Gram-negative bacteria are responsible for some of the deadliest and more widespread pandemics in the world. Helicobacter pylori is now recognized as the primary cause of peptic ulcer disease and gastric cancer, Camphorbacter jejuni and Vibrio cholera remain among the most common causes of diarrheal illness, and Acinetobacter baumannii is responsible for a growing number of the antibiotic-resistant infections in hospitals. Understanding the machinery of these bacteria used for initiation of pathogenesis, for recognition and activation of the immune system, and for development of antibiotic resistance are vital issues that require multi-disciplinary research strategies. The proposed work focuses on the development of advanced mass spectrometric approaches for characterization of the complex lipopolysaccharides (LPS), including the endotoxic lipid A sub-unit, that comprise the key constituents of the outer membrane of Gram-negative bacteria. Given the diversity seen in LPS and particularly lipid A structures, the structural characterizatio of LPS is a challenging task. We have begun to develop three photodissociation methods, including infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), and activated-electron photodetachment dissociation (a-EPD), for the characterization of lipid A and LPS structures. Specific objectives include: 1) Photodissociation and hybrid MS/MS methods for characterization of lipid A and core oligosaccharide/O-antigens.
The first aim entails systematic examination of the fragmentation patterns obtained by photodissociation and hybrid MS/MS methods for lipid A and oligosaccharide compounds. 2) Top-down characterization of LPS. Characterization of intact LPS will build by combining the fragmentation patterns obtained from bottom-up approaches (lipids and oligosaccharides) with information obtained from increasingly larger portions of the lipopolysaccharides via a middle-down strategy, then progressing to an integrated top-down workflow. 3) Development of an in silico database search algorithm. The complexity of the MS/MS spectra which contain an array of fragment ions from both the lipid and sugar portions makes their interpretation challenging. An in silico database search algorithm, MassMatrixLPS, will be developed by collaborator Hua Xu to facilitate automated, higher-throughput data analysis. 4) Applications to lipid A and LPS of H. pylori, C. jejuni, V. cholera, and E. coli. The collaboration forged between the Brodbelt and Trent groups is aimed at elucidating the LPS modification systems of three proteobacteria and elaborating their structure/function relationships. Specific biological problems include correlation of the structura changes of lipid A with the inflammatory response, unraveling the biosynthetic pathway of LPS, and elucidating the mechanism of resistance to antimicrobial peptides.
Gram-negative bacteria are responsible for some of the deadliest and more widespread pandemics in the world. The proposed work focuses on the development of advanced mass spectrometric approaches for characterization of the complex lipopolysaccharides, including the endotoxic lipid A domains that comprise the key outer membrane of Gram-negative bacteria.
|Williams, Peggy E; Klein, Dustin R; Greer, Sylvester M et al. (2017) Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. J Am Chem Soc 139:15681-15690|
|Crittenden, Christopher M; Akin, Lucas D; Morrison, Lindsay J et al. (2017) Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains. J Am Soc Mass Spectrom 28:1118-1126|
|Klein, Dustin R; Brodbelt, Jennifer S (2017) Structural Characterization of Phosphatidylcholines Using 193 nm Ultraviolet Photodissociation Mass Spectrometry. Anal Chem 89:1516-1522|
|Klein, Dustin R; Holden, Dustin D; Brodbelt, Jennifer S (2016) Shotgun Analysis of Rough-Type Lipopolysaccharides Using Ultraviolet Photodissociation Mass Spectrometry. Anal Chem 88:1044-51|
|Morrison, Lindsay J; Parker, W Ryan; Holden, Dustin D et al. (2016) UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures. Anal Chem 88:1812-20|
|Brodbelt, Jennifer S (2016) Ion Activation Methods for Peptides and Proteins. Anal Chem 88:30-51|
|Nowicki, Emily M; O'Brien, John P; Brodbelt, Jennifer S et al. (2015) Extracellular zinc induces phosphoethanolamine addition to Pseudomonas aeruginosa lipid A via the ColRS two-component system. Mol Microbiol 97:166-78|
|Boll, Joseph M; Tucker, Ashley T; Klein, Dustin R et al. (2015) Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival. MBio 6:e00478-15|
|Brodbelt, Jennifer S (2014) Photodissociation mass spectrometry: new tools for characterization of biological molecules. Chem Soc Rev 43:2757-83|
|Henderson, Jeremy C; Fage, Christopher D; Cannon, Joe R et al. (2014) Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases. ACS Chem Biol 9:2382-92|
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