J Infect Dis

J Infect Dis. wild-type in experimental disease in rabbits. is the etiologic agent of chancroid, a sexually transmitted disease characterized by genital ulcers and, in more than 50% of cases, inguinal Rabbit Polyclonal to SERPINB4 lymphadenopathy (14, 29). This disease is frequently diagnosed in developing countries, where it CB-184 is often the most common cause of genital ulcers (47). However, outbreaks of chancroid occur in the United States, particularly in inner cities and among those who exchange sex for drugs or money (14, 29). In Africa, chancroid has been shown to increase the risk of acquiring human immunodeficiency computer virus contamination (6, 38, 53), possibly by creating a portal of access in its host by disrupting the epithelium and/or by increasing the local concentration of CD4+ cells that are targets for infection by the computer virus (44). Chancroidal ulcers contain disintegrating epithelial cells, fibroblasts, and inflammatory cells, including macrophages, polymorphonuclear leukocytes, and lymphocytes, as well as viable (25). The tissue destruction and the ability to survive in the presence of an inflammatory cell infiltrate are consistent with the production of toxins. Several toxins including a cell-associated hemolysin (35, 50) and a secreted cytotoxin, the cytolethal distending toxin (10, 40), have been recognized in 35000. Other virulence factors include lipooligosaccharide (LOS), which may contribute to ulcer formation by enhancing the migration of inflammatory cells to the lesion site and increasing the resistance of to phagocytosis, and proteins that allow the organism to acquire heme, a nutritional requirement of this organism (7, 15, 45). As with other organisms, the virulence of is probably multifactorial and dependent on the presence, relative expression, and cell range of several different virulence factors. The hemolysin has been cloned and found to be homologous to the pore-forming, calcium-independent hemolysins of (22, 35, 50). The hemolysin requires at least two genes for expression, hemolysin is usually encoded by two genes, termed and (35), which presumably have functions analogous to those of the homologous genes. The cell types sensitive to hemolysin include human epithelial cells, fibroblasts, macrophages, T lymphocytes, and B lymphocytes (1, 34, 54). This target cell range may enable to cause the tissue destruction characteristic of chancroidal ulcers as well as inhibit the inflammatory and specific immune responses to this organism. 35000 is able to invade epithelial cells (16, 48), and hemolysin has been shown to enhance invasion by this organism (54), suggesting another role for hemolysin in virulence. In this study, we surveyed isolates in our international strain collection for the presence of genes homologous to and and for expression of hemolytic activity. We decided that hemolysin is usually immunogenic in both animal models and chancroid patients. We also evaluated the effectiveness of immunization with purified hemolysin in attenuating ulcer formation and growth of in CB-184 rabbits challenged with this organism. MATERIALS AND METHODS Bacterial strains, plasmids, media, and growth conditions. Bacterial strains and plasmids used in this study are outlined in Furniture ?Furniture11 and ?and2.2. The construction of strain 35000APC is described elsewhere (54). This strain is nonhemolytic due to a deletion in CB-184 the internal gene, replaced by a (chloramphenicol acetyltransferase) cassette as pictured in Fig. ?Fig.1.1. TABLE 1 stock cultures and clinical?isolates DH5Host for cloning experimentsGibcoBRL, Gaithersburg, Md. ?BL21(DE3)Host for protein expressionNovagen, Madison, Wis. ?35000-TcAStrain 35000 with Tninserted in 35000-KmAStrain 35000 with Tninserted in 35000APCStrain 35000 with cassette in gene, nonhemolytic54?RdMarilyn Roberts ?cloning vector, AprGibcoBRL ?pCR2.1TA cloning vector for CB-184 cloning PCR products, Ampr KanrInvitrogen, San Diego, Calif. ?pET24+Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, KanrNovagen ?pET24a+Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, KanrNovagen ?pBCKSCmr plasmid derived from pUC19 with KS multiple cloning siteStratagene, La Jolla, Calif. ?pPT384-ETagenes cloned into pET24a+ genes from pLS88 and multiple cloning sites and gene from pBluescript SK54?pPT384gene region in pTZ18 in same orientation as promoter50?pPT376BCKS5.8-kb and all of cloned into pBCKS genes cloned into pET24+ genes cloned into pLSSK in same orientation as promoter. His tag sequence from pET24+ is usually fused to 3 end of hemolysin gene region from strain 35000, the hemolysin-negative derivative of strain 35000, constructs made up of the and genes, and locations of primers and probes used in this study. Restriction sites utilized for cloning that were added as a result of PCR amplification or from multiple cloning sites are underlined. The cassette, used to replace the gene in 35000APC, is necessarily not drawn to scale..