Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb 3 receptors which present the P k trisaccharide Gal␣(1؊4)Gal␤(1-4)Glc␤. Crystallographic studies have identified three P k trisaccharide (P k -glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P k -glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P k -glycoside and pentavalent P k trisaccharide (penta-STARFISH) in solution and Gb 3 presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb 3 involved all three sites. Binding of all single site mutants to liposomal Gb 3 was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb 3 in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb 3, even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membranelocalized Gb 3 . Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb 3 trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.Verotoxins produced by Escherichia coli have been associated with diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome in humans (1, 2). There is evidence that verotoxins play a central role in the pathogenesis of these diseases, causing microvascular damage by direct toxicity to endothelial cells. Verotoxins consist of an enzymatic A subunit (32 kDa) that is noncovalently associated with a homopentameric receptor-binding B subunit (37.5 kDa). After holotoxin internalization by the cell, the A subunit causes catalytic inactivation of the 28 S ribosomal RNA, disrupting protein synthesis and inducing apoptosis (3, 4). Receptor binding is the primary event leading to toxin internalization, which is required for cellular toxicity (5).The B subunit oligomer is responsible for toxin binding to the carbohydrate portion of the specific cell-surface glycolipid receptor and it has been shown that the susceptibility to VT1 toxin is correlated with Gb 3 1 receptor presence on the cell surface (6). In the absence of the A subunit, B subunit monomers form pentamers that are fully functional in terms of binding to Gb 3 (7). Determination of the crystal structure o...

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastro-intestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA. Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T*6) DNA is taken up almost as effectively as homologous DNA. Both T6 and T4 richia coli 15T-and phages T4 and T6, provided by Irena Pietrzykowska of this Institute. The phages were cultivated on E. coli BB, obtained from W. Szybalski of the University of Wisconsin. Nonglucosylated phage T6 DNA (T*6 DNA) was prepared from phage T6 cultivated on the uridine diphosphoglucose phosphorylase-defective mutant of E. coli B-4°Luria no. 56 (6), provided by S. E. Luria of the Massachusetts Institute of Technology. The nonglucosylated phage T6 (T*6) titer was determined with the aid of the strain Shigella dysenteriae, supplied by N. Symonds of the University of Sussex, England. Absence of glucosylation was confirmed by 1429 on August 3, 2020 by guest

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thytrp-. Shortly after uptake, most of the radioactive E. coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5-photoreceptorspecific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin ␤␥ subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.In response to a single photon, mammalian photoreceptors produce an electrochemical signal that is integrated with many others, transmitted to higher visual centers in the brain, and ultimately perceived as vision. This elegant system depends on the coordinated expression of a large set of genes and on the interaction of their protein products. Many components of this system are expressed only in photoreceptors, and several are associated with retinal degeneration when defective. Mutations in genes encoding proteins involved in the phototransduction pathway (e.g. rhodopsin (Refs. 1 and 2), transducin (Refs. 3 and 4), or phosphodiesterase (Refs. 5-8)), photoreceptor structure (e.g. peripherin/RDS and ROM1 (Refs. 9 -12)), interactions between photoreceptors and RPE 1 (e.g. ABCR (Refs. 13-15)), or in photoreceptor-specific gene expression (e.g. CRX) (Refs. 16 -18)) result in inherited retinal degenerations (19,20). Accordingly, we reasoned that genes expressed preferentially or exclusively in retina would be important for the normal retinal structure and function, and would be likely candidates for retinal degenerations. To identify genes with these expression characteristics, we implemented a differential hybridization screen (9, 21). One of the clones identified in this screen, designated PHR1 (for pleckstrin homology domain retinal protein) was of interest because it showed preferential expression in retina and brain. We subsequently cloned and sequenced human and murine full-length PHR1 cDNAs and found that they encode proteins with an N-terminal PH domain and a hydrophobic, putative transmembrane domain at the C terminus. PHR1 is the first PH domain-containing protein found to be...