Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

<i>Clostridium difficile</i> is the major cause of intestinal infections in hospitals. The major virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong to the group of clostridial glucosylating toxins (CGT) that inactivate small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic cells (Caco-2) were analyzed using high-resolution LC–MS/MS and the SILAC technique. The changes in abundance of more than 5100 proteins were quantified. Nearly 800 toxin-responsive proteins were identified that were involved in cell cycle, cell structure, and adhesion as well as metabolic processes. Several proteins localized to mitochondria or involved in lipid metabolism were consistently of higher abundance after TcdA treatment. All changes of protein abundance depended on the glucosyltransferase activity of TcdA. Glucosylation of the known targets of TcdA such as RhoA, RhoC, RhoG was detected by LC–MS/MS. In addition, an almost complete glucosylation of Rap1­(A/B), Rap2­(A/B/C) and a partial glucosylation of Ral­(A/B) and (H/K/N)­Ras were detected. The glucosylation pattern of TcdA was compared to that of other CGT like TcdB, the variant TcdB from <i>C. difficile</i> strain VPI 1470 (TcdBF), and lethal toxin from <i>C. sordellii</i> (TcsL)

Contextual Dynamics of Immigration Attitudes: Regional Differences in Southern Europe

215 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.Research in the area of attitudes towards immigration will benefit from a more thorough discussion of the relationship between degrees of political engagement and the variance of political and social tolerance towards immigrants. Drawing upon institutional theory and realistic conflict theory, I further refine a theory of ethnic competition and prejudice in the Southern European context. I argue that popular attitudes towards immigration are correlated with a set of individual level factors (e.g. perceptions of personal and collective threat, as well as measures of political socialization), which are shaped and determined by the contextual characteristics (e.g. economic conditions and demographic characteristics) as well as the type of institutional environment (e.g. the presence or absence of support towards civic institutions) in which inter-group relations are embedded. The characterization of these environments determines the type of in-group/out-group social relations. I first, empirically characterize the type of "civic communities" existing in 50 Southern European regions and then, empirically test its significance in preventing inter-group hostility and the fostering of tolerance towards minority groups. Results show that there is strong significant effect between trust in institutions (such as NGOs and voluntary organizations) and decreased levels of anti-immigrant sentiment and intergroup conflict in Southern Europe. This dissertation provides evidence for the widespread effects that local minority group size and types of institutional trust have on political and social tolerance towards immigrants. Furthermore, evidence is provided that anti-immigrant sentiment has an extensive impact on Southern Europeans' policy opinions.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

<i>Clostridium difficile</i> is the major cause of intestinal infections in hospitals. The major virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong to the group of clostridial glucosylating toxins (CGT) that inactivate small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic cells (Caco-2) were analyzed using high-resolution LC–MS/MS and the SILAC technique. The changes in abundance of more than 5100 proteins were quantified. Nearly 800 toxin-responsive proteins were identified that were involved in cell cycle, cell structure, and adhesion as well as metabolic processes. Several proteins localized to mitochondria or involved in lipid metabolism were consistently of higher abundance after TcdA treatment. All changes of protein abundance depended on the glucosyltransferase activity of TcdA. Glucosylation of the known targets of TcdA such as RhoA, RhoC, RhoG was detected by LC–MS/MS. In addition, an almost complete glucosylation of Rap1­(A/B), Rap2­(A/B/C) and a partial glucosylation of Ral­(A/B) and (H/K/N)­Ras were detected. The glucosylation pattern of TcdA was compared to that of other CGT like TcdB, the variant TcdB from <i>C. difficile</i> strain VPI 1470 (TcdBF), and lethal toxin from <i>C. sordellii</i> (TcsL)

Proteome Alterations of Hippocampal Cells Caused by <i>Clostridium botulinum</i> C3 Exoenzyme

C3bot from <i>Clostridium botulinum</i> is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells

Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

<i>Clostridium difficile</i> is the major cause of intestinal infections in hospitals. The major virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong to the group of clostridial glucosylating toxins (CGT) that inactivate small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic cells (Caco-2) were analyzed using high-resolution LC–MS/MS and the SILAC technique. The changes in abundance of more than 5100 proteins were quantified. Nearly 800 toxin-responsive proteins were identified that were involved in cell cycle, cell structure, and adhesion as well as metabolic processes. Several proteins localized to mitochondria or involved in lipid metabolism were consistently of higher abundance after TcdA treatment. All changes of protein abundance depended on the glucosyltransferase activity of TcdA. Glucosylation of the known targets of TcdA such as RhoA, RhoC, RhoG was detected by LC–MS/MS. In addition, an almost complete glucosylation of Rap1­(A/B), Rap2­(A/B/C) and a partial glucosylation of Ral­(A/B) and (H/K/N)­Ras were detected. The glucosylation pattern of TcdA was compared to that of other CGT like TcdB, the variant TcdB from <i>C. difficile</i> strain VPI 1470 (TcdBF), and lethal toxin from <i>C. sordellii</i> (TcsL)

Effect of full length and CROP-deleted C. difficile toxins on intestinal barrier function following apical or basolateral uptake.

<p>A) 1 nM of TcdA (□,▪) and TcdA^1–1874 (Δ,▴) and B) 100 µM of TcdB (□,▪) and TcdB^1–1852 (Δ,▴) were applied to the apical (filled symbols) or basal (open symbols) compartment of CaCo-2 cells grown on Transwell filter inserts. Transepithelial electrical resistance (TER) was monitored over time as marker for toxin-uptake and activity. Untreated cells were used as control. Values are given as % of initial value after equilibration as means ± SD, n = 3.</p

Binding of TcdA and TcdA^1–1874 to host cells.

