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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912961

RESUMO

Septins colocalize with membrane sterol-rich regions and facilitate recruitment of cell wall synthases during wall remodeling. We show that null mutants missing an Aspergillus nidulans core septin present in hexamers and octamers (ΔaspAcdc11, ΔaspBcdc3 or ΔaspCcdc12) are sensitive to multiple cell wall-disturbing agents that activate the cell wall integrity MAPK pathway. The null mutant missing the octamer-exclusive core septin (ΔaspDcdc10) showed similar sensitivity, but only to a single cell wall-disturbing agent and the null mutant missing the noncore septin (ΔaspE) showed only very mild sensitivity to a different single agent. Core septin mutants showed changes in wall polysaccharide composition and chitin synthase localization. Mutants missing any of the five septins resisted ergosterol-disrupting agents. Hexamer mutants showed increased sensitivity to sphingolipid-disrupting agents. Core septins mislocalized after treatment with sphingolipid-disrupting agents, but not after ergosterol-disrupting agents. Our data suggest that the core septins are involved in cell wall integrity signaling, that all five septins are involved in monitoring ergosterol metabolism, that the hexamer septins are required for sphingolipid metabolism and that septins require sphingolipids to coordinate the cell wall integrity response.

2.
Front Immunol ; 12: 670578, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084170

RESUMO

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

3.
Fungal Biol ; 124(5): 235-252, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32389286

RESUMO

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

5.
mSphere ; 4(1)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760610

RESUMO

Aspergillus species cause pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients at the greatest risk of developing life-threatening aspergillosis have weakened immune systems and/or various lung disorders. Patients are treated with antifungals such as amphotericin B (AmB), caspofungin acetate, or triazoles (itraconazole, voriconazole, etc.), but these antifungal agents have serious limitations due to lack of sufficient fungicidal effect and human toxicity. Liposomes with AmB intercalated into the lipid membrane (AmB-LLs; available commercially as AmBisome) have severalfold-reduced toxicity compared to that of detergent-solubilized drug. However, even with the current antifungal therapies, 1-year survival among patients is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian innate immune receptor in the membrane of some leukocytes that binds as a dimer to beta-glucans found in fungal cell walls, signaling fungal infection. Using a novel protocol, we coated AmB-LLs with Dectin-1's beta-glucan binding domain to make DEC-AmB-LLs. DEC-AmB-LLs bound rapidly, efficiently, and with great strength to Aspergillus fumigatus and to Candida albicans and Cryptococcus neoformans, highly divergent fungal pathogens of global importance. In contrast, untargeted AmB-LLs and bovine serum albumin (BSA)-coated BSA-AmB-LLs showed 200-fold-lower affinity for fungal cells. DEC-AmB-LLs reduced the growth and viability of A. fumigatus an order of magnitude more efficiently than untargeted control liposomes delivering the same concentrations of AmB, in essence decreasing the effective dose of AmB. Future efforts will focus on examining pan-antifungal targeted liposomal drugs in animal models of disease.IMPORTANCE The fungus Aspergillus fumigatus causes pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients are often treated with antifungal drugs such as amphotericin B (AmB) loaded into liposomes (AmB-LLs), but all antifungal drugs, including AmB-LLs, have serious limitations due to human toxicity and insufficient fungal cell killing. Even with the best current therapies, 1-year survival among patients with invasive aspergillosis is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-AmB-LLs bound strongly to fungal cells, while AmB-LLs had little affinity. DEC-AmB-LLs killed or inhibited A. fumigatus 10 times more efficiently than untargeted liposomes, decreasing the effective dose of AmB. Dectin-1-coated drug-loaded liposomes targeting fungal pathogens have the potential to greatly enhance antifungal therapeutics.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Lectinas Tipo C/química , Lipossomos/química , Animais , Aspergilose/tratamento farmacológico , Candida albicans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/química , Cryptococcus neoformans/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Ligação Proteica
6.
BMC Evol Biol ; 19(1): 4, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30616529

