Isolation, Characterization and Targeted Metabolic Evaluation of Endophytic Fungi Harbored in 14 Seed-Derived Hypericum Species.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträger's test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.

Spatial chemo-profiling of hypericin and related phytochemicals in Hypericum species using MALDI-HRMS imaging.

Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees.

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n = 2) eating low-fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high-fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P <0.001). On the other hand, HFD inoculum increased the production of total short-chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet-to-be-sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production.

Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli.

The heterologous expression of enterolysin A (EnlA), heat-labile class III bacteriocin from Enterococcus faecalis II/1 with anti-listerial activity, was studied in Escherichia coli. The PCR amplified products of enterolysin A structural gene, N-terminal part of EnlA with endopeptidase-like activity and C-terminal part of EnlA similar to a lysis gene of bacteriophage, were cloned in prelinearized pQE-30UA expression vector. The expression of EnlA structural gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein, which was purified to homogeneity using His-Select Cartridge and was shown to be fully active against the indicator strain. The expression of N-terminal or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of biologically non-active proteins.

Bacteriocin-like activity production and resistance in selected enterococci and streptococci of animal origin.

Antimicrobial proteins and peptides produced by bacteria, termed bacteriocins, are widely acknowledged to be important contributors to their producer organism survival. Enterocin A, enterocin B, enterocin P and enterolysin A belong to the best studied enterocins, i.e., bacteriocins produced by enterococci and streptococci. Twenty-one enterococcal and seven streptococcal isolates were analysed for bacteriocin-like activity production and resistance by overlay test. Up to 50% of tested strains showed antibacterial activity at least against one indicator strain. The occurrence of enterocin B structural gene in several isolates was confirmed by PCR method. The results of this study should broaden knowledge of bacteriocin-like activity production and resistance among gram-positive bacteria.