Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling.

BACKGROUND: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. METHODS: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman's algorithm. RESULTS: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. CONCLUSION: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

Light-Quality-Adapted Carotenoid Photoprotection in the Photosystem of Roseiflexus castenholzii.

The photosystem of filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii comprises a light-harvesting (LH) complex encircling a reaction center (RC), which intensely absorbs blue-green light by carotenoid (Car) and near-infrared light by bacteriochlorophyll (BChl). To explore the influence of light quality (color) on the photosynthetic activity, we compared the pigment compositions and triplet excitation dynamics of the LH-RCs from Rfl. castenholzii was adapted to blue-green light (bg-LH-RC) and to near-infrared light (nir-LH-RC). Both LH-RCs bind γ-carotene derivatives; however, compared to that of nir-LH-RC (12%), bg-LH-RC contains substantially higher keto-γ-carotene content (43%) and shows considerably faster BChl-to-Car triplet excitation transfer (10.9 ns vs 15.0 ns). For bg-LH-RC, but not nir-LH-RC, selective photoexcitation of Car and the 800 nm-absorbing BChl led to Car-to-Car triplet transfer and BChl-Car singlet fission reactions, respectively. The unique excitation dynamics of bg-LH-RC enhances its photoprotection, which is crucial for the survival of aquatic anoxygenic phototrophs from photooxidative stress.

Carotenoid-Mediated Long-Range Energy Transfer in the Light Harvesting-Reaction Center Complex from Photosynthetic Bacterium Roseiflexus castenholzii.

The light harvesting-reaction center complex (LH-RC) of Roseiflexus castenholzii binds bacteriochlorophylls a (BChls a), B800 and B880, absorbing around 800 and 880 nm, respectively. We comparatively investigated the interband excitation energy transfer (EET) dynamics of the wild-type LH-RC (wt-LH-RC) of Rfl. castenholzii and its carotenoid (Car)-less mutant (m-LH-RC) and found that Car can boost the B800 → B880 EET rate from (2.43 ps)-1 to (1.75 ps)-1, accounting for 38% acceleration of the EET process. Interestingly, photoexcitation of wt-LH-RC at 800 nm induced pronounced excitation dynamics of Car despite the insufficient photon energy for direct Car excitation, a phenomenon which is attributed to the BChl-Car exciplex 1[B800(↑↑)···Car(↓↓)]*. Such an exciplex is suggested to play an essential role in promoting the B800 → B880 EET process, as corroborated by the recently reported cryo-EM structures of wt-LH-RC and m-LH-RC. The mechanism of Car-mediated EET will be helpful to deepen the understanding of the role of Car in bacterial photosynthesis.

Metformin inhibits EV71­induced pyroptosis by upregulating DEP domain­containing mTOR­interacting protein.

Enterovirus 71 (EV71) infection is one of the main causes of severe hand, foot and mouth disease (HFMD), which is usually accompanied by a marked inflammatory response. The excessive inflammatory response has been implicated to serve an important role in EV71-caused HFMD. Pyroptosis is a type of inflammatory programmed cell death. Therefore, a novel treatment strategy against EV71 infection could aim to alleviate the inflammatory response through inhibition of EV71-induced pyroptosis. The present study revealed that metformin had this therapeutic potential. A cell model of EV71 infection was established, cell viability was measured by CCK8 assay, cell damage was measured by LDH release kit, and the dead and dying cells were excluded by propidium iodide staining. The intracellular levels of DEP domain-containing mTOR interacting protein (DEPTOR) and pyroptosis-associated molecules were measured by western blot analysis, the NLRP3 expression was assessed by immunofluorescence labeling, and virus titers in cell culture supernatants were determined by a cell culture infectious dose 50 assay. The results demonstrated that EV71 infection could induce pyroptosis in a time- and dose-dependent manner, and metformin could inhibit EV71-induced pyroptosis. The mechanism of metformin inhibiting EV71-induced pyroptosis was explored next. Subsequent experiments indicated that metformin could increase the levels of DEPTOR, which were decreased by EV71. Finally, overexpression of DEPTOR in cells could reduce EV71-induced pyroptosis. Overall, the present study demonstrated that metformin could exert a novel pharmacodynamic anti-pyroptosis effect in the treatment of EV71 infection by upregulating DEPTOR expression.

Comparative genomics with evolutionary lineage in Streptomyces bacteria reveals high biosynthetic potentials.