RESUMO

BACKGROUND: Septins are cytoskeletal proteins important in cell division and in establishing and maintaining cell polarity. Although septins are found in various eukaryotes, septin genes had the richest history of duplication and diversification in the animals, fungi and protists that comprise opisthokonts. Opisthokont septin paralogs encode modular proteins that assemble into heteropolymeric higher order structures. The heteropolymers can create physical barriers to diffusion or serve as scaffolds organizing other morphogenetic proteins. How the paralogous septin modules interact to form heteropolymers is still unclear. Through comparative analyses, we hoped to clarify the evolutionary origin of septin diversity and to suggest which amino acid residues were responsible for subunit binding specificity. RESULTS: Here we take advantage of newly sequenced genomes to reconcile septin gene trees with a species phylogeny from 22 animals, fungi and protists. Our phylogenetic analysis divided 120 septins representing the 22 taxa into seven clades (Groups) of paralogs. Suggesting that septin genes duplicated early in opisthokont evolution, animal and fungal lineages share septin Groups 1A, 4 and possibly also 1B and 2. Group 5 septins were present in fungi but not in animals and whether they were present in the opisthokont ancestor was unclear. Protein homology folding showed that previously identified conserved septin motifs were all located near interface regions between the adjacent septin monomers. We found specific interface residues associated with each septin Group that are candidates for providing subunit binding specificity. CONCLUSIONS: This work reveals that duplication of septin genes began in an ancestral opisthokont more than a billion years ago and continued through the diversification of animals and fungi. Evidence for evolutionary conservation of ~ 49 interface residues will inform mutagenesis experiments and lead to improved understanding of the rules guiding septin heteropolymer formation and from there, to improved understanding of development of form in animals and fungi.


Assuntos
Sequência Conservada/genética , Eucariotos/genética , Variação Genética , Septinas/química , Septinas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Fungos/genética , Duplicação Gênica , Guanosina Trifosfato/metabolismo , Filogenia , Domínios Proteicos , Septinas/classificação
7.
Cytoskeleton (Hoboken) ; 76(1): 33-44, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30171672

RESUMO

Septins are highly conserved guanosine triphosphate (GTP)-binding proteins that are a component of the cytoskeletal systems of virtually all eukaryotes (except higher plants). Septins play important roles in a multitude of cellular processes, including cytokinesis, establishment of cell polarity, and cellular partitioning. The ease of genetic screens and a fully sequenced genome have made Saccharomyces cerevisiae one of the most extensively studied and well-annotated model organisms in eukaryotic biology. Here, we present a synopsis of the known point mutations in the seven S. cerevisiae septin genes: CDC3, CDC10, CDC11, CDC12, SHS1, SPR3, and SPR28. We map these mutations onto septin protein structures, highlighting important conserved motifs, and relating the functional consequences of mutations in each domain.


Assuntos
Septinas/genética , Mutação/genética , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
PLoS One ; 13(8): e0201828, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30114268

RESUMO

Histone H1 is an evolutionarily conserved linker histone protein that functions in arranging and stabilizing chromatin structure and is frequently fused to a fluorescent protein to track nuclei in live cells. In time-lapse analyses, we observed stochastic exchange of photoactivated Dendra2-histone H1 protein between nuclei within the same cellular compartment. We also observed exchange of histones between genetically distinct nuclei in a heterokaryon derived from fusion of strains carrying histone H1-RFP or H1-GFP. Subsequent analysis of the resulting uninucleate conidia containing both RFP- and GFP-labeled histone H1 proteins showed only parental genotypes, ruling out genetic recombination and diploidization. These data together suggest that the linker histone H1 protein can diffuse between non-daughter nuclei in the filamentous fungus Aspergillus nidulans.


Assuntos
Aspergillus nidulans/metabolismo , Núcleo Celular/metabolismo , Histonas/metabolismo , Aspergillus nidulans/citologia , Aspergillus nidulans/crescimento & desenvolvimento , Difusão , Esporos Fúngicos/citologia , Esporos Fúngicos/metabolismo , Processos Estocásticos
10.
Sci Rep ; 8(1): 11433, 2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061727

RESUMO

Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.


Assuntos
Aspergillus nidulans/citologia , Aspergillus nidulans/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Parede Celular/ultraestrutura , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genótipo , Hifas/ultraestrutura , Mutação/genética , Fenótipo , Proteômica , Temperatura , Resposta a Proteínas não Dobradas , Regulação para Cima
11.
Front Cell Dev Biol ; 5: 33, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28424773

