Anita Kecskeméti

Candida parapsilosis produces prostaglandins from exogenous arachidonic acid and OLE2 is not required for their synthesis

Prostaglandins are C20 fatty acid metabolites with diverse biological functions. In mammalian cells, prostaglandins are produced from arachidonic acid (AA) via cyclooxygenases (COX1 and COX2). Although fungi do not possess cyclooxygenase homologues, several pathogenic species are able to produce prostaglandins from host-derived arachidonic acid. In this study, we characterized the prostaglandin profile of the emerging human pathogen Candida parapsilosis with HPLC-MS and compared it to that of C. albicans. We found that both species synthesized prostaglandins (mainly PGD2 and PGE2) from exogenous AA. Furthermore, as OLE2 has been associated with prostaglandin synthesis in C. albicans, we generated homozygous OLE2 deletion mutants in C. parapsilosis and examined their PGE2 production. However, the PGE2 production of the OLE2 KO strain was similar to that of wild type (WT), indicating that OLE2 is not required for prostaglandin synthesis in C. parapsilosis. Interestingly, analyses of the fatty acid composition of WT and OLE2 KO cells by gas chromatography (GC) highlighted the accumulation of palmitoleic and oleic acid in the OLE2 deletion mutant. The OLE2 KO cells were killed more efficiently by human monocytes-derived macrophages (MDMs) as well as induced higher interleukin-10 (IL-10) secretion, indicating that OLE2 affects the virulence of C. parapsilosis. Taken together, these results contribute to the better understanding of fatty acid biosynthesis pathways in C. parapsilosis.

Species diversity and cytotoxic potency of airborne sterigmatocystin-producing Aspergilli from the section Versicolores

This study presents the distribution and species diversity of sterigmatocystin-producing Aspergilli from the section Versicolores in the indoor air of apartment-AP, basements-BS and grain mill-GM in Croatia, as well as the cytotoxic potency of isolates. The species comprised 0.7-20% of total airborne fungi detected in the AP, 11-55% in the BS, and 0-2% in the GM. Based on CaM sequences, seven species were identified; dominant were Aspergillus jensenii and Aspergillus creber, followed by Aspergillus protuberus, Aspergillus venenatus, Aspergillus tennesseensis, Aspergillus amoenus, Aspergillus griseoaurantiacus and three undescribed species. All of the identified species produced sterigmatocystin-STC (HPLC/UV-VIS); A. griseoaurantiacus (208.29μg/mL) and A. jensenii (1.192-133.63μg/mL) produced the highest levels, the lowest were detected in A. protuberus and A. tennesseensis (0.117-2.749μg/mL). Lower species diversity was obtained in the GM due to overgrowth with more propulsive fungi. Relatively high STC levels (0.06-2.35μg/g) detected in 52% of GM dust samples confirmed the presence of STC-producers, although this STC cannot be exclusively attributed to Aspergilli (Versicolores). STC and the majority of STC-producing Aspergilli were cytotoxic to human lung A549 cells (IC50 0.9-2.3μg/mL) and THP-1 macrophage-like cells (IC50 0.3-0.6μg/mL) in relatively low concentrations suggesting that humans can be at high risk during chronic exposure.

Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe.

Ion trap mass spectrometry of surfactins produced by Bacillus subtilis SZMC 6179J reveals novel fragmentation features of cyclic lipopeptides.

