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NMR Metabolomic Analysis of Bacterial Resistance Pathways Using Multivalent Quaternary Ammonium Functionalized Macromolecules

ABSTRACT: Multivalent antimicrobial dendrimers are an exciting new system that is being developed to address the growing problem of drug resistant bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is a quantitative and reproducible method for the determination of bacterial response to environmental stressors and for visualization of perturbations to biochemical pathways. NMR metabolomics is used to elucidate metabolite differences between wild type and antimicrobially mutated Escherichia coli (E. coli) samples. Proton (1H) NMR hydrophilic metabolite analysis was conducted on samples of E. coli after 33 growth cycles of a minimum inhibitory challenge to E. coli by poly(amidoamine) dendrimers functionalized with mannose and with C16-DABCO quaternary ammonium endgroups and compared to the metabolic profile of wild type E. coli. The wild type and mutated E. coli samples were separated into distinct sample sets by hierarchical clustering, principal component analysis (PCA) and sparse partial least squares discriminate analysis (sPLS-DA). Metabolite components of membrane fortification and energy related pathways had a significant p-value and fold change between the wild type and mutated E. coli. Amino acids commonly associated with membrane fortification from cationic antimicrobials, such as lysine, were found to have a higher concentration in the mutated E. coli than the wild type E. coli. N-acetylglucosamine, a major component of peptidoglycan synthesis, was found to have a 25 fold higher concentration in the mid log phase of the mutated E. coli than the mid log phase of the wild type.The metabolic profile suggests that E. coli change their peptidoglycan composition in order to garner protection from the highly positively charged and multivalent C16-DABCO and mannose functionalized dendrimer.

ORGANISM(S): Escherichia Coli E. Coli

TISSUE(S): Bacterial Cells

SUBMITTER: Michelle Aries

PROVIDER: ST001351 | MetabolomicsWorkbench | Fri Mar 20 00:00:00 GMT 2020

REPOSITORIES: MetabolomicsWorkbench

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Project description:Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both gram-positive and gram-negative bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is an important method for studying resistance development in bacteria since it is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. Determine the likely metabolic differences between antimicrobially challenged and unchallenged growth and wild type and antimicrobially mutated Bacillus cereus (B. cereus) samples by using NMR hydrophilic metabolomics. Proton (1H) NMR hydrophilic metabolite analysis was conducted using B. cereus wild type and B. cereus that was mutated with C16-DABCO and mannose functionalized poly(amidoamine) dendrimers (DABCOMD). Both the wild type and the mutated sample types were grown in low levels of DABCOMD (challenged samples) or without the addition of DABCOMD to the growth media (unchallenged samples) for sample collection at the mid log and stationary phases and for growth curve procurement. Hierarchical clustering of only the challenged sample type showed that both the stationary phase sample types (mutant and wild type) clustered together while the both the mid log phase sample types were distinct. Hierarchical clustering of the unchallenged samples showed complete separation of all sample types. There were statistically significant (p-value and fold change) changes in the concentrations of metabolites in both energy related pathways and peptidoglycan synthesis between all sample types, especially with mutants and especially the challenged sample types have more N-acetylglucosamine (as much as a 94.2-fold increase). The mid log phase sample types showed a larger difference between sample types than their stationary phase counter parts. The challenged and unchallenged mutant samples showed a larger difference between sample types in comparison to the differences between the challenged and unchallenged wild type sample types. There was a larger metabolite difference when comparing the challenged mutant samples to the challenged wild type samples than when comparing the unchallenged mutant samples to the unchallenged wild type samples. The metabolomic analysis of wild type and multivalent DABCOMD mutated B. cereus under both challenged and unchallenged conditions indicated that the mutants, especially the challenged mutants, are likely changing their peptidoglycan layer to protect themselves from the high positive charge on the membrane disrupting DABCOMD. This membrane fortification most likely led to the slow growth curve of the mutated and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for these changes to occur as well as to the decreased diffusion of nutrients across the membrane.

Project description:Transcription profiling of wild type E. coli MG1655, intestine-adapted E. coli MG1655star, and E. coli MG1655 flhD mutant grown on glucose, mannose, and mucus. We previously isolated a spontaneous mutant of E. coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, which has colonization traits superior to the wild-type parent strain (Leatham, et. al., 2005, Infect Immun 73:8039-49) The intestine-adapted strain (E. coli MG1655star) grew faster on several different carbon sources compared to the wild-type and was non-motile due to deletion of the flhD gene. To further characterize E. coli MG1655star, we used several high-throughput genomic approaches. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655star. Microarray analysis revealed modest, yet significant induction of catabolic gene systems across the genome in both E. coli MG1655star and the isogenic flhD mutant. Catabolome analysis with Biolog GN2 Microplates revealed an enhanced ability of both E. coli MG1655star and the isogenic flhD mutant to oxidize a wide variety of carbon sources. The results show that intestine-adapted E. coli MG1655star is more fit than the wild-type for intestinal colonization because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, leading to more efficient carbon source utilization, which results in a higher population size in the intestine. Hence mutations that enhance metabolic efficiency confer a colonization advantage. Three strains were profiled: E. coli MG1655 wildtype, E. coli flhD, and an intestine adapted strain, MG1655star, derived from the wildtype and isolated from feces after 15 days in the streptomycin treated mouse intestine, which proved to be a better colonizer than the wildtype, were grown on MOPS minimal medium containing 0.2% glucose or mannose, or mucus (10 mg/ml) and RNA was extracted from logarithmic phase cultures, and also from mucus grown cells in late log phase.

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NMR Metabolomic Analysis of Bacterial Resistance Pathways Using Multivalent Quaternary Ammonium Functionalized Macromolecules

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