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At least 451 records · Page 25
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus

Lysine acylation is a posttranslational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine antibodies, which are costly and time-consuming and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to explore the biological significance of acylation in each organism. We characterized the acetylome, propionylome, and butyrylome in P. aeruginosa UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed unique PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Chemoproteomic Elucidation of β-Lactam Drug Targets in Mycobacterium abscessus

The pathogen Mycobacterium abscessus (Mab) can cause severe and difficult-to-treat chronic lung infections. Despite the rising incidence and clinical concern of Mab infections, treatment options are limited and often ineffective. Treatment is complicated by Mab’s ability to persist in a nonreplicating, drug-resistant state. Several β-lactam antibiotics are potently bactericidal against Mab but are underutilized because their molecular mechanisms of action against Mab are incompletely understood. In the current study, we used β-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of Mab enzymes targeted by β-lactams. We compared β-lactam targets across two Mab subspecies in actively replicating and nonreplicating cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind β-lactams. Lastly, we characterized the β-lactamase activity and β-lactam inhibition profiles of nine Mab enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings demonstrate β-lactam target engagement in persistent Mab and provide clarity on the mechanisms of action of clinically relevant β-lactams in Mab, crucial steps toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

Mycobacterium abscessus↗
DOE OSTI2023

Organic Matrix Derived from Host–Microbe Interplay Contributes to Pathological Renal Biomineralization

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular Dynamics Simulation of Atomic Interactions in the Vancomycin Binding Site

Vancomycin is a glycopeptide antibiotic produced by Amycolaptopsis orientalis used to treat serious infections by Grampositive pathogens including methicillin-resistant Staphylococcus aureus. Vancomycin inhibits cell wall biosynthesis by targeting lipid II, which is the membrane-bound peptidoglycan precursor. The heptapeptide aglycon structure of vancomycin binds to the D-Ala-D-Ala of the pentapeptide stem structure in lipid II. The third residue of vancomycin aglycon is asparagine, which is not directly involved in the dipeptide binding. Nonetheless, asparagine plays a crucial role in substrate recognition, as the vancomycin analogue with asparagine substituted by aspartic acid (V D ) shows a reduction in antibacterial activities. To characterize the function of asparagine, binding of vancomycin and its aspartic-acid-substituted analogue V D to L-Lys-D-Ala-D-Ala and L-Lys-D-Ala-D-Lac was investigated using molecular dynamic simulations. Binding interactions were analyzed using root-mean-square deviation (RMSD), two-dimensional (2D) contour plots, hydrogen bond analysis, and free energy calculations of the complexes. The analysis shows that the aspartate substitution introduced a negative charge to the binding cleft of V D , which altered the aglycon conformation that minimized the repulsive lone pair interaction in the binding of a depsipeptide. Our findings provide new insight for the development of novel glycopeptide antibiotics against the emerging vancomycin-resistant pathogens by chemical modification at the third residue in vancomycin to improve its binding affinity to the D-Ala-D-Lac-terminated peptidoglycan in lipid II found in vancomycin-resistant enterococci and vancomycinresistant S. aureus.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is both round and elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structural insights into the mechanism of overcoming Erm-mediated resistance by macrolides acting together with hygromycin-A

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Colistin resistance plasmids dually enhance bacterial virulence and antibiotic resistance via surface polysaccharide biosynthesis

Plasmids carrying the mobilized colistin-resistance gene mcr-1 are prevalent among multidrug-resistant Gram-negative pathogens, yet their broad impact on bacterial physiology and virulence remains unclear. Here, we demonstrate that acquisition of an mcr-1 plasmid concurrently increases antimicrobial resistance and pathogenicity in Escherichia coli. On the same plasmid, the XRE-family transcriptional regulator EcaR cooperates with MCR-1 to activate the wec operon, driving biosynthesis of two surface polysaccharides: enterobacterial common antigen (ECA) and a high-molecular-weight O-chain. Expression of these surface polysaccharides increases bile resistance and virulence in a murine model and further elevates colistin resistance. MCR-1 enhances transcription of upstream genes in the wec operon, whereas EcaR directly activates an internal promoter (PwecE) to induce downstream gene expression. Thus, both components are required for surface polysaccharide expression, and deletion of either abolishes the phenotype. Genomic analysis of publicly available mcr plasmids reveals widespread co-occurrence of mcr-1 and ecaR on IncI2 and IncX4 plasmids, indicating their functional complementarity. These findings uncover a mechanism by which resistance plasmids remodel the bacterial surface, linking horizontal gene transfer to coordinated regulation of antimicrobial resistance and virulence.

