scholarly journals The Volatome of Aspergillus fumigatus

2014 ◽  
Vol 13 (8) ◽  
pp. 1014-1025 ◽  
Author(s):  
C. Heddergott ◽  
A. M. Calvo ◽  
J. P. Latgé
Keyword(s):  
Aspergillus Fumigatus ◽  
Growth Conditions ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
Volatile Organic ◽  
Volatile Terpenes ◽  
Nutritional Environment ◽  
Volatile Production ◽  
Related Compounds

ABSTRACT Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatus in vitro , and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β- trans -bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus . The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus.

scholarly journals The Glycosylphosphatidylinositol-Anchored DFG Family Is Essential for the Insertion of Galactomannan into the β-(1,3)-Glucan–Chitin Core of the Cell Wall of Aspergillus fumigatus

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Laetitia Muszkieta ◽  
Thierry Fontaine ◽  
Rémi Beau ◽  
Isabelle Mouyna ◽  
Marian Samuel Vogt ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Gene Family ◽  
Protein Complexes ◽  
Growth Conditions ◽  
Fungal Cell Wall ◽  
Cross Linking ◽  
Fungal Cell ◽  
Content Type

ABSTRACT The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatus. IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Pharmacodynamics of Itraconazole against Aspergillus fumigatus in anIn VitroModel of the Human Alveolus: Perspectives on the Treatment of Triazole-Resistant Infection and Utility of Airway Administration

2012 ◽  
Vol 56 (8) ◽  
pp. 4146-4153 ◽  
Author(s):  
Zaid Al-Nakeeb ◽  
Ajay Sudan ◽  
Adam R. Jeans ◽  
Lea Gregson ◽  
Joanne Goodwin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Therapeutic Strategies ◽  
Wild Type ◽  
Content Type ◽  
Exposure Response ◽  
Prevention And Treatment ◽  
Resistant Infection ◽  
Resistant Strains ◽  
Type Strains

ABSTRACTItraconazole is used for the prevention and treatment of infections caused byAspergillus fumigatus. An understanding of the pharmacodynamics of itraconazole against wild-type and triazole-resistant strains provides a basis for innovative therapeutic strategies for treatment of infections. Anin vitromodel of the human alveolus was used to define the pharmacodynamics of itraconazole. Galactomannan was used as a biomarker. The effect of systemic and airway administration of itraconazole was assessed, as was a combination of itraconazole administered to the airway and systemically administered 5FC. Systemically administered itraconazole against the wild type induced a concentration-dependent decline in galactomannan in the alveolar and endothelial compartments. No exposure-response relationships were apparent for the L98H, M220T, or G138C mutant. The administration of itraconazole to the airway resulted in comparable exposure-response relationships to those observed with systemic therapy. This was achieved without detectable concentrations of drug within the endothelial compartment. The airway administration of itraconazole resulted in a definite but submaximal effect in the endothelial compartment against the L98H mutant. The administration of 5FC resulted in a concentration-dependent decline in galactomannan in both the alveolar and endothelial compartments. The combination of airway administration of itraconazole and systemically administered 5FC was additive. Systemic administration of itraconazole is ineffective against Cyp51 mutants. The airway administration of itraconazole is effective for the treatment of wild-type strains and appears to have some activity against the L98H mutants. Combination with other agents, such as 5FC, may enable the attainment of near-maximal antifungal activity.

scholarly journals Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

2015 ◽  
Vol 197 (8) ◽  
pp. 1478-1491 ◽  
Author(s):  
Gustavo G. Caballero-Flores ◽  
Matthew A. Croxen ◽  
Verónica I. Martínez-Santos ◽  
B. Brett Finlay ◽  
José L. Puente
Keyword(s):  
Escherichia Coli ◽  
Intestinal Epithelium ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Growth Conditions ◽  
Citrobacter Rodentium ◽  
Content Type ◽  
Gut Colonization ◽  
Successful Infection

ABSTRACTThe Gram-negative enteric bacteriumCitrobacter rodentiumis a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagicEscherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. ForC. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novelC. rodentiumcolonization factor, calledgutcolonizationfimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. AgcfAmutant shows a severe colonization defect within the first 10 days of infection. Thegcfpromoter is not active inC. rodentiumunder severalin vitrogrowth conditions; however, it is readily expressed in aC. rodentiumΔhns1mutant lacking the closest ortholog of theEscherichia colihistone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region ofgcf, further supporting its direct role as a repressor of thegcfpromoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. Thegcfoperon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures.IMPORTANCEFimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection byC. rodentiumin mice, despite being poorly expressed underin vitrogrowth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.

scholarly journals Eosinophil Deficiency Compromises Lung Defense against Aspergillus fumigatus

