Agro-industrial wastes for production of paclitaxel by irradiated Aspergillus fumigatus under solid-state fermentation.

AIMS: Paclitaxel is the most profitable drug ever developed in cancer chemotherapy; however, the yield of paclitaxel from microbial platforms is still far from the commercial purpose. Thus, this study was conducted to explore the possibility of solid-state fermentation (SSF) for production of paclitaxel by fungal fermentation. METHODS AND RESULTS: Different agro-industrial wastes were screened as solid substrates for production of paclitaxel by the endophytic Aspergillus fumigatus TXD105 under SSF. Sugarcane bagasse followed by wheat bran, and rice bran were the most suitable substrates for maximum production of paclitaxel. In the effort to increase the paclitaxel production, selection of the most proper moistening agent that supports the production of paclitaxel by the fungal strain was investigated. The effect of varying inoculum concentrations on the production of paclitaxel was also studied. Moreover, optimization of SSF conditions (moisture level, substrate concentrations and nutrients concentration) was adopted using response surface methodology. SSF carried out under the optimum conditions of 20 g sugarcane bagasse, twofold nutrients concentration of the MM1D broth, 80% moisture level and inoculum concentration of 107 spores per ml intensified the paclitaxel concentration to 145·61 mg kg-1 which represents a 10-fold increase. The production of paclitaxel by the fungal strain was further improved via exposure to UV and gamma radiation at specific doses. The paclitaxel concentrations were intensified following UV and gamma radiation to 209·91 and 351·82 mg kg-1 . CONCLUSIONS: This is the first report on the production of paclitaxel using agro-industrial wastes as cheap source that may contribute in lowering the cost of producing paclitaxel. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer new and alternate sources with excellent biotechnological potential for paclitaxel production by fungal fermentation.

A high throughput method for rapid screening of chitosanase-producing fungal strain under acidic conditions.

A novel high-throughput method was established for rapid screening of a large numbers of Aspergillus fumigatus (A. fumigatus) mutants with high chitosanase production under acidic culture condition by exploiting the fact that iodine can be used as the indicator to stain chitosan but is ineffective for chitooligosaccharides. The mutant population was generated by irradiating A. fumigatus CICC 2434 with Co(60)-γ rays. Mutants were cultured on acidic plates containing colloidal chitosan and preliminary screened according to diameter of haloes formed around colonies. Then, chitosanase production of the isolates were verified by dinitrosalicylic acid assay. Lastly, molecular masses on enzymolysis products of isolated mutants were rapidly compared by aniline blue plate assay. Using this method, the mutant strain Co-8 was selected, which had chitosanase activity of 24.87 U/mL (increased by 369.2 % as compared to that of its parental strain).Taking together, the method is easy, efficient and particularly suited to rapid screen acidophilic fungal strains with high chitosanase-production.

Hsp90-dependent regulatory circuitry controlling temperature-dependent fungal development and virulence.

The pathogenic fungi Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans are an increasing cause of human mortality, especially in immunocompromised populations. During colonization and adaptation to various host environments, these fungi undergo morphogenetic alterations that allow for survival within the host. One key environmental cue driving morphological changes is external temperature. The Hsp90 chaperone protein provides one mechanism to link temperature with the signalling cascades that regulate morphogenesis, fungal development and virulence. Candida albicans is a model system for understanding the connections between morphogenesis and Hsp90. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy.

Distribution, expression and expansion of Aspergillus fumigatus LINE-like retrotransposon populations in clinical and environmental isolates.

