RESUMO
Fungi have been observed to exhibit resistance to high levels of ionizing radiation despite sharing most DNA repair mechanisms with other eukaryotes. Radioresistance, in fact, is such a common feature in fungi that it is difficult to identify species that exhibit widely different radiosensitivities, which in turn has hampered the identification of genetic elements responsible for this resistance phenotype. Due to the inherent mutagenic properties of radiation exposure, however, this can be addressed through adaptive laboratory evolution for increased ionizing radiation resistance. Here, using the black yeast Exophiala dermatitidis, we demonstrate that resistance to γ-radiation can be greatly increased through repeated rounds of irradiation and outgrowth. Moreover, we find that the small genome size of fungi situates them as a relatively simple functional genomics platform for identification of mutations associated with ionizing radiation resistance. This enabled the identification of genetic mutations in genes encoding proteins with a broad range of functions from 10 evolved strains. Specifically, we find that greatly increased resistance to γ-radiation is achieved in E. dermatitidis through disruption of the non-homologous end-joining pathway, with three individual evolutionary paths converging to abolish this DNA repair process. This result suggests that non-homologous end-joining, even in haploid cells where homologous chromosomes are not present during much of the cell cycle, is an impediment to repair of radiation-induced lesions in this organism, and that the relative levels of homologous and non-homologous repair in a given fungal species may play a major role in its radiation resistance.
Assuntos
Exophiala , Radiação Ionizante , Reparo do DNA/genética , Tolerância a Radiação/genéticaRESUMO
The melanized yeast Exophiala dermatitidis is resistant to many environmental stresses and is used as a model for understanding the diverse roles of melanin in fungi. Here, we describe the extent of resistance of E. dermatitidis to acute γ-radiation exposure and the major mechanisms it uses to recover from this stress. We find that melanin does not protect E. dermatitidis from γ-radiation. Instead, environmental factors such as nutrient availability, culture age and culture density are much greater determinants of cell survival after exposure. We also observe a dramatic transcriptomic response to γ-radiation that mobilizes pathways involved in morphological development, protein degradation and DNA repair, and is unaffected by the presence of melanin. Together, these results suggest that the ability of E. dermatitidis to survive γ-radiation exposure is determined by the prior and the current metabolic state of the cells as well as DNA repair mechanisms, and that small changes in these conditions can lead to large effects in radiation resistance, which should be taken into account when understanding how diverse fungi recover from this unique stress.
Assuntos
Exophiala/metabolismo , Exophiala/efeitos da radiação , Melaninas/metabolismo , Reparo do DNA/efeitos da radiação , DNA Fúngico/efeitos da radiação , Exophiala/genética , Tolerância a Radiação , Estresse Fisiológico , Transcrição Gênica/efeitos da radiação , TranscriptomaRESUMO
The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suite of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, respectively, is presented, along with a comparison to well-studied clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.
Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma , Astronave , Aspergillus fumigatus/isolamento & purificação , Metabolismo dos Carboidratos , Micotoxinas/metabolismo , Metabolismo Secundário , Estresse Fisiológico , Ausência de PesoRESUMO
The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.
Assuntos
Metabolismo dos Carboidratos/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Metabolismo Secundário/genética , Estresse Fisiológico/genética , Antraquinonas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Meio Ambiente , Genômica , Metabolômica , Proteômica , Metabolismo Secundário/fisiologia , Voo Espacial , Astronave , Xantonas/metabolismoRESUMO
Acetylaranotin is an epipolythiodiketopiperazine (ETP) secondary metabolite with a broad range of bioactivities. We demonstrated that ATEG_01465.1 located outside of acetylaranotin gene cluster is responsible for catalyzing the S-methylation of its biosynthetic pathway. Combining the previous characterization of acetylaranotin biosynthetic gene cluster together with the identification of its S-methyltransferase provides a means to obtain second-generation acetylaranotin derivatives previously inaccessible. By permutations of targeted deletions of ATEG_01465.1, acetyltransferase (AtaH), and benzoate hydroxylase (AtaY), three novel acetylaranotin derivatives were produced by Aspergillus terreus.
