Haemophilus ducreyi is a Gram-negative bacterium that infects the genital epidermis in humans, causing the sexually transmitted disease (STD) 'chancroid'. This pathogen disrupts the epithelium and forms genital ulcers. Chancroid can act synergistically with other genital diseases to effect an overall rise in STDs and AIDS. This disease continues to be endemic to large parts of the world, and appears disproportionately among minority heterosexuals and in the southern US, where it may now be endemic. This proposal seeks to define the structures and functional roles of surface lipooligosaccharides (LOS) in the pathogenesis of H. ducreyi. The investigator proposes to address the relationship of LOS structures (or chemotypes) to specific phenotypic traits (adherence, invasion, etc.) that are critical in defining the molecular determinants of H. ducreyi virulence. Given these goals, the investigator defines four major aims for this proposal:
Specific Aim 1 is proposed to identify and characterize novel LOS-oligosaccharide structures and biosynthetic pathways as expressed among wild-type strains of H. ducreyi and assay for functional differences in these LOS-oligosaccharide structures.
Specific aim 2 is proposed to construct and characterize the structures of LOS produced by defined mutants defective in LOS biosynthesis as tools to elucidate the synthetic pathways and functional roles of LOS in adhesion and invasion mechanisms. These studies will include work on the heptosyl and galactosyl transferase mutants of strain 35000 (losA-and losB-), as well as mutants defective in sialic acid (neuA) and lipid A biosynthesis.
Specific aim 3 is proposed to examine the specificity, binding affinities and valence of LOS-lectin interactions as a model for bacterial cell adhesion to human epithelial cells. The investigator proposes to identify the receptors involved in LOS binding, and to construct and test multi-valent carbohydrate ligands as biological and chemical models of the LOS-receptor interaction.
Specific aim 4 is proposed to characterize key enzymes involved in the biosynthesis of LOS that contain sialic acid, such as the CMP-NeuAc synthetase and H. ducreyi sialyltransferase. The investigator hypothesizes that data obtained from these studies will allow the investigator to determine the roles of LOS in H. ducreyi infection at the molecular level. The investigator's long-term goals are directed towards understanding the basic chemistry and biology of bacterial pathogenesis, as well as to exploit this information to develop novel therapeutics such as LOS-based vaccines, carbohydrate inhibitors, and/or inhibitors targeting LOS biosynthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI031254-09
Application #
6373227
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Quackenbush, Robert L
Project Start
1994-09-01
Project End
2003-03-31
Budget Start
2001-09-01
Budget End
2003-03-31
Support Year
9
Fiscal Year
2001
Total Cost
$248,064
Indirect Cost
Name
Buck Institute for Age Research
Department
Type
DUNS #
800772162
City
Novato
State
CA
Country
United States
Zip Code
94945
Post, Deborah M B; Gibson, Bradford W (2007) Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles. Proteomics 7:3131-42
Post, Deborah M B; Munson Jr, Robert S; Baker, Beth et al. (2007) Identification of genes involved in the expression of atypical lipooligosaccharide structures from a second class of Haemophilus ducreyi. Infect Immun 75:113-21
Post, Deborah M B; Mungur, Rachna; Gibson, Bradford W et al. (2005) Identification of a novel sialic acid transporter in Haemophilus ducreyi. Infect Immun 73:6727-35
Edwards, Katie J; Allen, Simon; Gibson, Bradford W et al. (2005) Characterization of a cluster of three glycosyltransferase enzymes essential for Moraxella catarrhalis lipooligosaccharide assembly. J Bacteriol 187:2939-47
Greiner, L L; Watanabe, H; Phillips, N J et al. (2004) Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans. Infect Immun 72:4249-60
Pupo, Elder; Phillips, Nancy J; Gibson, Bradford W et al. (2004) Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vib Electrophoresis 25:2156-64
Luke, Nicole R; Allen, Simon; Gibson, Bradford W et al. (2003) Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant. Infect Immun 71:6426-34
Goon, Scarlett; Schilling, Birgit; Tullius, Michael V et al. (2003) Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides. Proc Natl Acad Sci U S A 100:3089-94
Scheffler, N Karoline; Falick, Arnold M; Hall, Steven C et al. (2003) Proteome of Haemophilus ducreyi by 2-D SDS-PAGE and mass spectrometry: strain variation, virulence, and carbohydrate expression. J Proteome Res 2:523-33
Schilling, Birgit; Gibson, Bradford W; Filiatrault, Melanie et al. (2002) Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats. J Am Soc Mass Spectrom 13:724-34

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