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Analysis of Secondary Metabolites from Aspergillus fumigatus and Penicillium nalgiovense: Antimicrobial Compounds from Filamentous Fungi Isolated from Extreme Environments
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the cultivation and extraction of filamentous fungi isolated from extreme environments in the search for new antibiotic compounds. Filamentous fungi are a rich source of medicines including antibiotics, and it is believed that many currently unknown fungal species and bioactive fungal metabolites remain to be discovered.

Aspergillus fumigatus and Penicillium nalgiovense strains were isolated from an antibiotic-contaminated riverbed near Hyderabad, India, and soil taken from a penguin’s nest on Paulete Island, Antarctica, respectively. It was anticipated that the extreme conditions within these environments would exert unusual selective pressures on their filamentous fungi, possibly causing the secretion of new bioactive compounds.

The cultivation, extraction and analysis of metabolites from the A. fumigatus strain resulted in the isolation of the antimicrobial substance gliotoxin. Subsequent investigations revealed that this strain’s secretion of gliotoxin was increased by as much as 65 % when it was cultivated in the presence of pathogen-associated molecular patterns. These results indicate the existence of a fungal receptor/signaling system for detecting nearby bacteria. The scope for using gliotoxin and the related metabolite bis(methyl)gliotoxin as biomarker metabolites for diagnosing the lethal pulmonary condition invasive aspergillosis was also investigated. Bronchoalveolar lavage fluid from 42 patients with and without possible invasive aspergillosis was extracted and analyzed. The results obtained suggest that gliotoxin and bis(methyl)gliotoxin are not suitable markers for diagnosing invasive aspergillosis.

Studies on the P. nalgiovense strain from Antarctica resulted in the isolation of the antifungal agent amphotericin B. The secretion of this compound increased when P. nalgiovense was cultured on a potato-dextrose agar enriched with coconut flakes rather than liquid RPMI 1640 medium. This was the first time amphotericin B was isolated from any organism other than the bacterium Streptomyces nodosus.

The results presented in this thesis will be useful in the continuing search for novel bioactive compounds, the diagnosis of fungal infections, and as a source of insight into the interactions between microorganisms. Moreover, they show that even extensively studied fungal genera such as Aspergillus and Penicillium are not completely understood and may produce unexpected or previously unknown bioactive metabolites under appropriate conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 195
Keyword [en]
Aspergillus fumigatus, Penicillium nalgiovense, secondary metabolites, invasive aspergillosis, elicitation, gliotoxin, bis(methyl)gliotoxin, amphotericin B
National Category
Medicinal Chemistry
Research subject
Pharmacognosy
Identifiers
URN: urn:nbn:se:uu:diva-242611ISBN: 978-91-554-9154-3 (print)OAI: oai:DiVA.org:uu-242611DiVA: diva2:784360
Public defence
2015-03-20, BMC sal B22, Husargatan 4, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-02-25 Created: 2015-01-28 Last updated: 2015-03-11
List of papers
1. Antimicrobial activity of filamentous fungi isolated from highly antibiotic-contaminated river sediment
Open this publication in new window or tab >>Antimicrobial activity of filamentous fungi isolated from highly antibiotic-contaminated river sediment
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2012 (English)In: Infection ecology & epidemiology, ISSN 2000-8686, Vol. 2, 11591- p.Article in journal (Refereed) Published
Abstract [en]

Background:

Filamentous fungi are well known for their production of substances with antimicrobial activities, several of which have formed the basis for the development of new clinically important antimicrobial agents. Recently, environments polluted with extraordinarily high levels of antibiotics have been documented, leading to strong selection pressure on local sentinel bacterial communities. In such microbial ecosystems, where multidrug-resistant bacteria are likely to thrive, it is possible that certain fungal antibiotics have become less efficient, thus encouraging alternative strategies for fungi to compete with bacteria.

Methods:

In this study, sediment of a highly antibiotic-contaminated Indian river was sampled in order to investigate the presence of cultivable filamentous fungi and their ability to produce substances with antimicrobial activity.

Results:

Sixty one strains of filamentous fungi, predominantly various Aspergillus spp. were identified. The majority of the Aspergillus strains displayed antimicrobial activity against methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus faecalis and Candida albicans. Bioassay-guided isolation of the secondary metabolites of A. fumigatus led to the identification of gliotoxin.

Conclusion:

This study demonstrated proof of principle of using bioassay-guided isolation for finding bioactive molecules

Keyword
secondary metabolites, Aspergillus, gliotoxin
National Category
Microbiology
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-188192 (URN)10.3402/iee.v2i0.11591 (DOI)
Funder
Vinnova
Available from: 2012-12-13 Created: 2012-12-13 Last updated: 2015-03-11Bibliographically approved
2. Penicillium nalgiovense Laxa isolated from Antarctica is a new source of the antifungal metabolite amphotericin B
Open this publication in new window or tab >>Penicillium nalgiovense Laxa isolated from Antarctica is a new source of the antifungal metabolite amphotericin B
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2015 (English)In: Fungal biology and biothechnology, Vol. 2, no 1Article in journal (Refereed) Published
Abstract [en]

Background: The need for new antibiotic drugs increases as pathogenic microorganisms continue to develop resistance against current antibiotics. We obtained samples from Antarctica as part of a search for new antimicrobial metabolites derived from filamentous fungi. This terrestrial environment in the South Pole is hostile and extreme due to a sparsely populated food web, low temperatures, and insufficient liquid water availability. We hypothesize that this environment could cause the development of fungal defense or survival mechanisms not found elsewhere.

