanobodies enabled visualisation of candidalysin in the invasion pocket.
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http://dx.doi.org/10.1111/cmi.13378DOI Listing
July 2021

Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Antigens.

J Fungi (Basel) 2021 Jun 8;7(6). Epub 2021 Jun 8.

Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to antigens. Dual co-stimulation strongly enhanced -induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with -associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens.
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http://dx.doi.org/10.3390/jof7060462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230040PMC
June 2021

Inositol Signaling in the Basidiomycete Fungus .

J Fungi (Basel) 2021 Jun 10;7(6). Epub 2021 Jun 10.

Institute of Microbiology, Friedrich Schiller University Jena, Microbial Communication, Neugasse 25, 07743 Jena, Germany.

Intracellular signaling is conserved in eukaryotes to allow for response to extracellular signals and to regulate development and cellular functions. In fungi, inositol phosphate signaling has been shown to be involved in growth, sexual reproduction, and metabolic adaptation. However, reports on mushroom-forming fungi are lacking so far. In , an inositol monophosphatase has been found up-regulated during sexual development. The enzyme is crucial for inositol cycling, where it catalyzes the last step of inositol phosphate metabolism, restoring the inositol pool from the monophosphorylated inositol monophosphate. We overexpressed the gene in this model basidiomycete and verified its involvement in cell wall integrity and intracellular trafficking. Strong phenotypes in mushroom formation and cell metabolism were evidenced by proteome analyses. In addition, altered inositol signaling was shown to be involved in tolerance towards cesium and zinc, and increased metal tolerance towards cadmium, associated with induced expression of kinases and repression of phosphatases within the inositol cycle. The presence of the heavy metals Sr, Cs, Cd, and Zn lowered intracellular calcium levels. We could develop a model integrating inositol signaling in the known signal transduction pathways governed by Ras, G-protein coupled receptors, and cAMP, and elucidate their different roles in development.
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http://dx.doi.org/10.3390/jof7060470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230515PMC
June 2021

The Termite Fungal Cultivar Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

mBio 2021 06 15;12(3):e0355120. Epub 2021 Jun 15.

Group of Chemical Biology of Microbe-Host Interactions, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.

Macrotermitine termites have domesticated fungi in the genus as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that induces hydroquinone-mediated Fenton chemistry (Fe + HO + H → Fe + OH + HO) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MHQ, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.
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http://dx.doi.org/10.1128/mBio.03551-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262964PMC
June 2021

CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to .

Front Immunol 2021 27;12:659752. Epub 2021 May 27.

Department of Internal Medicine II, University Hospital of Wuerzburg, Wuerzburg, Germany.

causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti- defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an lysate and two  proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4 and CD8 T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with germ tubes. Taken together, our data suggest that CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA.
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http://dx.doi.org/10.3389/fimmu.2021.659752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192083PMC
May 2021

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nat Commun 2021 03 12;12(1):1631. Epub 2021 Mar 12.

Department of Biomedicine, University of Bergen, Bergen, Norway.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.
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http://dx.doi.org/10.1038/s41467-021-21307-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955114PMC
March 2021

Serological Proteome Analysis for the Characterization of Secreted Fungal Protein Antigens.

Methods Mol Biol 2021 ;2260:15-26

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany.

Defining the humoral immune response to infectious agents is important for gaining insights into infectious diseases and the response of the immune system. It can further aid development of serodiagnostic tests, discovery of vaccine antigen candidates, and immuno-epidemiological research. During the last three decades, serological proteome analyses (SERPAs) have played a significant role in characterizing the antibody response of humans or animals to fungal pathogens. SERPA combines 2D-gel electrophoresis with Western blotting. The introduction of multiplexing approaches by means of fluorescent dyes has greatly improved the reliability of the 2D technique and has boosted also the qualitative capabilities of the SERPA approach. In this chapter, we detail a SERPA protocol using fungal extracellular proteins from a fungal culture, here as an example the mold Aspergillus fumigatus.
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http://dx.doi.org/10.1007/978-1-0716-1182-1_2DOI Listing
March 2021

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA.

mBio 2021 01 5;12(1). Epub 2021 Jan 5.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto, Brazil

Filamentous fungi of the genus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the CH transcription factor CreA has been described as the major CC repressor in spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.
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http://dx.doi.org/10.1128/mBio.03146-20DOI Listing
January 2021

The Role of RodA-Conserved Cysteine Residues in the Conidial Surface Organization.