2013 ◽  
Vol 82 (3) ◽  
pp. 1315-1325 ◽  
Author(s):  
Lauren M. Lilly ◽  
Michaella Scopel ◽  
Michael P. Nelson ◽  
Ashley R. Burg ◽  
Chad W. Dunaway ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Cell Contact ◽  
Mrna Levels ◽  
Colony Stimulating Factor ◽  
Content Type ◽  
Eosinophil Peroxidase ◽  
Stimulating Factor ◽  
Deficient Mice

ABSTRACTExposure to the moldAspergillus fumigatusmay result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated withA. fumigatusexposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance ofA. fumigatusfrom the lung. AcuteA. fumigatuschallenge in normal mice induced the recruitment of CD11b+Siglec F+Ly-6GloLy-6CnegCCR3+eosinophils to the lungs, which was accompanied by an increase in lungEpx(eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impairedA. fumigatusclearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression ofEpx(eosinophil peroxidase) andPrg2(major basic protein) as well as lower interleukin 1β (IL-1β), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity againstA. fumigtausin vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response afterA. fumigatusexposure.

scholarly journals Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance inAspergillus fumigatus

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Fabio Gsaller ◽  
Takanori Furukawa ◽  
Paul D. Carr ◽  
Bharat Rash ◽  
Christoph Jöchl ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Regulatory Protein ◽  
Transcriptional Regulators ◽  
Primary Factor ◽  
Ph Sensing ◽  
Content Type ◽  
Ph Dependent ◽  
Mechanistic Basis

ABSTRACTThe antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treatAspergillusinfection. Despite being potent against other fungal pathogens, 5FC has limited activity againstAspergillus fumigatuswhen standardin vitroassays are used to determine susceptibility. However, in modifiedin vitroassays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence thatfcyB, a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression offcyBand, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repressfcyBexpression, such as PacC or other components of the pH-sensing machinery.

scholarly journals Antischistosomal Activities of Mefloquine-Related Arylmethanols

2012 ◽  
Vol 56 (6) ◽  
pp. 3207-3215 ◽  
Author(s):  
Katrin Ingram ◽  
William Ellis ◽  
Jennifer Keiser
Keyword(s):  
Body Weight ◽  
Worm Burden ◽  
Structural Features ◽  
Content Type ◽  
Antischistosomal Activity ◽  
Oral Doses ◽  
Related Compounds ◽  
Insight Into

ABSTRACTInteresting antischistosomal properties have been documented for the antimalarial mefloquine, a 4-quinolinemethanol. We evaluated the antischistosomal activities of nine mefloquine-related compounds belonging to the 4-pyridinemethanols, 9-phenanthrenmethanols, and 4-quinolinemethanols. Eight compounds revealed high activities againstSchistosoma mansoni in vitro, with two drugs (the 4-quinolinemethanols WR7573 and WR7930) characterized by significantly lower half-maximal inhibitory concentrations (IC50s) (2.7 and 3.5 μM, respectively) compared to mefloquine (11.4 μM). Mefloquine and WR7930 showed significantly decreased IC50s when incubated in the presence of hemoglobin. High worm burden reductions (WBR) were obtained with enpiroline (WBR, 82.7%; dosage, 200 mg/kg of body weight) and itsthreoisomers (+)-threo(WBR, 100%) and (−)-threo(WBR, 89%) and with WR7930 (WBR, 87%; dosage, 100 mg/kg) against adultS. mansoniin mice. Furthermore, excellentin vitroandin vivoantischistosomal activity was observed for two WR7930-related structures (WR29252 and WR7524). In addition, mefloquine (WBR, 81%), enpiroline (WBR, 77%), and WR7930 (WBR, 100%) showed high activities againstS. haematobiumharbored in mice following single oral doses of 200 mg/kg. These results provide a deeper insight into the structural features of the arylmethanols that rule antischistosomal activity. Further studies should be launched with enpiroline and WR7930.

scholarly journals Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

2012 ◽  
Vol 78 (9) ◽  
pp. 3166-3176 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Lorna Gallagher ◽  
Markus Schrettl ◽  
Christoph Jöchl ◽  
Kevin Kavanagh ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Peptide Synthetases ◽  
Increased Sensitivity

ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

scholarly journals In VitroInteraction between Alginate Lyase and Amphotericin B against Aspergillus fumigatus Biofilm Determined by Different Methods

2012 ◽  
Vol 57 (3) ◽  
pp. 1275-1282 ◽  
Author(s):  
Francesca Bugli ◽  
Brunella Posteraro ◽  
Massimiliano Papi ◽  
Riccardo Torelli ◽  
Alessandro Maiorana ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Extracellular Polymeric Substances ◽  
Alginate Lyase ◽  
Antifungal Drugs ◽  
Antibiofilm Activity ◽  
Content Type ◽  
Time Kill

ABSTRACTAspergillus fumigatusbiofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treatAspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study,in vitrointeractions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, againstA. fumigatusbiofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that whenA. fumigatusbiofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed inA. fumigatusbiofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

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