The main virulence factors of C. difficile are the toxins TcdA and TcdB that inactivate Rho proteins in eukaryotic cells. The toxins act in the cytosol of target cells to catalyze the transfer of a glucose moiety onto a crucial threonine residue of Rho proteins. Delivery of the toxins into the cytosol and their effects on Rho proteins and Rho-dependent signaling in host cells is investigated [4, 7] but proteins and pathways involved in processes downstream of Rho proteins are largely unclear at present. In particular, the question whether large clostridial cytotoxins affect target cells independently of the intrinsic glucosyltransferase activity and thus independently of Rho proteins is controversially discussed [4, 16]. Hence we initiated a toxicoproteomic study to analyze cellular effects of large clostridial cytotoxins using recombinant wild type or mutant TcdA. A similar study has been conducted to elucidate the function of the C3 exoenzyme of C. botulinum where several proteins were identified to be responsive to C3 treatment in neuronal cells . Thus, a comprehensive proteome analysis was conducted using the ICPL technique and LC-MALDI-MS. ICPL labeling occurs at lysine residues and prevents trypsin cleavage after these residues. Thus, the tryptic digestion was altered to an ArgC specificity leading to the generation of slightly larger peptides on average. However, quantification was only possible for half of the identified proteins. Although lysine is overrepresented in human proteins (>7%), several of the identified peptides did not contain lysine residues. Additionally, few lysine residues remained unlabeled at all or escaped labeling. For these peptides containing unlabeled lysine residues no corresponding peptides with a labeled lysine were detected. Also the opposite was not observed: for all identified ICPL-labeled peptides no unlabeled counterpart was detected. From this observation we concluded that ICPL labeling is either complete or completely lacking for a particular peptide.
Overall 30 proteins responded to the treatment with rTcdA wt or mutant rTcdA (Figure 4). Short time effects (5 h) were responsible for up- or down-regulation of sixteen proteins due to treatment with rTcdA wt; seven proteins were identified by mass spectrometry as up-regulated by mutant rTcdA (Figure 4A, B). Moreover, two proteins, Rac1 and clathrin heavy chain 1, were up-regulated by both rTcdA wt and mutant rTcdA as determined by MS and western blot techniques (Figure 5E, F). After long time treatment (24 h) with rTcdA wt, the concentration of eleven proteins was altered (Figure 4C). Additionally, one of the latter proteins, filamin A (FLNA), was significantly down-regulated after treatment with mutant rTcdA. These data strongly indicate particular effects of the catalytic inactive mutant rTcdA. This provides further evidence that large clostridial cytotoxins alter cellular processes of target cells independently of the intrinsic glucosyltransferase activity. Additionally, cellular processes independent from cytoskeleton homeostasis are influenced by the clostridial cytotoxins.
Short-time transferase-independent effects of toxins include changes in concentration of proteins involved in energy metabolism and signal transduction. Interestingly, annexin A3 (ANXA3) and the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) were up-regulated, which are known to be involved in response to bacterial pathogens [18, 19]. These proteins were not up-regulated in the cells treated with rTcdA wt for 5 h, but rTcdA wt induces down regulation of annexin A3 after 24 h (Figure 4C). MIF suppresses pro-oxidative stress-induced apoptosis and contributes to the regulation of cell survival . Thus, mutant rTcdA signaling further facilitates cell proliferation of Caco-2 cells and should be clearly different from that of rTcdA wt. Rac1 is a major target of rTcdA wt and was up-regulated after treatment with wild type as well as with mutant toxin. Thus, Rac1 up-regulation facilitated via a Rho-independent signaling pathway that might also adjust up-regulation of MIF, annexin A3, clathrin heavy chain 1, peroxiredoxins, metabolic enzymes, and probably other so far unrecognized proteins. Up-regulation of Rac1 and other proteins by the catalytic inactive toxin might be interpreted as a protection response of the colonic cells that is induced by so far unknown mechanisms or a general effect related to endocytotic uptake of toxin. Thus, Rho-independent signal transduction mechanism might be responsible to detect and respond to bacterial infections. Another cellular target of rTcdA wt, RhoB, is well known to be up regulated in response to TcdA wt treatment but is not induced by the catalytic inactive rTcdA mutant. RhoB induction is thought to have a proapoptotic function [11, 21].
