RESUMEN
BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.
Asunto(s)
Antraquinonas , Endófitos , Hypericum , Familia de Multigenes , Policétidos , Hypericum/microbiología , Hypericum/genética , Hypericum/metabolismo , Policétidos/metabolismo , Endófitos/genética , Endófitos/metabolismo , Antraquinonas/metabolismo , Hongos/genética , Genoma Fúngico , Simulación por Computador , Sintasas Poliquetidas/genética , Perileno/análogos & derivados , Perileno/metabolismo , Antracenos/metabolismo , Genómica , FilogeniaRESUMEN
Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w). KEY POINTS: ⢠The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS. ⢠HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.
Asunto(s)
Ascomicetos , Señalización del Calcio , Perileno , Perileno/análogos & derivados , Quinonas , Ascomicetos/metabolismo , Ascomicetos/genética , Ascomicetos/crecimiento & desarrollo , Quinonas/metabolismo , Perileno/metabolismo , Óxido Nítrico/metabolismo , Respuesta al Choque Térmico , Calcio/metabolismo , CalorRESUMEN
Plant-pathogenic fungi produce toxins as virulence factors in many plant diseases. In Cercospora leaf blight of soybean caused by Cercospora cf. flagellaris, symptoms are a consequence of the production of a perylenequinone toxin, cercosporin, which is light-activated to produce damaging reactive oxygen species. Cercosporin is universally toxic to cells, except to the cells of the producer. The current model of self-resistance to cercosporin is largely attributed to the maintenance of cercosporin in a chemically reduced state inside hyphae, unassociated with cellular organelles. However, in another perylenequinone-producing fungus, Phaeosphaeria sp., the toxin was specifically sequestered inside lipid droplets (LDs) to prevent reactive oxygen species production. This study hypothesized that LD-based sequestration of cercosporin occurred in C. cf. flagellaris and that lipid-inhibiting fungicides could inhibit toxin production. Confocal microscopy using light-cultured C. cf. flagellaris indicated that 3-day-old hyphae contained two forms of cercosporin distributed in two types of hyphae. Reduced cercosporin was uniformly distributed in the cytoplasm of thick, primary hyphae, and, contrary to previous studies, active cercosporin was observed specifically in the LDs of thin, secondary hyphae. The production of hyphae of two different thicknesses, a characteristic of hemibiotrophic plant pathogens, has not been documented in C. cf. flagellaris. No correlation was observed between cercosporin production and total lipid extracted, and two lipid-inhibiting fungicides had little effect on fungal growth in growth-inhibition assays. This study lays a foundation for exploring the importance of pathogen lifestyle, toxin production, and LD content in the pathogenicity and symptomology of Cercospora.
Asunto(s)
Cercospora , Hifa , Perileno , Enfermedades de las Plantas , Perileno/análogos & derivados , Perileno/metabolismo , Enfermedades de las Plantas/microbiología , Hifa/efectos de los fármacos , Hifa/crecimiento & desarrollo , Cercospora/metabolismo , Glycine max/microbiología , Ascomicetos/efectos de los fármacos , Ascomicetos/fisiología , Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fungicidas Industriales/farmacología , Gotas Lipídicas/metabolismo , Hojas de la Planta/microbiología , Microscopía ConfocalRESUMEN
The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.