Antimicrobial resistance↗
DOE OSTI2025

Unlocking expanded flagellin perception through rational receptor engineering

Abstract The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologues possess narrow perception ranges, but four with expanded perception have been identified. Using diversity analyses, AlphaFold modelling and amino acid properties, key residues enabling expanded recognition were mapped to FLS2’s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) into a homologue with canonical perception. A similar approach enabled transfer ofAgrobacteriumperception from FLS2 XL (Vitis riparia) into VrFLS2. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22’s C terminus, suggesting more alleles with expanded perception exist. Our experimental data enabled the identification of specific receptor amino acid properties and AlphaFold3 metrics that facilitate predicting FLS2–flg22 recognition. This study provides a framework for rational receptor engineering to enhance pathogen restriction.

Plant Sciences↗
DOE OSTI2020

Mathematical model of broadly reactive plasma cell production

Strain-specific plasma cells are capable of producing neutralizing antibodies that are essential for clearance of challenging pathogens. These neutralizing antibodies also function as a main defense against disease establishment in a host. However, when a rapidly mutating pathogen infects a host, successful control of the invasion requires shifting the production of plasma cells from strain-specific to broadly reactive. In this study, we develop a mathematical model of germinal center dynamics and use it to predict the events that lead to improved breadth of the plasma cell response. We examine scenarios that lead to germinal centers that are composed of B-cells that come from a single strain-specific clone, a single broadly reactive clone or both clones. We find that the initial B-cell clonal composition, T-follicular helper cell signaling, increased rounds of productive somatic hypermutation, and B-cell selection strength are among the mechanisms differentiating between strain-specific and broadly reactive plasma cell production during infections. Understanding the contribution of these factors to emergence of breadth may assist in boosting broadly reactive plasma cells production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A multi-scale pipeline linking drug transcriptomics with pharmacokinetics predicts in vivo interactions of tuberculosis drugs

Tuberculosis (TB) is the deadliest infectious disease worldwide. The design of new treatments for TB is hindered by the large number of candidate drugs, drug combinations, dosing choices, and complex pharmaco-kinetics/dynamics (PK/PD). Here we study the interplay of these factors in designing combination therapies by linking a machine-learning model, INDIGO-MTB, which predicts in vitro drug interactions using drug transcriptomics, with a multi-scale model of drug PK/PD and pathogen-immune interactions called GranSim. We calculate an in vivo drug interaction score (iDIS) from dynamics of drug diffusion, spatial distribution, and activity within lesions against various pathogen sub-populations. The iDIS of drug regimens evaluated against non-replicating bacteria significantly correlates with efficacy metrics from clinical trials. Our approach identifies mechanisms that can amplify synergistic or mitigate antagonistic drug interactions in vivo by modulating the relative distribution of drugs. Our mechanistic framework enables efficient evaluation of in vivo drug interactions and optimization of combination therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2022

Synthetic antibacterial minerals: harnessing a natural geochemical reaction to combat antibiotic resistance

The overuse of antibiotics in clinical and livestock settings is accelerating the selection of multidrug resistant bacterial pathogens. Antibiotic resistant bacteria result in increased mortality and financial strain on the health care and livestock industry. The development of new antibiotics has stalled, and novel strategies are needed as we enter the age of antibiotic resistance. Certain naturally occurring clays have been shown to have antimicrobial properties and kill antibiotic resistant bacteria. Harnessing the activity of compounds within these clays that harbor antibiotic properties offers new therapeutic opportunities for fighting the potentially devastating effects of the post antibiotic era. However, natural samples are highly heterogenous and exhibit variable antibacterial effectiveness, therefore synthesizing minerals of high purity with reproducible antibacterial activity is needed. Here we describe for the first time synthetic smectite clay minerals and Fe-sulfide microspheres that reproduce the geochemical antibacterial properties observed in natural occurring clays. We show that these mineral formulations are effective at killing the ESKAPE pathogens ( Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter sp., Pseudomonas aeruginosa and Enterobacter sp.) by maintaining Fe 2+ solubility and reactive oxygen species (ROS) production while buffering solution pH, unlike the application of metals alone. Our results represent the first step in utilizing a geochemical process to treat antibiotic resistant topical or gastrointestinal infections in the age of antibiotic resistance.

