scholarly journals Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

2004 ◽  
Vol 72 (3) ◽  
pp. 1402-1408 ◽  
Author(s):  
A. H. T. Hissen ◽  
J. M. T. Chow ◽  
L. J. Pinto ◽  
M. M. Moore
Keyword(s):  
Aspergillus Fumigatus ◽  
Human Serum ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Invasive Disease ◽  
Proteolytic Degradation ◽  
Logarithmic Phase ◽  
Late Logarithmic Phase ◽  
Triacetylfusarinine C

ABSTRACT Aspergillus fumigatus is a filamentous fungus which can cause invasive disease in immunocompromised individuals. A. fumigatus can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which A. fumigatus obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM), A. fumigatus growth was supported by the addition of holotransferrin (holoTf) or FeCl3 but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by A. fumigatus occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by A. fumigatus incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by A. fumigatus. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by A. fumigatus after 8 h of growth in MEM-serum and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by A. fumigatus even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from A. fumigatus culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that A. fumigatus survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.

scholarly journals Influence of Human Sera on the In Vitro Activity of the Echinocandin Caspofungin (MK-0991) against Aspergillus fumigatus

2000 ◽  
Vol 44 (12) ◽  
pp. 3302-3305 ◽  
Author(s):  
Tom Chiller ◽  
Kouros Farrokhshad ◽  
Elmer Brummer ◽  
David A. Stevens
Keyword(s):  
Aspergillus Fumigatus ◽  
Human Serum ◽  
Hyphal Growth ◽  
Significant Inhibition ◽  
Dependent Inhibition ◽  
Human Sera ◽  
Dose Dependent Inhibition ◽  
Synthase Inhibitor ◽  
Dose Dependent

ABSTRACT There have been several reports that the activity of echinocandin antifungal agents is not affected or decreased in the presence of human sera. It is known that these drugs are bound >80% in animal and human sera. The activity of the echinocandin caspofungin (MK-0991), a 1,3-β-d-glucan synthase inhibitor, againstAspergillus fumigatus with and without human sera was studied. Conidia of A. fumigatus in microtest plate wells formed germlings after overnight culture in RPMI 1640. Caspofungin was then added with or without serum, and the germlings were incubated at 37°C for 24 h. Human serum (5%) in RPMI 1640 alone did not significantly inhibit the growth of A. fumigatus in vitro. Caspofungin in RPMI 1640 exhibited dose-dependent inhibition, with concentrations of 0.1 and 0.05 μg/ml inhibiting 24.9% +/− 10.4% and 11.7% +/− 3.6%, respectively (n = 10;P < 0.01). The addition of 5% human serum to caspofungin at 0.1 or 0.05 μg/ml increased the inhibition to 78.6% +/− 5.8% or 58.3% +/− 19.2%, respectively (n = 10; P < 0.01 versus controls and versus the drug without serum). Lower concentrations of serum also potentiated drug activity. The effect of human sera was further seen when using caspofungin that had lost activity (e.g., by storage) against A. fumigatus at 0.1 μg/ml. Inactive caspofungin alone demonstrated no significant inhibition of hyphal growth, whereas the addition of 5% human serum to the inactive drug showed 83% +/− 16.5% inhibition (n = 5; P < 0.01). The restoration of activity of caspofungin was seen at concentrations as low as 0.05% human serum. In contrast to prior reports, this study suggests that human serum acts synergistically with caspofungin to enhance its inhibitory activity in vitro against A. fumigatus.

scholarly journals Immunological Characterisation of A∝ Chain Fragments of Human Fibrinogen

1977 ◽  
Author(s):  
J. A. Conkie ◽  
J. F. Davidson
Keyword(s):  
Human Serum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Normal Human Serum ◽  
Small Peptides ◽  
Early Cleavage ◽  
Human Fibrinogen ◽  
Normal Human ◽  
A Chain

