The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif. Cystic fibrosis (CF)1 is the most frequent lethal genetic disease associated with a single gene in Caucasians (1). CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ATP binding cassette (ABC) transporter that functions as a phosphorylation and nucleotide-regulated chloride channel located in the apical membrane of epithelial cells (2, 3). The ABC transporters constitute a large family of ubiquitously expressed proteins, mostly involved in ATP-driven translocation of diverse substrates across biological membranes (4, 5). It has been proposed that a functional ABC transporter has a minimal structural requirement of two membrane-spanning domains and two nucleotide binding domains (NBDs) (5). The NBDs, or ABC cassettes, share 30 -50% sequence identity (6) and are characterized by the presence of three conserved motifs; Walker A and Walker B motifs are present in several nucleotide binding and hydrolyzing proteins (7), and the ABC-signature motif, located just upstream of the Walker B, is diagnostic of ABC cassettes (5, 6).The deletion of the Phe-508 (⌬F508) in the first nucleotide binding domain (NBD1) of CFTR is the most frequent CFcausing mutation, present in 90% of CF chromosomes. ⌬F508 impairs normal protein maturation and trafficking to the plasma membrane (8, 9), presumably through a localized effect on the folding of the NBD1 domain (10 -12). This misfolding results in retention of CFTR⌬F508 by the endoplasmic reticulum-associated quality control and in subsequent degradation with the participation of the cytoplasmic proteasome (13). The CFTR...

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Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin O157 ) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of <100 were vaccinated twice with purified intimin O157 or shamvaccinated with sterile buffer. Intimin O157 -specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin O157 . Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10 6 CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin O157 -specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin O157 as a viable candidate for an EHEC O157:H7 antitransmission vaccine.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a common cause of bloody diarrhea and the most common cause of hemolytic-uremic syndrome in humans in the United States (1). EHEC strains can be foodborne pathogens, and cattle are important reservoirs of EHEC O157:H7 strains. All EHEC strains produce cytotoxins called Shiga toxins (Stx1 and Stx2), previously called Shiga-like toxins (SLT-1 and SLT-2) or verotoxins (VT1 and VT2). Many EHEC strains, including EHEC O157:H7, can attach intimately to host cell membranes and efface microvilli and cytoplasm in a pattern referred to as an attaching-and-effacing (A/E) lesion (18). EHEC intimin is an outer membrane protein adhesin encoded by the eae gene of EHEC (16) that is required for intestinal colonization and for A/E activity of EHEC O157:H7 in piglets (6,8,9,19) and neonatal calves (6). Antibodies against intimin O157 significantly reduce the adherence of E. coli O157:H7 strains, as well as certain related strains, to HEp-2 cells (11). Based on this observation, we hypothesize that a vaccine that contains intimin O157 may induce an immune response in cattle that will prevent or decrease the level of intestinal colonization by E. coli O157:H7. We speculate that the resultant diminution in fecal shedding of EHEC O157:H7 by these animals could, in turn, lead to a decrease in the transmission of EHEC O157:H7 and other A/E E. coli strains to humans.In the study reported here, we tested the concept that antiintimin antibodies can protect animals from colonization with EHEC O157:H7. For that purpose, we selected neonata...

Analysis of more than 100 extragenic suppressors of the lamB14D signal-sequence mutation {changes Val in the hydrophobic core region at position 14 to Asp) has revealed alterations that appear to lie at prlA (secYJ and secA {prlD), two loci known to be mutable to suppressor alleles, and a new suppressor termed priG. One allele of the new suppressor class, priG1, has been characterized in some detail. This suppressor counteracts, to some degree, the export defect conferred by a variety of signal-sequence mutations in two different genes, lamB and malE. Genetic analysis shows that the dominant suppressor mutations are linked tightly to, and probably allelic with, the gene secE. This result, coupled with data obtained with conditional-lethal alleles of secE (see accompanying paper by Schatz et al.), argues strongly that SecE is an important component of the cellular protein export machinery in Escherichia coli.

Enterohemorrhagic Escherichia coli produce an attaching and effacing lesion upon adhering to the intestinal epithelium. Bacterial factors involved in this histopathology include the intimin adhesin and E. coli secreted proteins (Esps) A and B. In this study we investigated the serum antibody responses to recombinant E. coli O157:H7 intimin, EspA, and EspB by immunoblotting. Canadian patients with O157:H7 infection (n=10), Swedish patients with O157:H7 (n=21), non-O157 (n=18), or infection from which the serotype was not available (n=3), and asymptomatic household members (n=25) were studied and compared with Canadian (n=20) and Swedish controls (n=52). In Canadian patients, IgG antibodies to intimin, EspA, and EspB were analyzed, in Swedish patients and their household members IgA, IgG, and IgM antibodies to EspA and EspB were studied. Patients and household members mounted an antibody response to the antigens. Significantly more patients developed an acute response to EspB compared with controls (P<0.01 Canadian patients, P<0.0001 Swedish patients). EspB IgA, IgG, and IgM had a specificity of 100%, 86%, and 86%, positive predictive value of 100%, 83%, and 81%, and sensitivity of 57%, 69%, and 63%, respectively, and appear to be an appropriate assay for the detection of EHEC infection. In cases of hemolytic uremic syndrome or hemorrhagic colitis this assay may be useful when a fecal strain has not been isolated, or in epidemics of non-O157 infection.