Mass Spectrometry of Carbohydrates as a Tool for Characterization of Bacterial Vaccines and Pathogens

Principal Investigator: John F. Cipollo
Office / Division / Lab: OVRR / DBPAP / LBP

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Overview

Public Health Issue: Glycoconjugates are present on the surface of all cells and their recognition is involved in pathogen invasion as well as host immune response. It is clear that the identification and structural characterization of these glycoconjugates and their protein interactors is crucial to the understanding of these immunogenic and pathogenic responses and interactions. The structural characterization of these pathogen glycoconjugates and adhesion molecules will reveal vaccine candidates as they are often abundant cell surface components.

Regulatory Contribution: The mass spectrometry methods developed herein will be used for the characterization of oligo- and polysaccharide vaccine components from a variety of bacterial species. The C. elegans surrogate host will be used to assay for vaccine candidates as well as study immune responses to potential vaccine components.

Research Approach: Mass spectrometry isused to identify glycoconjugates from host cells, pathogenic bacteria, and naturally and transgenically produced glycoconjugates and adhesin molecules. Several platforms are under development for use in these studies. Methods are developed for the analysis of bacterial LPS including the short or rough oligosaccharides and larger smooth polysaccharides. A major project involves the development of C. elegans as a surrogate host for bacterial infection. This nematode is infected by many human pathogens and it is expected that many require glycoconjugate specific ligands to infect. C. elegans strains, deficient in the synthesis of specific glycoconjugates as well as resistant to bacterial infection, will be characterized. Further glycoconjugate specific requirements of infection will be mapped out. Affinity proteomics approaches will be used to isolate and identify the bacterial adhesins in a carbohydrate specific manner. A glycan array will be constructed in collaboration with the David Smith at Emory University to study the binding characteristics of isolated adhesins. Studies to investigate the carbohydrate dependant infection of C. elegans by (?) the Coryneform bacteria Microbacterium nematophilum are already underway. M. nematophilum is related to several Coryneforms that infect humans including Corynebacterium diphtheriae.

Mission Relevance & Outcomes: CBER aims to facilitate the development of safe and effective products and promising new technologies. Mass spectrometry is a new and continuously developing technology, which is now being utilized in vaccine discovery and analysis. It is capable of generating large amounts of information in a very short time, which can then be used in structural characterization. The development and implementation of mass spectrometric based approaches to the analysis of oligosaccharides, polysaccharides, other glycoconjugates and their binding partners, will facilitate better characterization of vaccine components and discovery of new vaccine component candidates. C. elegans has recently been utilized as a surrogate host for identification of bacterial virulence factors. Implementation and use of the C. elegans surrogate host for the investigation of carbohydrate dependant host - pathogen interactions will allow identification of vaccine candidates and allow use of the assay for immune response to those candidates, both of which should aid in bringing a safer more effective vaccine product to the public.

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Publications

Appl Microbiol Biotechnol 2008 Oct;80(5):757-65
Bacterial CMP-sialic acid synthetases: production, properties, and applications.
Mizanur RM, Pohl NL

J Biol Chem 2008 Jun 27;283(26):18355-64
Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica.
Magnelli P, Cipollo JF, Ratner DM, Cui J, Kelleher D, Gilmore R, Costello CE, Robbins PW, Samuelson J

J Am Soc Mass Spectrom 2007 Oct;18(10):1799-812
A glycomics platform for the analysis of permethylated oligosaccharide alditols.
Costello CE, Contado-Miller JM, Cipollo JF