Lipid A structural diversity among members of the genus Leptospira.

Lipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the Leptospira genus was explored. The individual Leptospira species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 Leptospira reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of Leptospira membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on Leptospira lipid A activity.

Structure Determination of Lipid A with Multiple Glycosylation Sites by Tandem MS of Lithium-Adducted Negative Ions.

FLATn is a tandem mass spectrometric technique that can be used to rapidly generate spectral information applicable for structural elucidation of lipids like lipid A from Gram-negative bacterial species from a single bacterial colony. In this study, we extend the scope and capability of FLATn by tandem MS fragmentation of lithium-adducted molecular lipid A anions and fragments (FLATn-Li) that provides additional structural and diagnostic data from FLATn samples allowing for the discrimination of terminal phosphate modifications in a variety of pathogenic and environmental species. Using FLATn-Li, we elucidated the lipid A structure from several bacterial species, including novel structures from arctic bacterioplankton of the Duganella and Massilia genera that favor 4-amino-4-deoxy-l-arabinopyranose (Ara4N) modification at the 1-phosphate position and that demonstrate double glycosylation with Ara4N at the 1 and 4' phosphate positions simultaneously. The structures characterized in this work demonstrate that some environmental psychrophilic species make extensive use of this structural lipid A modification previously characterized as a pathogenic adaptation and the structural basis of resistance to cationic antimicrobial peptides. This observation extends the role of phosphate modification(s) in environmental species adaptation and suggests that Ara4N modification can functionally replace the positive charge of the phosphoethanolamine modification that is more typically found attached to the 1-phosphate position of modified lipid A.

Rapid excision of oxidized adenine by human thymine DNA glycosylase.

Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood. Human DNA polymerases incorporate dGTP or dCTP opposite oxoA, producing mispairs that can cause A→C or A→G mutations. oxoA also perturbs the activity of enzymes acting on DNA and causes interstrand crosslinks. To inform mechanisms for oxoA repair, we characterized oxoA excision by human thymine DNA glycosylase (TDG), an enzyme known to remove modified pyrimidines, including deaminated and oxidized forms of cytosine and 5-methylcystosine. Strikingly, TDG excises oxoA from G⋅oxoA, A⋅oxoA, or C⋅oxoA pairs much more rapidly than it acts on the established pyrimidine substrates, whereas it exhibits comparable activity for T⋅oxoA and pyrimidine substrates. The oxoA activity depends strongly on base pairing and is 370-fold higher for G⋅oxoA versus T⋅oxoA pairs. The intrinsically disordered regions of TDG contribute minimally to oxoA excision, whereas two conserved residues (N140 and N191) are catalytically essential. Escherichia coli mismatch-specific uracil DNA-glycosylase lacks significant oxoA activity, exhibiting excision rates 4 to 5 orders of magnitude below that of its ortholog, TDG. Our results reveal oxoA as an unexpectedly efficient purine substrate for TDG and underscore the large evolutionary divergence of TDG and mismatch-specific uracil DNA-glycosylase.

A Sensitive GC-MS Method for Quantitation of Lipid A Backbone Components and Terminal Phosphate Modifications.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria, serves as a target for cationic antimicrobial peptides, such as polymyxins. Membrane stress from polymyxins results in activation of two-component regulatory systems that produce lipid A modifying enzymes. These enzymes add neutral moieties, such as aminoarabinose (AraN) and ethanolamine (EtN) to lipid A terminal phosphates that mask the phosphate's negative charge and inhibit electrostatic interaction with the cationic polymyxins. Currently, these modifications may be detected by MALDI-TOF MS; however, this analysis is only semiquantitative. Herein we describe a GC-MS method to quantitate lipid A backbone components, glucosamine (GlcN) and inorganic phosphate (Pi), along with terminal phosphate modifications AraN and EtN. In this assay, lipid A is isolated from Gram-negative bacterial samples, hydrolyzed into its individual moieties, and derivatized via methoximation followed by silylation prior to analysis via GC-MS. Changes in AraN and EtN quantity were characterized using a variety of regulatory mutants of Salmonella, revealing differences that were not detected using MALDI-TOF MS analysis. Additionally, an increase in the abundance of AraN and EtN modifications were observed when resistant Enterobacter and Escherichia coli strains were grown in the presence of colistin (polymyxin E). Lastly, increased levels of Pi were found in bisphosphorylated lipid A compared to monophosphorylated lipid A samples. Because lipid A modifications serve as indicators of polymyxin resistance in Gram-negative bacteria, this method provides the capacity to monitor polymyxin resistance by quantification of lipid A modification using GC-MS.

A novel use of centrifugal force for cell seeding into porous scaffolds.

A novel rotor was constructed to allow for the seeding of porous scaffolds via centrifugal force. Using cell seeding times of 10 min, this method placed significantly (roughly 3-fold) more cells into poly(glycolic acid) scaffolds than 24 h of spinner flask seeding or static seeding. There were no significant differences in the mitochondrial activity per cell between the 3 seeding methods. Cell distribution was noted to be homogeneous throughout the scaffold thickness for the centrifugation method, as opposed to surface seeding for the spinner flask method. Centrifugation was especially efficient at low cell concentrations (1.33 x 10(5) cells/ml). This system is useful for the seeding of biomaterials having cylindrical or planar geometries, and may be used under conditions that require low cell numbers and/or short seeding time periods.