RESUMO
The drug-impermeable bacterial membrane in Gram-negative pathogens limits antibiotic access to intracellular drug targets. To expand our rapidly waning antibiotic arsenal, one approach is to improve the intracellular delivery of drugs with historically poor accumulation in Gram-negative bacteria. To do so, we engineered macromolecular potentiators to permeabilize the Gram-negative membrane to facilitate drug influx. Potentiators, known as WD40, were synthesized by grafting multiple copies of a cationic α-helical antimicrobial peptide, WLBU2, onto a dextran polymer scaffold. WD40 enabled drug uptake in the model pathogen P. aeruginosa, a capability that was not observed with unmodified WLBU2 peptide. WD40 was able to reduce minimum inhibitory concentrations of a drug panel by up to 3 orders of magnitude. Hydrophobic and highly three-dimensional antibiotics exhibited the greatest potentiation. Antibiotic activity was potentiated in several clinical strains and resulted in sensitization of drug-resistant strains to rifampin, a drug not previously used for Gram-negative infections.
Assuntos
Antibacterianos , Bactérias Gram-Negativas , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , RifampinaRESUMO
Harnessing the microbiota for beneficial outcomes is limited by our poor understanding of the constituent bacteria, as the functions of most of their genes are unknown. Here, we measure the growth of a barcoded transposon mutant library of the gut commensal Bacteroides thetaiotaomicron on 48 carbon sources, in the presence of 56 stress-inducing compounds, and during mono-colonization of gnotobiotic mice. We identify 516 genes with a specific phenotype under only one or a few conditions, enabling informed predictions of gene function. For example, we identify a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a 3-keto-glucoside hydrolase for disaccharide utilization, and a tripartite multidrug resistance system specifically for bile salt tolerance. Furthermore, we show that B. thetaiotaomicron uses alternative enzymes for synthesizing nitrogen-containing metabolic precursors based on ammonium availability and that these enzymes are used differentially in vivo in a diet-dependent manner.
Assuntos
Bacteroides thetaiotaomicron/genética , Dieta , Metabolismo Energético/genética , Microbioma Gastrointestinal/genética , Intestinos/microbiologia , Adaptação Fisiológica , Compostos de Amônio/metabolismo , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides thetaiotaomicron/efeitos dos fármacos , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Ácidos e Sais Biliares/metabolismo , Bases de Dados Genéticas , Dissacarídeos/metabolismo , Farmacorresistência Bacteriana/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Mutação , Especificidade por Substrato , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismoRESUMO
Human breath contains many volatile metabolites. However, few breath tests are currently used in the clinic to monitor disease due to bottlenecks in biomarker identification. Here we engineered breath biomarkers for respiratory disease by local delivery of protease-sensing nanoparticles to the lungs. The nanosensors shed volatile reporters upon cleavage by neutrophil elastase, an inflammation-associated protease with elevated activity in lung diseases such as bacterial infection and alpha-1 antitrypsin deficiency. After intrapulmonary delivery into mouse models with acute lung inflammation, the volatile reporters are released and expelled in breath at levels detectable by mass spectrometry. These breath signals can identify diseased mice with high sensitivity as early as 10 min after nanosensor administration. Using these nanosensors, we performed serial breath tests to monitor dynamic changes in neutrophil elastase activity during lung infection and to assess the efficacy of a protease inhibitor therapy targeting neutrophil elastase for the treatment of alpha-1 antitrypsin deficiency.