Biology subjects

Brown, E. D.

Publications and source records attributed to Brown, E. D..

12 recordsLinked to original sources

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

SyntheMol-RL: a flexible reinforcement learning framework for designing novel and synthesizable antibiotics

The rise of antibiotic-resistant pathogens such as Staphylococcus aureus has created an urgent need for new antibiotics. Generative artificial intelligence (AI) has shown promise in drug discovery, but existing models often fail to propose compounds that are both effective and synthetically tractable. To address these challenges, we introduce SyntheMol-RL, a reinforcement learning-based generative model that can rapidly design synthetically accessible small molecule drug candidates from a massive chemical space of 46 billion compounds. SyntheMol-RL improves upon our prior Monte Carlo Tree Search (MCTS)-based SyntheMol model by generalizing across chemically similar building blocks and enabling multi-parameter optimization. We applied SyntheMol-RL to generate candidate antibiotics against S. aureus by optimizing for both antibacterial activity and aqueous solubility, and we found that SyntheMol-RL generated molecules with improved predicted properties compared to both the previous MCTS version of SyntheMol as well as an AI-based virtual screening baseline. We synthesized 79 SyntheMol-RL compounds that were unique relative to the training dataset and found that 13 showed potent in vitro activity, of which seven were structurally novel after detailed literature searches. Furthermore, one hit compound, synthecin, demonstrated efficacy in a murine wound infection model of methicillin-resistant S. aureus (MRSA). These results validate SyntheMol-RLs ability to generate novel and synthetically accessible candidate antibiotics and position SyntheMol-RL as a powerful tool for drug design across therapeutic domains.

biochemistry↗

bioRxiv2025

Identifying Modulators of the Post-Antibiotic Effect

The post-antibiotic effect (PAE) is the delay in bacterial regrowth following antibiotic removal. It has important implications for dosing regimens since drugs that have extended activity following their elimination can be dosed less frequently, widening the therapeutic window. While the PAE has been associated with target vulnerability and the rate of target turnover, little is known about the genetic components that modulate the PAE. Here, we developed a high-throughput assay to screen the Escherichia coli Keio collection of [~]4000 deletion strains, identifying genes that enhance the PAE for CHIR-090, an inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). This screen revealed approximately 400 gene knockouts that enhanced the PAE of CHIR-090. The list of PAE enhancers was enriched for genes involved in transmembrane transport and outer membrane synthesis. Notably, deletion of the rfaE gene, which is involved in lipopolysaccharide (LPS) biosynthesis, increased the PAE of the LpxC inhibitors CHIR-090 and LPC-058 by 2 h and 3 h, respectively. Consistent with this phenotype, co-treatment of wild-type E. coli with an RfaE inhibitor increased the PAE of CHIR-090 or LPC-058 by 1 h. To probe the mechanism of this interaction, we measured the rate of LpxC turnover and found that knocking out rfaE reduced its half-life by 2-fold, suggesting that disrupting RfaE increases the stability of LpxC, increasing target vulnerability and enhancing the PAE of LpxC inhibitors. SIGNIFICANCEAntibiotics are generally dosed at very high levels leading to unwanted side effects and non-compliance, which in turn results in the emergence of drug-resistant bacterial infections. The goal of this work was to develop strategies that will enable antibiotics to be dosed at lower levels, thereby improving safety and compliance. In the present work, we have screened 4,000 strains of Escherichia coli to identify compounds that result in an increase in the post-antibiotic effect (PAE), which is the delay in bacterial regrowth following antibiotic exposure and removal. Drugs that cause a PAE are dosed less frequently, and the method we describe will provide a new approach to developing safer drugs.

microbiology↗

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

Micro-plaque assays: A high-throughput method to detect, isolate, and characterize bacteriophages

The gold standard for the isolation and characterization of bacteriophages (phages), the plaque assay, has remained almost unchanged for over 100 years. The need for improvements to its scalability has been driven home by successes with personalized phage therapy requiring large phage libraries and rapid sensitivity testing. Using a robotic pinning platform, we miniaturized plaque assays from bacterial lawns to micro-colonies from 100 nl of inoculant, increasing throughput by >1000 fold without compromising sensitivity. A comparable manual workflow with one quarter the throughput maintained the same sensitivity. These micro-plaque assays can replace plaque assays as a new gold standard in phage biology. As proof of principle, we used our technique to isolate and de-replicate 21 unique Pseudomonas aeruginosa phages from a single environmental sample. We then demonstrated - using the same assay - that of 17 multi-drug resistant clinical P. aeruginosa strains, 15 were susceptible to infection by one or more of the 21 phages tested. Our method allows rapid isolation and de-replication of phages, as well as enabling screening of large phage libraries against bacterial isolates of interest.

microbiology↗

bioRxiv2023

A mobile CRISPRi collection enables genetic interaction studies for the essential genes of Escherichia coli

Ordered collections of precise gene deletions in E. coli and other model microbes have proven to be invaluable resources to study the dispensability of the non-essential genome in different environmental and genetic contexts. The dispensability of canonically essential genes, however, has been largely unstudied in such contexts. Advances in gene editing, in particular CRISPR interference (CRISPRi), have enabled depletion of essential cellular machinery to study the downstream effects on bacterial physiology. Here, we describe the construction of an ordered E. coli CRISPRi collection, designed to knock down the expression of 356 essential genes with the induction of a catalytically inactive Cas9, harbored on the conjugative plasmid pFD152, that also encodes for expression of specific guide RNA scaffolds. This mobile CRISPRi library can be conjugated into other ordered genetic libraries, to assess combined effects of essential gene knockdowns with non-essential gene deletions. As proof of concept, we probed cell envelope synthesis with two crosses. First, we crossed a deletion in the gene lpp with the entire CRISPRi library. Similarly, we crossed the CRISPRi knockdown of lolA with [~]4,000 deletion strains of the Keio collection. These experiments revealed a number of notable genetic interactions for the essential phenotype probed and, in particular, showed supressing interactions for the loci in question.

