scholarly journals Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis

2008 ◽  
Vol 74 (22) ◽  
pp. 7023-7035 ◽  
Author(s):  
Matthias Brock ◽  
Grégory Jouvion ◽  
Sabrina Droin-Bergère ◽  
Olivier Dussurget ◽  
Marie-Anne Nicola ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Light Emission ◽  
Screening Method ◽  
Growth Conditions ◽  
Antifungal Drugs ◽  
In Vivo Studies ◽  
Drug Efficiency

ABSTRACT Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.

In vitro and in vivo characterization of two nonsporulating Aspergillus fumigatus clinical isolates from immunocompetent patients

2019 ◽  
Vol 58 (4) ◽  
pp. 543-551
Author(s):  
Zheng Zhang ◽  
Yuan Jiang ◽  
Jun Chen ◽  
Peiying Chen ◽  
Qingtao Kong ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Causative Role ◽  
Immunocompromised Patients ◽  
Pulmonary Aspergillosis ◽  
Brla Gene

Abstract Aspergillus fumigatus is a pathogenic fungus responsible for invasive aspergillosis (IA). Typically, it can produce abundant conidia to survive and spread. The infection by A. fumigatus usually occurs in immunocompromised patients due to failed clearance of inhaled conidia. However, the incidence of aspergillosis in immunocompetent hosts has been increasing, the pathogenesis of which is still unknown. Our team previously obtained two clinical nonsporulating A. fumigatus isolates from non-immunocompromised patients, which only have the form of hyphae. This present study demonstrated the in vitro and in vivo characteristics of the two nonsporulating A. fumigatus isolates and verified that their conidiation defects are associated to abolished expression of the sporulation-related central regulatory pathway brlA gene. In addition, we confirmed the mutation site of brlA gene (c.657_660delTCCT) contributes to the nonsporulating phenotype in one clinical isolate. Plate assay showed that the two nonsporulating isolates have a similar resistance to antifungal drugs, cell wall disturbing substances, and oxidative stress compared with the wild-type reference Af293. Most important of all, we employed an immunocompetent mouse model to mimic the pathogenesis of pulmonary aspergillosis in non-immunocompromised patients. It revealed that the hyphae of two nonsporulating isolates and Af293 have similar virulence in immunocompetent hosts. Interestingly, the hyphae fragments of Af293 but not conidia are able to induce invasive aspergillosis in immunocompetent mice. In conclusion, our study indicate that the form of hyphae may play a dominant causative role in pulmonary aspergillosis of immunocompetent hosts rather than conidia.

scholarly journals Marine Bioactive Compounds against Aspergillus fumigatus: Challenges and Future Prospects

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 813
Author(s):  
Chukwuemeka Samson Ahamefule ◽  
Blessing C. Ezeuduji ◽  
James C. Ogbonna ◽  
Anene N. Moneke ◽  
Anthony C. Ike ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Bioactive Compounds ◽  
Antifungal Agents ◽  
Antifungal Drugs ◽  
Marine Resources ◽  
Future Prospects ◽  
Speed Up

With the mortality rate of invasive aspergillosis caused by Aspergillus fumigatus reaching almost 100% among some groups of patients, and with the rapidly increasing resistance of A. fumigatus to available antifungal drugs, new antifungal agents have never been more desirable than now. Numerous bioactive compounds were isolated and characterized from marine resources. However, only a few exhibited a potent activity against A. fumigatus when compared to the multitude that did against some other pathogens. Here, we review the marine bioactive compounds that display a bioactivity against A. fumigatus. The challenges hampering the discovery of antifungal agents from this rich habitat are also critically analyzed. Further, we propose strategies that could speed up an efficient discovery and broaden the dimensions of screening in order to obtain promising in vivo antifungal agents with new modes of action.

scholarly journals Efficient Clearance of Aspergillus fumigatus in Murine Lungs by an Ultrashort Antimicrobial Lipopeptide, Palmitoyl-Lys-Ala-dAla-Lys

2008 ◽  
Vol 52 (9) ◽  
pp. 3118-3126 ◽  
Author(s):  
Alexandra Vallon-Eberhard ◽  
Arik Makovitzki ◽  
Anne Beauvais ◽  
Jean-Paul Latgé ◽  
Steffen Jung ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Microscopic Analysis ◽  
Current Standard ◽  
New Family ◽  
Therapeutic Properties ◽  
Survival Assay ◽  
Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.

