Haemophilus ducreyi is a gram-negative human pathogen and causes the genital ulcer disease 'chancroid'. Although uncommon in the U.S, outbreaks continue in some regional areas and chancroid remains a significant risk factor for the transmission of HIV in developing countries. Moreover, the emergence of antibiotic resistant strains suggest that a molecular-based understanding of the events that enable this human pathogen to infect, survive, form lesions, and disseminate is needed if we are to contain or eliminate this disease worldwide. Outer-membrane lipooligosaccharides (LOS) have been identified as a major virulence factor in several mucosal pathogens. LOS has been implicated in the adherence and invasion of human tissues, serum resistance, and resistance to phagocytosis and complement mediated killing. The previous application was concerned with elucidating the structures of these complex glycolipids and assessing biological activities. Ultimately, our goal is to provide the molecular underpinnings for the development of new carbohydrate-based vaccines and drugs targeting critical host-pathogen interactions. To do this, we need to understand the repertoire of LOS-glycoforms expressed by this pathogen, the function(s) of these glycoforms, and how LOS interacts with host proteins. In this renewal, we propose to explore the pathway that leads to the expression of LOS terminating in sialic acid and its acceptor, N-acetyllactosamine, using chemistry and structural biology tools. Sialic acid is used by many human pathogens, including bacteria, viruses and trypanosomes, and is an important sugar in cell recognition mechanisms throughout biology. Given the relative simplicity of the disease and the well-defined structures for H. ducreyi LOS, this organism represents an ideal model system for studying the pathway(s) leading to sialic acid expression in these surface glycolipids and the role of sialylated LOS in host-pathogen interactions. The hypothesis we wish to examine in this proposal is that bacterial surface LOS containing sialic acid play important roles in the infection process and are key to reaching an understanding of how carbohydrates and sialic acid mediate host-pathogen interactions. To address this hypothesis, three specific aims will be explored as follows:
Aim #1, investigate the sialic acid pathway leading to the expression of outer-membrane sialylated LOS glycoforms;
Aim #2, examine the relationships among the polylactosamine, sialyllactosamine and asialo- oligosaccharide branch pathways in LOS biosynthesis;
Aim #3, develop and employ sialic acid analogs as inhibitor of the sialic acid pathway in H. ducreyi.
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