Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach

Abstract

Lipid A isolated from several bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and various strains of Yersinia) showed abundant formation of pyrophosphate anions upon ion dissociation. Pyrophosphate [H3P2O7] ؊ and/or [HP2O6] ؊ anions were observed as dominant fragments from diphosphorylated lipid A anions regardless of the ionization mode (matrix-assisted laser desorption ionization or electrospray ionization), excitation mode (collisional activation or infrared photoexcitation), or mass analyzer (time-of-flight/time-of-flight, tandem quadrupole, Fourier transform-ion cyclotron resonance mass spectrometry). Dissociations of anions from model lipid phosphate, pyrophosphate, and hexose diphosphates confirmed that pyrophosphate fragments were formed abundantly only in the presence of an intact pyrophosphate group in the analyte molecule and were not due to intramolecular rearrangement upon ionization, ion-molecule reactions, or rearrangement following activation. This indicated that pyrophosphate groups are present in diphosphorylated lipid A from a variety of Gram-negative bacteria.pyrophosphorylation ͉ tandem mass spectrometry ͉ Yersinia pestis L ipopolysaccharide (LPS) is the primary constituent of the outer leaflet of the outer membrane of Gram-negative bacteria (1). In addition to being the major surface molecule in Gram-negative bacteria, LPS is also considered a major pathogenic factor. Lipid A, also referred to as endotoxin, is the hydrophobic membrane anchor of LPS and is known to be a potent inducer of the host innate immune system (1, 2). Structurally, lipid A is characterized as a phosphoglycolipid defined by a conserved diglucosamine disaccharide with structural variations occurring by fatty acid position and identity, phosphorylation, and additional monosaccharide modification. Alteration of lipid A structure (i.e., changes in acylation, phosphorylation, and glycosylation) greatly affects the bacterium's virulence and can occur via a variety of environmental stimuli including divalent ion concentration, temperature, and other growth conditions (1, 3-6).The phosphorylation pattern of lipid A has been shown to be important for its biological activity. For example, removal of a phosphate group has been shown to substantially reduce lipid A toxicity (7, 8) and interleukin-1 induction capacity (9). By contrast, masking of lipid A phosphate groups (e.g., addition of aminoarabinose) has been shown to affect bacterial resistance to host cationic antimicrobial peptides (10). The biochemical effects of phosphate groups in lipid A have been attributed to their negative charge that affects recognition by the Toll-like receptor 4 and further LPS-induced signaling in the host immune response to bacterial infection (11). Furthermore, monosaccharide modification to lipid A is thought to occur via an ester linkage with the phosphate substituents.The biosynthesis of lipid A, as characterized in Escherichia coli, involves LPS intermediates that have a 1-position pyrophosphate and a 4Ј-position mo...

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

“…2, C and G). Peaks at m/z 1737.1 for a P PEA lipid A were detected that arise from facile loss of H 4 P 2 O 7 (178 Da) from 3P PEA lipid A (Table 2) as reported previously (29).…”

Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data

“…Thereby, each precursor ion had a hierarchical set of tandem mass spectra (similar to a surname genealogical tree that branches out from the progenitor parental line) associated with it ( Figure 3) that could be used to aid structure assignment. While lipid A structures were assigned manually in our prior efforts [7,10,11], the objective of the current work was to develop HiTMS, an automated structure assignment algorithm, and demonstrate its effectiveness on two structural different types of lipid A.…”

“…Ion population in the LTQ was set at 10,000 and collision energies employed for MS n ranged from 25%-35%. For Yp Lipid A, data were acquired on an LTQ-FT Ultra (Thermo Scientific) as described elsewhere [10,11].…”

“…In our prior work on Francisella tularensis subspecies novicida (Fn) grown under two different environmental conditions, this hierarchical MS n strategy revealed 30 unique lipid A species from only a half dozen unique precursor ions [8,9]. While we demonstrated that high-throughput (HTP) hierarchical MS n profiling of lipid A mixtures can accelerate our discovery of structural diversity, a new bottleneck was created by the large amounts of data that had to be manually interpreted [6,7,10,11]. Thus, a major challenge for further implementation of our hierarchical MS n lipidomic strategy lay in the computational and bioinformatic demands of handling the large amounts of data [12].…”

“…However, the general structure of lipid A is very different from the majority of lipid classes, e.g., glycerolipids, glycerophospholipids, and sphingolipids, which were well characterized by AMDMS-SL builtin database. In addition, the initial analysis of structures by tandem mass spectrometry alone is often not sufficient to decipher the structures of lipid A, which we have found often require exhaustive MS n analysis to successfully characterize the isobaric components [7,10,11,22,23].…”