Caco-2 cells treated for 5 h with rTcdA wt responded in a considerable decrease of ribosomal proteins that are involved in ribosome function and mRNA trafficking . However, ribosomal proteins possess extra-ribosomal functions [22, 23]. The 40S ribosomal protein S3 (RPS3) is involved in maturation of 40S ribosomal subunit and exhibits apoptotic function [24, 25]. Lee et.al. have shown, that RPS3 phosphorylated by Akt accumulates in the nucleus possesses reduced proapoptotic properties . The proteins glutamate dehydrogenase 1, mitochondrial (GLUD1), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and polypyrimidine tract-binding protein 1 (PTBP1) were down regulated after short term rTcdA wt treatment (Figure 4B). The latter two proteins are also involved in mRNA metabolism, processing, and transport [27–30]. HnRNP A1 becomes localized in the cytoplasm upon transcription inhibition and belongs to transcription-sensitive group of hnR proteins. The hnRNPA1 shuttling activity is required for survival and granulocytic differentiation of normal myeloid precursors [31, 32]. The cellular distribution upon transcription inhibition of PTBP1 is transcription-insensitive; the protein is localized in the nucleus . The reduced concentration of ribosomal proteins was identified in the cytoplasmic protein fraction and might indicate a more nuclear localization of these proteins that can reside in both, the nuclear and the cytoplasmic compartment. Thus, a disruption of the balanced shuttling between cytoplasm and nucleus can be assumed due to the treatment with rTcdA wt. However, after 24 h treatment of Caco-2 cells with rTcdA wt no changes in expression of the ribosomal proteins, hnRNPA1, and PTBP1 were observed. Thus, the down-regulation of these proteins in cytosol is terminated as an early effect of the clostridial cytotoxins.
Protein glucosidase 2 (alpha and beta subunit, GANAB and PRKCSH), the chaperons endoplasmin (HSP90B1), protein disulfide-isomerases (P4HB and PDIA4) were induced after 5 h treatment with rTcdA wt. (Figure 4B, 5A, B). Endoplasmin is a glycoprotein  that has been linked to the Toll-like receptor pathway , response to hypoxia , and this protein is required for innate immunity but not cell viability . It forms a large chaperone multiprotein complex with protein disulfide-isomerase A4 and other chaperonins and is essential for translocation of C. botulinum C2 toxin and uptake of C. perfringens iota toxin into eukaryotic cells [38, 39]. Indeed, endoplasmin has been reported to bind to TcdA at the cell surface and is translocated together with TcdA into the cytoplasm . We propose that toxin-dependent endoplasmin translocation from membrane into the cytoplasma led to an increased cytoplasmic concentration of this protein that was not observed after treatment with mutant rTcdA.
Glucosidase 2 beta catalyzes the removal of three glucose residues from the peptide-bound Glc3-Man9-GlcNAc2 oligosaccharide remodelling the asparagine-linked precursor  and UDP-glucose:glycoprotein glycosyltransferase. The latter plays a possible role in control of protein glycosylation  and regulation of N-glycoprotein transport or protein degradation [43, 44]. These data indicate that post-translational modifications like glycosylation are influenced by large clostridial toxins. However, after 24 h treatment with rTcdA wt glucosidase 2 beta decreased by 2-fold.
After the long-term treatment of cells with rTcdA wt several cytoskeletal proteins (beta-, gamma-actin, filamine A and B, villin 1) were down regulated. This is in good accordance to the cytopathic cell rounding of rTcdA wt treated cells. Interestingly, filamine A was also down-regulated by rTcdA mutant. Thus, a Rho-independent signaling should be involved in filamine regulation.
Peroxiredoxins 1, 2, 6 were found down-regulated after treatment with rTcdA wt for 24 h; peroxiredoxins 1 and 2 were up-regulated after short time incubation with mutant rTcdA (Figure 4A, C). Kim et.al. reported that C. difficile Toxin A induces the release of reactive oxygen species (ROS) and influences the regulation of cyclooxigenase-2 and prostaglandin E2 synthesis by ROS [9, 45]. The expression changes of peroxiredoxins are involved in redox control and might be induced after 5 h to protect cells from ROS. Down regulation of these protecting enzymes after 24 h of toxin treatment might be a hint that the cells have become more apoptotic.
The presented data indicate that large clostridial cytotoxins like TcdA act not only on Rho proteins and induce Rho-dependent pathways. Rho-independent effects evoked by a catalytic inactive toxin mutant point to new cellular target structures in eukaryotic cells and so far unknown ways of signaling. For the first time evidence has been provided that TcdA influences more cellular functions than cytoskeleton homeostasis and apoptosis. The response of colonic cells to TcdA is time-dependent and different protein patterns are evident after short (5 h) and long (24 h) incubation times. Activity and probably location of ribosomal proteins, metabolic enzymes, and protection enzymes like peroxiredoxin were modified due to action of TcdA.