RATIONALE Surfactins are mixtures of cyclic lipopeptides consisting of variants of a heptapeptide and a linked β-hydroxy fatty acid with various chain lengths of 13-15 carbon atoms. A lactone bridge between the β-hydroxy functional group of the fatty acid and the carboxy terminal functional component of the peptide chain form their cyclic structures. Such lipopeptides, produced mainly by Bacillus species, possess several remarkable biological effects such as antitumor and antimicrobial activities, some of which are highly promising for utilization in plant disease biocontrol. The strain Bacillus subtilis SZMC 6179J was previously shown to exert significant antifungal properties against various phytopathogenic filamentous fungi; therefore, we characterized the structural features of the surfactins produced by this strain in order to explore the origin of the observed antagonistic effects of this potential biocontrol organism. METHODS Bacillus subtilis SZMC 6179J was used to produce surfactins, which were characterized by high-performance liquid chromatography/electrospray ionisation ion trap mass spectrometry (HPLC/ESI-ITMS) techniques after precipitation and extraction steps. RESULTS The 26 isoforms separated and identified represent three types of known surfactin variants and a fourth, previously unknown group characterised by the replacement of the leucine residue by valine in position 2. The relative amounts of this newly identified surfactin group were below 1%, and their cyclic structures were closed by C13-C15 hydroxy fatty acids. The structural assessment of the isoforms by MS(2) measurements led to the characterisation and description of a new fragmentation mechanism of surfactins. CONCLUSIONS The detected new natural lipoheptapeptide compounds with modified structures have significant potential for biotechnological and biocontrol applications. The complementary ITMS(2) data as well as the described internal fragmentation mechanism obtained from the sodiated surfactin molecules may further facilitate the structural elucidation of cyclic lipopeptides in the future. Copyright

New sterigmatocystin-producing species of Aspergillus section Versicolores from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.

High-Frequency Occurrence of Surfactin Monomethyl Isoforms in the Ferment Broth of a Bacillus subtilis Strain Revealed by Ion Trap Mass Spectrometry

Surfactins are cyclic lipopeptides consisting of a β-hydroxy fatty acid of various chain length and a peptide ring of seven amino acids linked together by a lactone bridge, forming the cyclic structure of the peptide chain. These compounds are produced mainly by Bacillus species and possess numerous biological effects such as antimicrobial (antiviral, antibacterial, and antifungal) activities. A mixture of surfactins extracted from Bacillus subtilis strain SZMC 6179J was examined by HPLC-ESI-IT-MS technique, enhancing their separation to reveal novel lipopeptide varieties with higher masses and to characterize their structures. During the MS2 spectra analyses of their sodiated molecular ions [M + Na]+, a previously rarely encountered group of surfactins was detected and two novel types of the group were discovered containing methyl esterified aspartic acid residue in their fifth amino acid position. The relative amounts of these monomethyl isoforms exceeded 35% of the produced surfactin in total. In the m/z value of 1114, all the detected isoforms possessed aspartic acid 4-methyl ester residue in their fifth amino acid position (C17-[Lxx4, AME5], C18-[AME5]), offering an opportunity to separate a pure fraction of the compound and to study its biological activities in the future.

Fumonisin production and toxic capacity in airborne black Aspergilli

Abstract This study presents the distribution of fumonisin (FB)-producing and non-producing airborne Aspergilli (Nigri) in apartments (AP), basements (BS) and a grain mill (GM) in Croatia, and their cytotoxic, immunomodulation and genotoxic potency in comparison with FB1 and FB2. Concentration of black Aspergilli was 260-fold higher in GM than in living environment with domination of A. tubingensis and A. welwitschiae. FB2- but not FB1- was confirmed via HPLC-MS and detection of fum1 and fum8 genes for one isolate of A. niger (0.015 μg/mL) and 8/15 isolates of A. welwitschiae (0.128–13.467 μg/mL). After 24 h, both FB1 and FB2 were weakly cytotoxic (MTT assay) to human lung A549 cells and THP-1 macrophage-like cells. In THP-1 cells FB1 but not FB2 provoked a higher increase of IL-8, TNF-α and IL-1β (ELISA). In A549 cells DNA damaging effect of FB1 was slightly higher than that of FB2 (Comet assay). In THP-1 macrophage-like cells A. tubingensis and A. piperis evoked immunomodulatory effects which corresponded to their cytotoxicity (IC50 = 0.228–0.323 mg/mL); A. welwitschiae, A. tubingensis and A. piperis exerted cytotoxic (IC50 = 0.214–0.460 mg/mL) and genotoxic effects in A549 cells. Presumably, secondary metabolites other than FB2 may have contributed to the toxicity of black Aspergilli.

