ABSTRACT
We envisioned that the rumen of Kitasato Yakumo beef cattle would contain unique microorganisms which produce bioactive compounds as their defense response to the external environment. The variety of microorganisms were collected from the feces of Kitasato Yakumo beef cattle. We evaluated the biological activity of the culture broth of the isolated strains, proving the utility of our approach.
ABSTRACT
In this paper, we describe our discovery of burnettiene A (1) as an anti-malarial compound from the culture broth of Lecanicillium primulinum (Current name: Flavocillium primulinum) FKI-6715 strain utilizing our original multidrug-sensitive yeast system. This polyene-decalin polyketide natural product was originally isolated as an anti-fungal active compound from Aspergillus burnettii. However, the anti-fungal activity of 1 has been revealed in only one fungal species for and the mechanism of action of 1 remains unknown. After the validation of mitochondrial function inhibitory of 1, we envisioned a new anti-malarial drug discovery platform based on mitochondrial function inhibitory activity. We evaluated anti-malarial activity and 1 showed anti-malarial activity against Plasmodium falciparum FCR3 (chloroquine sensitive) and K1 strain (chloroquine resistant). Our study revealed the utility of our original screening system based on a multidrug-sensitive yeast and mitochondrial function inhibitory activity for the discovery of new anti-malarial drug candidates.
ABSTRACT
We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of 10-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.
Subject(s)
Drug Discovery , Saccharomyces cerevisiae , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Drug Discovery/methods , Candida albicans/drug effects , Secondary Metabolism , Fungicides, Industrial/pharmacology , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Microbial Sensitivity Tests , Ascomycota/drug effects , Ascomycota/genetics , Aspergillus/drug effects , Aspergillus/genetics , Aspergillus/metabolism , Drug Evaluation, Preclinical/methods , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolismABSTRACT
Utilizing a binding mode-based physicochemical screening method using d-Ala-d-Ala silica gel, two new macrolactams, named banglactams A (1) and B (2), were discovered from the culture broth of Nonomuraea bangladeshensis K18-0086. In the course of our investigation, we found that d-Ala-d-Ala silica gel precisely differentiated the chemical structures of banglactams and separated them. However, we were not able to obtain enough of 1 to elucidate the structure due to its instability and insolubility. To overcome this challenge, we chemically modified 1 to improve solubility, enabling us to obtain a sufficient material supply for the indirect determination of the structure. Antibacterial activity evaluation of banglactams revealed that 1 binding to d-Ala-d-Ala silica gel exhibited antibacterial activity against Staphylococcus aureus; however, this was not the case with 2. This research indicates the utility of our original binding mode-based PC screening method, and the combination strategy of PC and chemical modifications led us to discover novel antibacterial compounds.
ABSTRACT
Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.
Subject(s)
Aurodox , Type III Secretion Systems , Type III Secretion Systems/metabolism , Aurodox/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Gram-Negative Bacteria/metabolism , Bacterial Proteins/metabolismABSTRACT
The emergence and spread of antimicrobial resistance are global threats. Pseudomonas aeruginosa (P. aeruginosa) is responsible for a substantial proportion of this global health issue because of its intrinsic resistance to many antibiotics due to the impermeability of its outer membrane and its multidrug efflux pump systems. Therefore, therapeutic drugs are limited, and the development of new drugs is extremely challenging. As an alternative approach, we focused on a combinational treatment strategy and found that 5-O-mycaminosyltylonolide (OMT) showed potent antibacterial activity against P. aeruginosa in the presence of an efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide (PAßN). In this report, we prepared a PAßN derivative and compared the potentiation activity of OMT by PAßNs against multidrug-resistant P. aeruginosa clinical isolates.
Subject(s)
Anti-Bacterial Agents , Dipeptides , Drug Resistance, Multiple, Bacterial , Microbial Sensitivity Tests , Pseudomonas aeruginosa , Tylosin/analogs & derivatives , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Drug Resistance, Multiple, Bacterial/drug effects , Dipeptides/pharmacology , Dipeptides/chemistry , Drug Synergism , HumansABSTRACT
Fusaramin (1) was isolated as a mitochondrial inhibitor. However, the fungal producer stops producing 1, which necessitates us to supply 1 by total synthesis. We proposed the complete stereochemical structure based on the biosynthetic pathway of sambutoxin. We have established concise and robust total synthesis of 1, enabling us to determine the complete stereochemical structure and to elucidate the structure-activity relationship, and uncover the hidden antiplant pathogenic fungal activity.