In an investigation of the early cleavage fragments resulting from fibrin(ogen)olysis, we have examined the nature of small peptides arising from the C-terminal part of the Aα chain of human fibrinogen.Antiserum to the carboxymethylated Aα chain of human fibrinogen has been prepared, and used to study the Aα-related antigens from (i) a plasmin digest of human fibrinogen, (ii) human serum, obtained from urokinase-treated normal plasma, and (iii) normal human serum. Using immunodiffusion and immunoelectrophoresis with anti-Aα antiserum, along with Polyacrylamide gel electrophoresis, at least two non-identical Aα -related antigens have been detected in a final plasmin digest of human fibrinogen. The largest of these antigens (MW 26,000) has been isolated and named Aα.-RA (26,000). This may be similar to the previously-described fragments, Hi2-Ala, fragment A or fragment H. Aα -related antigen has been produced by in vitro fibrinogenolysis in plasma and has been detected in the serum. In addition, small quantities of Aα -related antigen have been found in untreated normal human serum.These results suggest that anti-Aα antiserum is likely to be a useful reagent for the study of the early plasmin-derived fragments of fibrin(ogen)olysis in vitro and in vivo.

scholarly journals Siderophore Scaffold as Carrier for Antifungal Peptides in Therapy of Aspergillus fumigatus Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 367
Author(s):  
Joachim Pfister ◽  
Roland Bata ◽  
Isabella Hubmann ◽  
Anton Amadeus Hörmann ◽  
Fabio Gsaller ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Targeted Delivery ◽  
Fungal Pathogens ◽  
Growth Promotion ◽  
Modern Medicine ◽  
Triacetylfusarinine C ◽  
Gallium 68 ◽  
Conjugated Compounds

Antifungal resistance of human fungal pathogens represents an increasing challenge in modern medicine. Short antimicrobial peptides (AMP) display a promising class of antifungals with a different mode of action, but lack target specificity and metabolic stability. In this study the hexapeptide PAF26 (Ac-dArg-dLys-dLys-dTrp-dPhe-dTrp-NH2) and the three amino acid long peptide NLF (H2N-Asn-Leu-dPhe-COOH) were coupled to diacetylfusarinine C (DAFC), a derivative of the siderophore triacetylfusarinine C (TAFC) of Aspergillus fumigatus, to achieve targeted delivery for treatment of invasive aspergillosis. Conjugated compounds in various modifications were labelled with radioactive gallium-68 to perform in vitro and in vivo characterizations. LogD, serum stability, uptake- growth promotion- and minimal inhibitory concentration assays were performed, as well as in vivo stability tests and biodistribution in BALB/c mice. Uptake and growth assays revealed specific internalization of the siderophore conjugates by A. fumigatus. They showed a high stability in human serum and also in the blood of BALB/c mice but metabolites in urine, probably due to degradation in the kidneys. Only PAF26 showed growth inhibition at 8 µg/ml which was lost after conjugation to DAFC. Despite their lacking antifungal activity conjugates based on a siderophore scaffold have a potential to provide the basis for a new class of antifungals, which allow the combination of imaging by using PET/CT with targeted treatment, thereby opening a theranostic approach for personalized therapy.

scholarly journals Construction and Characterization ofMoraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

1999 ◽  
Vol 67 (11) ◽  
pp. 5815-5819 ◽  
Author(s):  
Nicole R. Luke ◽  
Anthony A. Campagnari
Keyword(s):  
Solid Phase ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Pcr Analysis ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Isogenic Mutants

ABSTRACT We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, inMoraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpABmutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, theompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, theompB1 (tbpB) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the M. catarrhalisTbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis.

scholarly journals Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

2015 ◽  
Vol 59 (10) ◽  
pp. 6514-6520 ◽  
Author(s):  
Hasan Nazik ◽  
John C. Penner ◽  
Jose A. Ferreira ◽  
Janus A. J. Haagensen ◽  
Kevin Cohen ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Iron Acquisition ◽  
Inhibitory Effect ◽  
Metabolic Effects ◽  
Content Type ◽  
Reference Isolate ◽  
Broth Macrodilution

ABSTRACTIron acquisition is crucial for the growth ofAspergillus fumigatus.A. fumigatusbiofilm formation occursin vitroandin vivoand is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50against planktonicA. fumigatuswas 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming inA. fumigatusor preformed biofilms (P< 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P< 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3was lower than that in the controls (P< 0.05 to 0.001). FeCl3at ≥625 μM reversed the DFP inhibitory effect (P< 0.05 to 0.01), but the reversal was incomplete compared to the controls (P< 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P< 0.01 to 0.001). FeCl3at ≥625 μM overcame inhibition by 625 μM DFP (P< 0.001). FeCl3alone at ≥156 μM stimulated biofilm formation (P< 0.05 to 0.001). PreformedA. fumigatusbiofilm increased with 2,500 μM FeCl3only (P< 0.05). In a strain survey, various susceptibilities of biofilms ofA. fumigatusclinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy forA. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.

