The concept that pathogenic bacteria have long range (pilus) and short range (specific proteins or sugars) attachment organelles is now well accepted. This proposal relates to short range attachment and invasion by Haemophilus ducreyi. The lipooligosaccharide (LOS) of Neisseria gonorrhoeae and H. ducreyi share important common characteristics. Both species have a terminal trisaccharide consisting of Gal beta1-4GlcNAc beta 1-3Gal on the LOS oligosaccharide. Recent data demonstrated that gonococcal LOS is important in adherence and invasion and the Gal beta 1-4GlcNAc beta 1-3Gal is involved. The applicant has shown that H. ducreyi adheres and invades human cells. Dr. Campagnari's hypothesis is that the oligosaccharide of H. ducrei LOS binds to a surface structure on keratinocytes. He also hypothesizes that the eukaryotic structure mediating such binding is in the family of the Galactose binding proteins expressed by human cells. He postulates that the Gal beta 1-4GlcNAc beta 1-3Gal LOS structure is involved in adherence, which leads to host cell destruction and ulcer formation. This carbohydrate-ligand-like interaction is one of the important mechanisms allowing H. ducreyi to adhere to, survive on and perhaps invade human cells. These hypotheses will be examined by the following specific aims: (1) To determine the role of LOS in adherence and invasion of keratinocytes will be determined by (a) comparing H. ducreyi 35000 and an isogenic LOS mutant(s) in adherence/invasion studies; (b) using MAbs directed to specific LOS epitopes to inhibit adherence/invasion; (c) using specific carbohydrates to block bacterial binding to keratinocytes; (d) determining the effect of sialic acid on adherence/invasion of keratinocytes. (2) To determine the role of H. ducreyi LOS in lesion formation in vivo, using the chilled rabbit model to (a) compare the ability of H. ducreyi 35000 and the isogenic LOS mutant(s) to cause lesions; (b) compare purified LOS from the isogenic strains in lesion formation; (c) compare desialylated and sialylated forms of these LOS to evaluate NANA in lesion formation. (3) To identify and isolate the structure(s) on keratinocytes which interacts with H. ducreyi LOS by (a) using photoactivatable, radiolabelled H. ducreyi LOS; (b) using antibodies to the asialoglycoprotein receptor or alpha-1-acid glycoprotein; (c) carbohydrate based affinity chromatography; (d) developing monoclonal and polyclonal antibodies to these structures. By carefully analyzing this bacterial-host cell interaction at the cell to cell level, Dr. Campagnari can begin to decipher the early steps for initiating an active infection and thus provide a better understanding of H. ducreyi pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030006-05
Application #
2429389
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1991-09-30
Project End
2001-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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
Tullius, Michael V; Phillips, Nancy J; Scheffler, N Karoline et al. (2002) The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis. Infect Immun 70:2853-61
Filiatrault, M J; Munson Jr, R S; Campagnari, A A (2001) Genetic analysis of a pyocin-resistant lipooligosaccharide (LOS) mutant of Haemophilus ducreyi: restoration of full-length LOS restores pyocin sensitivity. J Bacteriol 183:5756-61
Young, R S; Filiatrault, M J; Fortney, K R et al. (2001) Haemophilus ducreyi lipooligosaccharide mutant defective in expression of beta-1,4-glucosyltransferase is virulent in humans. Infect Immun 69:4180-4
Filiatrault, M J; Gibson, B W; Schilling, B et al. (2000) Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and beta1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats. Infect Immun 68:3352-61
Palmer, K L; Thornton, A C; Fortney, K R et al. (1998) Evaluation of an isogenic hemolysin-deficient mutant in the human model of Haemophilus ducreyi infection. J Infect Dis 178:191-9
Gibson, B W; Campagnari, A A; Melaugh, W et al. (1997) Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis. J Bacteriol 179:5062-71
Spinola, S M; Orazi, A; Arno, J N et al. (1996) Haemophilus ducreyi elicits a cutaneous infiltrate of CD4 cells during experimental human infection. J Infect Dis 173:394-402
Melaugh, W; Campagnari, A A; Gibson, B W (1996) The lipooligosaccharides of Haemophilus ducreyi are highly sialylated. J Bacteriol 178:564-70
Spinola, S M; Wild, L M; Apicella, M A et al. (1994) Experimental human infection with Haemophilus ducreyi. J Infect Dis 169:1146-50

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