An Organic Solvent-Tolerant Lipase with Both Hydrolytic and Synthetic Activities from the Oleaginous Fungus Mortierella echinosphaera

Lipase enzymes of the oleaginous fungal group Mortierella are rarely studied. However, considering that most commercial lipases are derived from filamentous fungal sources, their investigation can contribute to the cost-effective development of new biotechnological processes. Here, an extracellular lipase with a molecular mass of 30 kDa was isolated from Mortierella echinosphaera CBS 575.75 and characterized. The purified lipase exhibited an optimal p-nitrophenyl palmitate (pNPP)-hydrolyzing activity at 25 °C and pH 6.6–7.0 and proved to be highly stable at temperatures up to 40 °C and under broad pH conditions. The enzyme was active under low temperatures, retaining 32.5% of its activity at 10 °C, and was significantly stable in polar and non-polar organic solvents. The Km, Vmax, and kcat for pNPP were 0.336 mM, 30.4 μM/min, and 45.7 1/min for pNPP and 0.333 mM, 36.9 μM/min, and 55.6 1/min for pNP-decanoate, respectively. The pNPP hydrolysis was inhibited by Hg2+, N-bromosuccinimide, and sodium dodecyl sulfate, while ethylenediaminetetraacetic acid and metal ions, such as Ca2+, Mg2+, Na+, and K+ enhanced the activity. The purified lipase had non-regioselective activity and wide substrate specificity, showing a clear preference for medium-chained p-nitrophenyl esters. Besides its good transesterification activity, the enzyme appeared as a suitable biocatalyst to operate selective esterification reactions to long-chained alkyl esters. Adsorption to Accurel MP1000 improved the storage stability of the enzyme at 5 °C. The immobilized lipase displayed tolerance to a non-aqueous environment and was reusable for up to five cycles without significant loss in its synthetic and hydrolytic activities. These findings confirm the applicability of both the free and the immobilized enzyme preparations in future research.

Combined genotyping strategy reveals structural differences between Aspergillus flavus lineages from different habitats impacting human health

Aspergillus flavus is a filamentous fungus which is widespread on agricultural products and also able to cause various human diseases. This species is frequently isolated from indoor air as well, furthermore, it is known as a common causal agent of keratomycosis, particularly in subtropical and tropical areas. It is also able to produce aflatoxins, one of the most carcinogenic mycotoxins which are harmful to animals and humans. In this study, 59 A. flavus isolates from four different habitats and 1 A. minisclerotigenes isolate were investigated. The isolates were identified and confirmed at the species level by the sequence analysis of a part of their calmodulin gene. Applying a combined analysis of UP‐PCR, microsatellite, and calmodulin sequence data, the four group of isolates formed separate clusters on the phylogenetic tree. Examining the distribution of mating type genes MAT1‐1 and MAT1‐2, a ratio of approximately 3:1 was determined, and no correlation was found between the carried mating type gene and the aflatoxin production capability. HPLC analysis revealed that none of the examined isolates collected from indoor air or maize in Central Europe were able to produce aflatoxins, while about half of the isolates from India produced these mycotoxins under the test conditions.

Dietary supplements on the domestic market adulterated with sildenafil and tadalafil

Mandatory requirements regulating the manufacture and sale of dietary supplements are much less stringent than those related to pharmaceuticals. Hence, the sheer number and diversity of marketed products in this category has shown an unprecedented increase Europe-wide. Not surprising, that cases for incorrect marketing/promotion, incorrect recommendations for product use, as well as reported incidents of questionable product quality and/or deliberate adulterations have also become frequent in recent years. Typical adulterations consist of admixtures of synthetic pharmaceuticals to the matrix fraudulently declared to consist exclusively of extracts of various (medicinal) plants. In the present paper, the results of qualitative investigations of ten plant-based preparations, marketed in Hungary, and recommended as (or alleged to be) natural aphrodisiacs, are reported. Sildenafil and/or tadalafil or related analogs were detected in six of the ten products. These results highlight, once more, the unacceptable risks for the consumers of such adulterated dietary supplements.