scholarly journals Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Joachim Pfister ◽  
Alexander Lichius ◽  
Dominik Summer ◽  
Hubertus Haas ◽  
Thines Kanagasundaram ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Iron Acquisition ◽  
Live Cell ◽  
Subcellular Localisation ◽  
Gallium 68

Abstract Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

scholarly journals Divergent Targets of Aspergillus fumigatus AcuK and AcuM Transcription Factors during GrowthIn Vitroversus Invasive Disease

2014 ◽  
Vol 83 (3) ◽  
pp. 923-933 ◽  
Author(s):  
Monsicha Pongpom ◽  
Hong Liu ◽  
Wenjie Xu ◽  
Brendan D. Snarr ◽  
Donald C. Sheppard ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Aspergillus Fumigatus ◽  
Target Genes ◽  
Iron Acquisition ◽  
Altered Expression ◽  
Content Type ◽  
Regulatory Pathways ◽  
Transcriptional Profiles

InAspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. InAspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisitionin vitroand virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Throughin vitrostudies, we found thatA. fumigatusΔacuKsingle and ΔacuKΔacuMdouble mutants had impaired gluconeogenesis and iron acquisition, similar to the ΔacuMmutant. Also, the ΔacuK, ΔacuM, and ΔacuKΔacuMmutants had similar virulence defects in mice. However, the ΔacuKmutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damagein vitrothan did the ΔacuMmutant. Moreover, overexpression ofacuMin the ΔacuKmutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ΔacuKand ΔacuMmutants had very similar transcriptional profilesin vitro, their transcriptional profiles during murine pulmonary infection differed both from theirin vitroprofiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genesin vivo, they influenced the expression of other virulence-related genes, such ashexAanddvrA. Therefore, inA. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genesin vivothanin vitro, suggesting thatin vivoinfection stimulates unique transcriptional regulatory pathways inA. fumigatus.

scholarly journals Pharmacodynamics of Trovafloxacin, Ofloxacin, and Ciprofloxacin against Streptococcus pneumoniae in an In Vitro Pharmacokinetic Model

1999 ◽  
Vol 43 (5) ◽  
pp. 1118-1123 ◽  
Author(s):  
Philip D. Lister ◽  
Christine C. Sanders
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Human Serum ◽  
Clinical Data ◽  
Pharmacokinetic Model ◽  
The Other ◽  
Logarithmic Phase ◽  
Bacterial Counts ◽  
Oral Doses

ABSTRACT An in vitro pharmacokinetic model was used to simulate the pharmacokinetics of trovafloxacin, ofloxacin, and ciprofloxacin in human serum and to compare their pharmacodynamics against eightStreptococcus pneumoniae strains. The MICs of ofloxacin and ciprofloxacin ranged from 1 to 2 μg/ml. Trovafloxacin was 8- to 32-fold more potent, with MICs of 0.06 to 0.12 μg/ml. Logarithmic-phase cultures were exposed to peak concentrations of trovafloxacin, ofloxacin, or ciprofloxacin achieved in human serum after 200-, 400-, and 750-mg oral doses, respectively. Trovafloxacin was dosed at 0 and 24 h, and ofloxacin and ciprofloxacin were dosed at 0, 12, and 24 h. Human elimination pharmacokinetics were simulated, and viable bacterial counts were measured at 0, 2, 4, 6, 8, 12, 24, and 36 h. Trovafloxacin was rapidly and significantly bactericidal against all eight strains evaluated, with viable bacterial counts decreasing at least 5 logs to undetectable levels. Times to 99.9% killing were only 1 to 3 h. Although the rate of killing with ofloxacin was substantially slower than that with trovafloxacin, ofloxacin was also able to eradicate all eight strains from the model, despite a simulated area under the inhibitory curve/MIC ratio (AUC/MIC) of only 49. In contrast, ciprofloxacin eradicated only five strains (AUC/MIC = 44) from the model. Against the other three strains (AUC/MIC = 22), the antibacterial activity of ciprofloxacin was substantially diminished. These data corroborate clinical data and suggest that trovafloxacin has a pharmacodynamic advantage over ciprofloxacin and ofloxacin against S. pneumoniae in relation to its enhanced antipneumococcal activity.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

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