RESUMO

One of the key challenges faced by microbial pathogens is invasion of host tissue. Fungal pathogens adopt a number of distinct strategies to overcome host cell defenses, including the development of specialized infection structures, the secretion of proteins that manipulate host responses or cellular organization, and the ability to facilitate their own uptake by phagocytic mechanisms. Key to many of these adaptations is the considerable morphogenetic plasticity displayed by pathogenic species. Fungal pathogens can, for example, shift their growth habit between non-polarized spores, or yeast-like cells, and highly polarized hyphal filaments. These polarized filaments can then elaborate differentiated cells, specialized to breach host barriers. Septins play fundamental roles in the ability of diverse fungi to undergo shape changes and organize the F-actin cytoskeleton to facilitate invasive growth. As a consequence, septins are increasingly implicated in fungal pathogenesis, with many septin mutants displaying impairment in their ability to cause diseases of both plants and animals. In this mini-review, we show that a common feature of septin mutants is the emergence of extra polar outgrowths during morphological transitions, such as emergence of germ tubes from conidia or branches from hyphae. We propose that because septins detect and stabilize membrane curvature, they prevent extra polar outgrowths and thereby focus fungal invasive force, allowing substrate invasion.

12.
Med Mycol ; 55(4): 445-452, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664992

RESUMO

Aspergillus fumigatus is the most common airborne pathogen causing fatal mycoses in immunocompromised patients. During the first 8 hours of development A. fumigatus conidia break dormancy, expand isotopically, establish an axis of polarity, and begin to extend germ tubes in a polar manner. The transition from isotropic to polar growth is critical for tissue invasion and pathogenesis. In the current work, we used two-color microarrays to examine the A. fumigatus transcriptome during early development, focusing on the isotropic to polar switch. The most highly regulated transcripts in the isotropic to polar switch did not include known polarity genes. Transcripts encoding the Cdc42 module, polarisome components, and septins, known to be critical players in polarity, showed relatively steady levels during the isotropic to polar switch. Indeed, these transcripts were present in dormant conidia, and their levels changed little from dormancy through germ tube emergence. Not only did the isotropic to polar switch show little change in the expression of key polarity genes of the Cdc42 module, polarisome, and septins, it also showed the lowest overall levels of both up- and downregulation in early development.


Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/genética , Perfilação da Expressão Gênica , Complexos Multienzimáticos/análise , Septinas/biossíntese , Proteína cdc42 de Ligação ao GTP/biossíntese , Análise em Microsséries , Complexos Multienzimáticos/genética , Septinas/genética
14.
J Interferon Cytokine Res ; 36(8): 488-98, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27142572

RESUMO

Invasive aspergillosis is a major threat to patients suffering from impaired neutrophil function, with Aspergillus fumigatus being the most common species causing this life-threatening condition. Patients with chronic granulomatous disease (CGD) not only develop infections with A. fumigatus, but also exhibit a unique susceptibility to infection with the normally nonpathogenic species Aspergillus nidulans. In this study, we compared the inflammatory cytokine response of peripheral blood mononuclear cells (PBMCs) from healthy and CGD patients to these two fungal species. CGD patients displayed evidence for a chronic hyperinflammatory state as indicated by elevated plasma IL-1ß and TNF-α levels. PBMCs isolated from CGD patients secreted higher levels of IL-1ß and TNF-α in response to A. nidulans as compared with A. fumigatus. The presence or absence of melanin in the cell wall of A. nidulans did not alter the cytokine release by healthy or CGD PBMCs. In contrast, A. fumigatus mutants lacking melanin stimulated higher levels of proinflammatory cytokine release from healthy, but not CGD PBMCs. Purified cell wall polysaccharides of A. nidulans induced a much higher level of IL-1ß secretion by CGD PBMCs than did cell wall polysaccharides isolated from A. fumigatus. Using modified A. nidulans strains overexpressing galactosaminogalactan, we were able to show that the increased secretion of inflammatory cytokines by CGD PBMCs in response to A. nidulans are a consequence of low levels of cell wall-associated galactosaminogalactan in this species.


Assuntos
Aspergillus nidulans/metabolismo , Parede Celular/metabolismo , Doença Granulomatosa Crônica/microbiologia , Interações Hospedeiro-Patógeno , Polissacarídeos/metabolismo , Biomarcadores , Estudos de Casos e Controles , Citocinas/sangue , Citocinas/metabolismo , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Melaninas/metabolismo , Melaninas/farmacologia
15.
Microbiol Spectr ; 4(6)2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-28087934

