[Emergence of clostridium difficile ribotype 027 in Germany: epidemiological and clinical characteristics]. / Clostridium-difficile-Ribotyp 027: Epidemiologie und Klinik des erstmaligen endemischen Auftretens in Deutschland.

INTRODUCTION: In September 2007 an increase of severe Clostridium difficile-associated infections (CDI) was noticed in a hospital in the city of Trier, Germany. It was assumed that a new, possibly hypervirulent strain (PCR ribotype 027) was related to these events. An outbreak investigation was initiated by the local health authorities and the Robert Koch Institute to describe the epidemiology of the possible outbreak and to identify and control the possible sources. METHODS: In addition to retrospective case-finding of severe CDI and ribotype 027 infections by analysis of patient documents and certificates of death, an active surveillance system for severe CDI and ribotype 027 infections was established in the 6 hospitals of the affected region. In all suspected cases, a test for toxin A/B and a stool culture for C. difficile were conducted simultaneously. Bacterial isolates were further characterised by PCR ribotyping. Data on the course of disease, case fatality, and possible risk factors for CDI-related deaths were assessed using a standardised questionnaire. Environmental investigations were done. RESULTS: By 31 January 2008, 27 cases of severe CDI and 21 cases with C. difficile ribotype 027 infections were found in the area under investigation. Active surveillance found 76 of 399 (19 %) patients positive for C. difficile. In 20 patients, PCR ribotyp 027 could be proven. In total, 9 deaths occurred (19 %). An existing immunosupressive therapy (OR 35.8; 95 % CI 2.8 - 464.5) was related to case fatality in the multivariate analysis. Severe cases of CDI were also observed in non-ribotype 027 infections. In the screening of hospital personnel (n = 161), 6 % were found positive for toxin A/B. DISCUSSION: This investigation demonstrated the endemicity of C. difficile PCR ribotype 027 in Germany for the first time. As a consequence from this study, severe CDI became a reportable disease in Germany at the end of 2007. In addition to hygienic measures, the critical use of antibiotics is an important measure to prevent a further increase of CDI.

Increased number of Clostridium difficile infections and prevalence of Clostridium difficile PCR ribotype 001 in southern Germany.

In recent years, Clostridium difficile infection (CDI) has emerged as an increasing problem, both in in- and outpatients. In a rural region of southern Germany, the annual number of C. difficile toxin (Tcd)-positive patients has increased from 95 to 796 in the period from 2000 to 2007. Simultaneously, the proportion of positive tests among all Tcd examinations has risen from 7.0% to 12.8%, indicating that the higher number of affected patients was not solely due to an increase in the number of assays. Elevated numbers of CDI have recently been associated with outbreaks of the ribotype 027 strain, particularly in North America. This strain has also been isolated in Europe, including in Germany. Ribotyping and PCR testing for binary toxin genes of C. difficile strains isolated from in- and outpatients demonstrate a predominance (59%) of C. difficile ribotype 001, which exhibits antibiotic resistance to erythromycin, ciprofloxacin, and moxifloxacin, but lacks binary toxin genes. In summary, in our region of Germany, the number of patients affected by CDI has increased, probably due to spread of C. difficile ribotype 001.

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

Variant toxin B and a functional toxin A produced by Clostridium difficile C34.

A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first 'S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a 'D-type' enterotoxin. Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect.

[Nosocomial epidemiology and transmission of Clostridium difficile infection]. / Nosokomiale Epidemiologie und Transmission der Clostridium-difficile-Infektion.

BACKGROUND: Clostridium difficile is of growing importance as a hospital-acquired pathogen. Pseudomembraneous colitis is the main clinical disease. Transmission and epidemiological features are not yet fully understood. PATIENTS AND METHODS: Stool samples from 1164 individuals (571 women and 593 men) attending were examined for the presence of C. difficile. Follow-up examinations and molecular typing methods were used for the detection nosocomial transmissions. Additionally, hospital-borne environmental samples as well as staff samples were tested. RESULTS: Incidence of C. difficile infection was 8.4%. Nearly all patients (92.9%) had antibiotics given. Using molecular typing nosocomial transmission was evident. Though, environmental samples in general had a low positivity, toilet chairs were contaminated in 15.4% and may be a potential source of transmission. Staff was positive in only one case. CONCLUSIONS: Prevention of infections with C. difficile becomes to be a major threat for the clinical and hygienic management.

Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.

The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.

Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.

