EXCEED THE SPACEPROVIDED. Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates HIV transmission. Lacking specimens from naturally infected patients, we developed an experimental model of infection of the skin in human subjects, which resembles natural disease in both its clinical course and histopathology. Throughout the course of experimental infection, the H. ducreyi colocalizes with PMNs and macrophages in the epidermis and upper dermis, and with collagen and fibrin in the dermis. Despite its association with phagocytic cells, H. ducreyi remains predominantly extracellular during experimental infection. The relevance of these findings to natural infection is unknown. We have tested 10 isogenic mutant/parent pairs in the model. Many of these mutants were evaluated because in vitro studies suggested that the gene of interest encoded a virulence determinant. However, only 3 of the mutants were impaired in their ability to progress to pustule formation. Thus, genes that have functions in vitro frequently do not contribute to pustule formation in vivo. Identification of bacterial genes that are exclusively or differentially induced in vivo has led to many fundamental observations about host-pathogen interactions. We recently amplified //. ducreyi transcripts from biopsies of experimental lesions. With the completion of the H. ducreyi genome sequence, we wish to address hypotheses about expression of bacterial genes during human infection, where the bacteria are exposed to a relevant tissue (human skin) and a relevant host response. Our first hypothesis is that H. ducreyi continues to colocalize with PMNs, macrophages and collagen and fibrin during the ulcerative stage of disease and remains primarily extracellular throughout infection. Our second hypothesis is that specific virulence determinants are differentially upregulated by H. ducreyi during infection, and that isogenic mutants in these upregulated genes will be attenuated in the model. To test these hypotheses our aims include: localization of the bacteria in naturally occurring lesions; capture of bacterial transcripts differentially upregulated in vivo; construction of isogenic mutants in selected differentially upregulated genes; evaluation of the mutants in the model. PERFORMANCE SITE ========================================Section End===========================================
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