Publications by authors named "Timothy C Cairns"

19 Publications

  • Page 1 of 1

Disruption or reduced expression of the orotidine-5'-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger.

Microb Cell Fact 2020 Mar 24;19(1):76. Epub 2020 Mar 24.

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.

Background: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited.

Results: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5'-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors.

Conclusions: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.
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http://dx.doi.org/10.1186/s12934-020-01334-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092557PMC
March 2020

Correction to: Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in submerged culture.

Fungal Biol Biotechnol 2019;6:26. Epub 2019 Dec 17.

1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 People's Republic of China.

[This corrects the article DOI: 10.1186/s40694-019-0081-x.].
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http://dx.doi.org/10.1186/s40694-019-0087-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918661PMC
December 2019

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in submerged culture.

Fungal Biol Biotechnol 2019 9;6:18. Epub 2019 Nov 9.

1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 People's Republic of China.

Background: Filamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. Protein secretion and filamentous growth are tightly coupled at the hyphal tip. Additionally, both these processes require ATP and amino acid precursors derived from the citric acid cycle. Despite this interconnection of organic acid production and protein secretion/filamentous growth, few studies in fungi have identified genes which may concomitantly impact all three processes.

Results: We applied a novel screen of a global co-expression network in the cell factory to identify candidate genes which may concomitantly impact macromorphology, and protein/organic acid fermentation. This identified genes predicted to encode the Golgi localized ArfA GTPase activating protein (GAP, AgeB), and ArfA guanine nucleotide exchange factors (GEFs SecG and GeaB) to be co-expressed with citric acid cycle genes. Consequently, we used CRISPR-based genome editing to place the titratable Tet-on expression system upstream of , , and in . Functional analysis revealed that and are essential whereas was dispensable for early filamentous growth. Next, gene expression was titrated during submerged cultivations under conditions for either protein or organic acid production. ArfA regulators played varied and culture-dependent roles on pellet formation. Notably, or expression levels had major impacts on protein secretion, whereas was dispensable. In contrast, reduced expression of each predicted ArfA regulator resulted in an absence of citric acid in growth media. Finally, titrated expression of either GEFs resulted in an increase in oxaloacetic acid concentrations in supernatants.

Conclusion: Our data suggest that the Golgi may play an underappreciated role in modulating organic acid titres during industrial applications, and that this is SecG, GeaB and AgeB dependent in . These data may lead to novel avenues for strain optimization in filamentous fungi for improved protein and organic acid titres.
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http://dx.doi.org/10.1186/s40694-019-0081-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842248PMC
November 2019

The ORFeome: A Functional Genomics Community Resource.

Mol Plant Microbe Interact 2019 Dec 14;32(12):1564-1570. Epub 2019 Oct 14.

Biosciences, University of Exeter, Exeter EX4 4QD, U.K.

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for , which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors ( = 410) and secondary metabolite biosynthetic proteins ( = 171) in addition to genes residing at dispensable chromosomes ( = 122) or those that are preferentially expressed during plant infection ( = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of .[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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http://dx.doi.org/10.1094/MPMI-05-19-0123-ADOI Listing
December 2019

A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using in .

Biotechnol Biofuels 2019 15;12:149. Epub 2019 Jun 15.

1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 People's Republic of China.

Background: Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.

Results: In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for to identify a putative gamma-adaptin encoding gene () that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional expression mutant under control of the titratable Tet-on system. Reduced expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.

Conclusion: The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene plays a crucial role in filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.
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http://dx.doi.org/10.1186/s13068-019-1473-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570962PMC
June 2019

Moulding the mould: understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories.

Biotechnol Biofuels 2019 2;12:77. Epub 2019 Apr 2.

1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.

Filamentous fungi are harnessed as cell factories for the production of a diverse range of organic acids, proteins, and secondary metabolites. Growth and morphology have critical implications for product titres in both submerged and solid-state fermentations. Recent advances in systems-level understanding of the filamentous lifestyle and development of sophisticated synthetic biological tools for controlled manipulation of fungal genomes now allow rational strain development programs based on data-driven decision making. In this review, we focus on spp. and other industrially utilised fungi to summarise recent insights into the multifaceted and dynamic relationship between filamentous growth and product titres from genetic, metabolic, modelling, subcellular, macromorphological and process engineering perspectives. Current progress and knowledge gaps with regard to mechanistic understanding of product secretion and export from the fungal cell are discussed. We highlight possible strategies for unlocking lead genes for rational strain optimizations based on omics data, and discuss how targeted genetic manipulation of these candidates can be used to optimise fungal morphology for improved performance. Additionally, fungal signalling cascades are introduced as critical processes that can be genetically targeted to control growth and morphology during biotechnological applications. Finally, we review progress in the field of synthetic biology towards chassis cells and minimal genomes, which will eventually enable highly programmable filamentous growth and diversified production capabilities. Ultimately, these advances will not only expand the fungal biotechnology portfolio but will also significantly contribute to a sustainable bio-economy.
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http://dx.doi.org/10.1186/s13068-019-1400-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446404PMC
April 2019

Comprehensive Improvement of Sample Preparation Methodologies Facilitates Dynamic Metabolomics of Aspergillus niger.