J Fungi (Basel) 2020 Aug 26;6(3). Epub 2020 Aug 26.

Molecular Mycology Unit, Institut Pasteur, CNRS-UMR-2000, 10098 Paris, France.

Immune inertness of conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the deletion mutant (∆). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrpmutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆ conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆ was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆. Ccrp-mutant conidia were less immunostimulatory compared to ∆ conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host-fungal interaction.
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http://dx.doi.org/10.3390/jof6030151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558875PMC
August 2020

Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection.

mSphere 2020 08 12;5(4). Epub 2020 Aug 12.

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, Jena, Germany

is one of the most common airborne molds capable of causing mycoses and allergies in humans. During infection, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established for surface proteomics (surfomics). Biotinylation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptides is a particularly efficient method to identify the surface-exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 231 different biotinylated surface proteins (including several well-known proteins such as RodA, CcpA, and DppV; allergens; and heat shock proteins [HSPs]), as well as some previously undescribed surface proteins. The dynamic change of the surface proteome was illustrated by detection of a relatively high number of proteins exclusively at one developmental stage. Using immunofluorescence microscopy, we confirmed the surface localization of several HSPs of the HSP70 family, which may have moonlighting functions. Collectively, by comparing our data with data representative of previously published surface proteomes, our study generated a comprehensive data set corresponding to the surfome and uncovered the surface-exposed regions of many proteins on the surface of conidia or hyphae. These surface-exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, our data sets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. is the most important airborne human-pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergy-inducing infections in individuals with atopic allergy. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of infection remain major challenges. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between and other organisms, sometimes even including the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.
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http://dx.doi.org/10.1128/mSphere.00535-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426169PMC
August 2020

Functional surface proteomic profiling reveals the host heat-shock protein A8 as a mediator of Lichtheimia corymbifera recognition by murine alveolar macrophages.

Environ Microbiol 2020 09 21;22(9):3722-3740. Epub 2020 Jul 21.

Jena Microbial Resource Collection, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), Jena, Germany.

Mucormycosis is an emergent, fatal fungal infection of humans and warm-blooded animals caused by species of the order Mucorales. Immune cells of the innate immune system serve as the first line of defence against inhaled spores. Alveolar macrophages were challenged with the mucoralean fungus Lichtheimia corymbifera and subjected to biotinylation and streptavidin enrichment procedures followed by LC-MS/MS analyses. A total of 28 host proteins enriched for binding to macrophage-L. corymbifera interaction. Among those, the HSP70-family protein Hspa8 was found to be predominantly responsive to living and heat-killed spores of a virulent and an attenuated strain of L. corymbifera. Confocal scanning laser microscopy of infected macrophages revealed colocalization of Hspa8 with phagocytosed spores of L. corymbifera. The amount of detectable Hspa8 was dependent on the multiplicity of infection. Incubation of alveolar macrophages with an anti-Hspa8 antibody prior to infection reduced their capability to phagocytose spores of L. corymbifera. In contrast, anti-Hspa8 antibodies did not abrogate the phagocytosis of Aspergillus fumigatus conidia by macrophages. These results suggest an important contribution of the heat-shock family protein Hspa8 in the recognition of spores of the mucoralean fungus L. corymbifera by host alveolar macrophages and define a potential immunomodulatory therapeutic target.
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http://dx.doi.org/10.1111/1462-2920.15140DOI Listing
September 2020

The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus.

Eur J Immunol 2020 11 13;50(11):1712-1728. Epub 2020 Jul 13.

Institute of Immunology, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4 T cells in blood are described to reflect the actual host-pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 10 cfu/m conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.
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http://dx.doi.org/10.1002/eji.201948524DOI Listing
November 2020

Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles.

Nat Commun 2020 05 11;11(1):2331. Epub 2020 May 11.

Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, 07745, Jena, Germany.

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-β1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble β-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-β1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-β1 to the TGF-β receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-β1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-β1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.
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http://dx.doi.org/10.1038/s41467-020-16241-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214408PMC
May 2020

Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans.

mBio 2020 04 28;11(2). Epub 2020 Apr 28.

Research Group Fungal Septomics, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute, Jena, Germany

The capacity of to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes and , which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of and In contrast to , overexpression of was found to inhibit neither filamentous growth nor transcription of and In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of and and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different morphologies. is a major human fungal pathogen and the leading cause of systemic infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of and in hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in .
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http://dx.doi.org/10.1128/mBio.00206-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188989PMC
April 2020

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.

mBio 2020 04 14;11(2). Epub 2020 Apr 14.

