Characterization of the hemolytic activity of Haemophilus ducreyi.

H. ducreyi is the causative agent of chancroid, a genital ulcer disease most prevalent in developing countries. Chancroid enhances the heterosexual transmission of human immunodeficiency virus and is identified in focal outbreaks in the United States, but little is known about its pathogenesis. We studied the hemolysin produced by H. ducreyi because this molecule might be an important virulence factor in the pathogenesis of chancroid. Ten strains of H. ducreyi were tested on newly devised blood agar plates and were found to have hemolytic activity. We examined the hemolytic activity of H. ducreyi 35000 further and found that it was heat labile, cell associated, greatest at pH 7.0, and produced in logarithmic- but not stationary-phase cultures. Using transposons Tn916 and Tn1545-delta 3, we have isolated three classes of transposon mutants of strain 35000: those with no detectable hemolytic activity, those with reduced hemolytic activity, and those with enhanced hemolytic activity. Transposon insertions in the nonhemolytic mutants were located in a DNA sequence which hybridized to the Proteus mirabilis hemolysin gene. Analysis of clones containing overlapping sections of this region served to further localize the H. ducreyi hemolysin gene and allow its expression in Escherichia coli and complementation of the nonhemolytic defect in an H. ducreyi mutant. These experiments indicate that H. ducreyi 35000 produces a hemolysin that is related to the calcium-independent hemolysin produced by P. mirabilis. Further experiments are needed to define the similarity of the H. ducreyi hemolysin to other calcium-independent hemolysins and to determine its role in the pathogenesis of chancroid.