Genome mining in silico approaches allow scientists to proficiently evaluate the genomic potency of secondary bioactive chemical producers and find new bioactive compounds in different bacteria. Streptomyces is one of the most ubiquitous bacterial genera in the environments, and well-known as prolific producers of diverse and valuable natural products (NPs) with significant biological activities. Mining and prioritizing of NP biosynthetic gene clusters (BGCs) would be the most important stage in the identification of novel compounds. Comparative genomics and genetic similarity network analysis of 62 Streptomyces public reference genomes demonstrated that individuals of these species exhibit a huge number of distinct NP BGCs, the most of which are cryptic and unconnected to any reported NPs with high phylogenetic variation among individuals. It was assumed that substantial heterogeneity across the varieties of species of Streptomyces drives outstanding biosynthetic and metabolic potential, making them plausible candidates for the identification of novel molecules.

Isolation, complete genome sequencing and in silico genome mining of Burkholderia for secondary metabolites.

Recent years, Burkholderia species have emerged as a new source of natural products (NPs) with increasing attractions. Genome mining suggests the Burkholderia genomes include many natural product biosynthetic gene clusters (BGCs) which are new targets for drug discovery. In order to collect more Burkholderia, here, a strain S-53 was isolated from the soil samples on a mountain area in Changde, P.R. China and verified by comparative genetic analysis to belong to Burkholderia. The complete genome of Burkholderia strain S-53 is 8.2 Mbps in size with an average G + C content of 66.35%. Its taxonomy was both characterized by 16S rRNA- and whole genome-based phylogenetic trees. Bioinformatic prediction in silico revealed it has a total of 15 NP BGCs, some of which may encode unknown products. It is expectable that availability of these BGCs will speed up the identification of new secondary metabolites from Burkholderia and help us understand how sophisticated BGC regulation works.

Streptomyces: The biofactory of secondary metabolites.

Natural products derived from microorganisms serve as a vital resource of valuable pharmaceuticals and therapeutic agents. Streptomyces is the most ubiquitous bacterial genus in the environments with prolific capability to produce diverse and valuable natural products with significant biological activities in medicine, environments, food industries, and agronomy sectors. However, many natural products remain unexplored among Streptomyces. It is exigent to develop novel antibiotics, agrochemicals, anticancer medicines, etc., due to the fast growth in resistance to antibiotics, cancer chemotherapeutics, and pesticides. This review article focused the natural products secreted by Streptomyces and their function and importance in curing diseases and agriculture. Moreover, it discussed genomic-driven drug discovery strategies and also gave a future perspective for drug development from the Streptomyces.

Complete genome sequencing and in silico genome mining reveal the promising metabolic potential in Streptomyces strain CS-7.

Gram-positive Streptomyces bacteria can produce valuable secondary metabolites. Streptomyces genomes include huge unknown silent natural product (NP) biosynthetic gene clusters (BGCs), making them a potential drug discovery repository. To collect antibiotic-producing bacteria from unexplored areas, we identified Streptomyces sp. CS-7 from mountain soil samples in Changsha, P.R. China, which showed strong antibacterial activity. Complete genome sequencing and prediction in silico revealed that its 8.4 Mbp genome contains a total of 36 BGCs for NPs. We purified two important antibiotics from this strain, which were structurally elucidated to be mayamycin and mayamycin B active against Staphylococcus aureus. We identified functionally a BGC for the biosynthesis of these two compounds by BGC direct cloning and heterologous expression in Streptomyces albus. The data here supported this Streptomyces species, especially from unexplored habitats, having a high potential for new NPs.

Total astragalosides decrease apoptosis and pyroptosis by inhibiting enterovirus 71 replication in gastric epithelial cells.

Enterovirus 71 (EV71) is one of the primary pathogens involved in severe hand, foot and mouth disease in children. EV71 infection causes various types of programmed cell death. However, there are currently no clinically approved specific antiviral drugs for control of EV71 infection. Astragalus membranaceus (AM), a Traditional Chinese medicine, has been used in antiviral therapy in China. The aim of the present study was to determine whether total astragalosides (ASTs), bioactive components of AM, protect against EV. DAPI nuclear staining was used to observe morphological changes of the nucleus and the protective effect of ASTs, which revealed that the nucleus shrank following EV71 infection, while ASTs reversed it. Cell Counting Kit-8 assay found that human normal gastric epithelial cell (GES-1 cell) viability decreased following EV71 infection, while lactate dehydrogenase (LDH) assay showed that EV71 infection induced GES-1 cell damage. Western blotting was used to measure the expression levels of apoptosis and pyroptosis marker protein to determine whether EV71 infection induced apoptosis and pyroptosis in GES-1 cells. Reverse transcription-quantitative PCR was used to determine the anti-EV71 effect of ASTs. The results showed that ASTs protected GES-1 cells from EV71-induced cell apoptosis and pyroptosis. Furthermore, the present data demonstrated that the protective effect of ASTs was exerted by suppressing EV71 replication and release. These findings suggested that ASTs may represent a potential antiviral agent for the treatment of EV71 infection.