Subject(s)
Anti-Infective Agents , Fungi , Anti-Infective Agents/chemistry , Structure-Activity Relationship , Mycotoxins/chemistryABSTRACT
We discovered a new tetronomycin analog, C-32-OH tetronomycin (2) from the Streptomyces sp. K20-0247 strain, which produces tetronomycin (1). After NMR analysis of 2, we determined the planar structure. Futhermore, the absolute stereochemistry of 2 was deduced based on the biosynthetic pathway of 1 in the K20-0247 strain and a comparison of experimental electronic circular dichroism (ECD) results of 1 with 2. While 2 exihibits potent antibacterial activity aganist Gram-positive baceria including vancomycin-intermediate Staphylococcus aureus (VISA) strains and vancomycin-resistant Enterococci (VRE), the antibacterial activity of 2 shows 16-32-folds weaker than that of 1 suggesting that the C-34 methyl group in 1 is one of the very important functinal group. Moreover, we evaluated the ionophore activity of 1 and 2 and neither compound shows ionophore activity at reasonable concetrations. Our research suggests that 1 and 2 would have different target(s) from an ionophore mechanism in the antibacterial activity and tetronomycins are promising natural products for broad-spectrum antibiotics.
Subject(s)
Anti-Bacterial Agents , Ethers , Anti-Bacterial Agents/pharmacology , Gram-Positive Bacteria , Ionophores , Microbial Sensitivity TestsABSTRACT
Vancomycin is a potent and broad-spectrum antibiotic that binds to the d-Ala-d-Ala moiety of the growing bacterial cell wall and kills bacteria. This fascinating binding model prompted us to design and synthesize d-Ala-d-Ala silica gels for the establishment of a new physicochemical (PC) screening method. In this report, we confirmed that vancomycin binds to d-Ala-d-Ala silica gel and can be eluted with MeOH containing 50 mM TFA. Finally, d-Ala-d-Ala silica gel enables to purify vancomycin from the culture broth of a vancomycin-producing strain, Amycolatopsis orientalis.
ABSTRACT
BACKGROUND: Previous studies have revealed a nitric oxide (NO) metabolic cycle in which NO, nitrate (NO3-), and nitrite (NO2-) circulate. The NO produced in this cycle serves as a signalling molecule that regulates actinorhodin (ACT) production via the DevS/DevR NO-dependent two-component system (TCS) in Streptomyces coelicolor A3(2) M145. However, the mechanisms involved in the regulation of NO signalling in S. coelicolor have not yet been elucidated. Mycothiol (MSH), a thiol molecule produced by Actinomyces, is involved in the defence mechanisms against oxidative stress. Therefore, this study focused on the correlation between intracellular NO and MSH levels. RESULTS: To investigate the interaction of MSH with endogenously produced NO, we generated an S. coelicolor A3(2) strain deficient in MSH biosynthesis. This mutant strain exhibited a decrease in low-molecular-weight S-nitrosothiols and intracellular NO levels during culture compared to those of the wild-type strain. Moreover, the mutant strain exhibited reduced activity of the DevS/DevR TCS, a regulator of NO homeostasis and ACT production, from the early stage of culture, along with a decrease in ACT production compared to those of the wild-type strain. CONCLUSIONS: This study suggests that MSH maintains intracellular NO homeostasis by forming S-nitrosomycothiol, which induces NO signalling. Finally, we propose a metabolic model in which MSH from endogenously produced NO facilitates the maintenance of both NO homeostasis and signalling in S. coelicolor A3(2) M145.
Subject(s)
Streptomyces coelicolor , Streptomyces coelicolor/genetics , Streptomyces coelicolor/metabolism , Nitric Oxide/metabolism , Cysteine/metabolism , Homeostasis , Gene Expression Regulation, Bacterial , Anthraquinones/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Anti-Bacterial Agents/pharmacologyABSTRACT
Luminamicin (1) isolated in 1985, is a macrodiolide compound exhibiting selective antibacterial activity against anaerobes. However, the antibacterial activity of 1 was not fully examined. In this research, re-evaluation of the antibacterial activity of 1 revealed that 1 is a narrow spectrum and potent antibiotic againstClostridioides difficile(C. difficile) and effective against fidaxomicin resistantC. difficilestrain. This prompted us to obtain luminamicin resistantC. difficilestrains for the determination of the molecular target of 1 inC. difficile. Sequence analysis of 1-resistantC. difficileindicated that the mode of action of 1 differs from that of fidaxomicin. This is because no mutation was observed in RNA polymerase and mutations were observed in a hypothetical protein and cell wall protein. Furthermore, we synthesized derivatives from 1 to study the structure-activity relationship. This research indicated that the maleic anhydride and the enol ether moieties seem to be pivotal functional groups to maintain the antibacterial activity againstC. difficileand the 14-membered lactone may contribute to taking an appropriate molecular conformation.
