Mode of action of antimicrobial agents albofungins in eradicating penicillin- and cephalosporin-resistant Vibrio parahaemolyticus biofilm.

Albofungin is a promising broad-spectrum antimicrobial compound against multidrug-resistant bacteria. In the present study, we further investigated albofungin's biofilm eradication activity and its potential mode of action against drug-resistant Vibrio parahaemolyticus. Among all derivatives, albofungin exhibited the best antibiofilm and antibacterial activity with rapid killing effects at 0.12 µg mL-1. Confocal microscopy observation exhibited that albofungin disrupted V. parahaemolyticus biofilms by killing or dispersing biofilm cells. Meanwhile, scanning electron microscope and fluorescent staining experiments demonstrated that albofungin rapidly destroyed the integrity and permeability of the bacterial cell membrane. Moreover, this study revealed an antibiofilm mechanism of albofungin involving inhibition of peptidoglycan biosynthesis, flagella assembly pathways, and secretion system proteins in V. parahaemolyticus by quantitative proteomics and validation experiments. Our results highlighted albofungin's mechanism of action in planktonic cells and biofilms and suggested further development and potential applications of albofungin for treating infections caused by penicillins-and-cephalosporins-resistant V. parahaemolyticus. IMPORTANCE Infections caused by multidrug-resistant bacteria, as well as a scarcity of new antibiotics, have become a major health threat worldwide. To tackle the demand for new and effective treatments, we investigated the mechanism of action of albofungin, a natural product derived from Streptomyces, which exhibits potent antimicrobial activity against multidrug-resistant bacteria. Albofungin showed potent biofilm eradication activity against penicillins-and-cephalosporins-resistant Vibrio parahaemolyticus, which expresses a novel metallo-ß-lactamase and, thus, reduces their sensitivity to various antibiotics. We observed membrane disruption and permeation mechanisms in planktonic cells and biofilms after albofungin treatment, while albofungin had a weak interaction with bacterial DNA. Moreover, the antibiofilm mechanism of albofungin included inhibition of peptidoglycan biosynthesis, flagellar assembly pathways, and secretion system proteins. Our finding suggested potential applications of albofungin as an antibacterial and antibiofilm therapeutic agent.

Mode of action of antifouling compound albofungin in inhibiting barnacle larval settlement.

Marine biofouling causes huge economic losses to the marine industry every year. Albofungin is a potential antifoulant showing strong anti-macrofouling activities against larval settlement of major fouling organisms. In the present study, directed RNA-seq and proteomic analyses were used to investigate changes in the transcriptome and proteome of a major fouling barnacle Amphibalanus amphitrite cyprids in response to albofungin treatment. Results showed that albofungin treatment remarkably upregulated the metabolism of xenobiotics by the cytochrome P450 pathway to discharge the compound and downregulated energy metabolic processes. Intriguingly, immunostaining and whole-mount in situ hybridization (WISH) revealed the spatial expression patterns of selected differentially expressed genes (glutathione S-transferase [GST], nitric oxide synthase [NOS], and calmodulin [CaM]) distributed in the thorax and antennule of A. amphitrite. Our study provides new insights into the mechanism of albofungin in interrupting the larval settlement of A. amphitrite and suggests its potential application as an antifouling agent in marine environments.

Tau-aggregation inhibitors derived from Streptomyces tendae MCCC 1A01534 protect HT22 cells against okadaic acid-induced damage.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by tau aggregating into neurofibrillary tangles. Targeting tau aggregation is one of the most critical strategies for AD treatment and prevention. Herein, a high-throughput screening of tau-aggregation inhibitors was performed by thioflavin T (ThT) fluorescence assay and tauR3 peptides. According to bioactivity-guided isolation, homoprejadomycin (1) was obtained from the marine bacterium Streptomyces tendae MCCC 1A01534. Two new stable derivatives, 2 and 3, were yielded in a one-step reaction. By ThT assay, transmission electron microscopy, and circular dichroism, we demonstrated that the angucyclinones 2 and 3 inhibited tau aggregation and disaggregated tau fibrils. In the presence of 2, native tauR3 peptides maintained the disorder conformation, whereas the tauR3 aggregates reduced ß-sheet structures. And compound 2 was confirmed to inhibit the aggregation of full-length 2N4R tau protein. Furthermore, 2 with low cytotoxicity protected HT22 cells from okadaic acid-induced damage by suppressing tau aggregates. These results indicated that 2 was a promising lead structure with tau therapeutic potency for AD treatment.

Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models.

The G-quadruplex (G4) forming C9orf72 GGGGCC (G4C2) expanded hexanucleotide repeat (EHR) is the predominant genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Developing selective G4-binding ligands is challenging due to the conformational polymorphism and similarity of G4 structures. We identified three first-in-class marine natural products, chrexanthomycin A (cA), chrexanthomycin B (cB), and chrexanthomycin C (cC), with remarkable bioactivities. Thereinto, cA shows the highest permeability and lowest cytotoxicity to live cells. NMR titration experiments and in silico analysis demonstrate that cA, cB, and cC selectively bind to DNA and RNA G4C2 G4s. Notably, cA and cC dramatically reduce G4C2 EHR-caused cell death, diminish G4C2 RNA foci in (G4C2)29-expressing Neuro2a cells, and significantly eliminate ROS in HT22 cells. In (G4C2)29-expressing Drosophila, cA and cC significantly rescue eye degeneration and improve locomotor deficits. Overall, our findings reveal that cA and cC are potential therapeutic agents deserving further clinical study.

Proteomining-Based Elucidation of Natural Product Biosynthetic Pathways in Streptomyces.

The genus Streptomyces is known to harbor numerous biosynthetic gene clusters (BGCs) of potential utility in synthetic biology applications. However, it is often difficult to link uncharacterized BGCs with the secondary metabolites they produce. Proteomining refers to the strategy of identifying active BGCs by correlating changes in protein expression with the production of secondary metabolites of interest. In this study, we devised a shotgun proteomics-based workflow to identify active BGCs during fermentation when a variety of compounds are being produced. Mycelia harvested during the non-producing growth phase served as the background. Proteins that were differentially expressed were clustered based on the proximity of the genes in the genome to highlight active BGCs systematically from label-free quantitative proteomics data. Our software tool is easy-to-use and requires only 1 point of comparison where natural product biosynthesis was significantly different. We tested our proteomining clustering method on three Streptomyces species producing different compounds. In Streptomyces coelicolor A3(2), we detected the BGCs of calcium-dependent antibiotic, actinorhodin, undecylprodigiosin, and coelimycin P1. In Streptomyces chrestomyceticus BCC24770, 7 BGCs were identified. Among them, we independently re-discovered the type II PKS for albofungin production previously identified by genome mining and tedious heterologous expression experiments. In Streptomyces tenebrarius, 5 BGCs were detected, including the known apramycin and tobramycin BGC as well as a newly discovered caerulomycin A BGC in this species. The production of caerulomycin A was confirmed by LC-MS and the inactivation of the caerulomycin A BGC surprisingly had a significant impact on the secondary metabolite regulation of S. tenebrarius. In conclusion, we developed an unbiased, high throughput proteomics-based method to complement genome mining methods for the identification of biosynthetic pathways in Streptomyces sp.

Discovery, Yield Improvement, and Application in Marine Coatings of Potent Antifouling Compounds Albofungins Targeting Multiple Fouling Organisms.

Marine biofouling caused huge economic losses of maritime industries. We aim to develop high-efficient, less-toxic, and cost-effective antifoulants to solve the problems of biofouling. In this study, we described the antifouling compounds albofungin and its derivatives (albofungin A, chrestoxanthone A, and chloroalbofungin) isolated from the metabolites of bacterium Streptomyces chrestomyceticus BCC 24770, the construction of high-yield strains for albofungin production, and application of albofungin-based antifouling coatings. Results showed that these albofungins have potent antibiofilm activities against Gram-positive and Gram-negative bacteria and anti-macrofouling activities against larval settlement of major fouling organisms with low cytotoxicity. With the best antifouling activity and highest yield in bacterial culture, albofungin was subsequently incorporated with hydrolyzable and degradable copolymer to form antifouling coatings, which altered biofilm structures and prevented the settlement of macrofouling organisms in marine environments. Our results suggested that albofungins were promising antifouling compounds with potential application in marine environments.