Functional genomic analysis of the mould pathogen Aspergillus fumigatus has identified multiple secondary metabolism genes upregulated in the host niche. Intriguingly, transcriptomic analyses of infectious germlings, germinating spores and mutants lacking the LaeA methyltransferase reveal differential expression of transposable elements (TEs), which often flank secondary metabolite gene clusters. In this study we investigate, in clinical and environmental isolates, the structure and distribution of a specific class of A. fumigatus long interspersed nuclear element (LINE)-like retrotransposons occupying subtelomeric loci in the A. fumigatus genome, and probe their stability in response to laboratory- and host-imposed stresses. In silico analyses revealed that this class belongs to the Tad clade of LINE-like elements. Southern blotting with a LINE-specific probe in clinical and environmental isolates revealed a high variability in the insertion pattern between strains and active transcription of LINE-like element(s) was discernable, in the type strain Af293, by RT-PCR. One out of 14 tested clinical isolates did not contain any LINEs at all, arguing against an absolute requirement for LINE-mediated activities in human infections. Finally, we found preliminary evidence of an association between mycovirus-infection and the expansion of Tad-element populations in discrete A. fumigatus genomes.

Dual localization of Mdj1 in pathogenic fungi varies with growth temperature.

Paracoccidioides brasiliensis and P. lutzii are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis (PCM). Previously, we characterized the PbMDJ1 gene. This gene encodes P. brasiliensis chaperone Mdj1, which in yeast is a mitochondrial member of the J-domain family, whose main function is to regulate cognate Hsp70 activities. We produced rabbit polyclonal antibody antirecombinant PbMdj1 (rPbMdj1), which labeled the protein not only in mitochondria but also at the cell wall of P. brasiliensis yeasts of isolate Pb18. Here we used anti-rPbMdj1 in confocal microscopy to localize Mdj1 in Pb18 and other fungal isolates grown at different temperatures. Dual intracellular and cell surface pattern were initially seen in yeast-phase P. brasiliensis Pb3, Pb18 (control), P. lutzii Pb01, and Histoplasma capsulatum. Pb18 and Aspergillus fumigatus hyphae as well as Pb3 pseudo hyphae formed at 36°C were labeled predominantly along the cell surface. Preferential surface localization was observed by 72 h of yeast-mycelium thermotransition. It was interesting to observe that anti-rPbMdj1 concentrated at the surface tip and branching points of A. fumigatus hyphae grown at 36°C, suggesting a role in growth, whereas at 23°C, anti-rPbMdj1 was distributed along the hyphal surface. In Pb3, Pb18, and Pb01 mitochondrial extracts, the antibodies revealed a specific 55-kDa band, which corresponds to the processed Mdj1 size. The presence of Mdj1 on the fungal cell wall suggests that this protein could also play a role in the interaction with the host.

Neosartorya udagawae (Aspergillus udagawae), an emerging agent of aspergillosis: how different is it from Aspergillus fumigatus?

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55 degrees C but not at 10 degrees C, N. udagawae is able to grow at 10 degrees C but fails to grow at >42 degrees C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37 degrees C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91(phox-/-) mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains.

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress.IMPORTANCE Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.

Effective Control of Molds Using a Combination of Nanoparticles.

Molds are filamentous fungi able to grow on a variety of surfaces, including constructed surfaces, food, rotten organic matter, and humid places. Mold growth is characterized by having an unpleasant odor in enclosed or non-ventilated places and a non-aesthetic appearance. They represent a health concern because of their ability to produce and release mycotoxins, compounds that are toxic to animals and humans. The aim of this study was to evaluate commercial nanoparticles (NPs) that can be used as an additive in coatings and paints to effectively control the growth of harmful molds. Four different NPs were screened for their antifungal activities against the mycotoxin producing mold strains Aspergillus flavus and A. fumigatus. The minimal inhibitory concentrations of the NPs were determined in broth media, whereas an agar diffusion test was used to assess the antimold activity on acrylic- and water-based paints. The cytotoxic activity and the inflammatory response of the NPs were also evaluated using the established human derived macrophage cell line THP-1. Results showed that a combination of mix metallic- and ZnO-NPs (50:10 µg/mL) effectively inhibited the fungal growth when exposed to fluorescent light. Neither cytotoxic effect nor inflammatory responses were recorded, suggesting that this combination can be safely used in humid or non-ventilated environments without any health concerns.