Asunto(s)
Ascomicetos , Perileno , Perileno/análogos & derivados , Fotoquimioterapia , Quinonas , Ascomicetos/metabolismo , Biología Sintética , Perileno/farmacología , Perileno/metabolismoRESUMEN
Hypocrellins are fungal perylenequinones (PQs) from Shiraia fruiting bodies and potential photosensitizers for cancer photodynamic therapy. Shiraia fruiting bodies harbor diverse bacterial communities dominated by Pseudomonas. The present study was to characterize the exopolysaccharide (EPS) of P. fulva SB1 which acted as an elicitor to stimulate the PQ accumulation of the host Shiraia. A bacterial EPS named EPS-1 was purified from the culture broth of P. fulva SB1, which consisted of mannose (Man) and glucose (Glc) with an average molecular weight of 9.213 × 104 Da. EPS-1 had (1 â 2)-linked α-mannopyranose (Manp) backbone and side chains of α-D-Manp-(1â and α-D-Manp-(1 â 6)-ß-D-Glcp-(1 â 6)-α-D-Manp(1 â group attached to the O-6 positions of (1 â 2)-α-D-Manp. EPS-1 at 30 mg/L stimulated both intracellular and extracellular hypocrellin A (HA) by about 3-fold of the control group. The EPS-1 treatment up-regulated the expression of key genes for HA biosynthesis. The elicitation of HA biosynthesis by EPS-1 was strongly dependent on the induced reactive oxygen species (ROS) generation. The results may provide new insights on the role of bacterial EPS in bacterium-fungus interactions and effective elicitation strategy for hypocrellin production in mycelial cultures.
Asunto(s)
Ascomicetos , Perileno , Fotoquimioterapia , Humanos , Quinonas/farmacología , Quinonas/metabolismo , Fenol/metabolismo , Perileno/farmacología , Perileno/metabolismo , Ascomicetos/genéticaRESUMEN
BACKGROUND: Hypocrellin A (HA) is a perylene quinone pigment with high medicinal value that is produced by Shiraia bambusicola Henn. (S. bambusicola) and Hypocrella bambusae (Berk. & Broome) Sacc. (Ascomycetes) with great potential in clinical photodynamic therapy. Submerged cultivation of S. bambusicola is a popular technique for HA production. However, there is not much research on how temperature changes lead to differential yields of HA production. RESULTS: The temperature regulation of submerged fermentation is an efficient approach to promote HA productivity. After a 32 °C fermentation, the HA content in the mycelia S. bambusicola (GDMCC 60438) was increased by more than three- and fivefold when compared to that at 28 °C and 26 °C, respectively. RNA sequencing (RNA-seq) analysis showed that the regulation of the expression of transcription factors and genes essential for HA biosynthesis could be induced by high temperature. Among the 496 differentially expressed genes (DEGs) explicitly expressed at 32 °C, the hub genes MH01c06g0046321 and MH01c11g0073001 in the coexpression network may affect HA biosynthesis and cytoarchitecture, respectively. Moreover, five genes, i.e., MH01c01g0006641, MH01c03g0017691, MH01c04g0029531, MH01c04g0030701 and MH01c22g0111101, potentially related to HA synthesis also exhibited significantly higher expression levels. Morphological observation showed that the autolysis inside the mycelial pellets tightly composted intertwined mycelia without apparent holes. CONCLUSIONS: The obtained results provide an effective strategy in the submerged fermentation of S. bambusicola for improved HA production and reveal an alternative regulatory network responsive to the biosynthesis metabolism of HA in response to environmental signals.
Asunto(s)
Ascomicetos , Perileno , Ascomicetos/metabolismo , Fermentación , Perileno/análogos & derivados , Perileno/metabolismo , Fenol , Quinonas/metabolismo , TemperaturaRESUMEN
Blue light is a crucial environmental cue for fungi. Hypocrellin A (HA) is a photoactive perylenequinone from Shiraia with strong antimicrobial and anticancer properties. In this study, effects of the illumination of blue-light-emitting diode (LED) at 470 nm on Shiraia sp. S8 were investigated. Blue light at 50-200 lx and 4-6 h day-1 could enhance HA content in the mycelia, but suppress it at 300-400 lx or with longer exposure (8-24 h day-1 ). The intermittent blue light (6 h day-1 ) at 200 lx not only enhanced the fungal conidiation but also stimulated HA production without any growth retardation. The generation of fungal reactive oxygen species was induced to upregulate HA biosynthetic gene expressions. When the culture was maintained under the intermittent blue light for 8 days, HA production reached 242.76 mg L-1 , 2.27-fold of the dark control. On the other hand, both the degradation of HA and downregulation of HA biosynthetic genes occurred under long exposure time (8-24 h day-1 ), leading to the suppression of HA production. These results provide a basis for understanding the regulation of blue light on the biosynthesis of fungal photoactivated perylenequinones, and the application of a novel light elicitation to Shiraia mycelium cultures for enhanced HA production.
