Campylobacter jejuni is the leading cause of foodborne illness in North America and is among the major causes of bacterial diarrhea worldwide. Flagella and motility are required for intestinal colonization and invasion of intestinal epithelial cells by C. jejuni, and flagellin is an immunodominant and possibly a protective antigen. Flagellin from C. jejuni strain 81-176 and Campylobacter coli strain VC167 are glycosylated at 19 and 16 serine or threonine residues, respectively, with a 9 carbon sugar called pseudaminic acid and derivatives of pseudaminic acid. The modifications, which account for approximately 10% of the weight of these glycoproteins, are surface exposed on the flagella filament and are likely involved in interaction of flagellin with the eukaryotic host. Genetic analyses indicate that the pathway for biosynthesis of pseudaminic acid is conserved in both 81-176 and VC167. Flagellins from both strains contain minor modifications that are acetamidino forms of pseudaminic acid (mass 315 Da). However, the 315 Da group synthesized by 81-176 and VC 167 are structurally and immunologically distinct and are synthesized by independent pathways in each organism. The data suggest that campylobacter flagellin needs to be glycosylated in order to be exported and/or assembled into a filament. A mutant in 81-176 that is unable to synthesize the acetamidino form of pseudaminic acid appears to be attenuated in virulence.
The aim of this study is to further elucidate the pathways by which the different forms of pseudaminic acid are synthesized and to study unique aspects of the regulation of these glycosylation genes. Site-specific mutagenesis will be done on flagellin to eliminate modification sites sequentially in order to determine sites that are critical for flagella function and the rules of site occupancy. The biological role of flagella glycosylation will be studied by examining a series of mutants in in vitro and in vivo assays of virulence.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI043559-04A1
Application #
6630815
Study Section
Special Emphasis Panel (ZRG1-VR (02))
Program Officer
Schmitt, Clare K
Project Start
1999-07-01
Project End
2008-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
4
Fiscal Year
2003
Total Cost
$234,600
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Rockville
State
MD
Country
United States
Zip Code
20817
Ewing, Cheryl P; Andreishcheva, Ekaterina; Guerry, Patricia (2009) Functional characterization of flagellin glycosylation in Campylobacter jejuni 81-176. J Bacteriol 191:7086-93
Yokoyama, Takeshi; Paek, Seonghee; Ewing, Cheryl P et al. (2008) Structure of a sigma28-regulated nonflagellar virulence protein from Campylobacter jejuni. J Mol Biol 384:364-76
Galkin, Vitold E; Yu, Xiong; Bielnicki, Jakub et al. (2008) Divergence of quaternary structures among bacterial flagellar filaments. Science 320:382-5
Guerry, Patricia (2007) Campylobacter flagella: not just for motility. Trends Microbiol 15:456-61
Poly, Frederic; Ewing, Cheryl; Goon, Scarlett et al. (2007) Heterogeneity of a Campylobacter jejuni protein that is secreted through the flagellar filament. Infect Immun 75:3859-67
Guerry, Patricia; Ewing, Cheryl P; Schoenhofen, Ian C et al. (2007) Protein glycosylation in Campylobacter jejuni: partial suppression of pglF by mutation of pseC. J Bacteriol 189:6731-3
McNally, David J; Aubry, Annie J; Hui, Joseph P M et al. (2007) Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans. J Biol Chem 282:14463-75
Guerry, Patricia; Ewing, Cheryl P; Schirm, Michael et al. (2006) Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence. Mol Microbiol 60:299-311
Goon, Scarlett; Ewing, Cheryl P; Lorenzo, Maria et al. (2006) A sigma28-regulated nonflagella gene contributes to virulence of Campylobacter jejuni 81-176. Infect Immun 74:769-72
McNally, David J; Hui, Joseph P M; Aubry, Annie J et al. (2006) Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach. J Biol Chem 281:18489-98

Showing the most recent 10 out of 16 publications