Streptomyces extracellular vesicles are a broad and permissive antimicrobial packaging and delivery system.

Streptomyces are the primary source of bioactive specialized metabolites used in research and medicine, including many antimicrobials. These are presumed to be secreted and function as freely soluble compounds. However, increasing evidence suggests that extracellular vesicles are an alternative secretion system. We assessed environmental and lab-adapted Streptomyces (sporulating filamentous actinomycetes) and found frequent production of antimicrobial vesicles. The molecular cargo included actinomycins, anthracyclines, candicidin, and actinorhodin, reflecting both diverse chemical properties and diverse antibacterial and antifungal activity. The levels of packaged antimicrobials correlated with the level of inhibitory activity of the vesicles, and a strain knocked out for the production of anthracyclines produced vesicles that lacked antimicrobial activity. We demonstrated that antimicrobial containing vesicles achieve direct delivery of the cargo to other microbes. Notably, this delivery via membrane fusion occurred to a broad range of microbes, including pathogenic bacteria and yeast. Vesicle encapsulation offers a broad and permissive packaging and delivery system for antimicrobial specialized metabolites, with important implications for ecology and translation.IMPORTANCEExtracellular vesicle encapsulation changes our picture of how antimicrobial metabolites function in the environment and provides an alternative translational approach for the delivery of antimicrobials. We find many Streptomyces strains are capable of releasing antimicrobial vesicles, and at least four distinct classes of compounds can be packaged, suggesting this is widespread in nature. This is a striking departure from the primary paradigm of the secretion and action of specialized metabolites as soluble compounds. Importantly, the vesicles deliver antimicrobial metabolites directly to other microbes via membrane fusion, including pathogenic bacteria and yeast. This suggests future applications in which lipid-encapsulated natural product antibiotics and antifungals could be used to solve some of the most pressing problems in drug resistance.

A contractile injection system is required for developmentally regulated cell death in Streptomyces coelicolor.

Diverse bacterial species produce extracellular contractile injection systems (eCISs). Although closely related to contractile phage tails, eCISs can inject toxic proteins into eukaryotic cells. Thus, these systems are commonly viewed as cytotoxic defense mechanisms that are not central to other aspects of bacterial biology. Here, we provide evidence that eCISs appear to participate in the complex developmental process of the bacterium Streptomyces coelicolor. In particular, we show that S. coelicolor produces eCIS particles during its normal growth cycle, and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Furthermore, eCIS-deficient mutants display reduced levels of cell death and altered morphology during growth in liquid media. Our results suggest that the main role of eCISs in S. coelicolor is to modulate the developmental switch that leads to aerial hyphae formation and sporulation, rather than to attack other species.

PRKN/parkin-mediated mitophagy is induced by the probiotics Saccharomyces boulardii and Lactococcus lactis.

Mitochondrial impairment is a hallmark feature of neurodegenerative disorders, such as Parkinson disease, and PRKN/parkin-mediated mitophagy serves to remove unhealthy mitochondria from cells. Notably, probiotics are used to alleviate several symptoms of Parkinson disease including impaired locomotion and neurodegeneration in preclinical studies and constipation in clinical trials. There is some evidence to suggest that probiotics can modulate mitochondrial quality control pathways. In this study, we screened 49 probiotic strains and tested distinct stages of mitophagy to determine whether probiotic treatment could upregulate mitophagy in cells undergoing mitochondrial stress. We found two probiotics, Saccharomyces boulardii and Lactococcus lactis, that upregulated mitochondrial PRKN recruitment, phospho-ubiquitination, and MFN degradation in our cellular assays. Administration of these strains to Drosophila that were exposed to paraquat, a mitochondrial toxin, resulted in improved longevity and motor function. Further, we directly observed increased lysosomal degradation of dysfunctional mitochondria in the treated Drosophila brains. These effects were replicated in vitro and in vivo with supra-physiological concentrations of exogenous soluble factors that are released by probiotics in cultures grown under laboratory conditions. We identified methyl-isoquinoline-6-carboxylate as one candidate molecule, which upregulates mitochondrial PRKN recruitment, phospho-ubiquitination, MFN degradation, and lysosomal degradation of damaged mitochondria. Addition of methyl-isoquinoline-6-carboxylate to the fly food restored motor function to paraquat-treated Drosophila. These data suggest a novel mechanism that is facilitated by probiotics to stimulate mitophagy through a PRKN-dependent pathway, which could explain the potential therapeutic benefit of probiotic administration to patients with Parkinson disease.

A defect in cell wall recycling confers antibiotic resistance and sensitivity in Staphylococcus aureus.

