?(Fig.8E),8E), immunized animals appeared completely guarded against vascular changes induced by the challenge strain in the submucosa and serosa. by 81 C-terminal residues (860 to 939 amino acids) made up of a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1eae860-939) was well tolerated, as observed by the absence of clinical indicators of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1eae860-939 as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1eae860-939 followed by challenge with the virulent strain RDEC-H19A, an Stx1-generating derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and exhibited intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1eae860-939-immunized rabbits showed no clinical indicators of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1eae860-939, indicating that truncation of the intimin functional domain name not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it GW4064 is not possible to gauge the exact contribution of residual intimin immunity to protection, this GW4064 attenuation strategy for A/E strains shows promise for the development of effective vaccines to prevent EHEC contamination in humans and animals. Strains of Shiga toxin (Stx)-generating (EHEC), are important food-borne pathogens GW4064 for humans. The clinical manifestations of EHEC infections range from watery diarrhea, bloody diarrhea, or hemorrhagic colitis to life-threatening hemolytic-uremic syndrome (HUS) (29, 40). The majority of EHEC strains induce attaching and effacing (A/E) lesions characterized by intimate bacterial attachment to the epithelial cells with destruction of microvilli. Genes encoding the A/E phenotype are clustered on a pathogenicity island termed the locus of enterocyte effacement (LEE), the complete nucleotide sequences of which have been obtained for human EHEC O157:H7 strain EDL933 (43), and other related A/E organisms, including human enteropathogenic (EPEC) O127:H6 strain E2348/69 (17), rabbit EPEC (REPEC) O15:H? GW4064 strain RDEC-1 (50), and share a 34-kb conserved region made up of 40 (RDEC-1) or 41 (EPEC, EHEC, and (17). The operons encode components of the type III secretion system (and genes) and the (LEE-encoded regulator) gene. The operon encodes the adhesin intimin, Tir (translocated intimin receptor), and CesT (the chaperone for Tir) (27, 31). The operon encodes the secreted proteins EspA, -D, and -B involved in delivering Tir and other proteins to host cells, and EspF, which is usually translocated into host epithelial cells and alters their tight junction permeability (37). Intimin is the outer membrane protein (OMP) which is crucial for the A/E phenotype because bacterial romantic adherence to host epithelial cells is usually mediated by binding of intimin to Tir, which is usually delivered by A/E organisms to eukaryotic cells (19-21, 24, 25, 31). All intimin alleles (1, 42, 45, 48) currently known demonstrate high homology in their N-terminal regions, but great diversity in their C-terminal regions, which are essential for binding to Tir. Intimins of A/E (AEEC), including human EPEC O127:H6 (intimin-), EHEC O157:H7 (intimin-), or REPEC O15:H? (intimin-), show greater than 94% amino acid (aa) identity over the N-terminal two-thirds of the molecule while showing only 55% homology over the remaining one-third at the C terminus (50), which includes the region responsible for Tir binding. The crystal structure of GW4064 the EPEC intimin C-terminal fragment (residues 658 to 939) has been solved and reveals three adjacent domains with individual functions (18, 34): the Rabbit Polyclonal to PMS2 immunoglobulin (Ig)-like D1 (residues 658 to 751) and D2 (residues 752 to 841) domains and the C-type lectin-like D3 (residues 842 to 929) domain. Binding of intimin to Tir is usually mediated primarily by interaction of the lectin-like D3 domain name of intimin with the intimin-binding domain name of Tir (34). Within the Tir-binding region of intimin, two conserved cysteine residues (aa 860 and 937 of EPEC intimin) form a disulfide loop essential for intimin function (18, 25, 34). This disulfide loop is usually conserved in invasin and all the intimin variants (17, 42, 45, 48, 50). The immunodominant regions of the molecule have been demonstrated to be in the domains D1 and D2, as shown by reaction with intimin-specific antiserum (1). Attenuation of virulence by deletion of intimin has been demonstrated for human EPEC (O127:H6) (14), human EHEC (O157:H7) (15), REPEC (O103:H2) (35), and (12). Further evidence of the crucial role of intimin in bacterial virulence is usually provided by studies showing that anti-intimin immune responses directed against the C-terminal Tir-binding fragment can favorably modulate the outcome of contamination by.