Christoph von Eichel-Streiber

Large clostridial cytotoxins — a family of glycosyltransferases modifying small GTP-binding proteins

Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

Autocatalytic cleavage of Clostridium difficile toxin B

Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.

Comparative sequence analysis of the Clostridium difficile toxins A and B.

SummaryThe six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCdl3 cover thetox locus ofClostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the completetoxB andtoxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPS (combinedrepetitiveoligopeptides of 20–50 as length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison of the as sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved as in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa. The probable function of these domains is discussed. Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).

Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells.

Clostridium difficile toxins A and B together are responsible for the symptoms of pseudomembranous colitis. Both toxins intoxicate cultured cells by the same mechanism but they differ in cytotoxic potency, toxin B being generally 1,000 times more potent than toxin A. Don and T84 cells were used to determine differences in the intoxication process exerted by both toxins. Three main differences were identified: (a) the specific binding of radiolabeled toxins to the cell surfaces correlated with the cytotoxic potency, (b) toxin B was found to have a 100-fold higher enzymatic activity than toxin A, and (c) toxin A was found to modify an additional substrate, Rap. The relative contribution of (a) and (b) to the difference in cytotoxic potency was determined by microinjection of the toxins. The differing enzymatic activities turned out to be the main determinant of the difference in cytotoxic potency, whereas the difference in binding contributes to a lesser degree. These findings are discussed in the context of the pathophysiological role of the toxins.

Rac1 and PAK1 are upstream of IKK-ε and TBK-1 in the viral activation of interferon regulatory factor-3

The anti‐viral type I interferon (IFN) response is initiated by the immediate induction of IFNβ, which is mainly controlled by the IFN‐regulatory factor‐3 (IRF‐3). The signaling pathways mediating viral IRF‐3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF‐3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFNβ promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF‐3, we have identified the kinase p21‐activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF‐3 activators, IκB kinase‐ε and TANK‐binding kinase‐1, establishing a first canonical virus‐induced IRF‐3 activating pathway.

A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins.

The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between “reference” TcdB-10463 andClostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and β3-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras.

Expression and prognostic relevance of annexin A3 in prostate cancer.

OBJECTIVES By differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. METHODS Immunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. RESULTS All samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p<0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n=969) into high- and low-risk subgroups. CONCLUSIONS ANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.

Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA‐1470 and toxB‐1470. A polymerase chain reaction (PCR)‐based approach to the sequencing of the total toxB‐1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VPI10463, the toxB‐1470 gene has 16 additional nucleotides, 13 within the 5′‐untranslated region and three within the coding region. The Mr of ToxB‐1470 is 269 262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are Mr 269 709 and IP 4.13. In comparison with ToxB, ToxB‐1470 differs primarily in the N‐terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C‐terminal region between residues 869–2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB‐1470 with ToxA and ToxB reveals the highest homology between ToxB‐1470 and ToxB. Thus ToxB‐1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB‐1470 is less potent than ToxB. The cytopathic effects of ToxB‐1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii, but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or α‐toxin (Tcnα) of Clostridium novyi. The LT‐like action of ToxB‐1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB‐1470 are clustered within the N‐terminal third of the respective proteins, we conclude that these domains carry the toxic determinants. A three‐domain structure is proposed for the family of large clostridal cytotoxins.

Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades.

Yersinia enterocolitica triggers activation of the nuclear factor (NF)‐κB and production of the proinflammatory chemokine interleukin (IL)‐8 in intestinal epithelial cells. This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells. Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho‐GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF‐κB by invasin. Invasin activated the mitogen activated protein kinases (MAPK) p38 and c‐Jun N‐terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK). The functional relevance of these pathways for invasin‐mediated IL‐8 expression was assessed by protein kinase inhibitors and dominant‐negative kinase mutants. While NF‐κB and JNK contribute to IL‐8 transcription, p38 MAPK also acts through stabilization of IL‐8 mRNA, as confirmed by quantitative RT‐PCR and electrophoretic mobility shift assays. Transfection experiments with I‐κB kinase (IKK)1 and IKK2 mutants indicate that the release of NF‐κB from its cytoplasmic inhibitor I‐κB and its translocation into the nucleus is mediated by these kinases. Our data identify Rac1 as a key intermediate in invasin‐triggered IL‐8 synthesis and demonstrate that maximum IL‐8 induction involves several MAP kinase cascades.

A chimeric ribozyme in Clostridium difficile combines features of group I introns and insertion elements

CdISt1, a DNA insertion of 1975 bp, was identified within tcdA‐C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1‐C34, CdISt1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase‐like proteins tlpA and tlpB nucleotides 435–1975 represent the remainder of a IS605‐like insertion element. We show that the entire CdISt1 is accurately spliced from tcdA‐C34 primary transcripts and that purified TcdA‐C34 toxin is of regular size and catalytic activity. A search for CdISt1‐related sequences demonstrates that the element is widespread in toxinogenic and non‐toxinogenic C. difficile strains, indicating the mobility of CdISt1. In strain C34, we characterize 10 CdISt1 variants; all are highly homologous to CdISt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdISt1 and are precisely spliced from their primary transcripts. CdISt1‐like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.