This project is to study the attachment to and invasion of eukaryotic cells of human origin by Haemophilus ducreyi, a gram-negative bacillus that causes the genital ulcer disease chancroid. The long-term scientific objectives are to understand virulence factors of H. ducreyi and the pathogenesis of chancroid. Genital ulcer disease, especially chancroid, is a very important cofactor in the transmission of HIV. A better understanding of the pathogenesis of chancroid will help further the understanding of how chancroid enhances the transmission of HIV. Study of the morphology of chancroid suggests that H. ducreyi adherence to host cells at the base of the lesions and entry into the cells are important in the pathogenesis of chancroid. We have developed in vitro models of H. ducreyi infection of human foreskin fibroblast cells (HFF) and of keratitized epithelium-like cells. We will study factors that are important in the adherence to and entry into the eukaryotic cells and the mechanisms whereby H. ducreyi infect human epithelium. Our studies will enhance the understanding of virulence factors of H. ducreyi and help clarify the pathogenesis of chancroid. Our preliminary data show that H. ducreyi adheres to and invades HFF cells. H. ducreyi appears to be invasive based on multiple factors: (1) the rapid entry into HFF cells; (2) the morphology when H. ducreyi is inside the HFF cells; (3) the presence of H. ducreyi in a high percentage if not all the HFF cells; (4) the increase in the numbers of intracellular H. ducreyi over 24 hours; and, (5) observations with cytochalasin B and D and colchicine indicate that drugs active on microfilaments and microtubules block or inhibit the entry of H. ducreyi into HFF cells. The HFF cells with H. ducreyi inside them remain viable over greater than or equal to 24 hours, which also is consistent with a pathogenic role for intracellular infection. These preliminary data will be expanded by additional studies. To accomplish our goals we have outlined three Specific Aims: 1) To characterize the in vitro model of H. ducreyi adherence to and invasion into human foreskin fibroblasts; 2) To examine the biological activity of H. ducreyi on the keratinized in vitro model of epithelium (""""""""floating rafts""""""""); 3) To study the molecular mechanisms by which H. ducreyi adheres to and invades eukaryotic cells. The results of these studies will add significantly to the understanding of virulence factors of H. ducreyi and the pathogenesis of chancroid.
