共查询到20条相似文献,搜索用时 31 毫秒
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Ana Camila Oliveira Souza Adela Martin-Vicente Ashley V. Nywening Wenbo Ge David J. Lowes Brian M. Peters Jarrod R. Fortwendel 《PLoS pathogens》2021,17(8)
Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae. 相似文献
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Mark J. Lee Hong Liu Bridget M. Barker Brendan D. Snarr Fabrice N. Gravelat Qusai Al Abdallah Christina Gavino Shane R. Baistrocchi Hanna Ostapska Tianli Xiao Benjamin Ralph Norma V. Solis Mélanie Lehoux Stefanie D. Baptista Arsa Thammahong Robert P. Cerone Susan G. W. Kaminskyj Marie-Christine Guiot Jean-Paul Latgé Thierry Fontaine Donald C. Vinh Scott G. Filler Donald C. Sheppard 《PLoS pathogens》2015,11(10)
Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs. 相似文献
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Margherita Bertuzzi Markus Schrettl Laura Alcazar-Fuoli Timothy C. Cairns Alberto Mu?oz Louise A. Walker Susanne Herbst Maryam Safari Angela M. Cheverton Dan Chen Hong Liu Shinobu Saijo Natalie D. Fedorova Darius Armstrong-James Carol A. Munro Nick D. Read Scott G. Filler Eduardo A. Espeso William C. Nierman Hubertus Haas Elaine M. Bignell 《PLoS pathogens》2014,10(10)
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Eleftheria Mavridou Joseph Meletiadis Pavol Jancura Saiden Abbas Maiken C. Arendrup Willem J. G. Melchers Tom Heskes Johan W. Mouton Paul E. Verweij 《PloS one》2013,8(8)
Understanding resistance to antifungal agents in Aspergillus fumigatus is of increasing importance for the treatment of invasive infections in immunocompromised patients. Although a number of molecular resistance mechanisms are described in detail, the potential accompanying virulence changes and impact on clinical outcome have had little attention. We developed a new measure of survival, the composite survival index (CSI) to use as a measure of the virulence properties of A. fumigatus. Using a novel mathematical model we found a strong correlation between the in vitro growth characteristics and virulence in vivo expressed as CSI. Our model elucidates how three critical parameters (the lag phase (τ), decay constant (λ), and growth rate (ν)) interact with each other resulting in a CSI that correlated with virulence. Hence, strains with a long lag phase and high decay constant were less virulent in a murine model of invasive aspergillosis, whereas high virulence for isolates with a high CSI was associated in vitro with rapid growth and short lag phases. Resistant isolates with cyp51A mutations, which account for the majority of azole resistant aspergillosis cases, did not show a lower virulence compared to azole-susceptible isolates. In contrast, the CSI index revealed that a non-cyp51A-mediated resistance mechanism was associated with a dramatic decrease in CSI. Because of its predictive value, the mathematical model developed may serve to explore strain characteristics in vitro to predict virulence in vivo and significantly reduce the number of experimental animals required in such studies. The proposed measure of survival, the CSI can be used more in a general form in survival studies to explore optimal treatment options. 相似文献
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Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation,secondary metabolite production,and virulence
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Adriana Oliveira Manfiolli Patrícia Alves de Castro Thaila Fernanda dos Reis Stephen Dolan Sean Doyle Gary Jones Diego M. Riaño Pachón Mevlüt Ulaş Luke M. Noble Derek J. Mattern Axel A. Brakhage Vito Valiante Rafael Silva‐Rocha Ozgur Bayram Gustavo H. Goldman 《Cellular microbiology》2017,19(12)
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Sven D. Willger E. Jean Cornish Dawoon Chung Brittany A. Fleming Margaret M. Lehmann Srisombat Puttikamonkul Robert A. Cramer 《Eukaryotic cell》2012,11(12):1557-1567
Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function. 相似文献
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High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence
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Lizziane K. Winkelströter Vinícius Leite Pedro Bom Patrícia Alves de Castro Leandra Naira Zambelli Ramalho Maria Helena S. Goldman Neil Andrew Brown Ranjith Rajendran Gordon Ramage Elodie Bovier Thaila Fernanda dos Reis Marcela Savoldi Daisuke Hagiwara Gustavo H. Goldman 《Molecular microbiology》2015,96(1):42-54
Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β‐1,3‐glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus. 相似文献
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Katie Falloon Praveen R. Juvvadi Amber D. Richards José M. Vargas-Mu?iz Hilary Renshaw William J. Steinbach 《PloS one》2015,10(9)
Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease. 相似文献
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Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen. 相似文献
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Fabrice N. Gravelat Daniele E. Ejzykowicz Lisa Y. Chiang Josée C. Chabot Mirjam Urb K. Denyese Macdonald Nadia Al‐Bader Scott G. Filler Donald C. Sheppard 《Cellular microbiology》2010,12(4):473-488
In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro‐inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis. 相似文献
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Jason D. Oliver Sarah J. Kaye Danny Tuckwell Anna E. Johns Darel A. Macdonald Joanne Livermore Peter A. Warn Mike Birch Michael J. Bromley 《PloS one》2012,7(9)
Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC. 相似文献
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The greater wax moth Galleria mellonella has been widely used as
a heterologous host for a number of fungal pathogens including Candida
albicans and Cryptococcus neoformans. A positive
correlation in pathogenicity of these yeasts in this insect model and animal
models has been observed. However, very few studies have evaluated the
possibility of applying this heterologous insect model to investigate virulence
traits of the filamentous fungal pathogen Aspergillus
fumigatus, the leading cause of invasive aspergillosis. Here, we have
examined the impact of mutations in genes involved in melanin biosynthesis on
the pathogenicity of A. fumigatus in the G.
mellonella model. Melanization in A. fumigatus confers
bluish-grey color to conidia and is a known virulence factor in mammal models.
Surprisingly, conidial color mutants in B5233 background that have deletions in
the defined six-gene cluster required for DHN-melanin biosynthesis caused
enhanced insect mortality compared to the parent strain. To further examine and
confirm the relationship between melanization defects and enhanced virulence in
the wax moth model, we performed random insertional mutagenesis in the Af293
genetic background to isolate mutants producing altered conidia colors. Strains
producing conidia of previously identified colors and of novel colors were
isolated. Interestingly, these color mutants displayed a higher level of
pathogenicity in the insect model compared to the wild type. Although some of
the more virulent color mutants showed increased resistance to hydrogen
peroxide, overall phenotypic characterizations including secondary metabolite
production, metalloproteinase activity, and germination rate did not reveal a
general mechanism accountable for the enhanced virulence of these color mutants
observed in the insect model. Our observations indicate instead, that
exacerbated immune response of the wax moth induced by increased exposure of
PAMPs (pathogen-associated molecular patterns) may cause self-damage that
results in increased mortality of larvae infected with the color mutants. The
current study underscores the limitations of using this insect model for
inferring the pathogenic potential of A. fumigatus strains in
mammals, but also points to the importance of understanding the innate immunity
of the insect host in providing insights into the pathogenicity level of
different fungal strains in this model. Additionally, our observations that
melanization defective color mutants demonstrate increased virulence in the
insect wax moth, suggest the potential of using melanization defective mutants
of native insect fungal pathogens in the biological control of insect
populations. 相似文献