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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM103655-03
Application #
8721979
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Sheeley, Douglas
Project Start
2012-09-18
Project End
2016-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
3
Fiscal Year
2014
Total Cost
$279,104
Indirect Cost
$79,104
Name
University of Texas Austin
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
O'Brien, John P; Needham, Brittany D; Brown, Dusty B et al. (2014) Top-Down Strategies for the Structural Elucidation of Intact Gram-negative Bacterial Endotoxins. Chem Sci 5:4291-4301
O'Brien, John P; Needham, Brittany D; Henderson, Jeremy C et al. (2014) 193 nm ultraviolet photodissociation mass spectrometry for the structural elucidation of lipid A compounds in complex mixtures. Anal Chem 86:2138-45
Rubin, Erica J; O'Brien, John P; Ivanov, Petko L et al. (2014) Identification of a broad family of lipid A late acyltransferases with non-canonical substrate specificity. Mol Microbiol 91:887-99
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
Nowicki, Emily M; O'Brien, John P; Brodbelt, Jennifer S et al. (2014) Characterization of Pseudomonas aeruginosa?LpxT reveals dual positional lipid A kinase activity and co-ordinated control of outer membrane modification. Mol Microbiol 94:728-41
Brodbelt, Jennifer S (2014) Photodissociation mass spectrometry: new tools for characterization of biological molecules. Chem Soc Rev 43:2757-83
Henderson, Jeremy C; O'Brien, John P; Brodbelt, Jennifer S et al. (2013) Isolation and chemical characterization of lipid A from gram-negative bacteria. J Vis Exp :e50623
O'Brien, John P; Brodbelt, Jennifer S (2013) Structural characterization of gangliosides and glycolipids via ultraviolet photodissociation mass spectrometry. Anal Chem 85:10399-407
Cullen, Thomas W; O'Brien, John P; Hendrixson, David R et al. (2013) EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence. Infect Immun 81:430-40
Hankins, Jessica V; Madsen, James A; Needham, Brittany D et al. (2013) The outer membrane of Gram-negative bacteria: lipid A isolation and characterization. Methods Mol Biol 966:239-58