Genome Mining of Pseudomonas Species: Diversity and Evolution of Metabolic and Biosynthetic Potential.

Microbial genome sequencing has uncovered a myriad of natural products (NPs) that have yet to be explored. Bacteria in the genus Pseudomonas serve as pathogens, plant growth promoters, and therapeutically, industrially, and environmentally important microorganisms. Though most species of Pseudomonas have a large number of NP biosynthetic gene clusters (BGCs) in their genomes, it is difficult to link many of these BGCs with products under current laboratory conditions. In order to gain new insights into the diversity, distribution, and evolution of these BGCs in Pseudomonas for the discovery of unexplored NPs, we applied several bioinformatic programming approaches to characterize BGCs from Pseudomonas reference genome sequences available in public databases along with phylogenetic and genomic comparison. Our research revealed that most BGCs in the genomes of Pseudomonas species have a high diversity for NPs at the species and subspecies levels and built the correlation of species with BGC taxonomic ranges. These data will pave the way for the algorithmic detection of species- and subspecies-specific pathways for NP development.

Mining and Biosynthesis of Bioactive Lanthipeptides From Microorganisms.

Antimicrobial resistance is one of the most serious public health issues in the worldwide and only a few new antimicrobial drugs have been discovered in recent decades. To overcome the ever-increasing emergence of multidrug-resistant (MDR) pathogens, discovery of new natural products (NPs) against MDR pathogens with new technologies is in great demands. Lanthipeptides which are ribosomally synthesized and post-translationally modified peptides (RiPPs) display high diversity in their chemical structures and mechanisms of action. Genome mining and biosynthetic engineering have also yielded new lanthipeptides, which are a valuable source of drug candidates. In this review we cover the recent advances in the field of microbial derived lanthipeptide discovery and development.

Strategies for Natural Products Discovery from Uncultured Microorganisms.

Microorganisms are highly regarded as a prominent source of natural products that have significant importance in many fields such as medicine, farming, environmental safety, and material production. Due to this, only tiny amounts of microorganisms can be cultivated under standard laboratory conditions, and the bulk of microorganisms in the ecosystems are still unidentified, which restricts our knowledge of uncultured microbial metabolism. However, they could hypothetically provide a large collection of innovative natural products. Culture-independent metagenomics study has the ability to address core questions in the potential of NP production by cloning and analysis of microbial DNA derived directly from environmental samples. Latest advancements in next generation sequencing and genetic engineering tools for genome assembly have broadened the scope of metagenomics to offer perspectives into the life of uncultured microorganisms. In this review, we cover the methods of metagenomic library construction, and heterologous expression for the exploration and development of the environmental metabolome and focus on the function-based metagenomics, sequencing-based metagenomics, and single-cell metagenomics of uncultured microorganisms.

Synthetic biology-inspired strategies and tools for engineering of microbial natural product biosynthetic pathways.

Microbial-derived natural products (NPs) and their derivative products are of great importance and used widely in many fields, especially in pharmaceutical industries. However, there is an immediate need to establish innovative approaches, strategies, and techniques to discover new NPs with novel or enhanced biological properties, due to the less productivity and higher cost on traditional drug discovery pipelines from natural bioresources. Revealing of untapped microbial cryptic biosynthetic gene clusters (BGCs) using DNA sequencing technology and bioinformatics tools makes genome mining possible for NP discovery from microorganisms. Meanwhile, new approaches and strategies in the area of synthetic biology offer great potentials for generation of new NPs by engineering or creating synthetic systems with improved and desired functions. Development of approaches, strategies and tools in synthetic biology can facilitate not only exploration and enhancement in supply, and also in the structural diversification of NPs. Here, we discussed recent advances in synthetic biology-inspired strategies, including bioinformatics and genetic engineering tools and approaches for identification, cloning, editing/refactoring of candidate biosynthetic pathways, construction of heterologous expression hosts, fitness optimization between target pathways and hosts and detection of NP production.