Confirmation of the structure of lipid A fromEnterobacter agglomerans by electrospray ionization tandem mass spectrometry

Boué, Stephen M.; Cole, Richard B.

doi:10.1002/(sici)1096-9888(200003)35:3<361::aid-jms943>3.0.co;2-d

Cited by 31 publications

(28 citation statements)

References 23 publications

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“…Accurate mass measurement of the hepta-acylated structure (m/z 1,910) plus the 123 mass unit modification precursor ion at m/z 2,033 revealed the elemental composition of the 123 mass unit modification to be C 2 H 6 NPO 3 , which corresponded to pEtN (C 2 H 6 NPO 3 ; accurate mass, 123.0085; experimental mass, 123.0161; 7.6 millimass units [mmu]). The pEtN modification has been reported previously, yet evidence for localizing the pEtN group was suggestive (22,23). We determined the location of the pEtN modification via a series of tandem mass spectrometric experiments aimed at highlighting diagnostic product ions that provided convincing evidence for pEtN localization.…”

Section: Displayed Not Only the [M-h]mentioning

confidence: 51%

“…2C of four primary fatty acids, i.e., two 3-hydroxylauric acid [C 12 (3-OH)] acyl chains and two 3-hydroxymyristic acid [C 14 (3-OH)] acyl chains and of three secondary fatty acids, i.e., one C 12 (3-OH) acyl chain and two lauric acid (C 12 ) acyl chains. A similar acylation pattern has been reported previously, yet conclusive evidence for assignment of each fatty acid to a specific location was not determined (22,23).…”

Section: Displayed Not Only the [M-h]mentioning

confidence: 99%

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Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

Pelletier

Casella

Jones

et al. 2013

Antimicrob Agents Chemother

159

136

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f Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.

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Section: Displayed Not Only the [M-h]mentioning

confidence: 51%

Section: Displayed Not Only the [M-h]mentioning

confidence: 99%

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

Pelletier

Casella

Jones

et al. 2013

Antimicrob Agents Chemother

159

136

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“…In ESI-MS/MS investigations of the lipid A molecules from Enterobacter agglomerans, Salmonella minnesota, and Shigella flexneri, low-mass fragment ions corresponding to those seen in the 1291 lipid A spectrum (m/z 691 and 709) were also observed. However, in those species, the fragment ions were assigned as nonreducing terminal acylium ions (5,8). As acylium ions of that composition could not arise from the N. gonorrhoeae 1291 lipid A structure, we assigned the fragments differently.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A

et al. 2002

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Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec.Neisseria gonorrhoeae is a strict human pathogen, which invades and colonizes the epithelia of the urogenital tracts of both males and females (2, 12, 18). The lipooligosaccharide (LOS) of N. gonorrhoeae has been shown to play a role in the pathogenesis of human infections (19,40). The LOS is composed of three major components: the oligosaccharide chain extensions, the core region, and lipid A. The oligosaccharide extensions of the LOS contain determinants which resemble human glycosphingolipid antigens that play a role in molecular mimicry. Studies have also shown that the oligosaccharide region of the LOS can be involved in receptor-mediated interactions (17,19,37). Lipid A of N. gonorrhoeae is similar in structure to lipid A from other gram-negative bacteria (31,44,46). htrB (alternatively lpxL or waaM) is involved in the biosynthesis of lipid A of lipopolysaccharides (LPSs) and LOSs. HtrB is one of the 2-keto-3-deoxyoctulosonic acid (Kdo)-dependent acyl transferases responsible for the addition of a secondary acyl substitution on the lipid A portion of LPS or LOS. The htrB gene has been well characterized for Escherichia coli, Haemophilus influenzae, and Salmonella enterica serovar Typhimurium (6,36,44). Mutations in this gene have been shown to have a number of effects on the organism. One such effect is temperature sensitivity. E. coli, H. influenzae, and S. enterica serovar Typhimurium htrB mutants have all been show...

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“…Of particular interest, the use of mass spectrometry (MS) has proven to be an effective technique for determination of lipid A structure (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). Negative-ion mode matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS has proven to be very useful in initial characterization of lipid A structures (30-32, 38, 39).…”

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confidence: 99%

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

Jones

Shaffer

Ernst

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Confirmation of the structure of lipid A fromEnterobacter agglomerans by electrospray ionization tandem mass spectrometry

Cited by 31 publications

References 23 publications

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A

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

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