scholarly journals Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2492-2506 ◽  
Author(s):  
Sara J. Blosser ◽  
Brittney Merriman ◽  
Nora Grahl ◽  
Dawoon Chung ◽  
Robert A. Cramer
Keyword(s):  
Aspergillus Fumigatus ◽  
Growth Defect ◽  
Ergosterol Biosynthesis ◽  
Mammalian Host ◽  
Primary Role ◽  
Single Deletion ◽  
Methyl Sterol ◽  
Genes Encoding ◽  
Methyl Sterols

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

scholarly journals SREBP-Dependent Triazole Susceptibility in Aspergillus fumigatus Is Mediated through Direct Transcriptional Regulation oferg11A(cyp51A)

2011 ◽  
Vol 56 (1) ◽  
pp. 248-257 ◽  
Author(s):  
Sara J. Blosser ◽  
Robert A. Cramer
Keyword(s):  
Aspergillus Fumigatus ◽  
Regulatory Element ◽  
Critical Role ◽  
Clinical Importance ◽  
Growth Defect ◽  
Ergosterol Biosynthesis ◽  
Wild Type ◽  
Transcript Levels ◽  
Content Type ◽  
Conditional Expression

ABSTRACTAs triazole antifungal drug resistance during invasiveAspergillus fumigatusinfection has become more prevalent, the need to understand mechanisms of resistance inA. fumigatushas increased. The presence of twoerg11(cyp51) genes inAspergillusspp. is hypothesized to account for the inherent resistance of this mold to the triazole fluconazole (FLC). Recently, anA. fumigatusnull mutant of a transcriptional regulator in the sterol regulatory element binding protein (SREBP) family, the ΔsrbAstrain, was found to have increased susceptibility to FLC and voriconazole (VCZ). In this study, we examined the mechanism engendering the observed increase inA. fumigatustriazole susceptibility in the absence of SrbA. We observed a significant reduction in theerg11Atranscript in the ΔsrbAstrain in response to FLC and VCZ. Transcript levels oferg11Bwere also reduced but not to the extent oferg11A. Interestingly,erg11Atranscript levels increased upon extended VCZ, but not FLC, exposure. Construction of anerg11Aconditional expression strain in the ΔsrbAstrain was able to restoreerg11Atranscript levels and, consequently, wild-type MICs to the triazole FLC. The VCZ MIC was also partially restored upon increasederg11Atranscript levels; however, total ergosterol levels remained significantly reduced compared to those of the wild type. Induction of theerg11Aconditional strain did not restore the hypoxia growth defect of the ΔsrbAstrain. Taken together, our results demonstrate a critical role for SrbA-mediated regulation of ergosterol biosynthesis and triazole drug interactions inA. fumigatusthat may have clinical importance.

scholarly journals Differential Effects of Inhibiting Chitin and 1,3-β-d-Glucan Synthesis in Ras and Calcineurin Mutants of Aspergillus fumigatus

2008 ◽  
Vol 53 (2) ◽  
pp. 476-482 ◽  
Author(s):  
Jarrod R. Fortwendel ◽  
Praveen Rao Juvvadi ◽  
Nadthanan Pinchai ◽  
B. Zachary Perfect ◽  
J. Andrew Alspaugh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Hyphal Growth ◽  
Invasive Disease ◽  
Growth Defect ◽  
Compensatory Response ◽  
Ras Protein ◽  
Genes Encoding ◽  
Glucan Synthesis ◽  
Calcineurin Pathway

ABSTRACT Aspergillus fumigatus must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools. Strains deleted for three genes encoding proteins of these pathways, rasA (the Ras protein), cnaA (calcineurin), or crzA (the zinc finger transcription factor downstream of calcineurin), all displayed decreased cell wall 1,3-β-d-glucan content. Echinocandin treatment further decreased the levels of 1,3-β-d-glucan for all strains tested yet also partially corrected the hyphal growth defect of the ΔrasA strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active rasA, and ΔrasA strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (ΔcnaA and ΔcrzA). Taken together, our data suggest that the Ras and calcineurin pathways act in parallel to regulate cell wall formation and hyphal growth. Additionally, the calcineurin pathway elements cnaA and crzA play a major role in proper chitin and glucan incorporation into the A. fumigatus cell wall.

scholarly journals Aspergillus fumigatus C-5 Sterol Desaturases Erg3A and Erg3B: Role in Sterol Biosynthesis and Antifungal Drug Susceptibility

2006 ◽  
Vol 50 (2) ◽  
pp. 453-460 ◽  
Author(s):  
Laura Alcazar-Fuoli ◽  
Emilia Mellado ◽  
Guillermo Garcia-Effron ◽  
Maria J. Buitrago ◽  
Jordi F. Lopez ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Gene Knockout ◽  
Drug Susceptibility ◽  
Nucleotide Sequences ◽  
Ergosterol Biosynthesis ◽  
Null Mutant ◽  
Targeted Disruption ◽  
Genes Encoding ◽  
Mutant Phenotypes

ABSTRACT Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A − erg3B −, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination.