In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly. Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production. Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

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

CdlSt1, 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, Cd/St1 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 CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic and non-toxinogenic C. difficile strains, indicating the mobility of CdlSt1. In strain C34, we characterize 10 CdlSt1 variants; all are highly homologous to CdlSt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdlSt1 and are precisely spliced from their primary transcripts. CdlSt1-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.

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.

Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins.

Primary cultures of rat cortical astrocytes express phospholipase D (PLD) isoforms 1 and 2 as determined by RT-PCR and Western blot. Basal PLD activity was strongly (10-fold) increased by 4beta-phorbol-12beta,13alpha-dibutyrate (PDB) (EC(50): 56 nM), an effect which was inhibited by Ro 31-8220 (0.1-1 microM), an inhibitor of protein kinase C (PKC), and by brefeldin A (10-100 microg/ml), an inhibitor of ADP-ribosylating factor (ARF) activation. Pretreatment of the cultures with Clostridium difficile toxin B-10463 (0.1-1 ng/ml), which inactivates small G proteins of the Rho family, led to a breakdown of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50%. In contrast, inactivation of proteins of the Ras family by Clostridium sordellii lethal toxin 1522 did not affect PLD activation. In parallel experiments, serum-induced PLD activation was sensitive to brefeldin A, but not to Ro 31-8220 and not to clostridial toxins. We conclude that, in astrocytes, the PLD isoform which is activated by phorbol ester requires PKC, ARF and Rho proteins for full activity and probably represents PLD1.

Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864.

Here we report data describing some principles of the interaction between small GTP-binding proteins and large Clostridial cytotoxins (LCTs). Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL-1522, TcdB-1470 and TcdB-8864. Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates. Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N-terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated. Within this stretch, three regions differ significantly in Rac1 and RhoA. Regions containing amino acids 41-45 and 50-54 had no effect on toxin induced glucosylation, whereas amino acids 22-27 had a drastic impact on the potential of all three toxins to covalently modify the GTPases. Point mutations K25T of RhoA (numbering according to Rac1) and K27A of Cdc42 significantly increased glucosylation by the cytotoxins; introduction of lysines at the equivalent positions of Rac1 hindered modification. Our experiments demonstrate the influence of this charged residue on GTPase-LCT interactions. Amino acids 22-27 are part of the transition between the alpha1-helix to the switch I region of small GTP-binding proteins; both are known structures for specificity determination of the interactions with physiologic partners. Comparing these structures with data from our investigation we suggest that TcsL-1522, TcdB-1470 and TcdB-8864 mimic aspects of the physiologic interactions of small GTP-binding proteins.

A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X.

Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no glucosylation activity. We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing. Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47. Identical mutations were found in the two serogroup X strains 17663 and 10355. The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies.

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

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 and Clostridium 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 beta3-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.

UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C.

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli.

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.

A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates.

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10,463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864--implications for transcription, expression and enzymatic activity of toxins A and B.

The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3' ends of tcdA and tcdC. Transcription of the tcdA-E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA-E genes that confirms our previous findings in strain 10463. Recombinant TcdA-8864 had UDP-glucose-glucosyltransferase activity, proving that the N-terminal 698 amino acids of the polypeptide represent the catalytic domain. However, this truncated TcdA molecule lacks a ligand and translocation domain. To assess the catalytic domain of TcdB-8864, the sequence of the 5' end of its gene was determined. TcdB-8864 shows high homology to TcdB-1470 but lower homology to TcdB-10463 within this domain. This fits well with the altered glucosylation specificity of TcdB-8864 (Rac1, Rap2 and Ra1). Having defined the variations of transcription, expression and enzymatic activity of toxins A and B, implications for the pathogenic potential of strain 8864 are discussed.

Rho protein inhibition blocks protein kinase C translocation and activation.

Small GTP-binding proteins of the Ras and Rho family participate in various important signalling pathways. Large clostridial cytotoxins inactivate GTPases by UDP-glucosylation. Using Clostridium difficile toxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Rac/Cdc42) and Clostridium sordellii lethal toxin-1522 (TcsL) for inactivation of Ras-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kinase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) induced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western blots for PKC alpha. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., cells without Ras activity), suggesting that Rho GTPases (RhoA and/or Cdc42) are the most likely GTP-binding proteins responsible for PKC activation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimulated endothelial cells. In summary, the data presented indicate that Rho protein inhibition blocked PKC translocation/activation in endothelial and epithelial cells.