ABSTRACT
Our previous studies revealed that a two-component system (TCS), DevS, and DevR, regulate both nitric oxide (NO) signaling and NO homeostasis in the actinobacterium Streptomyces coelicolor A3(2) M145, suggesting a reasonable system for NO-dependent metabolism. In this study, sequence alignment of DevR and DevR homologs found Asp66 (D66) and Thr196 (T196) as predicted phosphorylation sites of DevR. Phos-tag gel electrophoretic mobility shift assay suggested that D66 and T196 are involved in the phosphorylation of DevR. The respective point mutations of D66 and T196 significantly decreased the transcriptional activity of DevR, which affected nitrite production and aerial mycelium formation. These results suggested that both D66 and T196 of DevR are important for the regulation of NO homeostasis and signaling in S. coelicolor A3(2) M145.
Subject(s)
Streptomyces coelicolor , Phosphorylation , Streptomyces coelicolor/genetics , Streptomyces coelicolor/metabolism , Nitric Oxide/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Sequence Alignment , Gene Expression Regulation, BacterialABSTRACT
Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes. IMPORTANCE Eukaryotic and prokaryotic cells utilize nitric oxide (NO) to regulate physiological functions. Besides its role as a producer of different bioactive substances, Streptomyces is suggested to be involved in mycelial development regulated by endogenously produced NO. However, the regulatory mechanisms are unclear. In this study, we proposed that NO signaling is involved in aerial mycelium formation in S. coelicolor A3(2) M145. NO serves as a signaling molecule for the regulation of intracellular cyclic 3',5'-diguanylate (c-di-GMP) levels, resulting in aerial mycelium formation controlled by a c-di-GMP receptor, BldD. As the abundant production of valuable secondary metabolites is closely related to the initiation of morphological differentiation in Streptomyces, NO may provide value for application in industrial fermentation by serving as a tool for regulating secondary metabolism.
Subject(s)
Streptomyces coelicolor , Streptomyces , Streptomyces coelicolor/genetics , Streptomyces coelicolor/metabolism , Nitric Oxide/metabolism , Gene Expression Regulation, Bacterial , Bacterial Proteins/genetics , Streptomyces/metabolism , Mycelium/metabolismABSTRACT
Nitric oxide (NO) is an important signaling molecule in eukaryotic and prokaryotic cells. A previous study revealed an NO synthase-independent NO production metabolic cycle in which the three nitrogen oxides, nitrate (NO3-), nitrite (NO2-), and NO, were generated in the actinobacterium Streptomyces coelicolor A3(2). NO was suggested to act as a signaling molecule, functioning as a hormone that regulates secondary metabolism. Here, we demonstrate the NO-mediated regulation of the production of the blue-pigmented antibiotic actinorhodin (ACT), via the heme-based DevS/R two-component system (TCS). Intracellular NO controls the stabilization or inactivation of DevS, depending on the NO concentration. An electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR analysis revealed the direct binding between DevR and the promoter region of actII-ORF4, resulting in gene expression. Our results indicate that NO regulates the DevS/R TCS, thereby strictly controlling the secondary metabolism of S. coelicolor A3(2). IMPORTANCE Diverse organisms, such as mammals, plants, and bacteria, utilize NO via well-known signal transduction mechanisms. Many useful secondary metabolite-producing bacteria of the Streptomyces genus had been also suggested for the metabolism regulated by endogenously produced NO; however, the regulatory mechanisms remain to be elucidated. In this study, we demonstrated the molecular mechanism by which endogenously produced NO regulates antibiotic production via the DevS/R TCS in S. coelicolor A3(2). NO serves as both a stabilizer and a repressor in the regulation of antibiotic production. This report shows the mechanism by which Streptomyces utilizes endogenously produced NO to modulate its normal life cycle. Moreover, this study implies that studying NO signaling in actinobacteria can help in the development of both clinical strategies against pathogenic actinomycetes and the actinobacterial industries.