The enzymatically active component i, of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet Ply-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle or-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.Several bacterial toxins modify functions in eukaryotic cells by an ADP-ribosylation of intracellular target proteins. Diphtheria toxin and Pseudomonas exotoxin A ADPribosylate elongation factor I1 and thereby inhibit protein synthesis [l]. Cholera toxin and pertussis toxin modify guanylnucleotide-binding regulatory (G) proteins involved in trans-Botulinum C2 toxin is another member of the family of microbial ADP-ribosyltransferases [5]. It has been shown that botulinum C2 toxin modifies actin in intact cells and isolated cytoplasmic /?/y actin in its monomeric G form, but not F-actin [6 -91. The covalent modification of purified actin by botulinum C2 toxin severely reduces its ability to polymerize Recently it has been demonstrated that perfringens iota toxin also possesses ADP-ribosyltransferase activity [lo]. Furthermore, it has been reported in a preliminary note that it also accepts actin as substrate [lo]. We were prompted to compare the molecular mode of action of these two toxins because of their striking similarities. Both toxins are clostridial in origin and are produced during opportunistic intestinal infections. They both cause increased fluid transport across membranes [ll, 121. They both are binary in structure and consist of a high and low-molecular-mass component, which are components I and I1 of botulinum C2 toxin and components i, and ib of perfringens iota toxin. The respective components are linked neither by covalent nor non-covalent bonds [13-151. In each case the smaller components of botulinum C2 toxin (C21, molecular mass 50 kDa) and of perfringens iota toxin (i,, molecular mass 48 kDa) are enzymatically active as ADP-ribosyltransferases [5,6, lo]. The larger components have a molecular mass of 72 kDa in iota toxin [14] and 88 kDa in trypsin-activated C2 toxin [16] and are respectively responsible for the binding of the toxin to the eukaryotic cell surface. Thus, to elicit toxic effects, both components of either toxin have to act together [13, 151. Here we report on studies on the ADP-r...

Infections of the hip joint are usually of bacterial etiology. Only rarely, an infectious arthritis is caused in this localization by viruses or fungi. Native joint infections of the hip are less common than infections after implantation of prosthetic devices. Difficulties in prosthetic joint infections are, (I) a higher age of patients, and, thus an associated presence of other medical risk factors, (II) often long courses of treatment regimes depending on the bacterium and its antibiotic resistance, (III) an increased mortality, and (IV) a high economic burden for removal and reimplantation of an infected prosthetic device. The pathogenic mechanisms responsible for articular infections are well studied only for some bacteria, e.g. Staphylococcus aureus, while others are only partially understood. Important known bacterial properties and microbiological characteristics of infection are the bacterial adhesion on the native joint or prosthetic material, the bacterial biofilm formation, the development of small colony variants (SCV) as sessile bacterial types and the increasing resistance to antibiotics.

Institut fur Pharmakologie und Toxikologie der Justus-Liebig-Universitat Giefjen ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound [Y-~~PIATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP [yS], much less with ADP but not with AMP or NAD. ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+(1 mM)-induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADPribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity.Clostridium perfringens iota and Clostridium botulinum C2 toxins belong to a newly discovered class of bacterial toxins which ADP-ribosylates actin [I]. Both clostridial toxins are binary in structure and consist of a high-M, and a low-M, component [2, 31. While the high-Mr component of both toxins is apparently involved in binding to the eukaryotic cell surface, the low-M, component possesses ADP-ribosyltransferase activity [4-71. Studies on the effect of botulinum C2 toxin on intact cells suggest that the microfilament protein is the pathobiochemical substrate of these toxins [I, 81. Both toxins differ in their substrate specificity. Whereas botulinum C2 toxin modifies non-muscle actin and (much less) skeletal muscle actin, perfringens iota toxin ADP-ribosylates both actin species readily [7]. Recently it has been shown that both toxins ADP-ribosylate actin at Arg-177 [9, 101, a modification which largely reduces the ability of actin to polymerize [5, 7, 1 I]. The functional properties of actin, e. g. polymerization and depolymerization reactions, depend largely on the ability of actin to bind and hydrolyze ATP [12]. In order to gain more insight into the functional consequences of this modification of actin, we studied the effects of the toxin-induced ADPribosylation on ATP exchange and ATPase activity. Here we report that the ADP-ribosylation of actin increases the rate of ATP exchange and inhibits 'basal' and Mg2+-induced hydrolysis of ATP. MATERIALS AND METHODS MaterialsClostridium botulinum C2 toxin and Clostridium perfringens iota toxin were purified from culture medium of C. botulinum type C strain 92-13, kindly donated by Dr. S.

Periprosthetic infection is a significant complication in joint replacement surgery and develops in 0.5-2% of cases. Staphylococcus aureus and commensal microorganisms of the skin, especially coagulase-negative staphylococci, as well as a broad spectrum of other potential pathogens typically already colonize the surface of the foreign body at the time of implantation. Specific mechanisms such as bacterial adhesion to host factors absorbed in the material, biofilm formation, and a metabolic adaptation of adherent microorganisms play a particularly important role in the pathogenesis and course of the disease. Microbiological diagnosis requires to some extent complex culture procedures of puncture specimens or tissue removed during surgery; this can be supplemented by modern molecular testing. Antimicrobial treatment must be conceived as a synopsis of clinical picture, confirmed pathogen, and the intended surgical procedure on an individual basis and is routinely administered as combination therapy for several weeks, sometimes also as sequential therapy. Validated preventive measures in joint replacement surgery include mandatory perioperative antibiotic prophylaxis and other additional measures.

Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D-stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 microM), at low concentrations of Mg2+ (50 microM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and that the inhibitory effect is not due to inhibition of the polymerization of actin.