Asunto(s)
Ascomicetos , Perileno , Perileno/metabolismo , Quinonas/metabolismo , Fenol , Micelio , Ascomicetos/genética , LuzRESUMEN
OBJECTIVE: Breast cancer (BC) currently has no effective treatment especially for the highly aggressive and metastatic triple negative breast cancer (TNBC). Here, we investigated the antitumoral and antimigratory effects of hypericin (HYP) encapsulated on Pluronic F127 (F127/HYP) photodynamic therapy (PDT) against TNBC cell line MDA-MB-231 compared to a nontumorigenic human breast ductal cell line (MCF-10A). METHODS: The phototoxicity/cytotoxicity was assessed by MTT assay, long-term cytotoxicity by clonogenic assay, cell uptake, subcellular distribution, and cellular oxidative stress by fluorescence microscopy, cell death with annexin V-FITC/propidium iodide, PDT mechanism using sodium azide and D-mannitol, and cell migration by wound-healing assay. RESULTS: The treatment promoted phototoxic effect on tumor cell line in a dose-dependent and selective manner. Internalization of F127/HYP was efficient and accumulation occurred in the endoplasmic reticulum and mitochondria, resulting in cellular oxidative stress mainly by the type II mechanism, induced by necrosis. Furthermore, F127/HYP decreased colony formation and reduced the cell migration ability in MDA-MB-231 cells. CONCLUSION: Our results suggest a potentially useful role of F127/HYP micelles as a platform for HYP delivery to more specifically and effectively treat TNBC.
Asunto(s)
Perileno , Fotoquimioterapia , Neoplasias de la Mama Triple Negativas , Antracenos , Humanos , Perileno/análogos & derivados , Perileno/metabolismo , Perileno/farmacología , Poloxámero , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia-to-industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin-structure-centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1â mg L-1 ), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.
Asunto(s)
Antracenos/metabolismo , Hongos/metabolismo , Hypericum/química , Perileno/análogos & derivados , Fitoquímicos/biosíntesis , Antracenos/química , Hongos/química , Estructura Molecular , Perileno/química , Perileno/metabolismo , Fitoquímicos/químicaRESUMEN
Elsinochromes (ESCs) are virulence factors produced by Elsinoë arachidis which is the cause of peanut scab. However, the biosynthesis pathway of ESCs in E. arachidis has not been elucidated and the potential pathogenic mechanism of E. arachidis is poorly understood. In this study, we report a high-quality genome sequence of E. arachidis. The size of the E. arachidis genome is 33.18Mb, which is comparable to the Ascomycota genome (average 36.91 Mb), encoding 9174 predicted genes. The self-detoxification family including transporters and cytochrome P450 enzymes were analysis, candidate effectors and cell wall degrading enzymes were investigated as the pathogenicity genes by using PHI and CAZy databases. Additionally, the E. arachidis genome contains 24 secondary metabolism gene clusters, in which ESCB1 was identified as the core gene of ESC biosynthesis. Taken together, the genome sequence of E. arachidis provides a new route to explore its potential pathogenic mechanism and the biosynthesis pathway of ESCs.