Discovery, Bioactivity Evaluation, Biosynthetic Gene Cluster Identification, and Heterologous Expression of Novel Albofungin Derivatives.

The crude extract of Streptomyces chrestomyceticus exhibited strong and broad activities against most "ESKAPE pathogens." We conducted a comprehensive chemical investigation for secondary metabolites from the S. chrestomyceticus strain and identified two novel albofungin (alb) derivatives, i.e., albofungins A (1) and B (2), along with two known compounds, i.e., albofungin (3) and chloroalbofungin (4). The chemical structures of the novel compounds were elucidated using HRMS, 1D and 2D NMR, and electronic circular dichroism spectroscopy. The draft genome of S. chrestomyceticus was sequenced, and a 72 kb albofungin (alb) gene cluster with 72 open reading frames encoding type II polyketide synthases (PKSs), regulators, and transporters, and tailoring enzymes were identified using bioinformatics analysis. The alb gene cluster was confirmed using the heterologous expression in Streptomyces coelicolor, which successfully produced the compounds 3 and 4. Furthermore, compounds 1-4 displayed remarkable activities against Gram-positive bacteria and antitumor activities toward various cancer cells. Notably, compounds 1 and 3 showed potent activities against Gram-negative pathogenic bacteria. The terminal deoxynucleotidyl transferase (dUTP) nick-end labeling and flow cytometry analysis verified that compound 1 inhibited cancer cell proliferation by inducing cellular apoptosis. These results indicated that albofungins might be potential candidates for the development of antibiotics and antitumor drugs.

ATM loss disrupts the autophagy-lysosomal pathway.

ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A1; CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB: cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate; Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol binding protein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM: standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.

Albofungin and chloroalbofungin: antibiotic crystals with 2D but not 3D isostructurality.

The potent antibiotics albofungin [systematic name: (1S,4R,8aR)-13-amino-1,15,16-trihydroxy-4-methoxy-12-methyl-3,4,8a,13-tetrahydro-1H-xantheno[4',3',2':4,5][1,3]benzodioxino[7,6-g]isoquinoline-14,17(2H,9H)-dione, C27H24N2O9, 1] and its chlorinated analogue chloroalbofungin (the 11-chloro analogue, C27H23ClN2O9, 2) have been crystallized following their isolation from the bacterial strain Streptomyces chrestomyceticus and their structures determined by single-crystal X-ray diffraction. The novel N-aminoquinolone molecular arrangement shows N-N bond lengths of 1.4202 (16) and 1.424 (2) Šin 1 and 2, respectively. The regiochemistry of chloro substitution in the A-ring is para to the quinolone O atom, with a C-Cl bond length of 1.741 (2) Å. The absolute stereochemistry at three chiral centres of the xanthone rings (i.e. 10S, 13R and 19R) is confirmed. Both compounds crystallize in chiral Sohncke space groups consistent with enantiopurity, but are not fully isostructural. A preserved supramolecular construct (SC) confers two-dimensional (2D) isostructurality, but the SC self-associates via either a twofold screw operation in 1, giving a monoclinic P21 structure, or a twofold rotation in 2, affording a monoclinic C2 structure with a doubled unit-cell axis.

A novel chresdihydrochalcone from Streptomyces chrestomyceticus exhibiting activity against Gram-positive bacteria.

Microbial-derived natural products provide unique bioactivities and serve as a unique source of drug leads. In the present study, we isolated one new chresdihydrochalcone (1), one new chresphenylacetone (2), and one known streptimidone (3) from Streptomyces chrestomyceticus BCC 24770 using antibacterial activity-guided isolation and purification procedures. We determined their molecular weights using MS and HRMS and elucidated their chemical structures from their 1D and 2D NMR and electronic circular dichroism (ECD) spectra. Compound 1 showed moderate inhibitory activities against the Gram-positive bacteria Methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus. Cytotoxicity and hemolytic activity were not observed at a concentration of up to 100 µg ml-1. The specific antimicrobial activity and low toxicity of compound 1 indicate this compound to be a potential antibiotic candidate, especially as antibiotic resistance has become a significant public health threat.