Biology subjects

Wright, G. D.

Publications and source records attributed to Wright, G. D..

9 recordsLinked to original sources

bioRxiv2026

Vacidobactin A: An anti-Pseudomonas aeruginosa siderophore

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a significant clinical challenge due to its poorly permeable outer membrane, efflux systems, biofilm formation, and rapid acquisition of resistance genes. The lack of new treatments for P. aeruginosa infections underscores the necessity for innovative therapeutic solutions. Iron uptake is essential for bacterial survival, making it a promising target for the development of new antimicrobials. Iron-chelating siderophores are vital for bacterial iron acquisition, important agents for direct antimicrobial action, adjuvants to enhance the effectiveness of currently available antibiotics, and components of prodrugs that facilitate the transport of covalently linked antibiotics into the cell. Here, we report the anti-pseudomonal activity of vacidobactin A, a siderophore produced by the soil bacterium Variovorax paradoxus, identified through a screen of natural product extracts targeting a clinical MDR strain of P. aeruginosa. Vacidobactin A inhibits P. aeruginosa growth by limiting iron availability, particularly in strains that do not produce pyoverdine, their native siderophore. Expression of a TonB-dependent transporter sourced from the vacidobactin producer in a P. aeruginosa pyoverdine and pyochelin-null mutant restored its ability to acquire iron and grow in the presence of vacidobactin. Additionally, vacidobactin A synergized with thiostrepton, which hijacks pyoverdine receptors to enter the cell and inhibit protein synthesis. This study supports the therapeutic potential of targeting P. aeruginosa iron acquisition pathways and leveraging siderophores as adjuvants to enhance the efficacy of existing antimicrobials. These findings, along with recent advancements in siderophore-based research and combination therapies, offer innovative strategies to combat antibiotic-resistant infections. IMPORTANCEMultidrug-resistant Pseudomonas aeruginosa is a critical priority pathogen for which new therapeutic strategies are urgently needed. Iron acquisition is essential for P. aeruginosa survival and virulence, yet remains underexploited as a drug target. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. We further show that vacidobactin A enhances the activity of thiostrepton, an antibiotic that exploits siderophore uptake pathways. These findings highlight iron competition as a source of anti-pseudomonal agents and support the development of siderophore-based therapeutics and adjuvant strategies. Targeting iron acquisition networks offers a mechanistically distinct approach to combat antibiotic resistance and expands the repertoire of vulnerabilities that can be leveraged against this highly drug-resistant pathogen.

microbiology↗

bioRxiv2025

A ubiquitous Streptomyces biosynthetic megacluster encodes an arsenal of synergistic biotin-targeting antibiotics

The rise of multidrug-resistant pathogens underscores the urgent need for antibiotics that act through new targets and mechanisms. Biotin metabolism, essential in most bacteria, remains underexploited therapeutically. Here, we uncover a highly conserved, co-located biosynthetic megacluster in Streptomyces, a striking "cluster of clusters", that encodes four distinct natural product families: acidomycin, stravidins, dapamycins, and -methyl-KAPA, and is flanked by genes that encode streptavidin, a high-affinity biotin-binding protein. Remarkably, all molecules target different steps in bacterial biotin metabolism, revealing a multi-pronged natural strategy for biotin starvation. This arrangement of four functionally convergent biosynthetic gene clusters at a single genomic locus is without precedent. Even more surprisingly, we find that this anti-biotin megacluster is widespread across Streptomyces bacteria, suggesting a deeply conserved evolutionary solution to microbial competition. Mechanistically, the compounds inhibit biotin biosynthesis through enzyme blockade, prodrug activation, covalent cofactor mimicry, and biotin sequestration via co-expressed streptavidin. Stravidin S2 and -methyl-KAPA are effective in a murine model of multidrug-resistant E. coli infection. These findings expose a coordinated biosynthetic logic in microbial secondary metabolites and point to higher-order biosynthetic architectures as promising reservoirs of antibiotic innovation.

microbiology↗

bioRxiv2025

A Saccharomyces boulardii synthetic biotic platform for delivery of therapeutic nanobodies to ameliorate gastrointestinal inflammation