<p>A) The polyclonal antisera α-TcdA_1–2710, α-TcdA_1–1065 and α-TcdA_1–543 were tested by Western blot analyses to identify a specific antibody detecting full length and truncated TcdA with same sensitivity. Antiserum α-TcdA_1–1065 was selected for further studies showing strong and comparable recognition of TcdA and TcdA^1–1874. B) Binding of TcdA and TcdA^1–1874 to intact 3T3, HT29 and CHO-C6 cells was performed for 30 min at 4°C and analyzed by Western blot with α-TcdA_1–1065. C) Binding of fluorescent labeled TcdA-PE/Cy5 and TcdA^1–1874-Atto488 to HT29 and CHO-C6 cells was investigated by FACS analysis. Right shift of the black curve illustrates toxin binding which was detected through fluorescence emission at 667 nm for TcdA and at 523 nm for TcdA^1–1874, respectively. Due to different ratio of fluorophor and toxin, fluorescence intensity of TcdA-PE/Cy5 cannot directly be compared with TcdA^1–1874-Atto488.</p

Cellular uptake of TcdA, TcdA^1–1874 and TcdA^1875–2710.

<p>A) Efficiencies of TcdA and TcdA^1–1874 uptake into HT29 cells were indirectly determined by monitoring lysosomal toxin degradation following endosomal acidification. After binding at 4°C, endocytosis of toxin was allowed by temperature shift to 37°C. At the indicated time-points cells were lyzed and lysates were subjected to Western blot analysis to detect non-degraded toxins. β-Actin served as control protein excluding non-specific protein degradation. B) TcdA or TcdA^1–1874 was applied to 3T3 fibroblasts (time point 0) and Bafilomycin A1 was added at indicated times before or after toxin application. Cell rounding (CPE) was quantified 3 h after toxin treatment as rounded cells per total cells in %. Values are given as means ± SD (N = 3). C) Endocytosis of the isolated TcdA CROP domain (TcdA^1875–2710) was proven by immunofluorescence microscopy. Binding of EGFP or EGFP- labeled TcdA^1875–2710 to HT29 cells were performed on ice followed by a temperature-shift to 37°C allowing endocytotic processes. At indicated time points, cells were washed and fixed and immunofluorescence microscopy was performed to display nuclei (DAPI, blue), TcdA^1875–2710 (EGFP, green) and early endosomes (EEA1, red). Untreated (ctr) and EGFP-bound cells (EGFP) were used as controls. The lower panels illustrate magnification of an area indicated by rectangles in the upper panels.</p

Neutralization assay emphasizes the role of TcdA CROPs in toxin functionality.

<p>A) Dot blot showing specificity of generated polyclonal antiserum α-TcdA_1875–2710. The antiserum raised against the CROPs of TcdA only recognizes full length TcdA as well as the isolated CROP domain TcdA^1875–2710. No cross-reactivity was detected towards CROP-truncated TcdA^1–1874 or native TcdB, respectively. <i>B. megaterium</i> lysate was used as negative control. B) Recognition of full length toxin or toxin fragments by α-TcdA_1875–2710 was checked by ELISA. A 96-well plate was coated with full length TcdA, TcdA^1–1874 and TcdA^1875–2710, respectively. The bar diagram shows absorption at 405 nm after concentration-dependent binding of α-TcdA_1875–2710 to full length TcdA and the C-terminal repeats. Binding to TcdA^1–1874 was only observed to a small extent and after applying high amounts of antiserum (dilution factor 1∶100). Values are given as means ± standard deviation, n = 5. C) Neutralization of TcdA and TcdA^1–1874 with α-TcdA_1875–2710 antiserum was investigated in CHO-C6 cell rounding assay (left panel). Cytopathic effect (CPE) was quantified as round cells per total cells in %. Values are given as means ± SD, n = 5 (right panel).</p

Competition of TcdA, TcdA^1–1874 and TcdA^1875–2710 for cellular binding structures.

<p>A) Binding competition of PE/Cy5-labeled TcdA, Atto488-labeled TcdA^1–1874 and EGFP-fused TcdA^1875–2710, respectively, for cellular receptor structures was analyzed by flow cytometry. Therefore, 4 nM of TcdA-PE/Cy5 (red curve, upper panel) or TcdA^1–1874-Atto488 (red curve, lower panel) were applied either separately, in combination (green curves) or simultaneously after pre-incubation with 150 nM non-labeled TcdA^1875–2710 (blue curves) to HT29 cells. Intensity of fluorescence emission (x-axis) at 667 nm and 523 nm illustrates binding of TcdA and TcdA^1–1874 to HT29 cells, respectively. B) HT29 cells were treated with 150 nM of TcdA^1875–2710 (black curve) or pre-incubated with either 150 nM of TcdA^1–1874 (green curve) or TcdA^1875–2710 (blue curve) followed by immediate incubation with 8 nM of EGFP-fused TcdA^1875–2710 in the presence of either toxin. FACS analysis of EGFP-induced fluorescence emission at 509 nm showed reduced binding of CROP-truncated TcdA^1–1874 (compare black and green curve). C) The same experiment as shown in B) except that excessive toxin was washed off before addition of EGFP-fused TcdA^1875–2710. These results indicate that excessive TcdA^1–1874 sequesters TcdA^1875–2710 in solution being falsely interpreted as competition. The yellow highlighted lanes in legends mark the fluorescence labeled toxins that were detected at wavelength shown in respective graphs.</p