Escherichia coli
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ABSTRACT:
PROVIDER: PRJNA121459 | ENA |
REPOSITORIES: ENA
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Project description:The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 microM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 microg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.
2007-01-01 | S-EPMC2709454 | BioStudies
Project description:CHIR-090 is an inhibitor of lipid A biosytnesis. CHIR-090 treatment on E. coli sinificantly increased the expression of fatty acid biosythesis gene fabA and fabB. Keywords: Inhibition response Overall design: The lipid A biosynthesis in E. coli was inhibited by CHIR-090, and the RNA was extracted after 10 minutes treatment to capture the effect of increased saturated fatty acid on global gene expression.
2009-10-20 | GSE18623 | GEO
Project description:CHIR-090 is an inhibitor of lipid A biosytnesis. CHIR-090 treatment on E. coli sinificantly increased the expression of fatty acid biosythesis gene fabA and fabB. Experiment Overall Design: The lipid A biosynthesis in E. coli was inhibited by CHIR-090, and the RNA was extracted after 10 minutes treatment to capture the effect of increased saturated fatty acid on global gene expression.
2009-10-27 | E-GEOD-18623 | ArrayExpress
Project description:The zinc-dependent enzyme LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the first committed step of lipid A biosynthesis. Lipid A is an essential component of the outer membranes of most Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, making LpxC an attractive target for antibiotic design. The inhibition of LpxC by a novel N-aroyl-l-threonine hydroxamic acid (CHIR-090) from a recent patent application (International Patent WO 2004/062601 A2 to Chiron and the University of Washington) is reported here. CHIR-090 possesses remarkable antibiotic activity against both E. coli and P. aeruginosa, comparable to that of ciprofloxacin. The biological activity of CHIR-090 is explained by its inhibition of diverse LpxC orthologues at low nanomolar concentrations, including that of Aquifex aeolicus, for which structural information is available. The inhibition of A. aeolicus LpxC by CHIR-090 occurs in two steps. The first step is rapid and reversible, with a K(i) of 1.0-1.7 nM, depending upon the method of assay. The second step involves the conversion of the EI complex with a half-life of about a minute to a tightly bound form. The second step is functionally irreversible but does not result in the covalent modification of the enzyme, as judged by electrospray ionization mass spectrometry. CHIR-090 is the first example of a slow, tight-binding inhibitor for LpxC and may be the prototype for a new generation of LpxC inhibitors with therapeutic applicability.
1000-01-01 | S-EPMC2742919 | BioStudies
Project description:The first committed step of lipid A biosynthesis is catalyzed by UDP-(3-O-((R)-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase, a metal-dependent deacetylase also known as LpxC. Because lipid A is essential for bacterial viability, the inhibition of LpxC is an appealing therapeutic strategy for the treatment of Gram-negative bacterial infections. Here we report the 1.79 Å resolution X-ray crystal structure of LpxC from Yersinia enterocolitica (YeLpxC) complexed with the potent hydroxamate inhibitor CHIR-090. This enzyme is a nearly identical orthologue of LpxC from Yersinia pestis (99.7% sequence identity), the pathogen that causes bubonic plague. Similar to the inhibition of LpxC from Escherichia coli, CHIR-090 inhibits YeLpxC via a two-step slow, tight-binding mechanism with an apparent K(i) of 0.54 ± 0.14 nM followed by conversion of the E·I to E·I* species with a rate constant of 0.11 ± 0.01 min(-1). The structure of the LpxC complex with CHIR-090 shows that the inhibitor hydroxamate group chelates the active site zinc ion, and the "tail" of the inhibitor binds in the hydrophobic tunnel in the active site. This hydrophobic tunnel is framed by a ??? subdomain that exhibits significant conformational flexibility as it accommodates inhibitor binding. CHIR-090 displays a 27 mm zone of inhibition against Y. enterocolitica in a Kirby-Bauer antibiotic assay, which is comparable to its reported activity against other Gram-negative species including E. coli and Pseudomonas aeruginosa. This study demonstrates that the inhibition of LpxC should be explored as a potential therapeutic and/or prophylatic response to infection by weaponized Yersinia species.
2011-01-01 | S-EPMC3070812 | BioStudies
Project description:BACKGROUND:Culturomics can ascertain traces of microorganisms to be cultivated using different strategies and identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry or 16S rDNA sequencing. However, to cater to all requirements of microorganisms and isolate as many species as possible, multiple culture conditions must be used, imposing a heavy workload. In addition, the fast-growing bacteria (e.g., Escherichia) surpass the slow-growing bacteria in culture by occupying space and using up nutrients. Besides, some bacteria (e.g., Pseudomonas) suppress others by secreting antibacterial metabolites, making it difficult to isolate bacteria with lower competence. Applying inhibitors to restrain fast-growing bacteria is one method to cultivate more bacterial species from human feces. RESULTS:We applied CHIR-090, an LpxC enzyme inhibitor that has antibacterial activity against most Gram-negative bacteria, to culturomics of human fresh feces. The antibacterial activity of CHIR-090 was first assessed on five Gram-negative species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, and Bacteroides vulgatus), all of which are commonly isolated from the human gut. Then, we assessed suitable concentrations of the inhibitor. Finally, CHIR-090 was applied in blood culture bottles for bacterial cultivation. In total, 102 species from five samples were identified. Of these, we found one new species, two species not reported previously in the human gut, and 11 species not previously isolated from humans. CONCLUSIONS:CHIR-090 can suppress E. coli, P. aeruginosa, K. pneumoniae, Pro. vulgaris, but not B. vulgatus. Compared with the non-inhibitor group, CHIR-090 increased bacteria isolation by 23.50%, including four species not reported in humans and one new species. Application of LpxC enzyme inhibitor in culturomics increased the number of species isolated from the human gut.