Protein-based pharmaceuticals, such as engineered antibodies, form a major drug class of steadily increasing market share. However, these biologic medicines are costly to manufacture, are subject to strict supply chain and storage constraints and often require invasive administration routes. Engineered microbes that secrete bioactive products directly within the microbiome milieu may mitigate these challenges. Here, we describe a cell microfactory platform based on the probiotic yeast Saccharomyces boulardii for the production of nanobody biologics in the gastrointestinal (GI) tract. High-level secretion of nanobodies by S. boulardii was achieved by optimizing promoters, secretion signals, and antibody formats. In mice, oral gavage of S. boulardii allowed efficient and transient colonization of the colonic compartment and in situ production of a therapeutic nanobody directed against tumor necrosis factor (TNF). In a mouse model of chemical-induced colitis, GI-delivery of anti-mTNF nanobody via live S. boulardii improved both survival and disease severity without causing overt perturbation of microbiome composition. These results position S. boulardii as a synthetic biotic platform for the in situ production and delivery of protein-based therapeutics to the GI tract.

synthetic biology↗

bioRxiv2025

The histidine kinase VraS orchestrates the cell-wall stress response in Staphylococcus aureus via its direct interaction with glycopeptides and β-lactams

Multidrug-resistant Staphylococcus aureus is a major global health threat, with the VraTSR three-component system playing a key role in sensing and conferring resistance to cell-wall active antibiotics, particularly vancomycin. VraTSR comprises the membrane histidine kinase VraS, the cytoplasmic response regulator VraR, and the uncharacterized membrane protein VraT, which regulate the cell wall stress stimulon. However, the molecular signals sensed by VraTSR remain unknown. To elucidate the activation mechanism of this regulatory system, we investigated interactions with {beta}-lactams and glycopeptides. Using a transcriptional reporter strain, we confirmed VraTSR activation by {beta}-lactams, glycopeptides, a vancomycin-derived photoprobe (VPP), and the previously unreported activators A47934 and moenomycin A. Photo-crosslinking assays with VPP and full-length VraS expressed in membranes revealed a direct interaction with vancomycin, which was further confirmed in purified VraS reconstituted in liposomes. VPP binding was concentration-dependent, saturable, and displaced by vancomycin. Saturation transfer difference (STD) Nuclear Magnetic Resonance (NMR) experiments confirmed vancomycin binding to VraS and demonstrated ampicillin interaction, highlighting the involvement of aryl protons from both antibiotics. These findings establish VraS as a receptor for vancomycin and ampicillin. In contrast, assays with membrane vesicles expressing only VraT or co-expressing VraS/VraT did not show covalent adduct formation between VraT and VPP. While VraTs exact role remains unclear, its participation in antibiotic sensing or signal transduction cannot yet be excluded. These results demonstrate that vancomycin and ampicillin directly activate VraS, providing critical insights into the activation of the cell wall stress stimulon and the mechanisms underlying antibiotic resistance. Disrupting VraTSR signaling is a promising strategy to combat multidrug resistance in S. aureus, and we provide invaluable in vitro platforms for identifying potential VraS inhibitors. Author SummaryMultidrug-resistant Staphylococcus aureus poses a major global health threat due to its resistance to cell-wall active antibiotics. Our study focuses on the VraTSR three-component system, a key regulator of the cell wall stress response in S. aureus, whose activation signals have remained unknown. We demonstrate that VraS, the membrane histidine kinase of the system, acts as a direct receptor for vancomycin and ampicillin--two structurally distinct antibiotics. These findings uncover the activation mechanism of VraTSR and position VraS as a central player in antibiotic sensing and resistance. By identifying VraS as a direct antibiotic receptor, we provide a promising target for developing inhibitors to disrupt VraTSR signaling and restore antibiotic efficacy. Additionally, the in vitro platforms we established enable the identification and testing of potential VraS inhibitors. This study highlights the importance of understanding bacterial stress-response pathways to combat antibiotic resistance, offering critical insights for developing new therapeutic strategies against multidrug-resistant S. aureus, a growing global health challenge.

biochemistry↗

bioRxiv2025

Butyrolactol A is a phospholipid flippase inhibitor that potentiates the bioactivity of caspofungin against resistant fungi