Asunto(s)
Ascomicetos/genética , Micotoxinas/metabolismo , Perileno/análogos & derivados , Quinonas/metabolismo , Factores de Virulencia/genética , Arachis/microbiología , Ascomicetos/metabolismo , Ascomicetos/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Perileno/metabolismo , Filogenia , Enfermedades de las Plantas/microbiología , Secuenciación Completa del GenomaRESUMEN
Hypericin (Hy) is a hydrophobic photosensitizer used in photodynamic therapy for cancer therapeutic. In this study, Hy-loaded oil-in-water (O/W) nanoemulsions (NEs) were produced by the ultrasonication method combing different biocompatible oils and surfactants to enhance Hy aqueous solubility and bioavailability. Experimental parameters were optimized by the characterization of droplet size, zeta potential, and physicochemical properties. In vitro studies based on the release profile, cytotoxicity, cell morphology, and Hy intracellular accumulation were assayed. Hy at 100 mg L-1 was incorporated into the low viscosity (~0.005 Pa s) NEs with spherical droplets averaging 20-40 nm in size and polydispersity index <0.02. Hy release from the NE was significantly higher (4-fold) than its suspension (p < 0.001). The NEs demonstrated good physical stability during storage at 5 °C for at least six months. The Hy-loaded NEs exhibited an IC50 value 6-fold lower than Hy suspension during PDT against breast cancer cell lines (MCF-7). Cell microscopy imaging confirmed the increased cytotoxic effects of Hy-loaded NEs, showing damaged and apoptotic cells. Confocal laser scanning microscopy evidenced greater Hy delivery through NE into MCF-7 cells followed by improved intracellular ROS generation. Our results suggest that the Hy-loaded NEs can improve hypericin efficacy and assist Hy-PDT's preclinical development as a cancer treatment.
Asunto(s)
Antracenos/química , Emulsiones/química , Nanoestructuras/química , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Sensibilizantes a Radiaciones/química , Antracenos/metabolismo , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos/efectos de la radiación , Estabilidad de Medicamentos , Humanos , Luz , Células MCF-7 , Aceites/química , Perileno/química , Perileno/metabolismo , Perileno/farmacología , Fármacos Sensibilizantes a Radiaciones/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sonicación , Temperatura , Agua/químicaRESUMEN
α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.
Asunto(s)
Antracenos/química , Proteínas Fúngicas/antagonistas & inhibidores , Inhibidores de Glicósido Hidrolasas/química , Hipoglucemiantes/química , Perileno/análogos & derivados , alfa-Glucosidasas/química , Antracenos/metabolismo , Sitios de Unión , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Inhibidores de Glicósido Hidrolasas/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Perileno/química , Perileno/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/enzimología , Resonancia por Plasmón de Superficie , alfa-Glucosidasas/metabolismoRESUMEN
BACKGROUND: Adenosine 5'-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. RESULTS: The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10-300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. CONCLUSIONS: Our results indicate that eATP release is an early event during the intimate bacterial-fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal-bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ascomicetos/metabolismo , Perileno/análogos & derivados , Fenol/metabolismo , Pseudomonas/metabolismo , Quinonas/metabolismo , Transducción de Señal/efectos de los fármacos , Adenosina Trifosfato/farmacología , Ascomicetos/efectos de los fármacos , Citosol/metabolismo , Perileno/análisis , Perileno/metabolismo , Fenol/análisis , Pseudomonas/efectos de los fármacos , Quinonas/análisis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production. RESULTS: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion. CONCLUSIONS: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.
Asunto(s)
Cercospora/metabolismo , Endófitos/metabolismo , Interacciones Microbianas/fisiología , Perileno/análogos & derivados , Enfermedades de las Plantas/microbiología , Bacillaceae/crecimiento & desarrollo , Bacillaceae/metabolismo , Bacillus/crecimiento & desarrollo , Bacillus/metabolismo , Cercospora/genética , Cercospora/crecimiento & desarrollo , Endófitos/genética , Endófitos/crecimiento & desarrollo , Regulación Fúngica de la Expresión Génica , Técnicas In Vitro , Perileno/análisis , Perileno/metabolismoRESUMEN
Hypericin (Hyp), well-known as an antidepressant, is mainly extracted from Hypericum perforatum. Although Hyp accumulation and biomass are greater at lower compared to higher temperature, the regulation mechanism has not been reported. Here, the physiological characteristics and transcriptome of H. perforatum grown at 15 and 22 °C were determined and analyzed by HPLC and de novo sequencing. The results showed that the stomatal density and opening percentages were 1.1- and 1.4-fold more, and the Hyp content was 4.5-fold greater at 15 °C compared to 22 °C. A total of 1584 differentially expressed genes (DEGs) were observed at 15 versus 22 °C, with 749 characterized genes, 421 upregulated (UR) and 328 downregulated (DR). Based on biological functions, 150 genes were associated with Hyp biosynthesis, plant growth and the stress response, including photosynthesis, carbohydrate metabolism, fatty acids metabolism, cytochrome P450 (CYPs), morpho-physiological traits, heat shock proteins (HSPs), cold-responsive proteins (CRPs) and transcription factors (TFs). The differential expression levels of the master genes were confirmed by qRT-PCR and almost consistent with their Reads Per kb per Million (RPKM) values. This physiological and transcriptomic analyses provided insight into the regulation mechanisms of low temperature enhancing Hyp biosynthesis in H. perforatum.