2019-01-01 | S-EPMC6937742 | BioStudies
Project description:Multi-drug resistant (MDR), pathogenic Gram-negative bacteria pose a serious health threat, and novel antibiotic targets must be identified to combat MDR infections. One promising target is the zinc-dependent metalloamidase UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyzes the committed step of lipid A (endotoxin) biosynthesis. LpxC is an essential, single copy gene that is conserved in virtually all Gram-negative bacteria. LpxC structures, revealed by NMR and X-ray crystallography, demonstrate that LpxC adopts a novel 'beta-alpha-alpha-beta sandwich' fold and encapsulates the acyl chain of the substrate with a unique hydrophobic passage. Kinetic analysis revealed that LpxC functions by a general acid-base mechanism, with a glutamate serving as the general base. Many potent LpxC inhibitors have been identified, and most contain a hydroxamate group targeting the catalytic zinc ion. Although early LpxC-inhibitors were either narrow-spectrum antibiotics or broad-spectrum in vitro LpxC inhibitors with limited antibiotic properties, the recently discovered compound CHIR-090 is a powerful antibiotic that controls the growth of Escherichia coli and Pseudomonas aeruginosa, with an efficacy rivaling that of the FDA-approved antibiotic ciprofloxacin. CHIR-090 inhibits a wide range of LpxC enzymes with sub-nanomolar affinity in vitro, and is a two-step, slow, tight-binding inhibitor of Aquifex aeolicus and E. coli LpxC. The success of CHIR-090 suggests that potent LpxC-targeting antibiotics may be developed to control a broad range of Gram-negative bacteria.
2008-01-01 | S-EPMC3022321 | BioStudies
Project description:The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising antibiotic target. CHIR-090, the most potent LpxC inhibitor discovered to date, displays two-step time-dependent inhibition and kills a wide range of Gram-negative pathogens as effectively as ciprofloxacin or tobramycin. In this study, we report the solution structure of the LpxC-CHIR-090 complex. CHIR-090 exploits conserved features of LpxC that are critical for catalysis, including the hydrophobic passage and essential active-site residues. CHIR-090 is adjacent to, but does not occupy, the UDP-binding pocket of LpxC, suggesting that a fragment-based approach may facilitate further optimization of LpxC inhibitors. Additionally, we identified key residues in the Insert II hydrophobic passage that modulate time-dependent inhibition and CHIR-090 resistance. CHIR-090 shares a similar, although previously unrecognized, chemical scaffold with other small-molecule antibiotics such as L-161,240 targeting LpxC, and provides a template for understanding the binding mode of these inhibitors. Consistent with this model, we provide evidence that L-161,240 also occupies the hydrophobic passage.
2007-01-01 | S-EPMC2141794 | BioStudies
Project description:With the rapid spread of antimicrobial resistance in Gram-negative pathogens, biofilm-associated infections are increasingly harder to treat and combination therapy with colistin has become one of the most efficient therapeutic strategies. Our study aimed to evaluate the potential for the synergy of colistin combined with CHIR-090, a potent LpxC inhibitor, against in vitro and in vivoPseudomonas aeruginosa biofilms. Four P. aeruginosa isolates with various colistin susceptibilities were chosen for evaluation. The tested isolates of P. aeruginosa exhibited MIC values ranging from 1 to 64 and 0.0625 to 0.5 ?g/ml for colistin and CHIR-090, respectively. Against 24-h static biofilms, minimum biofilm eradication concentration values ranged from 256 to 512 and 8 to >128 ?g/ml for colistin and CHIR-090, respectively. Interestingly, subinhibitory concentrations of CHIR-090 contributed to the eradication of subpopulations of P. aeruginosa with the highest colistin MIC values. The combination of colistin and CHIR-090 at subinhibitory concentrations demonstrated synergistic activity both in vivo and in vitro and prevented the formation of colistin-tolerant subpopulations in in vitro biofilms. In summary, our study highlights the in vivo and in vitro synergistic activity of the colistin and CHIR-090 combination against both colistin-susceptible and -nonsusceptible P. aeruginosa biofilms. Further studies are warranted to investigate the clinical relevance of the combination of these two antimicrobials and outline the underlying mechanism for their synergistic action.
1000-01-01 | S-EPMC5487635 | BioStudies
Project description:Upregulated expression of efflux pumps, lpxC target mutations, LpxC protein overexpression, and mutations in fabG were previously shown to mediate single-step resistance to the LpxC inhibitor CHIR-090 in P. aeruginosa Single-step selection experiments using three recently described LpxC inhibitors (compounds 2, 3, and 4) and mutant characterization showed that these mechanisms affect susceptibility to additional novel LpxC inhibitors. Serial passaging of P. aeruginosa wild-type and efflux pump-defective strains using the LpxC inhibitor CHIR-090 or compound 1 generated substantial shifts in susceptibility and underscored the interplay of efflux and nonefflux mechanisms. Whole-genome sequencing of CHIR-090 passage mutants identified efflux pump overexpression, fabG mutations, and novel mutations in fabF1 and in PA4465 as determinants of reduced susceptibility. Two new lpxC mutations, encoding A214V and G208S, that reduce susceptibility to certain LpxC inhibitors were identified in these studies, and we show that these and other target mutations differentially affect different LpxC inhibitor scaffolds. Lastly, the combination of target alteration (LpxCA214V) and upregulated expression of LpxC was shown to be tolerated in P. aeruginosa and could mediate significant decreases in susceptibility.
2019-01-01 | S-EPMC7187589 | BioStudies