Directed Evaluation of Enterotoxigenic Escherichia coli Autotransporter Proteins as Putative Vaccine Candidates

Diarrheal diseases are responsible for more than 1.5 million deaths annually in developing countries. Enterotoxigenic E. coli (ETEC) are among the most common bacterial causes of diarrhea, accounting for an estimated 300,000–500,000 deaths each year, mostly in young children. There unfortunately is not yet a vaccine that can offer sustained, broad-based protection against ETEC. While most vaccine development effort has focused on plasmid-encoded finger-like ETEC adhesin structures known as colonization factors, additional effort is needed to identify conserved target antigens. Epidemiologic studies suggest that immune responses to uncharacterized, chromosomally encoded antigens could contribute to protection resulting from repeated infections. Earlier studies of immune responses to ETEC infection had identified a class of surface-expressed molecules known as autotransporters (AT). Therefore, available ETEC genome sequences were examined to identify conserved ETEC autotransporters not shared by the commensal E. coli HS strain, followed by studies of the immune response to these antigens, and tests of their utility as vaccine components. Two chromosomally encoded ATs, identified in ETEC, but not in HS, were found to be immunogenic and protective in an animal model, suggesting that conserved AT molecules contribute to protective immune responses that follow natural ETEC infection and offering new potential targets for vaccines

The Beta3 499–513 Peptide Region is Required for AlphaIIb/Beta3 Active Complex Formation and Fibrinogen Binding

Background AlphaIIb/beta3 (αIIb/β3) complex is an important integrin that is involved in the final step of platelet aggregation. Peptides derived from either αIIb or β3 have demonstrated to have an effect on the activation of the complex and its ability to bind fibrinogen. We have previously defined a peptide from β3, which inhibits agonists-induced platelet aggregation. Methods We used standard methodologies for construction of clones and expression of cDNAs, establishment of stable cell lines that contained these cDNAs. Expression of proteins was detected with immunoblots. Flow cytometric analyses were used to verify the presence of the active and inactive complexes with different antibodies. In addition, a fibrinogen-binding assay was used to determine the inhibition of the active complex by the peptide. Results and discussion A stable cell line of the co-transfected cDNAs of αIIb, β3 wild type and mutants of β3 (scrambled sequence of the peptide region, replacement of C499A and C512A), expressing the inactive complex on CHO cells, has allowed us to examine the important role of the peptide sequence and the cysteine residues within the peptide. The peptide inhibits the active complex formation and thereby inhibits the binding of FITC-PAC-1 in a dose dependent manner by flow cytometric analyses, as well as binding of [ 3 H]-fibrinogen. In addition, creation of a second stable cell line containing wild type αIIb and the mutated region of β3 (residues 499–513) shows that the binding of FITC-PAC-1 and [ 3 H]-fibrinogen on the mutant activated complex was much lower than the wild type activated complex. Our results indicate that the region 499–513 in β3 is one of the important sites for αIIb/β3 active complex formation and the cysteines play an important role in the process

The Beta3 499–513 Peptide Region is Required for AlphaIIb/ Beta3 Active Complex Formation and Fibrinogen Binding

Background: AlphaIIb/beta3 (αIIb/β3) complex is an important integrin that is involved in the final step of platelet aggregation. Peptides derived from either αIIb or β3 have demonstrated to have an effect on the activation of the complex and its ability to bind fibrinogen. We have previously defined a peptide from β3, which inhibits agonists-induced platelet aggregation.Methods: We used standard methodologies for construction of clones and expression of cDNAs, establishment of stable cell lines that contained these cDNAs. Expression of proteins was detected with immunoblots. Flow cytometric analyses were used to verify the presence of the active and inactive complexes with different antibodies. In addition, a fibrinogen- binding assay was used to determine the inhibition of the active complex by the peptide.Results and discussion: A stable cell line of the co-transfected cDNAs of αIIb, β3 wild type and mutants of β3 (scrambled sequence of the peptide region, replacement of C499A and C512A), expressing the inactive complex on CHO cells, has allowed us to examine the important role of the peptide sequence and the cysteine residues within the peptide. The peptide inhibits the active complex formation and thereby inhibits the binding of FITC-PAC-1 in a dose dependent manner by flow cytometric analyses, as well as binding of [3H]-fibrinogen. In addition, creation of a second stable cell line containing wild type αIIb and the mutated region of β3 (residues 499–513) shows that the binding of FITC-PAC-1 and [3H]-fibrinogen on the mutant activated complex was much lower than the wild type activated complex. Our results indicate that the region 499–513 in β3 is one of the important sites for αIIb/β3 active complex formation and the cysteines play an important role in the process