Asunto(s)
Hypericum/química , Perileno/análogos & derivados , Transcriptoma/genética , Antracenos , Perfilación de la Expresión Génica , Perileno/metabolismo , TemperaturaRESUMEN
BACKGROUND: Nitric oxide (NO) is a ubiquitous signaling mediator in various physiological processes. However, there are less reports concerning the effects of NO on fungal secondary metabolites. Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from fungal perylenequinone pigments of Shiraia. NO donor sodium nitroprusside (SNP) was used as a chemical elicitor to promote hypocrellin biosynthesis in Shiraia mycelium cultures. RESULTS: SNP application at 0.01-0.20 mM was found to stimulate significantly fungal production of perylenequinones including hypocrellin A (HA) and elsinochrome A (EA). SNP application could not only enhance HA content by 178.96% in mycelia, but also stimulate its efflux to the medium. After 4 days of SNP application at 0.02 mM, the highest total production (110.34 mg/L) of HA was achieved without any growth suppression. SNP released NO in mycelia and acted as a pro-oxidant, thereby up-regulating the gene expression and activity of reactive oxygen species (ROS) generating NADPH oxidase (NOX) and antioxidant enzymes, leading to the increased levels of superoxide anion (O2-) and hydrogen peroxide (H2O2). Gene ontology (GO) analysis revealed that SNP treatment could up-regulate biosynthetic genes for hypocrellins and activate the transporter protein major facilitator superfamily (MFS) for the exudation. Moreover, SNP treatment increased the proportion of total unsaturated fatty acids in the hypha membranes and enhanced membrane permeability. Our results indicated both cellular biosynthesis of HA and its secretion could contribute to HA production induced by SNP. CONCLUSIONS: The results of this study provide a valuable strategy for large-scale hypocrellin production and can facilitate further understanding and exploration of NO signaling in the biosynthesis of the important fungal metabolites.
Asunto(s)
Ascomicetos/efectos de los fármacos , Ascomicetos/genética , Vías Biosintéticas/genética , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Perileno/análogos & derivados , Fenol/metabolismo , Quinonas/metabolismo , Transcripción Genética , Ascomicetos/metabolismo , Micelio/crecimiento & desarrollo , Perileno/metabolismo , Especies Reactivas de OxígenoRESUMEN
The present study reports the performance of the pigment hypericin (HYP)-loaded poloxamer-based mucoadhesive in situ gelling liquid crystalline precursor system (LCPS) for the treatment of vulvovaginal candidiasis (VVC) in mice. LCPS composed of 40% of ethoxylated and propoxylated cetyl alcohol, 30% of oleic acid and cholesterol (7:1), 30% of a dispersion of 16% poloxamer 407 and 0.05% of HYP (HYP-LCPS) was prepared and characterized by polarized light microscopy (PLM), small-angle X-ray scattering (SAXS) and ex vivo permeation and retention studies across vaginal porcine mucosa were performed. In addition, the antifungal properties of the HYP-LCPS were evaluated in a murine in vivo model; for this, infected C57BL female mice groups were treated with both HYP in solution and HYP-LCPS, and after 6 days colony forming unit (CFU)/ml count was performed. PLM and SAXS confirmed that HYP-LCPS is a microemulsion situated in boundary transition region confirming its action as an LCPS. When in contact with simulated vaginal fluid, HYP-LCPS became rigid and exhibited maltase crosses and bragg peaks characteristics of lamellar phase. Ex vivo permeation and retention studies showed that HYP-LCPS provides a localized treatment on the superficial layers of porcine vaginal mucosa. HYP-LCPS induced a significant reduction in the number of CFU/ml in the mice; thus this formulation indicated it is as effective as a commercial dosage form. It was concluded that LCPS maintains the biological activity of HYP and provides an adequate drug delivery system for this lipophilic molecule at the vaginal mucosa, being a promising option in cases of VVC.
