Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing bundle-forming pili.

Infection with enteropathogenic Escherichia coli (EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world. The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation. We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells. BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis. The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP induced significantly less cell death, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island. These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine.

Enteropathogenic Escherichia coli virulence factor bundle-forming pilus has a binding specificity for phosphatidylethanolamine.

The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP did not bind PE, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

In vitro binding of Helicobacter pylori to monohexosylceramides.

H. pylori is the major cause of human gastritis, duodenal ulcer and thus gastric adenocarcinoma. Many glycosphingolipid species have been postulated as receptors for H. pylori and it is likely that H. pylori attachment requires multiple, perhaps sequential receptor/ligand interactions. In this study, the binding of a number of H. pylori clinical isolates, as well as stock strains, to acid and neutral glycosphingolipids separated on thin-layer chromatograms was characterized under microaerobic conditions. All H. pylori clinical isolates, laboratory strains and type culture collection strains recognized galactosylceramide (Galbeta1Cer) with ceramide containing sphingosine and hydroxylated fatty acid (type I), or non-hydroxylated fatty acid (type II), on thin-layer chromatograms and when incorporated into liposomes. The clinical isolates bound stronger to Galbeta1Cer (type II) than Galbeta1Cer (type I) on TLC, whereas lab and culture collection strains showed the opposite binding preference. A clear preference in binding to Galbeta1Cer (type I) incorporated into liposome was shown by most tested strains. Clinical isolates bound well to glucosylceramide (Glcbeta1Cer) with hydroxylated fatty acid, whereas weak binding to this glycolipid was detected with the lab and type collection strains. None of the tested strains bound Glcbeta1Cer with non-hydroxylated fatty acid on the solid surface, but some strains of both clinical or type collection origins showed weak or very weak binding in the liposome assay. A clear distinction between the binding specificity of living organisms (under microaerobic conditions) as opposed to dying organisms (under normoxic conditions) illustrates the importance of cellular physiology in this process. These studies illustrate lipid modulation of the potential receptor function of monohexosylceramides and the distinction between the receptor repertoire of H. pylori clinical isolates and cultured strains commonly used to study host-cell adhesion.

Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet.

Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.

Modified on-line monitoring of total gaseous mercury in flue gases using Semtech Hg 2000 analyzer.

The Semtech Hg 2000 analyzer continuously monitors the Hg0 content in flue gas. An on-line measurement method of total gaseous mercury in flue gas developed in our laboratory is described, which uses the absorption cell of the Semtech Hg Analyzer connected to a converter that is located in a furnace heated up to 650 degrees C. The converter can be heated up to 800 degrees C by both the furnace and an extra heating of a Ni-Cr alloy heating wire. Both the absorption cell and the converter are made of quartz. All gaseous Hg2+ species in flue gas are thermally reduced to Hg0 by the converter and detected by the Semtech Hg 2000 analyzer. The thermal reduction efficiencies of different conversion materials, which were filled in the converter, such as quartz chips, granular MgO, Ni and CoO powder, were tested using different flue gas conditions. Studies have shown that HCI is the major factor to inhibit the thermal reduction of Hg2+ to Hg0, and in the converter and the absorption cell Hg0 will react readily with HCl to form HgCl2. Both MgO and Ni could be used in the converter to absorb HCl in the flue gas, but Ni has better absorption efficiency. By using an original Semtech and a modified one, both Hg0 and total gaseous Hg contents in flue gas could be monitored simultaneously and continuously.

Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment.

Using both solid phase and liposome aggregation assays, we screened a variety of glycolipids and phospholipids and found that EHEC and EPEC bind specifically and in a dose-dependent manner to PE. This binding was consistently observed whether the lipid was immobilized on a thin layer chromatography plate, in a microtitre well or incorporated into a unilamellar vesicle suspended in aqueous solution. There was no evidence of binding to other phospholipids such as phosphatidylcholine (PC) or phosphatidylserine (PS). Bacterial binding to two epithelial cell lines also correlated with the level of outer leaflet PE and was reduced following preincubation with anti-PE. The PE-binding phenotype of EPEC appeared to correlate with the bundle-forming pilus (bfp) genotype of a number of clinical isolates. These results provide evidence of a receptor role for PE in the adhesion of EHEC and EPEC to host cells.

Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography.

The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.

Influence of plasmids on bacterial susceptibility to the bactericidal activity of sera. I. Characteristic of R plasmids of enteropathogenic strains of Escherichia coli isolated from clinical cases.

The 47 enteropathogenic Escherichia coli strains isolated from the cases of infant diarrhoea were characterized from the point of view of antigenic structures and drug resistance. Six R plasmids were obtained from these strains and were tested for protective activity of E. coli K12 W 1485 against lethal action of sera. Out of three sera tested, the investigated plasmids showed the most evident protective activity upon lethal action of neonatal serum. One of the plasmids appeared even more efficient than R100.1.

Influence of plasmids on bacterial susceptibility to the bactericidal activity of sera. II. Relation between transfer frequency of R plasmids and their protective activity to the lethal effect of normal rabbit serum.

The plasmid pAM-Ia separated from the aggregate R588 protected E. coli K12 cells from the bactericidal action of normal rabbit serum much stronger when carried alone than in the aggregate. When aggregated with R100.1, its protective effect was also weakened. These decreases in protective effect are correlated qualitatively with inhibition of conjugational fertility. On the other hand the plasmid aggregate causing increase in protective activity showed no influence on their fertility.

Influence of interactions between plasmids R. Vir and antigen K31 on the susceptibility of Escherichia coli strains to the normal rabbit serum.

K31 antigen is the important element of outer membrane in resistance of E. coli H209 to the bactericidal activity of normal rabbit serum. The strains more resistant to the effect of serum are those containing simultaneously K31 and other factors, R plasmids (like R100. 1 and pAM588-Ia) or virulence plasmids (Vir). Mutant strains lacking of K31 antigen are strongly killed by the serum but this effect is delayed when these strains had one of the above plasmids.