RESUMO

All cells must accurately replicate DNA and partition it to daughter cells. The basic cell cycle machinery is highly conserved among eukaryotes. Most of the mechanisms that control the cell cycle were worked out in fungal cells, taking advantage of their powerful genetics and rapid duplication times. Here we describe the cell cycles of the unicellular budding yeast Saccharomyces cerevisiae and the multicellular filamentous fungus Aspergillus nidulans. We compare and contrast morphological landmarks of G1, S, G2, and M phases, molecular mechanisms that drive cell cycle progression, and checkpoints in these model unicellular and multicellular fungal systems.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Ciclo Celular , Saccharomyces cerevisiae/crescimento & desenvolvimento , Aspergillus nidulans/citologia , Replicação do DNA , Saccharomyces cerevisiae/citologia
16.
Mol Microbiol ; 98(4): 605-6, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26387769

RESUMO

In the filamentous fungus Aspergillus nidulans BrlA triggers the central developmental pathway that controls the transition from vegetative growth to asexual reproduction. Upstream regulators including the bZIP transcription factor FlbB activate the expression of brlA. Previous work has established that FlbB localizes to both the apex of the hypha, where it interacts with and is anchored by FlbE, and to nuclei, with highest levels in the nucleus closest to the apex and successively lower levels in nuclei further away from the apex. In this issue, Herrero-Garcia et al. dissect the roles of these two FlbB pools and the mechanisms underlying their localization and activity. Using a photoactivatable tag, they demonstrate that FlbB moves from the tip into the apical nucleus. Through a series of deletion constructs, they show that import of FlbB into the nucleus requires a bipartite NLS, that FlbB localization at the tip requires actin and that the FlbB tip-high gradient appears to be mass action dependent as the gradient is lost with FlbB constitutive upregulation. They show that while the pool of FlbB at the apex is required for triggering asexual development, the tip high nuclear gradient is not required.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Fúngicas/metabolismo
18.
FEBS Open Bio ; 4: 335-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24936400

RESUMO

Eukaryotic protein O-mannosyltransferases (Pmts) are divided into three subfamilies (Pmt1, Pmt2, and Pmt4) and activity of Pmts in yeasts and animals requires assembly into complexes. In Saccharomyces cerevisiae, Pmt1 and Pmt2 form a heteromeric complex and Pmt 4 forms a homomeric complex. The filamentous fungus Aspergillus nidulans has three Pmts: PmtA (subfamily 2), PmtB (subfamily 1), and PmtC (subfamily 4). In this study we show that A. nidulans Pmts form heteromeric complexes in all possible pairwise combinations and that PmtC forms homomeric complexes. We also show that MsbA, an ortholog of a Pmt4-modified protein, is not modified by PmtC.

19.
PLoS One ; 9(3): e92819, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24664283

RESUMO

Septins are important components of the cytoskeleton that are highly conserved in eukaryotes and play major roles in cytokinesis, patterning, and many developmental processes. Septins form heteropolymers which assemble into higher-order structures including rings, filaments, and gauzes. In contrast to actin filaments and microtubules, the molecular mechanism by which septins assemble is not well-understood. Here, we report that in the filamentous fungus Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a fifth septin lacking in unicellular yeasts, interacts with only one of the core septins, and only during multicellular growth. AspE is required for proper localization of three of the core septins, and requires this same subset of core septins for its own unique cortical localization. The ΔaspE mutant lacks developmentally-specific septin higher-order structures and shows reduced spore production and slow growth with low temperatures and osmotic stress. Our results show that at least two distinct septin heteropolymer populations co-exist in A. nidulans, and that while AspE is not a subunit of either heteropolymer, it is required for assembly of septin higher-order structures found in multicellular development.


Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/metabolismo , Complexos Multienzimáticos/metabolismo , Septinas/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Complexos Multienzimáticos/genética , Mutação , Pressão Osmótica/fisiologia , Transporte Proteico , Septinas/genética
20.
Curr Opin Microbiol ; 15(6): 660-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23116980

RESUMO

Septins are cytoskeletal elements that contain a highly conserved canonical G domain flanked by more divergent N- and C-termini. Septin monomers form heteropolymers that in turn associate into a variety of higher-order structures. SUMOylation, acetylation and phosphorylation of septins have all been reported; however, there are no examples of residues that are universally modified suggesting that posttranslational modifications of septins evolved relatively recently. Within the conserved G domain, posttranslational modifications cluster in regions near the G interface, consistent with roles in modulating heteropolymer assembly. Within the highly diverged N- and C-termini, posttranslational modifications are scattered randomly, consistent with roles in modulating assembly of higher-order structures that are unique to individual organisms.


Assuntos
Multimerização Proteica , Processamento de Proteína Pós-Traducional , Septinas/metabolismo , Acetilação , Eucariotos , Fosforilação , Sumoilação
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