systems biology↗

bioRxiv2022

Screening under infection-relevant conditions reveals chemical sensitivity in multidrug-resistant invasive non-typhoidal Salmonella (iNTS)

Bloodstream infections caused by invasive, non-typhoidal salmonellae (iNTS) are a major global health concern. These infections are especially problematic in sub-Saharan Africa, where the sequence type (ST) 313 of invasive non-typhoidal Salmonella Typhimurium (iNTS) is dominant. Unlike S. Typhimurium strains that cause mild gastroenteritis, iNTS strains are resistant to multiple first-line antibiotics and have higher extraintestinal invasiveness, limiting current treatment options. Here, we performed multiple small molecule screens under infection-relevant conditions to reveal chemical sensitivities in ST313 as entry points to drug discovery to combat the clinical burden of iNTS. By screening the invasive ST313 sequence type under host-mimicking conditions, we identified the antimicrobial activity of the nucleoside analog 3-azido-3-deoxythymidine, which required bacterial thymidine kinase activity for its antimicrobial activity. In a parallel macrophage-based screening platform, we also identified three host-directed compounds (amodiaquine, berbamine, and indatraline) that significantly restricted intracellular replication of ST313 in macrophages without directly impacting bacterial viability. This work provides evidence that despite elevated invasiveness and multidrug resistance, iNTS S. Typhimurium remains susceptible to unconventional drug discovery approaches.

microbiology↗

bioRxiv2022

Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened [~]4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon - controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a pre-activated phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions and failed to increase further with the addition of sub-MIC antibiotics. Supporting a role for respiratory stress, the biofilm stimulation response was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of genes encoding the nitrate respiratory machinery abolished its effects, and nitrate respiration increased during growth with sub-MIC antibiotics. In probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors was minimally affected by nitrate supplementation. Finally, using a metabolism-sensitive dye, we showed spatial co-localization of increased respiration with sub-MIC bactericidal antibiotics. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

microbiology↗

bioRxiv2022

Polynuclear ruthenium complexes are effective antibiotics against Pseudomonas aeruginosa

There is an urgent need to develop new antibiotics for the treatment of infections caused by drug-resistant Gram-negative bacteria. In particular, new and diverse chemical classes of antibiotics are needed, as most antibiotics in clinical development are derivatives of existing drugs. Despite a history of use as antimicrobials, metals and metal-based compounds have largely been overlooked as a source of new chemical matter for antibacterial drug discovery. In this work, we identify several ruthenium complexes, ruthenium red, Ru265, and Ru360, that possess potent antibacterial activity against both laboratory and clinical isolates of Pseudomonas aeruginosa. Suppressors with increased resistance were sequenced and found to contain mutations in the mechanosensitive ion channel mscS-1 or the colRS two component system. The antibacterial activity of these compounds translated in vivo to Galleria mellonella larvae and mouse infection models. Finally, we identify strong synergy between these compounds and the antibiotic rifampicin, with a dose-sparing combination therapy showing efficacy in both infection models. Our findings provide clear evidence that these ruthenium complexes are effective antibacterial compounds against a critical priority pathogen and show promise for the development of future therapeutics.

microbiology↗

bioRxiv2019

Uncovering the hidden antibiotic potential of Cannabis

The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against MRSA, inhibit its ability to form biofilms and eradicate pre-formed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multi-drug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids.

microbiology↗

bioRxiv2019

Armeniaspirols inhibit the ATP-dependent proteases ClpYQ and ClpXP in Gram-positive bacteria, dysregulating the divisome

Multi-drug resistant bacteria present an urgent threat to modern medicine, creating a desperate need for the discovery of antibiotics with new modes of action. Natural products whose unique highly diverse structures have been shaped by evolution to possess biologically relevant activity are an ideal discovery ground for new antibiotics with new mechanisms of action. In this study we elucidate the mechanism of action of the Gram-positive antibiotic armeniaspirol, a compound for which resistant bacteria could not be selected for. We show that armeniaspirol inhibits the ATP-dependent proteases ClpXP and ClpYQ in biochemical assays and in the Gram-positive bacteria Bacillus subtilis. We then show that this activity dysregulates key proteins involved in the divisome and elongasome including FtsZ, DivIVA, and MreB all of which are known to inhibit cell division when upregulated. Inhibition of ClpXP and ClpYQ leading to dysregulation of the divisome and elongasome represents a new mechanism of action and armeniaspirol is the first known natural product inhibitor of the coveted anti-virulence target ClpP. Thus armeniaspirol is the lead compound for a promising new class of antibiotics with a unique pharmacology and a novel mechanism for combating antimicrobial resistance, making it a highly promising candidate for further development.

microbiology↗

bioRxiv2018

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium

Salmonella Typhimurium (S. Tm) evades the innate immune response by residing within host phagocytes. To identify inhibitors of intracellular S. Tm growth, we performed parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. These screens identified novel antibacterials, and revealed that antibiotics with limited Gram-negative coverage are active against intracellular S. Tm. Screening of a S. Tm deletion library in the presence of one compound, metergoline, revealed that outer membrane perturbation enhanced activity in vitro. Combined with our observation of atypical cell surface characteristics of intracellular S. Tm, our work indicates that the bacterial outer membrane is permeabilized within macrophages. We show that metergoline targets the bacterial cytoplasmic membrane, and prolongs animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and uncovers new aspects of bacterial physiology of intracellular S. Tm.

microbiology↗