Biotechnol J 2019 Mar 20;14(3):e1800315. Epub 2018 Sep 20.

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.

Metabolomics is an essential discipline in industrial biotechnology. Sample preparation approaches dramatically influence data quality and, ultimately, interpretation and conclusions from metabolomic experiments. However, standardized protocols for highly reproducible metabolic datasets are limited, especially for the fungal cell factory Aspergillus niger. Here, an improved liquid chromatography-tandem mass spectrometry-based pipeline for A. niger metabolomics is developed. It is found that fast filtration with liquid nitrogen is more suitable for cell quenching, causing minimal disruption to cell integrity, and improved intracellular metabolite recovery when compared to cold methanol quenching approaches. Seven solutions are evaluated for intracellular metabolite extraction, and found acetonitrile/water (1:1, v/v) at -20 °C, combined with boiling ethanol extraction protocols, showed unbiased metabolite profiling. This improved methodology is applied to unveil the dynamic metabolite profile of one citrate over-producing A. niger isolate under citrate fermentation. Citrate precursors, especially pyruvate, oxaloacetate, and malate, are maintained at a relatively high intracellular level, which can be necessary for high citrate synthesis flux. Glutamine shows a similar trend compared to citrate production, suggesting glutamine may be involved in intracellular pH homeostasis. Taken together, this study delivers a highly standardized and improved metabolomics methodology and paves the way for systems metabolic engineering in biotechnologically important fungi.
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http://dx.doi.org/10.1002/biot.201800315DOI Listing
March 2019

Conditional Expression of the Small GTPase ArfA Impacts Secretion, Morphology, Growth, and Actin Ring Position in .

Front Microbiol 2018 8;9:878. Epub 2018 May 8.

Department of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.

In filamentous fungi, growth and protein secretion occurs predominantly at the tip of long, thread like cells termed hyphae. This requires coordinated regulation of multiple processes, including vesicle trafficking, exocytosis, and endocytosis, which are facilitated by a complex cytoskeletal apparatus. In this study, functional analyses of the small GTPase ArfA from demonstrate that this protein functionally complements the , and that this protein is essential for . Loss-of-function and gain-of-function analyses demonstrate that titration of expression impacts hyphal growth rate, hyphal tip morphology, and protein secretion. Moreover, localization of the endocytic machinery, visualized via fluorescent tagging of the actin ring, was found to be abnormal in ArfA under- and overexpressed conditions. Finally, we provide evidence that the major secreted protein GlaA localizes at septal junctions, indicating that secretion in may occur at these loci, and that this process is likely impacted by expression levels. Taken together, our results demonstrate that ArfA fulfills multiple functions in the secretory pathway of .
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http://dx.doi.org/10.3389/fmicb.2018.00878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952172PMC
May 2018

How a fungus shapes biotechnology: 100 years of research.

Fungal Biol Biotechnol 2018 24;5:13. Epub 2018 May 24.

Department of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.

In 1917, a food chemist named James Currie made a promising discovery: any strain of the filamentous mould would produce high concentrations of citric acid when grown in sugar medium. This tricarboxylic acid, which we now know is an intermediate of the Krebs cycle, had previously been extracted from citrus fruits for applications in food and beverage production. Two years after Currie's discovery, industrial-level production using began, the biochemical fermentation industry started to flourish, and industrial biotechnology was born. A century later, citric acid production using this mould is a multi-billion dollar industry, with additionally producing a diverse range of proteins, enzymes and secondary metabolites. In this review, we assess main developments in the field of biology over the last 100 years and highlight scientific breakthroughs and discoveries which were influential for both basic and applied fungal research in and outside the community. We give special focus to two developments of the last decade: systems biology and genome editing. We also summarize the current international research community, and end by speculating on the future of fundamental research on this fascinating fungus and its exploitation in industrial biotechnology.
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http://dx.doi.org/10.1186/s40694-018-0054-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966904PMC
May 2018

5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in .

ACS Synth Biol 2019 07 30;8(7):1568-1574. Epub 2018 Apr 30.

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences , Tianjin , 300308 , China.

The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, , to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in . We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.
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http://dx.doi.org/10.1021/acssynbio.7b00456DOI Listing
July 2019

A silver bullet in a golden age of functional genomics: the impact of -mediated transformation of fungi.

Fungal Biol Biotechnol 2017 26;4. Epub 2017 Sep 26.

College of Life and Applied Sciences, Yeungnam University, Gyeongsan, South Korea.

