Biology subjects

Nodwell, J. R.

Publications and source records attributed to Nodwell, J. R..

7 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle packaging of a supercluster-encoded antimicrobial combination with enhanced potency against fungal biofilms

Bacterial specialized metabolites are bioactive compounds that have been mined extensively as antibacterials, antifungals, and other drugs. They are widely used as experimental probes of complex biological systems. Since their discovery, their functional characteristics have typically been investigated as single, soluble compounds. The discoveries in this work expand this model. First, we report the co-packaging in extracellular vesicles (EVs) of two specialized metabolite complexes, candicidin and antimycin A, the products of a supercluster of biosynthetic genes, by a Streptomyces soil isolate. Secondly, we report that the products of this supercluster have synergistic antimicrobial activity against fungal biofilms, whereas biofilms are tolerant to the individual compounds. Finally, we show that co-packaging these compounds in EVs ensures simultaneous delivery with enhanced potency. This research advances understanding of the elegant complexity that shapes the evolution and function of bacterial specialized metabolites. In particular, the enhanced antibiofilm activity of co-packaged metabolites compared to individual soluble compounds is notable given the relevance of biofilms to microbial ecology and to pathogenesis. Our work supports the evolution of coordinated activity of specialized metabolites to counter conditional environmental challenges, and highlights co-packaging in EVs as one mechanism for secretion and co-delivery of combinations of bioactive molecules.

microbiology↗

bioRxiv2026

A Cationic Amphiphilic Drug (CAD) Defense System in the Nematode Caenorhabditis elegans

Cationic Amphiphilic Drugs (CADs) severely disrupt lysosomal function, which leads to a cellular pathology characterized by excess phospholipids called phospholipidosis. Through a forward genetic screen and mining of published datasets, we discovered that CADs induce the expression of the CYP-35B family of cytochrome P450s and the PGP-13 p-glycoprotein pump via the nuclear receptors NHR-70 and NHR-107 in the nematode C. elegans. A pgp-13 fluorescent reporter revealed hundreds of human drugs that upregulate the CAD defense system in vivo. Chemoinformatic analyses indicate that the pgp-13 reporter may be useful in identifying CADs that have pathogenic potential in humans. Mutant analyses coupled to metabolomics and structural modeling show that the CYP-35Bs are necessary and sufficient for CAD metabolism, and that CYP-35B2 D311 is key in mediating electrostatic interactions with the positively charged CADs. We also show that CAD metabolites are effluxed via PGP-13 acting partially redundantly with PGP-14 and that an intact defense system is necessary to resist CAD-induced pathology. Finally, we demonstrate that bacteria that likely cohabitate with C. elegans in nature trigger the CAD defense system, providing a plausible explanation for why a pathway that protects against anthropogenic small molecules exists in nematodes.

pharmacology and toxicology↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2024

Phosphatidylethanolamine is a phagocytic ligand implicated in the binding and removal of microbial extracellular vesicles

The efficient recognition and removal of apoptotic cells by phagocytes is critical to prevent secondary necrosis and maintain tissue homeostasis. Such detection involves receptors and bridging molecules that recognize lipids -normally restricted to the inner leaflet of healthy cells- which become exposed on the surface of dead cells and the vesicles they produce. A majority of studies focus on phosphatidylserine (PS) for which there are well-established receptors that either bind to the lipid directly or indirectly via intermediary proteins. Phosphatidylethanolamine (PE) is even more prevalent than PS in the inner leaflet of mammalian cells and also becomes exposed by the action of scramblases during cell death, though little is known about the effects of PE once scrambled. Here, we report that PE can itself serve as a phagocytic ligand for macrophages by engaging CD300 family receptors. CD300a and CD300b specifically modulated PE-mediated uptake, and this process involved ITAM-containing adaptors and the spleen tyrosine kinase (Syk). For bacteria, which contain PE but largely lack PS in their membranes, we report that PE engagement enabled the binding and uptake of spheroplasts and extracellular vesicles (EVs) that were unsheathed by the cell wall. The inflammatory responses of macrophages to PE particles containing LPS was also curtailed by CD300a expression. Based on these observations, we posit that the direct recognition of PE facilitates mechanisms of clearance that stand to have a broad impact on the immune response.

cell biology↗

bioRxiv2023

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 extracellular vesicles are an alternative secretion system. We assessed environmental and lab-adapted Streptomyces (sporulating filamentous actinomycetes) and found a majority of strains produce vesicles packaging antimicrobial compounds. The cargo included actinomycins, anthracyclines, candicidin, and actinorhodin, reflecting both diverse chemical properties and diverse antibacterial and antifungal activity. The production of the antimicrobials and vesicles was linked, although vesicle production continued in the absence of antimicrobials. 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/560488v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@56f1c6org.highwire.dtl.DTLVardef@1b33aeorg.highwire.dtl.DTLVardef@1ddf706org.highwire.dtl.DTLVardef@184e75e_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPORTANCEExtracellular vesicle encapsulation changes our picture of how antimicrobial metabolites function in the environment, and provides an alternative translational approach for delivery of antimicrobials. We find the majority of 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 secretion and action of specialized metabolites as soluble compounds. Importantly the vesicles deliver antimicrobial metabolites directly to other microbes via membrane fusion, including to 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.

microbiology↗

bioRxiv2022

A contractile injection system mediates developmentally regulated intra-strain cell death in Streptomyces coelicolor

Extracellular contractile injection systems (eCIS) are encoded in diverse clades of bacterial species. Although closely related to contractile phage tails, these entities can inject toxic proteins into eukaryotic cells. The roles of eCIS in mediating cytotoxic activities has led to a view of them as defense mechanisms that are not central to normal bacterial lifecycles. Here, we provide evidence that eCIS play an entirely distinct role in Streptomyces coelicolor (Sco), where they appear to participate in the complex developmental process of this species. In particular, we have shown that Sco produces eCIS particles as a part of its normal growth cycle and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Most intriguingly, eCIS-deficient mutants display significantly reduced levels of cell death and altered morphology during liquid growth. These data suggest that Sco eCIS function by inducing intra-strain lethality rather than by attacking foreign species.

microbiology↗

bioRxiv2019

Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2

Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces arsenal of signalling and antagonistic compounds.

microbiology↗