WalKR is a two-component system that is essential for viability in Gram-positive bacteria that regulates the all-important autolysins in cell wall homeostasis. Further investigation of this essential system is important for identifying ways to address antibiotic resistance. Here, we show that a T101M mutation in walR confers a defect in autolysis, a thickened cell wall, and decreased susceptibility to antibiotics that target lipid II cycle, a phenotype that is reminiscent of the clinical resistance form known as vancomycin intermediate-resistant Staphylococcus aureus. Importantly, this is accompanied by dramatic sensitization to tunicamycin. We demonstrate that this phenotype is due to partial collapse of a pathway consisting of autolysins, AtlA and Sle1, a transmembrane sugar permease, MurP, and GlcNAc recycling enzymes, MupG and MurQ. We suggest that this causes a shortage of substrate for the peptidoglycan biosynthesis enzyme MraY, causing it to be hypersensitive to competitive inhibition by tunicamycin. In conclusion, our results constitute a new molecular model for antibiotic sensitivity in S. aureus and a promising new route for antibiotic discovery.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

High-Throughput Chemical Screen Identifies a 2,5-Disubstituted Pyridine as an Inhibitor of Candida albicans Erg11.

Fungal infections contribute to over 1.5 million deaths annually, with Candida albicans representing one of the most concerning human fungal pathogens. While normally commensal in nature, compromise of host immunity can result in C. albicans disseminating into the human bloodstream, causing infections with mortality rates of up to 40%. A contributing factor to this high mortality rate is the limited arsenal of antifungals approved to treat systemic infections. The most widely used antifungal class, the azoles, inhibits ergosterol biosynthesis by targeting Erg11. The rise of drug resistance among C. albicans clinical isolates, particularly against the azoles, has escalated the need to explore novel antifungal strategies. To address this challenge, we screened a 9,600-compound subset of the University of Tokyo Core Chemical Library to identify molecules with novel antifungal activity against C. albicans. The most potent hit molecule was CpdLC-6888, a 2,5-disubstituted pyridine compound, which inhibited growth of C. albicans and closely-related species. Chemical-genetic, biochemical, and modeling analyses suggest that CpdLC-6888 inhibits Erg11 in a manner similar to the azoles despite lacking the canonical five-membered nitrogen-containing azole ring. This work characterizes the antifungal activity of a 2,5-disubstituted pyridine against C. albicans, supporting the mining of existing chemical collections to identify compounds with novel antifungal activity. IMPORTANCE Pathogenic fungi represent a serious but underacknowledged threat to human health. The treatment and management of these infections relies heavily on the use of azole antifungals, a class of molecules that contain a five-membered nitrogen-containing ring and inhibit the biosynthesis of the key membrane sterol ergosterol. By employing a high-throughput chemical screen, we identified a 2,5-disubstituted pyridine, termed CpdLC-6888, as possessing antifungal activity against the prominent human fungal pathogen Candida albicans. Upon further investigation, we determined this molecule exhibits azole-like activity despite being structurally divergent. Specifically, transcriptional repression of the azole target gene ERG11 resulted in hypersensitivity to CpdLC-6888, and treatment of C. albicans with this molecule blocked the production of the key membrane sterol ergosterol. Therefore, this work describes a chemical scaffold with novel antifungal activity against a prevalent and threatening fungal pathogen affecting human health, expanding the repertoire of compounds that can inhibit this useful antifungal drug target.

A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-ß-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-ß-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

Biology and applications of co-produced, synergistic antimicrobials from environmental bacteria.

Environmental bacteria, such as Streptomyces spp., produce specialized metabolites that are potent antibiotics and therapeutics. Selected specialized antimicrobials are co-produced and function together synergistically. Co-produced antimicrobials comprise multiple chemical classes and are produced by a wide variety of bacteria in different environmental niches, suggesting that their combined functions are ecologically important. Here, we highlight the exquisite mechanisms that underlie the simultaneous production and functional synergy of 16 sets of co-produced antimicrobials. To date, antibiotic and antifungal discovery has focused mainly on single molecules, but we propose that methods to target co-produced antimicrobials could widen the scope and applications of discovery programs.

The ARC2 response in Streptomcyes coelicolor requires the global regulatory genes afsR and afsS.

ARC2 is a synthetic compound, related in structure and mechanism to the antibiotic triclosan, that activates the production of many specialized metabolites in the Streptomyces genus of bacteria. In this work, we demonstrate that the addition of ARC2 to Streptomyces coelicolor cultures results in considerable alterations in overall gene expression including most notably the specialized metabolic genes. Using actinorhodin production as a model system, we show that the effect of ARC2 depends on the pleiotropic regulators afsR and afsS but not afsK. We find that the constitutive expression of afsS can bypass the need for afsR but not the reverse, while the constitutive expression of afsK had no effect on actinorhodin production. These data are consistent with a model in which ARC2 activates a cell stress response that depends on AfsR activating the expression of the afsS gene such that AfsS then triggers the production of actinorhodin.

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Chemical and biological studies on the soft coral Nephthea sp.

