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.
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