Rylux BSU stimulates spore germination in Trichophyton mentagrophytes and Aspergillus fumigatus and increases the survival rate after UV-irradiation.

Calcofluor-allied optical brightener Rylux BSU stimulated spore germination rate in Trichophyton mentagrophytes and Aspergillus fumigatus both if supplemented into Sabouraud glucose agar and if used for pretreatment of spore suspension prior to inoculation at low concentrations. Maximum stimulation of germination was obtained if 0.2% Rylux BSU was used for pretreatment in aqueous solution for 1 d prior to inoculation (130% in T. mentagrophytes and 150% in A. fumigatus, respectively). Pretreatment with 1% Rylux BSU provided strong protection against UV-irradiation and resulted in increased yields of cultural variants after UV-irradiation.

A comparative study of the effects of UVC irradiation on select procaryotic and eucaryotic wound pathogens.

Managing wounds infected with a mixture of several types of microorganisms such as bacteria (procaryotes) and fungi (eucaryotes) is a challenging clinical situation. The purpose of this study was to determine the effectiveness of ultraviolet light (UVC) in eradicating select procaryotic and eucaryotic organisms, in both pure culture and mixed cultures in vitro. Five replications of each organism or mixture of organisms (10(6) organisms/mL singly or 10(15) organisms/mL mixed culture) were plated. The cultures were treated with a UVC light 1 inch from the surface. Irradiation times were 0, 2, 3, 4, 5, 15, 30, 45, 60, 90, 120, and 180 seconds. Bacterial cultures were incubated and colony counts performed. Upon exposure to UVC, a 99.9% kill rate was obtained at 3 to 5 seconds for the procaryotic organisms (Pseudomonas aeruginosa and Mycobacterium abscessus) tested. However, 15 to 30 seconds of UVC treatment was required to obtain 99.9% kill of the eucaryotic organisms (Candida albicans, Aspergillus fumigatus) tested. This study demonstrates a decreasing sensitivity of evolutionarily more complex organisms to UVC. This study also provides further evidence that short exposure times to UVC are detrimental to procaryotic and simple unicellular eucaryotic organisms while sparing more complex multicellular organisms.

The fungal pathogen Aspergillus fumigatus regulates growth, metabolism, and stress resistance in response to light.

Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.

Light sensing in Aspergillus fumigatus highlights the case for establishing new models for fungal photobiology.

Microbes inhabit diverse environmental locations, and many species need to shift their physiology between different niches. To do this effectively requires the accurate sensing of and response to the environment. For pathogens, exposure to light is one major change between a free-living saprophyte lifestyle and causation of disease within the host. However, how light may act as a signal to influence pathogenesis, on the side of either the host or the pathogen, is poorly understood. Research during the last 2 decades has uncovered aspects about the machinery for light sensing in a small number of fungi. Now, Fuller et al. have initiated studies into the role that light and two photosensor homologs play in the behavior of the ubiquitous fungal pathogen Aspergillus fumigatus [K. K. Fuller, C. S. Ringelberg, J. J. Loros, and J. C. Dunlap, mBio 4(2):e00142-13, 2013, doi:10.1128/mBio.00142-13]. Light represses the germination of A. fumigatus spores and enhances resistance to ultraviolet light, oxidative stresses, and cell wall perturbations. The phenotypes of the strains with mutations in the LreA and FphA homologs revealed that these sensors control some, but not all, responses to light. Furthermore, interactions occur between blue and red light signaling pathways, as has been described for a related saprophytic species, Aspergillus nidulans. Genome-wide transcript analyses found that about 2.6% of genes increase or decrease their transcript levels in response to light. This use of A. fumigatus establishes common elements between model filamentous species and pathogenic species, underscoring the benefits of extending photobiology to new species of fungi.

Quantification of cyclobutane pyrimidine dimers induced by UVB radiation in conidia of the fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii.

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m(-2). CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m(-2)) of UVB radiation.