Results: We isolated a strain of Penicillium nalgiovense Laxa from a soil sample obtained from an abandoned penguin’s nest. Amphotericin B was the only metabolite secreted from P. nalgiovense Laxa with noticeable antimicrobial activity,with minimum inhibitory concentration of 0.125 µg/mL against Candida albicans. This is the first time that amphotericin B has been isolated from an organism other than the bacterium Streptomyces nodosus. In terms of amphotericin B production, cultures on solid medium proved to be a more reliable and favorable choice compared to a liquid.

Conclusions: These results encourage further investigation of the many unexplored sampling sites characterized by extreme conditions, and confirm filamentous fungi as potential sources of metabolites with antimicrobial activity.

Keyword
Amphotericin B, Penicillium nalgiovense Laxa, Antarctica
National Category
Medicinal Chemistry
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-242609 (URN)10.1186/s40694-014-0011-x (DOI)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2015-03-11Bibliographically approved
3. Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules
Open this publication in new window or tab >>Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, e93685- p.Article in journal (Refereed) Published
Abstract [en]

Aspergillus fumigatus is the most common causative agent of mold diseases in humans, giving rise to life-threatening infections in immunocompromised individuals. One of its secreted metabolites is gliotoxin, a toxic antimicrobial agent. The aim of this study was to determine whether the presence of pathogen-associated molecular patterns in broth cultures of A. fumigatus could induce gliotoxin production. Gliotoxin levels were analyzed by ultra-performance liquid chromatography and mass spectrometry. The presence of a bacteria-derived lipopolysaccharide, peptidoglycan, or lipoteichoic acid in the growth media at a concentration of 5 mu g/ml increased the gliotoxin concentration in the media by 37%, 65%, and 35%, respectively. The findings reveal a correlation between the concentrations of pathogen-associated molecular patterns and gliotoxin secretion. This shows that there is a yet uncharacterized detection system for such compounds within fungi. Inducing secondary metabolite production by such means in fungi is potentially relevant for drug discovery research. Our results also give a possible explanation for the increased virulence of A. fumigatus during bacterial co-infection, one that is important for the transition from colonization to invasiveness in this pulmonary disease.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-224740 (URN)10.1371/journal.pone.0093685 (DOI)000334107500056 ()
Available from: 2014-05-21 Created: 2014-05-19 Last updated: 2017-12-05Bibliographically approved
4. Bis(methyl)gliotoxin and gliotoxin in bronchoalveolar lavage fluids are not suitable markers for invasive aspergillosis
Open this publication in new window or tab >>Bis(methyl)gliotoxin and gliotoxin in bronchoalveolar lavage fluids are not suitable markers for invasive aspergillosis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Introduction: Invasive aspergillosis is challenging to diagnose partly due to shortcomings in sensitivity, reliability, and selectivity of current diagnostic methods, which rely on cultures, assays, and histopathology. This problem may be addressed by chemical analysis of metabolites in lung fluid from infected patients. Gliotoxin and bis(methyl)gliotoxin have been pinpointed as potential marker metabolites in serum and plasma for invasive aspergillosis patients, but whether lung fluid samples could be assessed for these markers is still unknown.

Methods: Bronchoalveolar lavage samples were taken from 42 individuals with a variety of pulmonary diseases whereof  20 were diagnosed with possible invasive aspergillosis. The samples were analyzed with ultra high performance liquid chromatography coupled to triple quadropole time-of-flight mass spectrometry to investigate the use of the Aspergillus fumigatus metabolites gliotoxin and bis(methyl)gliotoxin as marker metabolites for invasive aspergillosis.

Results: Gliotoxin was not detected in any of the 42 samples, but  bis(methyl)gliotoxin in 10 (24%). Bis(methyl)gliotoxin was detected in 5 (25%) of the 20 patients with possible IA and in 5 (23%) in the other 22 samples. One unknown compound (357.30 m/z) with a similar mass spectrum profile to bis(methyl)gliotoxin (357.09 m/z) was found in 32 (76%) of all samples.

Conclusions: Neither gliotoxin nor bis(methyl)gliotoxin appears to be an acceptable marker metabolite in bronchoalveolar lavage fluids for invasive aspergillosis. Further development of MS-based analyses should include chromatography. 

Keyword
Invasive aspergillosis, gliotoxin, bis(methyl)gliotoxin, Aspergillus fumigatus, diagnosis
National Category
Medicinal Chemistry
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-242610 (URN)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2015-03-11Bibliographically approved

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