scholarly journals Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites

Blood ◽  
2009 ◽  
Vol 114 (26) ◽  
pp. 5393-5399 ◽  
Author(s):  
Ronen Ben-Ami ◽  
Russell E. Lewis ◽  
Konstantinos Leventakos ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Capillary Tube ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Dependent Manner ◽  
Antiangiogenic Effect ◽  
Umbilical Vein Endothelial Cells

AbstractIn susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia, and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A fumigatus directly modulates angiogenesis. A fumigatus culture filtrates profoundly inhibited the differentiation, migration, and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo Matrigel assay in cyclophosphamide-treated BALB/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in Matrigel plugs implanted in A fumigatus–infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A fumigatus was completely abolished by deletion of the global regulator of secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis mediator–encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A fumigatus both in vitro and in a neutropenic mouse model is mediated through secondary metabolite production.

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.

scholarly journals Antifungal effect of all-trans retinoic acid against Aspergillus fumigatus in vitro and in a pulmonary aspergillosis in vivo model

2020 ◽  
Author(s):  
Elena Campione ◽  
Roberta Gaziano ◽  
Elena Doldo ◽  
Daniele Marino ◽  
Mattia Falconi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Aspergillus Fumigatus ◽  
Rat Model ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Antifungal Drugs ◽  
Pulmonary Aspergillosis ◽  
Fungistatic Activity

AIM: Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. Fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro. We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. MATERIALS AND METHODS: A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. RESULTS: ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of sub-inhibitory concentration of Amphotericin B (AmB) and Posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to Posaconazole. CONCLUSION: Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal Hsp90 expression and Hsp90-related genes. ATRA reduced mortality in a model of IPA in vivo. Those findings suggest ATRA as suitable fungistatic agent, also to reduce dosage and adverse reaction of classical antifungal drugs, and new therapeutic strategies against IPA and systemic fungal infections.

Preparation, characterizations and evaluation of antifungal activity of eugenol-linalool emulgel against anthrophilic dermatophytic trichophyton rubrum

2021 ◽  
pp. 1-15
Author(s):  
Abdullah Akram ◽  
Muhammad Khalid Khan ◽  
Barkat Ali Khan
Keyword(s):  
Antifungal Activity ◽  
Skin Infection ◽  
Trichophyton Rubrum ◽  
Sem Analysis ◽  
Antifungal Drugs ◽  
In Vivo Studies ◽  
Stability Studies ◽  
Antifungal Activities

Trichophyton rubrum (T. Rubrum) is responsible for chronic cases of dermatophytosis which have high rates of resistance to antifungal drugs worldwide. The aim of this study was to formulate an emulgel of Eugenol-Linalool for the treatment of T. Rubrum infections. The emulgel was prepared by slow emulsification method and characterized for physical examination, pH analysis, swelling index, stability studies, spreading coefficient, SEM analysis, thermal analysis and PXRD studies. In-vitro antifungal activities were performed by growing T. rubrum on specialized media in petri dishes. In-vivo antifungal activity was performed in rabbits by inducing the skin infection by application of fungal strain. Results indicated that the emulgel formulation is highly stable and the physical properties of the emulgel remained quite feasible. No deterioration was observed in the formulation and the pH remained the same as the pH of skin. The viscosity and spreadability of the emulgel remained highly compatible. The results of in vitro and in vivo studies indicated that the Eugenol and Linalool both inhibited the growth of T. rubrum. Eugenol was more effective in inhibition of zone (38±0.01 mm) of T. rubrum as compared to Linalool (32.9±0.03 mm). Similarly it was observed that when the combination of both Linalool and Eugenol was used, the growth of T. rubrum (42±0.01 mm) was significantly (P <  0.05) inhibited. It is hence concluded that the emulgel containing Eugenol and Linalool possess strong in vitro and in vivo antifungal activities against the commercial strains of anthrophilic dermophytic T. Rubrum.

scholarly journals Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung

2020 ◽  
Author(s):  
Hong Liu ◽  
Wenjie Xu ◽  
Vincent M. Bruno ◽  
Quynh T. Phan ◽  
Norma V. Solis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Secondary Metabolites ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Transcriptional Profiling ◽  
Transcriptional Response ◽  
Transcription Factor Genes