Institute of Microbiology, Friedrich Schiller University, Jena, Germany

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against Invasive fungal infections caused by the mold are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against not previously described. We found that extracellular vesicles produced by neutrophils in response to infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of host pathogenesis and opens up novel diagnostic and therapeutic possibilities.
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http://dx.doi.org/10.1128/mBio.00596-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157820PMC
April 2020

Dynamic Surface Proteomes of Allergenic Fungal Conidia.

J Proteome Res 2020 May 14;19(5):2092-2104. Epub 2020 Apr 14.

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute (Leibniz-HKI), Jena 07745, Germany.

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of to other allergy-inducing molds, including , , and , and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by . Ultimately, this study highlights the dynamic nature of the conidial surface and provides targets for future diagnostics and immunotherapy.
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http://dx.doi.org/10.1021/acs.jproteome.0c00013DOI Listing
May 2020

Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II.

Cells 2020 02 12;9(2). Epub 2020 Feb 12.

Institute for Biochemistry II, Jena University Hospital, Friedrich Schiller University, 07743 Jena, Germany.

Paracrine interactions between malignant estrogen receptor positive (ER) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD/NADH-ratio in BAFs. This suggests that cellular NAD/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.
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http://dx.doi.org/10.3390/cells9020427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072628PMC
February 2020

The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus.

PLoS Genet 2019 09 16;15(9):e1008379. Epub 2019 Sep 16.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.
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http://dx.doi.org/10.1371/journal.pgen.1008379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762210PMC
September 2019

Conidial surface proteins at the interface of fungal infections.

PLoS Pathog 2019 09 12;15(9):e1007939. Epub 2019 Sep 12.

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

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http://dx.doi.org/10.1371/journal.ppat.1007939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742213PMC
September 2019

Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus.

mBio 2019 06 4;10(3). Epub 2019 Jun 4.

Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, New Jersey, USA

is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the gene. Yet these clinical isolates did not contain mutations in , indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins. Resistance to first-line triazole antifungal agents among species has prompted the use of second-line therapy with echinocandins. As the number of -infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from spp., while little is known about resistance in Therefore, it is important to understand the specific cellular responses raised by against echinocandins. We discovered a new mechanism of resistance in that is independent of the well-characterized mutation mechanism observed in This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.
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http://dx.doi.org/10.1128/mBio.00779-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550521PMC
June 2019

Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

Cell 2019 03 21;176(6):1340-1355.e15. Epub 2019 Feb 21.

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute (HKI), Jena, Germany.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.
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http://dx.doi.org/10.1016/j.cell.2019.01.041DOI Listing
March 2019

Crosstalk between Ras and inositol phosphate signaling revealed by lithium action on inositol monophosphatase in Schizophyllum commune.

Adv Biol Regul 2019 05 3;72:78-88. Epub 2019 Jan 3.

Friedrich Schiller University Jena, Institute of Microbiology, Jena, Germany. Electronic address:

Mushroom forming basidiomycete Schizophyllum commune has been used as a tractable model organism to study fungal sexual development. Ras signaling activation via G-protein-coupled receptors (GPCRs) has been postulated to play a significant role in the mating and development of S. commune. In this study, a crosstalk between Ras signaling and inositol phosphate signaling by inositol monophosphatase (IMPase) is revealed. Constitutively active Ras1 leads to the repression of IMPase transcription and lithium action on IMPase activity is compensated by the induction of IMPase at transcriptome level. Astonishingly, in S. commune lithium induces a considerable shift to inositol phosphate metabolism leading to a massive increase in the level of higher phosphorylated inositol species up to the inositol pyrophosphates. The lithium induced metabolic changes are not observable in a constitutively active Ras1 mutant. In addition to that, proteome profile helps us to elucidate an overview of lithium action to the broad aspect of fungal metabolism and cellular signaling. Taken together, these findings imply a crosstalk between Ras and inositol phosphate signaling.
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http://dx.doi.org/10.1016/j.jbior.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520614PMC
May 2019

Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species during Hypoxia Adaptation of Aspergillus fumigatus.

Proteomics 2019 03 13;19(5):e1800339. Epub 2019 Feb 13.

