scholarly journals Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

2015 ◽  
Vol 14 (11) ◽  
pp. 1073-1080 ◽  
Author(s):  
Kevin K. Fuller ◽  
Shan Chen ◽  
Jennifer J. Loros ◽  
Jay C. Dunlap
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Disruption ◽  
Polyketide Synthase ◽  
High Efficiency ◽  
Constitutive Expression ◽  
Targeted Mutagenesis ◽  
Genomic Alteration ◽  
Loss Of Function ◽  
Content Type ◽  
Targeted Gene Disruption

ABSTRACTLow rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogenAspergillus 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 inA. 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 theA. fumigatuspolyketide 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 toA. fumigatusgrowth 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 inA. fumigatusand has the potential to bolster the genetic toolbox for this important pathogen.

scholarly journals High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

Biology Open ◽  
2013 ◽  
Vol 2 (5) ◽  
pp. 448-452 ◽  
Author(s):  
K.-i. T. Suzuki ◽  
Y. Isoyama ◽  
K. Kashiwagi ◽  
T. Sakuma ◽  
H. Ochiai ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Xenopus Laevis ◽  
Gene Disruption ◽  
High Efficiency ◽  
Targeted Gene Disruption ◽  
Xenopus Laevis Embryos

scholarly journals A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Qian Wang ◽  
Dimitrios P. Kontoyiannis ◽  
Ruoyu Li ◽  
Wei Chen ◽  
Dingfang Bu ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Gene Mutations ◽  
Health Concern ◽  
Content Type ◽  
Clinical Strains ◽  
Allele Specific ◽  
Resistant Strains

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

scholarly journals Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

2013 ◽  
Vol 13 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Elodie Bovier ◽  
Carole H. Sellem ◽  
Adeline Humbert ◽  
Annie Sainsard-Chanet
Keyword(s):  
Transcription Factors ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Constitutive Expression ◽  
Podospora Anserina ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Zinc Cluster ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

scholarly journals Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Bang Shen ◽  
Kevin M. Brown ◽  
Tobie D. Lee ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Gene Disruption ◽  
Biological Diversity ◽  
Type I ◽  
Reverse Genetic ◽  
Site Specific ◽  
Content Type ◽  
Targeted Gene Disruption ◽  
Efficient Gene ◽  
Natural Isolates

ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. However, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to such approaches. Unfortunately, such common lab isolates have also been passaged frequently in vitro and have thus lost many of the attributes of wild isolates, often affecting important traits, like virulence. On the other hand, wild isolates are often not amenable to standard genetic approaches, thus limiting inquiry about the genetic basis of biological diversity. Here we imported a new genetic system based on CRISPR/CAS9, which allows high efficiency of targeted gene disruption in natural isolates of T. gondii. This advance promises to bring the power of genetics to bear on the broad diversity of T. gondii strains that have been described recently.

scholarly journals The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02880-20
Author(s):  
E. Keats Shwab ◽  
Praveen R. Juvvadi ◽  
Greg Waitt ◽  
Shareef Shaheen ◽  
John Allen ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Protein Kinase ◽  
Aspergillus Fumigatus ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Targeted Mutagenesis ◽  
Human Pathogen ◽  
Content Type ◽  
Kinase A ◽  
Microbial Disease

ABSTRACTProtein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

scholarly journals Targeted Inactivation of Francisella tularensis Genes by Group II Introns

2008 ◽  
Vol 74 (9) ◽  
pp. 2619-2626 ◽  
Author(s):  
Stephen A. Rodriguez ◽  
Jieh-Juen Yu ◽  
Greg Davis ◽  
Bernard P. Arulanandam ◽  
Karl E. Klose
Keyword(s):  
Francisella Tularensis ◽  
Gene Disruption ◽  
Molecular Mechanisms ◽  
High Efficiency ◽  
Single Step ◽  
Group Ii Introns ◽  
Targeted Gene Disruption ◽  
Genetic Techniques ◽  
Group Ii ◽  
Targeted Inactivation

ABSTRACT Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and “F. tularensis subsp. novicida.” A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.

scholarly journals Genome-Based Cluster Deletion Reveals an Endocrocin Biosynthetic Pathway in Aspergillus fumigatus