scholarly journals Borrelia Host Adaptation Protein (BadP) Is Required for the Colonization of a Mammalian Host by the Agent of Lyme Disease

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Trever C. Smith ◽  
Sarah M. Helm ◽  
Yue Chen ◽  
Ying-Han Lin ◽  
S. L. Rajasekhar Karna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lyme Disease ◽  
Immunoblot Analysis ◽  
Growth Defect ◽  
Mammalian Host ◽  
Content Type ◽  
Magnesium Chelatase ◽  
Virulence Potential ◽  
Genes Encoding

ABSTRACT Borrelia burgdorferi , the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the B orrelia host ad aptation r egulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086 , shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi , were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the B orrelia host ad aptation p rotein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of B . burgdorferi .

scholarly journals Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae

2021 ◽  
Vol 7 (3) ◽  
pp. 237
Author(s):  
Mohammad Sayari ◽  
Magrieta A. van der Nest ◽  
Emma T. Steenkamp ◽  
Saleh Rahimlou ◽  
Almuth Hammerbacher ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Ergosterol Biosynthesis ◽  
Terpene Biosynthesis ◽  
Biosynthesis Gene Cluster ◽  
Spectrometry Analysis ◽  
Genes Encoding ◽  
Ergosterol Synthesis

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (Ceratocystis manginecans, C. adiposa, Huntiella moniliformis, Thielaviopsis punctulata, Bretziella fagacearum, Endoconidiophora polonica and Davidsoniella virescens), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.

scholarly journals A Regulator of Aspergillus fumigatus Extracellular Proteolytic Activity Is Dispensable for Virulence

2009 ◽  
Vol 77 (9) ◽  
pp. 4041-4050 ◽  
Author(s):  
Anna Bergmann ◽  
Thomas Hartmann ◽  
Timothy Cairns ◽  
Elaine M. Bignell ◽  
Sven Krappmann
Keyword(s):  
Aspergillus Fumigatus ◽  
Translational Control ◽  
Extracellular Proteases ◽  
Genes Encoding ◽  
Proteolytic Activities ◽  
Eif2α Kinase ◽  
Secreted Proteases ◽  
The Impact

ABSTRACT Virulence of the fungal pathogen Aspergillus fumigatus is in part based on the saprophytic lifestyle that this mold has evolved. A crucial function for saprophytism resides in secreted proteases that allow assimilation of proteinaceous substrates. The impact of extracellular proteolytic activities on the pathogenesis of aspergillosis, however, remains controversial. In order to address this issue, characterization of a conserved regulatory factor, PrtT, that acts on expression of secreted proteases was pursued. Expression of PrtT appears to be regulated posttranscriptionally, and the existence of an mRNA leader sequence implies translational control via eIF2α kinase signaling. Phenotypic classification of a prtTΔ deletion mutant revealed that expression of several major extracellular proteases is PrtT dependent, resulting in the inability to utilize protein as a nutritional source. Certain genes encoding secreted proteases are not regulated by PrtT. Most strikingly, the deletant strain is not attenuated in virulence when tested in a leukopenic mouse model, which makes a strong case for reconsidering any impact of secreted proteases in pulmonary aspergillosis.

Isolation and characterization of the genes encoding antimicrobial neutrophil cationic peptides, defensins.

Ensho ◽  
1995 ◽  
Vol 15 (1) ◽  
pp. 33-41
Author(s):  
Isao Nagaoka ◽  
Noriko Ishihara ◽  
Akimasa Someya ◽  
Kazuhisa Iwabuchi ◽  
Shin Yomogida ◽  
...  
Keyword(s):  
Cationic Peptides ◽  
Isolation And Characterization ◽  
Genes Encoding

scholarly journals Protein complex formation in methionine chain-elongation and leucine biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Li-Qun Chen ◽  
Shweta Chhajed ◽  
Tong Zhang ◽  
Joseph M. Collins ◽  
Qiuying Pang ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Complete Characterization ◽  
Bimolecular Fluorescence Complementation ◽  
Chain Elongation ◽  
Substrate Channeling ◽  
Leucine Biosynthesis ◽  
Protein Complex Formation ◽  
Genes Encoding ◽  
Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

scholarly journals Camphor and Eucalyptol—Anticandidal Spectrum, Antivirulence Effect, Efflux Pumps Interference and Cytotoxicity

2021 ◽  
Vol 22 (2) ◽  
pp. 483
Author(s):  
Marija Ivanov ◽  
Abhilash Kannan ◽  
Dejan S. Stojković ◽  
Jasmina Glamočlija ◽  
Ricardo C. Calhelha ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Liver Cells ◽  
Ergosterol Biosynthesis ◽  
Antifungal Compound ◽  
Porcine Liver ◽  
Fungal Virulence ◽  
Genes Encoding ◽  
Plant Constituents

Candidaalbicans represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different Candida species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps (CDR1 and CDR2), ergosterol biosynthesis (ERG11), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125–0.35 mg/mL while eucalyptol was active in the range of 2–23 mg/mL. The results showed camphor’s potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated CDR1;CDR2 was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on ERG11 expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.

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