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

Aspergillus fumigatus faces abrupt changes in oxygen concentrations at the site of infection. An increasing number of studies has demonstrated that elevated production of intracellular reactive oxygen species (ROS) under low oxygen conditions plays a regulatory role in modulating cellular responses for adaptation to hypoxia. To learn more about this process in A. fumigatus, intracellular ROS production during hypoxia has been determined. The results confirm increased amounts of intracellular ROS in A. fumigatus exposed to decreased oxygen levels. Moreover, nuclear accumulation of the major oxidative stress regulator AfYap1 is observed after low oxygen cultivation. For further analysis, iodoTMT labeling of redox-sensitive cysteine residues is applied to identify proteins that are reversibly oxidized. This analysis reveals that proteins with important roles in maintaining redox balance and protein folding, such as the thioredoxin Asp f 29 and the disulfide-isomerase PdiA, undergo substantial thiol modification under hypoxia. The data also show that the mitochondrial respiratory complex IV assembly protein Coa6 is significantly oxidized by hypoxic ROS. Deletion of the corresponding gene results in a complete absence of hypoxic growth, indicating the importance of complex IV during adaptation of A. fumigatus to oxygen-limiting conditions.
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http://dx.doi.org/10.1002/pmic.201800339DOI Listing
March 2019

Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins.

PLoS One 2018 27;13(12):e0209658. Epub 2018 Dec 27.

Plant Physiology, Matthias-Schleiden-Institute for Genetics, Bioinformatics and Molecular Botany, Faculty of Biological Science, Friedrich-Schiller-University Jena, Jena, Germany.

Plants interact with a wide variety of fungi in a mutualistic, parasitic or neutral way. The associations formed depend on the exchange of nutrients and signalling molecules between the partners. This includes a diverse set of protein classes involved in defence, nutrient uptake or establishing a symbiotic relationship. Here, we have analysed the secretomes of the mutualistic, root-endophytic fungus Piriformospora indica and Arabidopsis thaliana when cultivated alone or in a co-culture. More than one hundred proteins were identified as differentially secreted, including proteins associated with growth, development, abiotic and biotic stress response and mucilage. While some of the proteins have been associated before to be involved in plant-microbial interaction, other proteins are newly described in this context. One plant protein found in the co-culture is PLAT1 (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase). PLAT1 has not been associated with plant-fungal-interaction and is known to play a role in abiotic stress responses. In colonised roots PLAT1 shows an altered gene expression in a stage specific manner and plat1 knock-out plants are colonised stronger. It co-localises with Brassicaceae-specific endoplasmic reticulum bodies (ER-bodies) which are involved in the formation of the defence compound scopolin. We observed degraded ER-bodies in infected Arabidopsis roots and a change in the scopolin level in response to the presence of the fungus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209658PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307754PMC
May 2019

Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

Proteomics Clin Appl 2019 01 26;13(1):e1700173. Epub 2018 Nov 26.

Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany.

Purpose: The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI-MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level.

Experimental Design: MALDI-MSI is used on snap frozen and formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) to analyze m/z values localized in tumor and nontumor regions. Peptide identification is performed using LC-MS/MS and immunohistochemistry (IHC).

Results: In both FFPE and frozen tissue specimens, eight characteristic masses of the tumor's epithelial region are found. Using LC-MS/MS, the peaks are identified as vimentin, keratin type II, nucleolin, heat shock protein 90, prelamin-A/C, junction plakoglobin, and PGAM1. Lastly, vimentin, nucleolin, and PGAM1 are verified with IHC.

Conclusions And Clinical Relevance: The combination of MALDI-MSI, LC-MS/MS, and subsequent IHC furnishes a tool suitable for characterizing the molecular heterogeneity of tissue. It is also suited for use in identifying new representative biomarkers to enable a more individualized therapy.
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http://dx.doi.org/10.1002/prca.201700173DOI Listing
January 2019

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

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

Front Physiol 2018 17;9:921. Epub 2018 Jul 17.

Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae SAG 11-32b and NC64A, and in plant cell lines of . When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.
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http://dx.doi.org/10.3389/fphys.2018.00921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056685PMC
July 2018

The regulator of G-protein signalling Thn1 links pheromone response to volatile production in Schizophyllum commune.

Environ Microbiol 2018 10 17;20(10):3684-3699. Epub 2018 Sep 17.

Friedrich Schiller University Jena, Institute of Microbiology, Microbial Communication, Neugasse 25, 07743, Jena, Germany.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism.
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http://dx.doi.org/10.1111/1462-2920.14369DOI Listing
October 2018
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