AbstractTo gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which governs the ability of the organism to proliferate in the lung. The other was ace1, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the Δace1 mutant is due to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.Author summaryAlthough A. fumigatus causes the majority of cases of invasive aspergillosis, the function of most of the genes in its genome remains unknown. To identify genes encoding transcription factors that may be important for virulence, we used a NanoString nCounter to measure the mRNA levels of A. fumigatus transcription factor genes in the lungs of mice with invasive aspergillosis. The transcriptional profiling data indicate that the organism is exposed to nutrient limitation and stress during growth in the lungs, and that it responds by up-regulating genes that encode mycotoxins and secondary metabolites. In vitro, this response was most closely mimicked by growth in medium that was deficient in nitrogen, iron and/or zinc. Using the transcriptional profiling data, we identified two transcription factors that govern A. fumigatus virulence. These were RlmA, which is governs proliferation in the lung and Ace1, which controls the production of mycotoxins and secondary metabolites.

scholarly journals In vitro Inhibitory Action of the Essential Oils of Origanum Vulgare and Rosmarinus Officinalis against Aspergillus Fumigatus

2021 ◽  
Vol 8 (03) ◽  
pp. e143-e152
Author(s):  
Antonia Carolina Melo Monteiro ◽  
Aminata Doucoure Drame ◽  
Francisca Melo Nascimento ◽  
Ana Luisa Miranda-Vilela ◽  
Alexandre Vasconcelos Lima ◽  
...  
Keyword(s):  
Essential Oils ◽  
Aspergillus Fumigatus ◽  
Inhibitory Action ◽  
Rosmarinus Officinalis ◽  
Disk Diffusion ◽  
In Vivo Studies ◽  
Origanum Vulgare ◽  
Agar Dilution

Abstract Aspergillus fumigatus is the main etiological agent of aspergillosis. Considering azole antifungal drug resistance in A. fumigatus, which compromises treatment, new alternatives are needed. Among them, essential oils (EOs) can be an alternative treatment, having shown positive results in inhibiting phytopathogenic fungi in vitro. We aimed to determine the in vitro antifungal activity of Origanum vulgare L. subsp. hirtum (Link) (oregano) and Rosmarinus officinalis L. (rosemary) EOs alone and in association (O. vulgare+R. officinalis) against A. fumigatus. EOs were analyzed by gas chromatography (GC-FID and GC/MS systems), and analyses showed that the major components of O. vulgare EO were carvacrol (67.8%), p-cymene (14.8%), and thymol (3.9%); for R. officinalis, they were the monoterpenes 1,8-cineole (49.1%), camphor (18.1%) and α-pinene (8.1). For biological assays, five EO concentrations, 0.2; 0.4; 0.6; 0.8 and 1.0%, were used in disk diffusion and agar dilution tests for 21 days. In disk diffusion, O. vulgare EO alone and in association (O. vulgare+R. officinalis) showed fungicidal activity at all concentrations. In agar dilution, inhibitory action was demonstrated from 0.6% for O. vulgare EO and in association (O. vulgare+R. officinalis). R. officinalis EO at 1.0% showed no fungal growth, determining the minimum inhibitory concentration (MIC). The present study demonstrated inhibitory actions of O. vulgare and R. officinalis EOs in A. fumigatus. GC analyses corroborated the literature regarding their antibacterial and antifungal effects. However, further in vitro and in vivo studies are needed to evaluate EOs as alternative antifungals for treating aspergillosis.

scholarly journals In Vitro Activities at pH 5.0 and pH 7.0 and In Vivo Efficacy of Flucytosine against Aspergillus fumigatus

2008 ◽  
Vol 52 (12) ◽  
pp. 4483-4485 ◽  
Author(s):  
Paul E. Verweij ◽  
Debbie T. A. Te Dorsthorst ◽  
Willem H. P. Janssen ◽  
Jacques F. G. M. Meis ◽  
Johan W. Mouton
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Murine Model ◽  
Antifungal Agent ◽  
In Vivo Efficacy

ABSTRACT The antifungal agent flucytosine was found to be active in vitro against Aspergillus fumigatus isolates when the MIC was determined at pH 5.0 instead of pH 7.0. The in vitro MIC at pH 5.0 corresponded to the in vivo efficacy of flucytosine monotherapy in a murine model of invasive aspergillosis.

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