In vitro efficacy of antifungal treatment using riboflavin/UV-A (365 nm) combination and amphotericin B.

PURPOSE: To demonstrate the antimicrobial properties of riboflavin/UV-A (365 nm) against fungal pathogens. METHODS: The antimicrobial properties of riboflavin/UV-A (365 nm), with or without previous treatment with amphotericin B, were tested on three groups of fungi selected from severe cases of keratomycosis: Candida albicans, Fusarium sp, and Aspergillus fumigatus. They were tested by using Kirby-Bauer discs with empty disc (control), riboflavin 0.1% alone (R), UV-A alone (UV-A), riboflavin 0.1% and additional UV-A exposure (R+UV-A), amphotericin B alone (A), amphotericin B and riboflavin 0.1% (A+R), amphotericin B and UV-A (A+UV-A), amphotericin B and riboflavin 0.1%, and additional UV-A exposure (A+R+UV-A). The mean growth inhibition zone (GIZ) was measured around the discs. RESULTS: C. albicans, Fusarium sp, and A. fumigatus did not show any increased GIZ after treatment without previous amphotericin B medication. However, GIZ was significantly greater after pretreatment with amphotericin B and riboflavin/UV-A (A+R+UV-A) for C. albicans (P = 0.0005), Fusarium sp (P = 0.0023) and A. fumigatus (P = 0.0008) compared with A, A+R, and A+UV-A. CONCLUSIONS: Amphotericin B is believed to interact with fungi membrane sterols to produce aggregates that form transmembrane channels. Given that collagen is one of the principal components of the cornea, it is also probable that amphotericin B may diffuse easily after cross-linking. Previous treatment with amphotericin B allowed riboflavin/UV-A effectiveness against C. albicans, Fusarium sp, and A. fumigatus. This schema might be used in the future for the treatment of keratomycosis.

Disinfection of selected Aspergillus spp. using ultraviolet germicidal irradiation.

AIMS: The efficacy of ultraviolet germicidal irradiation (UVGI) and the UVGI dose necessary to inactivate fungal spores on an agar surface for cultures of Aspergillus flavus and Aspergillus fumigatus were determined. METHODS AND RESULTS: A four-chambered UVGI testing unit with a 9-W, Phillips, low pressure, mercury UVGI lamp in each chamber was used in this study. An aperture was adjusted to provide 50, 100, 150, and 200 micro W/cm2 of uniform flux to the surfaces of the Petri dish, resulting in a total UVGI dose to the surface of the Petri dishes ranging from 12 to 96 mJ/cm2. The UVGI dose necessary to inactivate 90% of the A. flavus and A. fumigatus was 35 and 54 mJ/cm2, respectively. CONCLUSIONS: UVGI can be used to inactivate culturable fungal spores. Aspergillus flavus was more susceptible than A. fumigatus to UVGI. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may not be directly correlated to the effect of UVGI on airborne fungal spores, but they indicate that current technology may not be efficacious as a supplement to ventilation unless it can provide higher doses of UVGI to kill spores traveling through the irradiated zone.

[Inactivation of viruses and bacteria in sewage sludge by gamma radiation (author's transl)]. / Inaktivierung von Viren und Bakterien in Klärschlamm durch Gammastrahlen.

The kinetics of inactivation and the resistance to gamma radiation of microorganisms usually to be found in raw sludge were examined with five viruses, three bacteria and a fungus serving as prototypes in comparative studies. All these infectious agents could reliably be inactivated by gamma rays in raw sewage sludge but they were clearly more resistant to gamma rays compared to irradiation in a liquid suspension. The reduction of the virus content required a much higher radiation dose compared to bacteria and the fungus used, excluding Streptococcus faecalis which was exceptionally resistant. Considering the content of pathogenic viruses and other agents in raw sewage sludge, the required radiation dose necessary to comply with average to strict demands for the hygienisation of sewage sludge is discussed. The radiation dose of 500 to 1,000 krad seems therefore to be sufficient.