58 GEOSCIENCES↗
DOE OSTI2022

Molecular dynamics simulations of the secondary-binding site in disaccharide-modified glycopeptide antibiotics

Oritavancin is a semisynthetic glycopeptide antibiotic used to treat severe infections by multidrug-resistant Gram-positive pathogens. Oritavancin is known to be a thousand times more potent than vancomycin against Gram-positive bacteria due to the additional interactions with bacterial peptidoglycan (PG) facilitated by a secondary-binding site. The presence of this secondary-binding site is evident in desleucyl-oritavancin, an Edman degradation product of oritavancin, still retaining its potency against Gram-positive bacteria, whereas desleucyl-vancomycin is devoid of any antimicrobial activities. Herein, using explicit solvent molecular dynamics (MD) simulations, steered MD simulations, and umbrella sampling, we show evidence of a secondary-binding site mediated by the disaccharide-modified hydrophobic sidechain of oritavancin interactions with the pentaglycyl-bridge segment of the PG. The interactions were characterized through comparison to the interaction of PG with chloroeremomycin, vancomycin, and the desleucyl analogs of the glycopeptides. Our results show that the enhanced binding of oritavancin to PG over the binding of the other complexes studied is due to an increase in the hydrophobic effect, electrostatic and van der Waals interactions, and not the average number of hydrogen bonds. Our ranking of the binding interactions of the biomolecular complexes directly correlates with the order based on their experimental minimum inhibitory concentrations. The results of our simulations provide insight into the modification of glycopeptides to increase their antimicrobial activities or the design of novel antibiotics against pathogenic Gram-positive bacteria.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds

Abstract Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A simple model of COVID-19 explains disease severity and the effect of treatments

Abstract Considerable effort has been made to better understand why some people suffer from severe COVID-19 while others remain asymptomatic. This has led to important clinical findings; people with severe COVID-19 generally experience persistently high levels of inflammation, slower viral load decay, display a dysregulated type-I interferon response, have less active natural killer cells and increased levels of neutrophil extracellular traps. How these findings are connected to the pathogenesis of COVID-19 remains unclear. We propose a mathematical model that sheds light on this issue by focusing on cells that trigger inflammation through molecular patterns: infected cells carrying pathogen-associated molecular patterns (PAMPs) and damaged cells producing damage-associated molecular patterns (DAMPs). The former signals the presence of pathogens while the latter signals danger such as hypoxia or lack of nutrients. Analyses show that SARS-CoV-2 infections can lead to a self-perpetuating feedback loop between DAMP expressing cells and inflammation, identifying the inability to quickly clear PAMPs and DAMPs as the main contributor to hyperinflammation. The model explains clinical findings and reveal conditions that can increase the likelihood of desired clinical outcome from treatment administration. In particular, the analysis suggest that antivirals need to be administered early during infection to have an impact on disease severity. The simplicity of the model and its high level of consistency with clinical findings motivate its use for the formulation of new treatment strategies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Antimicrobial properties of a multi-component alloy

High traffic touch surfaces such as doorknobs, countertops, and handrails can be transmission points for the spread of pathogens, emphasizing the need to develop materials that actively self-sanitize. Metals are frequently used for these surfaces due to their durability, but many metals also possess antimicrobial properties which function through a variety of mechanisms. This work investigates metallic alloys comprised of several metals which individually possess antimicrobial properties, with the target of achieving broad-spectrum, rapid sanitation through synergistic activity. An entropy-motivated stabilization paradigm is proposed to prepare scalable alloys of copper, silver, nickel and cobalt. Using combinatorial sputtering, thin-film alloys were prepared on 100 mm wafers with ≈50% compositional grading of each element across the wafer. The films were then annealed and investigated for alloy stability. Antimicrobial activity testing was performed on both the as-grown alloys and the annealed films using four microorganisms—Phi6, MS2, Bacillus subtilis and Escherichia coli —as surrogates for human viral and bacterial pathogens. Testing showed that after 30 s of contact with some of the test alloys, Phi6, an enveloped, single-stranded RNA bacteriophage that serves as a SARS-CoV-2 surrogate, was reduced up to 6.9 orders of magnitude (> 99.9999%). Additionally, the non-enveloped, double-stranded DNA bacteriophage MS2, and the Gram-negative E. coli and Gram-positive B. subtilis bacterial strains showed a 5.0, 6.4, and 5.7 log reduction in activity after 30, 20 and 10 min, respectively. Antimicrobial activity in the alloy samples showed a strong dependence on the composition, with the log reduction scaling directly with the Cu content. Concentration of Cu by phase separation after annealing improved activity in some of the samples. The results motivate a variety of themes which can be leveraged to design ideal antimicrobial surfaces.

36 MATERIALS SCIENCE↗