Identification of ferrichrome- and ferrioxamine B-mediated iron uptake by Aspergillus fumigatus

2016 ◽  
Vol 473 (9) ◽  
pp. 1203-1213 ◽  
Author(s):  
Yong-Sung Park ◽  
Ju-Yeon Kim ◽  
Cheol-Won Yun
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Virulence Factors ◽  
Membrane Transporters ◽  
Yeast Cells ◽  
Double Deletion ◽  
Ferrioxamine B ◽  
Uptake Activity ◽  
Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen for immunocompromised patients, and genes involved in siderophore metabolism have been identified as virulence factors. Recently, we identified the membrane transporters sit1 and sit2, which are putative virulence factors of A. fumigatus; sit1 and sit2 are homologous to yeast Sit1, and sit1 and sit2 gene expression was up-regulated after iron depletion. When expressed heterologously in Saccharomyces cerevisiae, sit1 and sit2 were localized to the plasma membrane; sit1 efficiently complemented ferrichrome (FC) and ferrioxamine B (FOB) uptake in yeast cells, whereas sit2 complemented only FC uptake. Deletion of sit1 resulted in a decrease in FOB and FC uptake, and deletion of sit2 resulted in a decrease in FC uptake in A. fumigatus. It is of interest that a sit1 and sit2 double-deletion mutant resulted in a synergistic decrease in FC uptake activity. Both sit1 and sit2 were localized to the plasma membrane in A. fumigatus. The expression levels of the sit1 and sit2 genes were dependent on hapX under low-but not high-iron conditions. Furthermore, mirB, and sidA gene expression was up-regulated and sreA expression down-regulated when sit1 and sit2 were deleted. Although sit1 and sit2 failed to affect mouse survival rate, these genes affected conidial killing activity. Taken together, our results suggest that sit1 and sit2 are siderophore transporters and putative virulence factors localized to the plasma membrane.

A copper transcription factor, AfMac1, regulates both iron and copper homeostasis in the opportunistic fungal pathogen Aspergillus fumigatus

2018 ◽  
Vol 475 (17) ◽  
pp. 2831-2845 ◽  
Author(s):  
Yong-Sung Park ◽  
Suzie Kang ◽  
Hyewon Seo ◽  
Cheol-Won Yun
Keyword(s):  
Gene Expression ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogen ◽  
Promoter Region ◽  
Regulation Of Gene Expression ◽  
Copper Homeostasis ◽  
Living Cells ◽  
The Other ◽  
Binding Motif ◽  
Opportunistic Fungal Pathogen

Although iron and copper are co-ordinately regulated in living cells, the homeostatic effects of each of these metals on the other remain unknown. Here, we show the function of AfMac1, a transcriptional activator of the copper and iron regulons of Aspergillus fumigatus, on the interaction between iron and copper. In addition to the copper-specific AfMac1-binding motif 5′-TGTGCTCA-3′ found in the promoter region of ctrC, the iron-specific AfMac1-binding motif 5′-AT(C/G)NN(A/T)T(A/C)-3′ was identified in the iron regulon but not in the copper regulon by ChIP sequence analysis. Furthermore, mutation of the AfMac1-binding motif of sit1 eliminated AfMac1-mediated sit1 up-regulation. Interestingly, the regulation of gene expression in the iron regulon by AfMac1 was not affected by copper and vice versa. AfMac1 localized to the nucleus under iron- or copper-depleted conditions, and AfMac1 was mostly detected in the cytoplasm under iron- or copper-replete conditions. Taken together, these results suggest that A. fumigatus independently regulates iron and copper homeostasis in a manner that involves AfMac1 and mutual interactions.

Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice

2002 ◽  
Vol 361 (3) ◽  
pp. 673-679 ◽  
Author(s):  
Oliver MÜLLER ◽  
Carmen SCHALLA ◽  
Jürgen SCHEIBNER ◽  
Eduard F. STANGE ◽  
Michael FUCHS
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Protein Expression ◽  
Bile Salt ◽  
Membrane Transporters ◽  
Biliary Lipid ◽  
Liver Plasma Membrane ◽  
Lithogenic Diet ◽  
Secretion Rates ◽  
Salt Secretion

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt—phospholipid uncoupling due to the increased bile flow.

scholarly journals Effect of Carum carvi essential oil on ERG6 gene expression and virulence factors in Candida albicans

2020 ◽  
Author(s):  
Samira Nasiri ◽  
Masoomeh Shams-Ghahfarokhi ◽  
Mehdi Razzaghi-Abyaneh
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Essential Oil ◽  
Virulence Factors ◽  
Mrna Level ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Yeast Cells ◽  
Minimum Fungicidal Concentration ◽  
Carum Carvi

Background and Purpose: The present study was conducted to investigate the inhibitory effects of Carum carvi essential oil (EO) against ERG6 gene expression in relation to fungal growth and some important virulence factors in Candida albicans. Materials and Methods: The minimum inhibitory concentration (MIC) of C. carvi EO against C. albicans was determined by the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 20-1280 μg/ml. Furthermore, the expression of ERG6 gene was studied at the 0.5× MIC concentration of C. carvi EO using real-time polymerase chain reaction. The proteinase and phospholipase activities, cell surface hydrophobicity (CSH), and cell membrane ergosterol (CME) content of C. albicans were also assessed at the 0.5× MIC concentration of the plant EO using the approved methods. In addition, fluconazole (FLC) was used as a control antifungal drug. Results: The results indicated that the MIC and minimum fungicidal concentration of C. carvi EO for C. albicans growth were 320 and 640 μg/ml, respectively. The expression of fungal ERG6 at an mRNA level and ergosterol content of yeast cells were significantly decreased by both C. carvi EO (640 μg/ml) and FLC (2 μg/ml). The proteinase and phospholipase activities were also reduced in C. carvi EO by 49.82% and 53.26%, respectively, while they were inhibited in FLC-treated cultures by 27.72% and 34.67%, respectively. Furthermore, the CSH was inhibited in EO- and FLC-treated cultures by 12.75% and 20.80%, respectively. Conclusion: Our findings revealed that C. carvi EO can be considered a potential natural compound in the development of an efficient antifungal agent against C. albicans.

scholarly journals Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

2012 ◽  
Vol 11 (11) ◽  
pp. 1333-1344 ◽  
Author(s):  
Isabelle Raymond-Bouchard ◽  
Cassandra S. Carroll ◽  
Jason R. Nesbitt ◽  
Kevin A. Henry ◽  
Linda J. Pinto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Mutant Proteins ◽  
Opportunistic Fungal Pathogen ◽  
Triacetylfusarinine C ◽  
Siderophore Transporter

ABSTRACTSiderophores have been identified as virulence factors in the opportunistic fungal pathogenAspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophoreN,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids ofA. fumigatusMirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in theSaccharomyces cerevisiaestrain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using55Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization inA. fumigatusshowed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

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