H. proteins were D-Luciferin potassium salt observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Much like other O-antigen deletion mutants, the mutant was pleiotropic for autoaggregation and motility (it was FliC unfavorable as determined by immunoblotting and flagellum unfavorable as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the mutant shed lower numbers of bacteria ( 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the mutant than of the wild type Rabbit polyclonal to WBP2.WW domain-binding protein 2 (WBP2) is a 261 amino acid protein expressed in most tissues.The WW domain is composed of 38 to 40 semi-conserved amino acids and is shared by variousgroups of proteins, including structural, regulatory and signaling proteins. The domain mediatesprotein-protein interactions through the binding of polyproline ligands. WBP2 binds to the WWdomain of Yes-associated protein (YAP), WW domain containing E3 ubiquitin protein ligase 1(AIP5) and WW domain containing E3 ubiquitin protein ligase 2 (AIP2). The gene encoding WBP2is located on human chromosome 17, which comprises over 2.5% of the human genome andencodes over 1,200 genes, some of which are involved in tumor suppression and in the pathogenesisof Li-Fraumeni syndrome, early onset breast cancer and a predisposition to cancers of the ovary,colon, prostate gland and fallopian tubes colonized the rectoanal junction mucosa, and the duration of the colonization was shorter ( 0.05). Our previous work showed that flagella do not influence O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization. The enterohemorrhagic D-Luciferin potassium salt (EHEC) strains are a subset of Shiga toxin-producing strains that have been D-Luciferin potassium salt associated with human illnesses, including self-limited watery diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (19, 27). Among the EHEC serotypes, O157:H7, which expresses somatic (O) antigen 157 and flagellar (H) antigen 7, causes severe morbidity and large disease outbreaks, making this bacterium one of the most important food-borne and waterborne pathogens worldwide (12, 27). Healthy cattle are the main reservoirs for O157:H7 and non-O157 EHEC pathogens (2, 41) and the most common source of food-borne and direct-animal-contact infections (6, 14). Cattle frequently carry O157:H7 for either transient or long periods without suffering from pathological symptoms (4). Previous studies have exhibited that this bovine terminal rectal mucosa is the main site of O157:H7 colonization (28, 32). The O-specific polysaccharide side chain or O antigen contains many repeats of an oligosaccharide unit and is part of the lipopolysaccharide (LPS) present in the outer membrane of gram-negative bacteria. The structure of the repeating units exhibits enormous antigenic variability and determines serological specificity. The O157 O side chain contains cluster. The O157 region is comprised of 12 genes, including four GDP-l-fucose pathway genes (O157:H7 are required for efficient bacterial colonization at the bovine terminal rectal mucosa (37). Other effector proteins associated with the type III secretion system (TTSS) may also play a role in bovine colonization (28). LPS and O antigen have been implicated in cell adherence and D-Luciferin potassium salt colonization in animals for numerous microorganisms, including 16M (13), serovar Typhi (26), serovar Typhimurium (8, 21), and O139 (29, 39). The O antigen of O157:H7 has been suggested to be important for bacterial survival in an infant rabbit intestinal disease model (15). However, the role of the O antigen of O157:H7 in persistence and colonization in the healthy ruminant host is not known. In this study, we (i) constructed a defined O-antigen-negative mutant by deleting the perosamine synthetase gene in the locus (mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa. MATERIALS AND METHODS Bacterial strains, plasmids, media, and growth conditions. Table ?Table11 lists the bacterial strains and plasmids used in this study. Bacteria were produced in Luria-Bertani (LB) (10 g/liter Bacto tryptone, 5 g/liter Bacto yeast extract, 5 g/liter NaCl) broth or agar (1.5% [wt/vol] agar), Eagle’s minimal essential medium (MEM), or sorbitol MacConkey agar (SMAC), as indicated below. Ampicillin (100 g/ml), kanamycin (50 g/ml), cefixime (50 ng/ml), potassium tellurite (2.5 g/ml), 4-methylumbelliferyl–d-glucuronide (0.1 mg/ml), and/or novobiocin (200 g/ml) was added when appropriate (Sigma Aldrich, St. Louis, MO). Oligonucleotide primers were purchased from Invitrogen (Carlsbad, CA). TABLE 1. Bacterial strains and plasmids used in this study O157:H7 ATCC 43894, clinical isolate, O157:H7 ATCC 43894, clinical isolate, transformed with pCRII::O157:H7, clinical isolate, plasmid vector (Apr Knr)Invitrogen????pCRII::cloned into pCRII (Apr Knr)This study Open in a separate windows aATCC, American Type Culture Collection, Manassas, VA. Gene manipulation. To make a mutant deficient in expression of the O157 antigen, the gene encoding a putative perosamine synthetase was selected for deletion based on its position in the cluster and previous reports of the effect of disruption of this gene (3, 23, 40). The Red recombinase system (9) was utilized for gene deletion, as previously described.