The implementation of as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. mediated transformation (MT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. MT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, MT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using MT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases MT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of MT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, MT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.
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http://dx.doi.org/10.1186/s40694-017-0035-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615635PMC
September 2017

Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper.

Fungal Biol Biotechnol 2016 31;3. Epub 2016 Aug 31.

Ceratium Limited, The Haven, West Kirby, CH48 8AP UK.

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.
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http://dx.doi.org/10.1186/s40694-016-0024-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611618PMC
August 2016

Secondary metabolite arsenal of an opportunistic pathogenic fungus.

Philos Trans R Soc Lond B Biol Sci 2016 12;371(1709)

Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA,

Aspergillus fumigatus is a versatile fungus able to successfully exploit diverse environments from mammalian lungs to agricultural waste products. Among its many fitness attributes are dozens of genetic loci containing biosynthetic gene clusters (BGCs) producing bioactive small molecules (often referred to as secondary metabolites or natural products) that provide growth advantages to the fungus dependent on environment. Here we summarize the current knowledge of these BGCs-18 of which can be named to product-their expression profiles in vivo, and which BGCs may enhance virulence of this opportunistic human pathogen. Furthermore, we find extensive evidence for the presence of many of these BGCs, or similar BGCs, in distantly related genera including the emerging pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats, and suggest such BGCs may be predictive of pathogenic potential in other fungi.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.
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http://dx.doi.org/10.1098/rstb.2016.0023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095546PMC
December 2016

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

Microb Cell Fact 2016 Aug 20;15(1):145. Epub 2016 Aug 20.

Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.

Background: The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.

Results: In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein.

Conclusion: Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.
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http://dx.doi.org/10.1186/s12934-016-0543-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992228PMC
August 2016

New and Improved Techniques for the Study of Pathogenic Fungi.

Trends Microbiol 2016 Jan 5;24(1):35-50. Epub 2015 Nov 5.

Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.

Fungal pathogens pose serious threats to human, plant, and ecosystem health. Improved diagnostics and antifungal strategies are therefore urgently required. Here, we review recent developments in online bioinformatic tools and associated interactive data archives, which enable sophisticated comparative genomics and functional analysis of fungal pathogens in silico. Additionally, we highlight cutting-edge experimental techniques, including conditional expression systems, recyclable markers, RNA interference, genome editing, compound screens, infection models, and robotic automation, which are promising to revolutionize the study of both human and plant pathogenic fungi. These novel techniques will allow vital knowledge gaps to be addressed with regard to the evolution of virulence, host-pathogen interactions and antifungal drug therapies in both the clinic and agriculture. This, in turn, will enable delivery of improved diagnosis and durable disease-control strategies.
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http://dx.doi.org/10.1016/j.tim.2015.09.008DOI Listing
January 2016

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

PLoS Pathog 2014 Oct 16;10(10):e1004413. Epub 2014 Oct 16.

Institute for Inflammation and Repair, University of Manchester, Manchester, United Kingdom.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.
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http://dx.doi.org/10.1371/journal.ppat.1004413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199764PMC
October 2014

Oligopeptide transport and regulation of extracellular proteolysis are required for growth of Aspergillus fumigatus on complex substrates but not for virulence.

Mol Microbiol 2011 Nov 24;82(4):917-35. Epub 2011 Oct 24.

Research Center for Infectious Diseases, Julius Maximilians University Würzburg, Germany.

Moulds are characterized by their saprophytic lifestyle that is based on osmotrophy. Among them, Aspergillus fumigatus has emerged as the leading cause of fungal infections in the presence of an underlying immunodeficiency. To assess the role of its nutritional versatility for virulence, transcriptional profiling studies in the presence of varying sources of nitrogen were carried out and revealed an extensive reprogramming of the fungal transcriptome when shifting to a proteinaceous growth substrate. Transcripts encoding metabolic activities were predominantly upregulated, as were proteinases and transport activities. To probe whether fundamental aspects of its osmotrophic lifestyle, that is, extracellular proteolysis and uptake of oligopeptides, are required for A. fumigatus pathogenicity, serial gene replacements were carried out, which eventually yielded an octuple deletion mutant ablated for the opt gene family. This strain displayed no growth defect on various substrates, but supplementary reduction of extracellular proteolytic activity by additional deletion of the prtT gene revealed a synthetic phenotype on porcine lung tissue agar. Virulence studies in a murine model of pulmonary aspergillosis did not disclose any attenuation in virulence of these deletants. Our data emphasize a high degree of redundancy encoded by the A. fumigatus genome that secures nutrient supply for growth and, therefore, virulence.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07868.xDOI Listing
November 2011

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus.

Fungal Genet Biol 2011 Nov 5;48(11):1071-5. Epub 2011 Aug 5.

Division of Biology, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, United Kingdom.

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.
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http://dx.doi.org/10.1016/j.fgb.2011.07.008DOI Listing
November 2011