Soft corals belonging to the family Nephtheidae have been appreciated as marine sources of diverse metabolites with promising anticancer potential. In view of that, the current work investigates the anti-proliferative potential of the crude extract, different fractions, and green synthesized silver nanoparticles (AgNPs) of the Red Sea soft coral, Nephthea sp. against a panel of tumor cell lines. The metabolic pool of the soft coral under study was also explored via an LC-HR-ESI-MS metabolomics approach, followed by molecular docking analysis of the characterized metabolites against the target proteins, EGFR, VEGFR, and HER2 (erbB2) that are known to be involved in cancer cell proliferation, growth, and survival. Overall, the n-butanol fraction of Nephthea sp. exhibited the highest inhibitory activities against MCF7 (breast cancer) and A549 (lung cancer) cell lines, with interesting IC50 values of 2.30 ± 0.07 and 3.12 ± 0.10 µg ml-1, respectively, whereas the maximum growth inhibition of HL60 (leukemia) cells was recorded by the total extract (IC50 = 2.78 ± 0.09 µg ml-1). More interestingly, the anti-proliferative potential of the total soft coral extract was evidently improved when packaged in the form of biogenic AgNPs, particularly against A549 and MCF7 tumor cells, showing IC50 values of 0.72 ± 0.06 and 9.32 ± 0.57 µg ml-1, respectively. On the other hand, metabolic profiling of Nephthea sp. resulted in the annotation of structurally diverse terpenoids, some of which displayed considerable binding affinities and molecular interactions with the studied target proteins, suggesting their possible contribution to the anti-proliferative properties of Nephthea sp. via inhibition of tyrosine kinases, especially the EGFR type. Taken together, the present findings highlighted the relevance of Nephthea sp. to future anticancer drug discovery and provided a base for further work on the green synthesis of a range of bioactive NPs from marine soft corals.

Metabolomic profiling and biological investigation of Tabebuia Aurea (Silva Manso) leaves, family Bignoniaceae.

Both ethyl acetate and aqueous fractions of Tabebuia aurea leaves exhibited noteworthy antioxidant and nephroprotective activities against carbon tetrachloride (CCl4)-induced nephrotoxicity in rats, as evidenced by the remarkable improvements of renal serum biomarkers and histopathological features. Additionally, the ethyl acetate fraction displayed a prominent in vitro antitrypanosomal activity against Trypanosoma brucei; consequently, the leaves were subjected to LC-HR-ESI-MS metabolomic profiling to discover the constituents that possibly underlie their bioactivities. Therefore, ten metabolites were characterized, mostly dominated by flavonoids. Interestingly, two identified constituents viz., 3,9,12,15-octadecatetraenoic acid (9) and 9,11,13-octadecatrienoic acid (10) are reported firstly herein from the genus Tabebuia. Furthermore, among the dereplicated constituents, rutin (5) and kaempferol 3-O-rutinoside (6) exhibited the highest docking scores as effective antitrypanosomal compounds.

An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris.

Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.

Chemical entrapment and killing of insects by bacteria.

Actinobacteria produce antibacterial and antifungal specialized metabolites. Many insects harbour actinobacteria on their bodies or in their nests and use these metabolites for protection. However, some actinobacteria produce metabolites that are toxic to insects and the evolutionary relevance of this toxicity is unknown. Here we explore chemical interactions between streptomycetes and the fruit fly Drosophila melanogaster. We find that many streptomycetes produce specialized metabolites that have potent larvicidal effects against the fly; larvae that ingest spores of these species die. The mechanism of toxicity is specific to the bacterium's chemical arsenal: cosmomycin D producing bacteria induce a cell death-like response in the larval digestive tract; avermectin producing bacteria induce paralysis. Furthermore, low concentrations of volatile terpenes like 2-methylisoborneol that are produced by streptomycetes attract fruit flies such that they preferentially deposit their eggs on contaminated food sources. The resulting larvae are killed during growth and development. The phenomenon of volatile-mediated attraction and specialized metabolite toxicity suggests that some streptomycetes pose an evolutionary risk to insects in nature.

Natural Products Repertoire of the Red Sea.

Marine natural products have achieved great success as an important source of new lead compounds for drug discovery. The Red Sea provides enormous diversity on the biological scale in all domains of life including micro- and macro-organisms. In this review, which covers the literature to the end of 2019, we summarize the diversity of bioactive secondary metabolites derived from Red Sea micro- and macro-organisms, and discuss their biological potential whenever applicable. Moreover, the diversity of the Red Sea organisms is highlighted as well as their genomic potential. This review is a comprehensive study that compares the natural products recovered from the Red Sea in terms of ecological role and pharmacological activities.

Dual-PKS Cluster for Biosynthesis of a Light-Induced Secondary Metabolite Found from Genome Sequencing of Hyphodiscus hymeniophilus Fungus.

Filamentous fungi are known producers of important secondary metabolites. In spite of this, the majority of these organisms have not been studied at the genome level, leaving many of the bioactive molecules they produce undiscovered. In this study, we explore the secondary metabolite potential of an understudied fungus, Hyphodiscus hymeniophilus. By sequencing and assembling the first genome from this genus, we show that this fungus has genes for at least 20 natural products and that many of these products are likely novel. One of these metabolites is identified: a new, red-pigmented member of the azaphilone class, hyphodiscorubrin. We show that this metabolite is only produced when the fungus is grown in the light. Furthermore, the biosynthetic gene cluster of hyphodiscorubrin is identified though homology to other known azaphilone producing clusters.