2012 ◽  
Vol 78 (12) ◽  
pp. 4117-4125 ◽  
Author(s):  
Fang Yun Lim ◽  
Yanpeng Hou ◽  
Yiming Chen ◽  
Jee-Hwan Oh ◽  
Inhyung Lee ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Polyketide Synthase ◽  
Wild Type Strain ◽  
Global Regulator ◽  
Content Type ◽  
In The Wild ◽  
Secondary Metabolite Gene Cluster

ABSTRACTEndocrocin is a simple anthraquinone frequently identified in extracts of numerous fungi. Several biosynthetic schemes for endocrocin synthesis have been hypothesized, but to date, no dedicated secondary metabolite gene cluster that produces this polyketide as its major metabolite has been identified. Here we describe our biosynthetic and regulatory characterization of the endocrocin gene cluster inAspergillus fumigatus. This is the first report of this anthraquinone in this species. The biosynthetic genes required for endocrocin production are regulated by the global regulator of secondary metabolism, LaeA, and encode an iterative nonreducing polyketide synthase (encA), a physically discrete metallo-β-lactamase type thioesterase (encB), and a monooxygenase (encC). Interestingly, the deletion of a gene immediately adjacent toencC, termedencDand encoding a putative 2-oxoglutarate-Fe(II) type oxidoreductase, resulted in higher levels of endocrocin production than in the wild-type strain, whereas overexpression ofencDeliminated endocrocin accumulation. We found that overexpression of theencAtranscript resulted in higher transcript levels ofencA-Dand higher production of endocrocin. We discuss a model of theenccluster as one evolutionary origin of fungal anthraquinones derived from a nonreducing polyketide synthase and a discrete metallo-β-lactamase-type thioesterase.

scholarly journals Aspergillus fumigatus Catalytic Glucokinase and Hexokinase: Expression Analysis and Importance for Germination, Growth, and Conidiation

2010 ◽  
Vol 9 (7) ◽  
pp. 1120-1135 ◽  
Author(s):  
Christian B. Fleck ◽  
Matthias Brock
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
High Efficiency ◽  
Isocitrate Lyase ◽  
Carbon Sources ◽  
Colony Growth ◽  
Biochemical Properties ◽  
Content Type ◽  
Double Deletion ◽  
A Minor

ABSTRACT Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.

scholarly journals Hemin Binding Protein C Is Found in Outer Membrane Vesicles and Protects Bartonella henselae against Toxic Concentrations of Hemin

2012 ◽  
Vol 80 (3) ◽  
pp. 929-942 ◽  
Author(s):  
Julie A. Roden ◽  
Derek H. Wells ◽  
Bruno B. Chomel ◽  
Rickie W. Kasten ◽  
Jane E. Koehler
Keyword(s):  
Outer Membrane ◽  
Protein C ◽  
High Efficiency ◽  
Binding Protein ◽  
Constitutive Expression ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mammalian Host ◽  
Content Type ◽  
Arthropod Vector

Bartonellaspecies are Gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential forBartonellasurvival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes inB. henselaeheme binding and uptake pathways. We identified aB. henselaetransposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription ofB. henselae hbpCwas upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to theB. henselaeouter membrane and outer membrane vesicles. Overexpression ofhbpCinB. henselaeincreased resistance to heme toxicity, implicating HbpC in protection ofB. henselaefrom the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats withB. henselaestrains demonstrated that both constitutive expression and deletion ofhbpCaffect the ability ofB. henselaeto infect the cat host. Modulation ofhbpCexpression appears to be a strategy employed byB. henselaeto survive in the arthropod vector and the mammalian host.

scholarly journals Identification and Regulation offusA, the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

2012 ◽  
Vol 78 (20) ◽  
pp. 7258-7266 ◽  
Author(s):  
Violeta Díaz-Sánchez ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Nitrogen Availability ◽  
Polyketide Synthase ◽  
High Sensitivity ◽  
Targeted Mutagenesis ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Wild Type ◽  
Sequence Comparisons ◽  
Content Type ◽  
Polyketide Synthase Gene

ABSTRACTFusarins are a class of mycotoxins of the polyketide family produced by differentFusariumspecies, including the gibberellin-producing fungusFusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in otherFusariumstrains, we have identified theF. fujikuroiorthologue, calledfusA. The participation offusAin fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by thefusAmRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of thefusAgene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, thefusAmutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions inFusarium oxysporum. ThefusAmutants, however, are not affected in their capacities to grow on plant tissues.

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