Pathways Involved in the Synergistic Activation of Macrophages by Lipoteichoic Acid and Hemoglobin

<div><p>Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.</p> </div

Macrophage dose response curves for IL-6 secretion induced by TLR2 ligands with and without Hb. HeNC2 cells were stimulated with various amounts of LTA (diamonds), Pam3CSK4 (triangles) or Pam2CSK4 (squares) with or without Hb (50 µg/ml, dashed lines or solid lines, respectively) for 18 hours at 37°C.

<p>Levels of IL-6 in the supernatants were analyzed by ELISA. Data are plotted on a log-log scale. These results denote mean +/− standard error (n = 3) and are representative of three independent experiments.</p

Degradation of IκBα during macrophage activation.

<p>HeNC2 cells were stimulated with LTA (1 µg/ml), LTA plus Hb (1 µg/ml and 50 µg/ml, respectively), Hb (50 µg/ml), Pam2CSK4 (100 ng/ml), Pam2CSK4 plus Hb (100 ng/ml and 50 µg/ml, respectively) or maintained in medium for the indicated times. Cell extracts were electrophoresed on SDS-PAGE gels, transferred to nitrocellulose and incubated with anti-IκBα. Similar results were obtained from each of two independent experiments.</p

qRT-PCR analysis of macrophage gene expression.

<p>Tlr4⁺/₊ HeNC2 cells were stimulated with either LTA (2 µg/ml, diamonds, solid line), Hb (50 µg/ml, squares, short dashes), LTA+Hb (2 µg/ml and 50 µg/ml, respectively, triangles, long dashes) or maintained in medium for the indicated time period and then analyzed by qRT-PCR for TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-1α (<b>C</b>), IL-6 (<b>D</b>), CCL5/RANTES (<b>E</b>) or IFN-β (<b>F</b>) mRNA levels compared to control. Tlr4^d/_d GG2EE cells were stimulated with LTA+Hb (2 µg/ml and 50 µg/ml, respectively; circles, dashes and dots) and assayed for IL-6 (D) or CCL5/RANTES (E). These results denote mean +/− standard error (n = 3) and are representative of three independent experiments.</p

Effect of Dynasore® on macrophage TNF-α and IL-6 secretion induced by LTA plus Hb. HeNC2 cells were preincubated with either 40 µM Dynasore® or an equivalent amount of the solvent, DMSO, for 30 min and then stimulated with LTA plus Hb (2 µg/ml and 50 µg/ml, respectively) for 6 hours.

<p>TNF-α and IL-6 concentrations were analyzed by ELISA. These results denote mean +/− standard error (n = 3) and are representative of three independent experiments.</p

Cellular localization of NF-κB p65 in HeNC2 cells.

<p>HeNC2 cells were stimulated with Pam2CSK4 (100 ng/ml, panel A), Pam2CSK4 plus Hb (100 ng/ml and 50 µg/ml, respectively, panel B), LTA (1 µg/ml, panel C), LTA plus Hb (1 µg/ml and 50 µg/ml, respectively, panel D), Hb (50 µg/ml, panel E) or maintained in medium (panel F) for 4 hours. Cells were then fixed, permeabilized, blocked and incubated with NF-κB p65 overnight at 4°C. After washing, cells were incubated with anti-rabbit IgG conjugated to Alexa Fluor-488 (green). Cells were also stained with SYTOX Orange (blue) to visualize the nuclei and then analyzed by confocal microscopy.</p