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Structural heterogeneity and environmentally regulated remodeling of Francisella tularensis subspecies novicida lipid A characterized by tandem mass spectrometry.

ABSTRACT: The structural characterization of environmentally-regulated lipid A derived from Francisella tularensis subspecies novicida (Fn) U112 is described using negative electrospray ionization with a linear ion trap Fourier transform ion cyclotron resonance (IT-FT-ICR) hybrid mass spectrometer. The results indicate that a unique profile of lipid A molecular structures are synthesized in response to Fn growth at 25 degrees C versus 37 degrees C. Molecular species were found to be tetra-acylated, sharing a conserved glucosamine disaccharide backbone, a galactosamine-1-phosphate linked to the reducing glucosamine, and multiple O- and N-linked fatty acyl groups. Deprotonated molecules were interrogated by MS(n) scanning techniques at both high and nominal mass resolution and were found to be complex heterogeneous mixtures where structures differed based on the positions and identities of the O- and N-linked fatty acyl substituents. For the dominant ion series, which consisted of five peaks, 30 unique lipid A structures were identified. Estimates for the relative abundance of each structure were derived from MS relative abundance ratios and fragment ion ratios from comparable dissociation pathways from MS(2) through MS(4) experiments. The results suggest a remodeling pathway in which the amide linked fatty acid of the reducing glucosamine favors a 3-hydroxyhexadecanoic acid substituent for growth conditions at 25 degrees C versus a 3-hydroxyoctadecanoic acid substituent for growth conditions at 37 degrees C.

SUBMITTER: Shaffer SA

PROVIDER: S-EPMC2743246 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Structural heterogeneity and environmentally regulated remodeling of Francisella tularensis subspecies novicida lipid A characterized by tandem mass spectrometry.

Shaffer Scott A SA Harvey Megan D MD Goodlett David R DR Ernst Robert K RK

Journal of the American Society for Mass Spectrometry 20070328 6

The structural characterization of environmentally-regulated lipid A derived from Francisella tularensis subspecies novicida (Fn) U112 is described using negative electrospray ionization with a linear ion trap Fourier transform ion cyclotron resonance (IT-FT-ICR) hybrid mass spectrometer. The results indicate that a unique profile of lipid A molecular structures are synthesized in response to Fn growth at 25 degrees C versus 37 degrees C. Molecular species were found to be tetra-acylated, sharin ...[more]

PMID: 17446084

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Project description:Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F. tularensis subsp. holarctica is highly pathogenic for humans, F. novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13C-labeling experiments with F. tularensis subsp. holarctica wild type (beaver isolate), F. tularensis subsp. holarctica strain LVS, or F. novicida strain U112 in complex media containing either [U-13C6]glucose, [1,2-13C2]glucose, [U-13C3]serine, or [U-13C3]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F. tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F. novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F. tularensis subsp. holarctica or metabolized, with higher rates in F. novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F. tularensis and F. novicida. Glycerol efficiently served as a gluconeogenetic substrate in F. novicida, but only less in the F. tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., via acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the F. tularensis subsp. holarctica strains. In summary, the data reflect differential metabolite fluxes in F. tularensis subsp. holarctica and F. novicida suggesting that the different utilization of substrates could be related to host specificity and virulence of Francisella.

Project description:Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of these strains for humans. This strategy could be extended to other closely-related bacterial species for inter-strain and inter-species analyses.

Dataset Information

Structural heterogeneity and environmentally regulated remodeling of Francisella tularensis subspecies novicida lipid A characterized by tandem mass spectrometry.

Publications

Structural heterogeneity and environmentally regulated remodeling of Francisella tularensis subspecies novicida lipid A characterized by tandem mass spectrometry.

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