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)

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)

MALDI-TOF MS analysis of desialylated N-glycans from purified exosomes of SKOV3 cells.

<p>The <i>m/z</i> values of the first detected isotopic mass for major peaks are shown. Proposed compositions and compatible structures are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078631#pone-0078631-t002" target="_blank">Table <b>2</b></a>.</p

Glycoproteins from membrane fractions of SKOV3 cells.

<p><b>A</b>. Lectin blotting of glycoproteins from cell extract (Ext), microsomal fraction (MF), plasma membrane (PM), intracellular fraction (IC) that consisted of non-biotinylated proteins, cytoplasmic fraction (Cyt) that consisted of the supernatant of the microsomal fraction and crude exosomes (Exos_c) were analysed. Three μg total protein were applied per lane. Detection was performed using the chemiluminescent method. * Correspond to most abundant sialoglycoproteins enriched in the exosomes. Results were representative of at least two independent experiments. <b>B</b>. SDS-PAGE analysis of vesicle proteins from SKOV3 cells for peptide mass fingerprinting. A. MAL-binding glycoproteins (bands 1 to 21 identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078631#pone-0078631-t001" target="_blank">Table <b>1</b></a>) and total vesicles proteins (bands <i>a</i> to <i>j</i> were identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078631#pone-0078631-t001" target="_blank">Table <b>1</b></a>). Detection was with Coomassie G-250. </p

NP-HPLC analysis of 2-AB labeled N-glycans from purified exosomes.

<p>N-glycans were digested with mannosidase (M), sialidase (S), galactosidase (G) and fucosidase (F) as indicated. M5 to M9 consist of high mannose glycans Man₅GlcNAc₂ to Man₉GlcNAc₂, respectively. Structures of components from peaks 1 to 10 were identified by comparison with retention times of standards (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078631#pone.0078631.s001" target="_blank">Figure S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078631#pone.0078631.s005" target="_blank">Table S1</a>) and after digestion with exoglycosidases. </p

Characterization of exosomes obtained from SKOV3 cell supernatants.

<p>A) Immunoblotting of LGALS3BP in purified exosomes and plasma membrane. SKOV3 cell extracts (Ext), crude exosomes (Exos_c), biotinylated plasma membrane proteins (PM), and fractions from the sucrose gradient used for Exos_c fractionation (F1, F2-5, purified exosomes, F6-7, F8-11 and F12). Three µg protein were applied per lane with the exception of F1, F6-7 and F12, where 20% of the amount obtained from the sucrose gradient was used. Detection was by the chemiluminescent method. Results were representative of two independent experiments. B) Electron microscopy visualization of crude exosomes (pellet collected after centrifugation at 100,000x<i>g</i> of pre-cleared supernatant). C) Electron microscopy visualization of purified exosomes (fractions 2 to 5 of sucrose gradient). Arrows, cup-shaped vesicles; open arrowhead, vesicles larger than 100 nm; closed arrowheads, vesicles approximately from 30 to 50 nm. The scale bar corresponds to 100 nm.</p

MS/MS analysis results of the peptide precursors shown in Figure 4.

<p>Sequence coverage 12%, Mascot score 170, 6 matched queries. Data analysis from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039848#pone-0039848-g004" target="_blank">Figure 4</a>.</p

MALDI-MS spectrum of RIIα peptide precursors from cell lysates of HEK293 cells.

<p>Peptides of the 46 kDa region of gels were digested and analyzed by MALDI-MS. A detailed analysis of the peptides is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039848#pone-0039848-t003" target="_blank">Table 3</a>. Peaks labelled by asterisk were subjected to MS/MS analysis.</p