Michael Moos

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.

A role for Rho in receptor- and G protein-stimulated phospholipase C Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B

Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated α or free βγ subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signalling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, AlFinf4sup−and guanosine 5′-[γ-thio]triphosphate (GTPγS), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPγS-stimulated PLC activity. Finally, both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, when PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca2+- as well as GTPγS- or A1Finf4sup−-stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.

Clostridium difficile IStron CdISt1: Discovery of a Variant Encoding Two Complete Transposase-Like Proteins

Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.

9.4 Activation and Inactivation of Ras-Like Gtpases by Bacterial Cytotoxins

Publisher Summary This chapter describes bacteria-induced alterations in animal cells, in particular their influence on the cytoskeleton. When pathogenic bacteria interact with eukaryotic cells, they use unique mechanisms to exploit host processes. Bacterial action is often focused on altering the cytoskeleton. This influence is exerted by close contact between bacteria and the cell, invasion of bacteria into the cytosol, or by soluble factors. Invasive bacteria induce their uptake into eukaryotic cells by stimulating macropinocytosis or using a “zipper”-like mechanism. Once inside the cell, these bacteria may remain within or they may escape from the vacuole and live in the cytoplasm. This process is controlled by prokaryote proteins but is executed by eukaryotic proteins. Thus, some bacteria with an intracellular lifestyle reprogram the cytoskeleton to their advantage. A variety of soluble bacterial toxins can induce changes in eukaryotic cell morphology. They are either secreted into the external medium even in the absence of cells and therefore can act at a distance, or are focused onto the cell surface after contact between a bacterium and a cell via a type III secretion system. The cytoskeleton is composed of a multitude of eukaryotic proteins. Thus, the chapter further discusses cytoskeletal targets of bacterial toxins.