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1.
Methods Mol Biol ; 2260: 15-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405028

RESUMO

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.

2.
mBio ; 12(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402538

RESUMO

Filamentous fungi of the genus Aspergillus 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 C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus 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 Aspergillus nidulans 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.IMPORTANCE 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 Aspergillus nidulans 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.


Assuntos
Aspergillus nidulans/metabolismo , Repressão Catabólica/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Carbono/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genética
3.
J Fungi (Basel) ; 6(3)2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32859091

RESUMO

Immune inertness of Aspergillus fumigatus 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 RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to ß-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA 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 ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA 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.

4.
mSphere ; 5(4)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32817453

RESUMO

Aspergillus fumigatus 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 A. fumigatus surface proteomes, our study generated a comprehensive data set corresponding to the A. fumigatus 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.IMPORTANCE Aspergillus fumigatus 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 A. fumigatus 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 A. fumigatus 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 A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.

5.
Eur J Immunol ; 50(11): 1712-1728, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32558930

RESUMO

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 106 cfu/m3 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.

6.
Environ Microbiol ; 22(9): 3722-3740, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32583550

RESUMO

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.

7.
Nat Commun ; 11(1): 2331, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393780

RESUMO

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-ß1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ß-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-ß1-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-ß1 to the TGF-ß receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-ß1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-ß1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.


Assuntos
Imunomodulação , Antígeno de Macrófago 1/metabolismo , Monócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Apoptose , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Regulação para Baixo , Difusão Dinâmica da Luz , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Monócitos/microbiologia , Monócitos/ultraestrutura , Transporte Proteico , Solubilidade , Transcrição Genética , Regulação para Cima , beta-Glucanas/metabolismo
8.
mBio ; 11(2)2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345638

RESUMO

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, 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 C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 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 ALS3 and ECE1 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 C. albicans morphologies.IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida 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 ALS3 and ECE1 in C. albicans 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 C. albicans.

9.
J Proteome Res ; 19(5): 2092-2104, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233371

RESUMO

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 Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus 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 A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, 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 A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

10.
mBio ; 11(2)2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32291301

RESUMO

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 Aspergillus fumigatus 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 A. fumigatus 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 A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus 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 A. fumigatus 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 A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus 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 A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

11.
Cells ; 9(2)2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32059481

RESUMO

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.

12.
PLoS Genet ; 15(9): e1008379, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525190

RESUMO

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.


Assuntos
Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Ferro/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Homeostase , Ferro/deficiência , Inanição , Fatores de Transcrição/genética , Virulência
14.
mBio ; 10(3)2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164462

RESUMO

Aspergillus fumigatus 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) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in ß-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, 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 fks1 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.IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-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 Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida 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.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Glucosiltransferases/genética , Estresse Fisiológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Humanos , Testes de Sensibilidade Microbiana , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
15.
Cell ; 176(6): 1340-1355.e15, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799037

RESUMO

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.


Assuntos
Candida albicans/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Candida albicans/patogenicidade , Reações Cruzadas/imunologia , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Humanos , Imunidade , Imunidade Heteróloga/imunologia , Células Th17/fisiologia
16.
Proteomics ; 19(5): e1800339, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30632700

RESUMO

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.


Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/citologia , Aspergillus fumigatus/crescimento & desenvolvimento , Hipóxia Celular , Humanos , Oxirredução , Estresse Oxidativo , Oxigênio/metabolismo , Proteômica/métodos
17.
Adv Biol Regul ; 72: 78-88, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30639095

RESUMO

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.


Assuntos
Proteínas Fúngicas/metabolismo , Fosfatos de Inositol/metabolismo , Lítio/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Schizophyllum/enzimologia , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Schizophyllum/química , Schizophyllum/genética , Schizophyllum/crescimento & desenvolvimento , Transdução de Sinais
18.
Proteomics Clin Appl ; 13(1): e1700173, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30411850

RESUMO

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.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Imagem Molecular , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cromatografia Líquida , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Espectrometria de Massas em Tandem , Fixação de Tecidos
19.
PLoS One ; 13(12): e0209658, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30589877

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Basidiomycota/fisiologia , Simbiose , Proteínas de Arabidopsis/genética , Biologia Computacional/métodos , Resistência à Doença , Retículo Endoplasmático/metabolismo , Interações Hospedeiro-Patógeno , Mutação , Desenvolvimento Vegetal , Proteoma , Proteômica/métodos
20.
mBio ; 9(5)2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279286

RESUMO

Aspergillus fumigatus 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 A. fumigatus 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 conidial cell wall protein A (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 ΔccpA resting conidia appeared normal. However, trypsin shaving of ΔccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpA 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 vitro In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus 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.IMPORTANCE 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 Aspergillus fumigatus, 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. A. fumigatus 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.


Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteoma/análise , Células A549 , Animais , Aspergilose/imunologia , Parede Celular/química , Cromatografia Líquida , Células Dendríticas/imunologia , Endocitose , Células Epiteliais/imunologia , Feminino , Proteínas Fúngicas/genética , Humanos , Hospedeiro Imunocomprometido , Proteínas de Membrana/genética , Camundongos , Ativação de Neutrófilo , Esporos Fúngicos/patogenicidade , Linfócitos T/imunologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
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