Assuntos
Vias Biossintéticas/genética , Metiltransferases/genética , Oxepinas/metabolismo , Piperazinas/metabolismo , Acetiltransferases/genética , Aspergillus/genética , Aspergillus/metabolismo , Genoma Fúngico/genética , Família Multigênica/genética , Oxigenases/genética , Deleção de Sequência/genéticaRESUMO
Due to immense phenotypic plasticity and adaptability, Aspergillus niger is a cosmopolitan fungus that thrives in versatile environments, including the International Space Station (ISS). This is the first report of genomic, proteomic, and metabolomic alterations observed in A. niger strain JSC-093350089 grown in a controlled experiment aboard the ISS. Whole-genome sequencing (WGS) revealed that ISS conditions, including microgravity and enhanced irradiation, triggered non-synonymous point mutations in specific regions, chromosomes VIII and XII of the JSC-093350089 genome when compared to the ground-grown control. Proteome analysis showed altered abundance of proteins involved in carbohydrate metabolism, stress response, and cellular amino acid and protein catabolic processes following growth aboard the ISS. Metabolome analysis further confirmed that space conditions altered molecular suite of ISS-grown A. niger JSC-093350089. After regrowing both strains on Earth, production of antioxidant-Pyranonigrin A was significantly induced in the ISS-flown, but not the ground control strain. In summary, the microgravity and enhanced irradiation triggered unique molecular responses in the A. niger JSC-093350089 suggesting adaptive responses.
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Melanized fungi have been isolated from some of the harshest radioactive environments, and their ability to thrive in these locations is in part due to the pigment melanin. Melanin imparts a selective advantage to fungi by providing a physical shield, a chemical shield, and possibly a signaling mechanism. In previous work we demonstrated that protracted exposure of the melanized yeast Exophiala dermatitidis to mixed alpha-, beta-, and gamma-emitting radiation resulted in an adapted strain able to mount a unique response to ionizing radiation in the environment in a melanin-dependent fashion. By exploring the genome and transcriptome of this adapted melanized strain relative to a non-irradiated control we determined the altered response was transcriptomic in nature, as whole genome sequencing revealed limited variation. Transcriptomic analysis indicated that of the adapted isolates analyzed, two lineages existed: one like the naïve, non-adapted strain, and one with a unique transcriptomic signature that exhibited downregulation of metabolic processes, and upregulation of translation-associated genes. Analysis of differential gene expression in the adapted strain showed an overlap in response between the control conditions and reactive oxygen species conditions, whereas exposure to an alpha particle source resulted in a robust downregulation of metabolic processes and upregulation of DNA replication and repair genes, and RNA metabolic processes. This suggest previous exposure to radiation primes the fungus to respond to subsequent exposures in a unique way. By exploring this unique response, we have expanded our knowledge of how melanized fungi interact with and respond to ionizing radiation in their environment.
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The black yeast Exophiala lecanii-corni of the order Chaetothyriales is notable for its ability to produce abundant quantities of DHN-melanin. While many other Exophiala species are frequent causal agents of human infection, E. lecanii-corni CBS 102400 lacks the thermotolerance requirements that enable pathogenicity, making it appealing for use in targeted functional studies and biotechnological applications. Here, we report the stress tolerance characteristics of E. lecanii-corni, with an emphasis on the influence of melanin on its resistance to various forms of stress. We find that E. lecanii-corni has a distinct stress tolerance profile that includes variation in resistance to temperature, osmotic, and oxidative stress relative to the extremophilic and pathogenic black yeast Exophiala dermatitidis. Notably, the presence of melanin substantially impacts stress resistance in E. lecanii-corni, while this was not found to be the case in E. dermatitidis. The cellular context, therefore, influences the role of melanin in stress protection. In addition, we present a detailed analysis of the E. lecanii-corni genome, revealing key differences in functional genetic content relative to other ascomycetous species, including a significant decrease in abundance of genes encoding ribosomal proteins. In all, this study provides insight into how genetics and physiology may underlie stress tolerance and enhances understanding of the genetic diversity of black yeasts.
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Secondary metabolite (SM) production in Aspergillus niger JSC-093350089, isolated from the International Space Station (ISS), is reported, along with a comparison to the experimentally established strain ATCC 1015. The analysis revealed enhanced production levels of naphtho-γ-pyrones and therapeutically relevant SMs, including bicoumanigrin A, aurasperones A and B, and the antioxidant pyranonigrin A. Genetic variants that may be responsible for increased SM production levels in JSC-093350089 were identified. These findings include INDELs within the predicted promoter region of flbA, which encodes a developmental regulator that modulates pyranonigrin A production via regulation of Fum21. The pyranonigrin A biosynthetic gene cluster was confirmed in A. niger, which revealed the involvement of a previously undescribed gene, pyrE, in its biosynthesis. UVC sensitivity assays enabled characterization of pyranonigrin A as a UV resistance agent in the ISS isolate.