Asunto(s)
Antracenos/administración & dosificación , Antifúngicos/administración & dosificación , Candida albicans/efectos de los fármacos , Candidiasis Vulvovaginal/tratamiento farmacológico , Perileno/análogos & derivados , Vagina/metabolismo , Adhesivos/administración & dosificación , Animales , Antracenos/metabolismo , Antifúngicos/metabolismo , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Microscopía de Polarización , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Membrana Mucosa/patología , Perileno/administración & dosificación , Perileno/metabolismo , Poloxámero/administración & dosificación , Fármacos Sensibilizantes a Radiaciones , Dispersión del Ángulo Pequeño , Porcinos , Vagina/microbiología , Vagina/patología , Difracción de Rayos XRESUMEN
Shiraia bambusicola has been used as a traditional Chinese medicine for a long history. Its major medicinal active metabolites are perylenequinones, including hypocrellin A, elsinochrome A and so on. At present, the fermentation yield of perylenequinones is low, and its complex biosynthesis and regulatory pathways are still unclear. In this study, nitric oxide, as a downstream signal molecule of hydrogen peroxide, regulates the biosynthesis of perylenequinones. Exogenous addition of 0.01 mM sodium nitroprusside (nitric oxide donor) can promote perylenequinones production by 156% compared with the control. Further research found that hydrogen peroxide and nitric oxide increased the transcriptional level of the biosynthetic genes of hypocrellin A. The results showed that nitric oxide is involved in the biosynthesis and regulation of perylenequinones in Shiraia bambusicola as a signal molecule. In the future, the yield of perylenequinones can be increased by adding exogenous nitric oxide in fermentation.
Asunto(s)
Ascomicetos/efectos de los fármacos , Ascomicetos/metabolismo , Peróxido de Hidrógeno/farmacología , Óxido Nítrico/farmacología , Perileno/análogos & derivados , Quinonas/metabolismo , Perileno/metabolismo , Fenol/metabolismoRESUMEN
The use of nanocarriers for intracellular transport of actives has been extensively studied in recent years and represents a central area of nanomedicine. The main novelty of this paper lies on the use of nanogels formed by a low-molecular-weight gelator (1). Here, non-polymeric, molecular nanogels are successfully used for intracellular transport of two photodynamic therapy (PDT) agents, Rose Bengal (RB) and hypericin (HYP). The two photosensitizers (PSs) exhibit different drawbacks for their use in clinical applications. HYP is poorly water-soluble, while the cellular uptake of RB is hindered due to its dianionic character at physiological pH values. Additionally, both PSs tend to aggregate precluding an effective PDT. Despite the different nature of these PSs, nanogels from gelator 1 provide, in both cases, an efficient intracellular transport into human colon adenocarcinoma cells (HT-29) and a notably improved PDT efficiency, as assessed by confocal laser scanning microscopy and flow cytometry. Furthermore, no significant dark toxicity of the nanogels is observed, supporting the biocompatibility of the delivery system. The developed nanogels are highly reproducible due to their non-polymeric nature, and their synthesis is easily scaled up. The results presented here thus confirm the potential of molecular nanogels as valuable nanocarriers, capable of entrapping both hydrophobic and hydrophilic actives, for PDT of cancer.
Asunto(s)
Antracenos/química , Nanogeles/química , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/química , Rosa Bengala/química , Antracenos/metabolismo , Antracenos/farmacología , Materiales Biocompatibles/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Humanos , Luz , Microscopía Confocal , Perileno/química , Perileno/metabolismo , Perileno/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/farmacología , Rosa Bengala/metabolismo , Rosa Bengala/farmacología , Oxígeno Singlete/metabolismoRESUMEN
Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.