title: Live-cell high resolution magic angle spinning magnetic resonance spectroscopy forin vivoanalysis ofPseudomonas aeruginosametabolomics.
abstract: Pseudomonas aeruginosa(PA) is a pathogenic gram-negative bacterium that is widespread in nature, inhabiting soil, water, plants and animals. PA is a prevalent cause of deleterious human infections, particularly in patients whose host defense mechanisms have been compromised. Metabolomics is an important tool used to study host-pathogen interactions and to identify novel therapeutic targets and corresponding compounds. The aim of the present study was to report the metabolic profile of live PA bacteria usingin vivohigh-resolution magic angle spinning (HRMAS) nuclear magnetic resonance spectroscopy (NMR), in combination with 1- and 2-dimensional HRMAS NMR. This methodology provides a new and powerful technique to rapidly interrogate the metabolome of intact bacterial cells and has several advantages over traditional techniques that identify metabolome components from disrupted cells. Furthermore, application of multidimensional HRMAS NMR, in combination with the novel technique total through-Bond correlation Spectroscopy (TOBSY), is a promising approach that may be used to obtainin vivometabolomics information from intact live bacterial cells and can mediate such analyses in a short period of time. Moreover, HRMAS1H NMR enables the investigation of the associations between metabolites and cell processes. In the present study, we detected and quantified several informative metabolic molecules in live PA cells, including N-acetyl, betaine, citrulline, alanine and glycine, which are important in peptidoglycan synthesis. The results provided a complete metabolic profile of PA for future studies of PA clinical isolates and mutants. In addition, thisin vivoNMR biomedical approach might have clinical utility and should prove useful in gene function validation, the study of pathogenetic mechanisms, the classification of microbial strains into functional/clinical groups, the testing of anti-bacterial agents and the determination of metabolic profiles of bacterial mutants.
authors.affiliation: Nuclear Magnetic Resonance Surgical Laboratory, Department of Surgery, Division of Burns, Massachusetts General Hospital and Shriners Burns Institute, Harvard Medical School, Boston, MA 02114, USA ; Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA ; Department for Life Quality, University of Bologna, Rimini 47921, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Righi
authors.affiliation: Nuclear Magnetic Resonance Surgical Laboratory, Department of Surgery, Division of Burns, Massachusetts General Hospital and Shriners Burns Institute, Harvard Medical School, Boston, MA 02114, USA ; Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Caterina
authors.initials: C
authors.lastname: Constantinou
authors.affiliation: Molecular Surgery Laboratory, Department of Surgery, Division of Burns, Massachusetts General Hospital and Shriners Burns Institute, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Meenu
authors.initials: M
authors.lastname: Kesarwani
authors.affiliation: Molecular Surgery Laboratory, Department of Surgery, Division of Burns, Massachusetts General Hospital and Shriners Burns Institute, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Laurence G
authors.initials: LG
authors.lastname: Rahme
authors.affiliation: Nuclear Magnetic Resonance Surgical Laboratory, Department of Surgery, Division of Burns, Massachusetts General Hospital and Shriners Burns Institute, Harvard Medical School, Boston, MA 02114, USA ; Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Aria A
authors.initials: AA
authors.lastname: Tzika
keywords: Pseudomonas aeruginosa; high resolution magic angle spinning; nuclear magnetic resonance; total correlation spectroscopy; total through bond correlation spectroscopy