Fungal infections cause millions of deaths annually and are challenging to treat due to limited therapeutic options and rising resistance. Cryptococci are intrinsically resistant to the latest generation of antifungals, echinocandins, while Candida auris, a notorious global threat, is also increasingly resistant. We performed a natural product extract screen to rescue caspofungin fungicidal activity against Cryptococcus neoformans H99 and identified butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including multidrug-resistant C. auris. Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryo-electron microscopy analysis of the Apt1*butyrolactol A complex revealed that the flippase is trapped in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies lipid flippases as promising antifungal targets and demonstrates the potential of revisiting natural products to expand the antifungal arsenal and combat resistance.

biochemistry↗

bioRxiv2024

CARPDM: cost-effective antibiotic resistome profiling of metagenomic samples using targeted enrichment

Better interrogation of antimicrobial resistance requires new approaches to detect the associated genes in metagenomic samples. Targeted enrichment is an ideal method for their sequencing and characterization. However, no open-source, up-to-date hybridization probe set targeting antimicrobial resistance genes exists. Here we describe CARPDM, a probe design software package made to run alongside all future Comprehensive Antibiotic Resistance Database releases. To test its efficacy, we have created and validated two separate probe sets: AllCARD, which enriches all genes encoded in the Comprehensive Antibiotic Resistance Databases protein homolog models (n = 4,661), and clinicalCARD, which focuses on a clinically relevant subset of resistance genes (n = 323). We demonstrate that allCARD increases the number of reads mapping to resistance genes by up to 594-fold. ClinicalCARD performs similarly when clinically relevant genes are present, increasing the number of resistance-gene mapping reads by up to 598-fold. In parallel with this development, we have established a protocol to synthesize any probe set in-house, saving up to 350 dollars per reaction. Together, these probe sets, their associated design program CARPDM, and the protocol for in-house synthesis will democratize metagenomic resistome analyses, allowing researchers access to a cost-effective and efficient means to explore the antibiotic resistome.

genomics↗

bioRxiv2021

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are widely used for the management of tuberculosis and other bacterial infections. These drugs inhibit prokaryotic RNA polymerase (RNAP) by preventing elongation of mRNA resulting in cell death. Rifamycin resistance in the clinic is manifested primarily through amino acid substitutions in RNAP that decrease target affinity for the antibiotics. In contrast, environmental bacteria possess a wide variety of highly specific rifamycin enzyme-mediated resistance mechanisms that modify and inactivate the antibiotics by glycosylation, phosphorylation, ADP-ribosylation, or hydroxylation. Expression of rifamycin resistance is controlled by a common 19bp cis-acting rifamycin associated element (RAE) upstream of inactivating genes. Guided by the presence of RAE sequences, we identify an unprecedented ATP-dependent mechanism of rifamycin resistance that acts not by antibiotic inactivation but by protecting the RNAP target. We show that Streptomyces venezuelae encodes a helicase-like protein, HelR, which confers broad spectrum rifamycin resistance. Furthermore, HelR is essential for promoting rifamycin tolerance at inhibitory concentrations, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP in vivo and rescues transcription inhibition by rifampin in vitro. We synthesized a rifamycin photoprobe and demonstrated that HelR directly displaces rifamycins from RNAP. HelR-encoding genes associated with RAEs are broadly distributed in actinobacteria, including many opportunistic Mycobacterial pathogens, which cannot currently be treated with rifamycins. This first report of an RNAP protection protein conferring antibiotic resistance and offers guidance for developing new rifamycin antibiotics that can avoid this mechanism.

biochemistry↗

bioRxiv2021

Desmosomal dualism: the core is stable while plakophilin is dynamic.

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calciumindependent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.

cell biology↗

bioRxiv2020

Human AKTIP interacts with ESCRT proteins and functions at the midbody in cytokinesis

To complete mitosis, the intercellular bridge that links daughter cells needs to be cleaved. This abscission step is carried out by the sequential recruitment of ESCRT proteins at the midbody. We report here that a new factor, named AKTIP, works in association with ESCRTs. We find that AKTIP binds to the ESCRT I subunit VPS28, and show by high resolution microscopy that AKTIP forms a ring in the dark zone of the intercellular bridge. This ring is positioned in between the circular structures formed by ESCRTs type III. Functionally, we observe that the reduction of AKTIP impinges on the recruitment of the ESCRT III member IST1 at the midbody and causes abscission defects. Taken together, these data indicate that AKTIP is a new factor that contributes to the formation of the ESCRT complex at the midbody and is implicated in the performance of the ESCRT machinery during cytokinetic abscission.

cell biology↗