RESUMO
The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies-many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains-the wild type and a hyper-resistant strain developed through adaptive laboratory evolution-before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi.
Assuntos
Exophiala/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Raios gama/efeitos adversos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Proteoma/metabolismo , Tolerância a Radiação , Transcriptoma/efeitos da radiação , DNA Fúngico/análise , DNA Fúngico/genética , Exophiala/genética , Exophiala/metabolismo , Exophiala/efeitos da radiação , Proteínas Fúngicas/genética , Melaninas/metabolismo , Proteoma/análiseRESUMO
Secondary metabolites (SMs) produced by filamentous fungi possess diverse bioactivities that make them excellent drug candidates. Whole genome sequencing has revealed that fungi have the capacity to produce a far greater number of SMs than have been isolated, since many of the genes involved in SM biosynthesis are either silent or expressed at very low levels in standard laboratory conditions. There has been significant effort to activate SM biosynthetic genes and link them to their downstream products, as the SMs produced by these "cryptic" pathways offer a promising source for new drug discovery. Further, an understanding of the genes involved in SM biosynthesis facilitates product yield optimization of first-generation molecules and genetic engineering of second-generation analogs. This review covers advances made in genome mining SMs produced by Aspergillus nidulans, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus in the past six years (2012-2018). Genetic identification and molecular characterization of SM biosynthetic gene clusters, along with proposed biosynthetic pathways, will be discussed in depth.
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The initial characterization of the Aspergillus niger isolate JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS), is reported, along with a comparison to the extensively studied strain ATCC 1015. Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement within the A. niger/welwitschiae/lacticoffeatus clade and revealed that the genome of JSC-093350089 is within the observed genetic variance of other sequenced A. niger strains. The ISS isolate exhibited an increased rate of growth and pigment distribution compared to a terrestrial strain. Analysis of the isolate's proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger on board the ISS and provide insight into the characteristics of melanized fungal species inhabiting spacecraft environments. IMPORTANCE A thorough understanding of how fungi respond and adapt to the various stimuli encountered during spaceflight presents many economic benefits and is imperative for the health of crew. As A. niger is a predominant ISS isolate frequently detected in built environments, studies of A. niger strains inhabiting closed systems may reveal information fundamental to the success of long-duration space missions. This investigation provides valuable insights into the adaptive mechanisms of fungi in extreme environments as well as countermeasures to eradicate unfavorable microbes. Further, it enhances understanding of host-microbe interactions in closed systems, which can help NASA's Human Research Program maintain a habitat healthy for crew during long-term manned space missions.
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The whole-genome sequences of eight fungal strains that were selected for exposure to microgravity at the International Space Station are presented here. These baseline sequences will help to understand the observed production of novel bioactive compounds.
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[This corrects the article DOI: 10.1039/C5SC01965F.].
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One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments. IMPORTANCE As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.
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Meroterpenoids are a class of secondary metabolites that are produced from polyketide and terpenoid precursors. 15-Deoxyoxalicine B (1) belongs to one structural group consisting of a unique pyridinyl-α-pyrone polyketide subunit and a diterpenoid subunit connected through a characteristic asymmetric spiro carbon atom. An understanding of the genes involved in the biosynthesis of this class of compounds should provide a means to facilitate engineering of second-generation molecules and increasing production of first-generation compounds. We found that the filamentous fungus Penicillium canescens produces 15-deoxyoxalicine B (1). Using targeted gene deletions, we have identified a cluster of 12 responsible contiguous genes. This gene cluster includes one polyketide synthase gene which we have designated olcA. Chemical analysis of wild-type and gene deletion mutant extracts enabled us to isolate and characterize 7 additional metabolites that are either intermediates or shunt products of the biosynthetic pathway. Two of the compounds identified have not been reported previously. Our data have allowed us to propose a biosynthetic pathway for 15-deoxyoxalicine B (1).
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Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.