Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.     

Disruption of Intrinsic Motions as a Mechanism for Enzyme Inhibition

Clostridium difficile (C. diff) is one of the most common and most severe hospital-acquired infections; its consequences range from lengthened hospital stay to outright lethality. C. diff causes cellular damage through the action of two large toxins TcdA and TcdB. Recently, there has been increased effort toward developing antitoxin therapies, rather than antibacterial treatments, in hopes of mitigating the acquisition of drug resistance. To date, no analysis of the recognition mechanism of TcdA or TcdB has been attempted. Here, we use small molecule flexible docking followed by unbiased molecular dynamics to obtain a more detailed perspective on how inhibitory peptides, exemplified by two species HQSPWHH and EGWHAHT function. Using principal component analysis and generalized masked Delaunay analysis, an examination of the conformational space of TcdB in its apo form as well as forms bound to the peptides and UDP-Glucose was performed. Although both species inhibit by binding in the active site, they do so in two very different ways. The simulations show that the conformational space occupied by TcdB bound to the two peptides are quite different and provide valuable insight for the future design of toxin inhibitors and other enzymes that interact with their substrates through conformational capture mechanisms and thus work by the disruption of the protein’s intrinsic motions.     

Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.     

Probing the local dynamics of nucleotide-binding pocket coupled to the global dynamics: myosin versus kinesin

Based on the elastic network model, we develop a new analysis for protein complexes, which probes the local dynamics of a subsystem that is elastically coupled to a fluctuating environment. This method is applied to a comparative dynamical analysis of the nucleotide-binding pocket of two motor proteins-myosins and kinesins. In myosins, the observed structural changes in the nucleotide-pocket from the transition state to the rigorlike state are dominated by the lowest normal mode that involves significant movements in both switch I and switch II; in kinesins, the measured conformational changes in the nucleotide-pocket are also dominated by the lowest mode, which, however, only involves large movement in switch I. We then compute the global structural changes induced by the nucleotide-pocket deformations as described by the dominant pocket-mode, which yield encouraging results: in myosins, multiple hinge motions involving the opening/closing of the cleft between the upper and lower 50 -kDa subdomains and the swinging movement of the converter are induced, which are dominated by precisely the same global mode that has been recently identified by us as important to the dynamical correlations among the nucleotide-pocket, the actin-binding site, and the converter; in kinesins, the induced global conformational changes are well described by a highly collective global mode which hints for a dynamical pathway spanning from the nucleotide-pocket to the neck-linker via the H6 helix.     

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556). Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.     

Counterbalance of ligand‐ and self‐coupled motions characterizes multispecificity of ubiquitin

Date hub proteins are a type of proteins that show multispecificity in a time‐dependent manner. To understand dynamic aspects of such multispecificity we studied Ubiquitin as a typical example of a date hub protein. Here we analyzed 9 biologically relevant Ubiquitin‐protein (ligand) heterodimer structures by using normal mode analysis based on an elastic network model. Our result showed that the self‐coupled motion of Ubiquitin in the complex, rather than its ligand‐coupled motion, is similar to the motion of Ubiquitin in the unbound condition. The ligand‐coupled motions are correlated to the conformational change between the unbound and bound conditions of Ubiquitin. Moreover, ligand‐coupled motions favor the formation of the bound states, due to its in‐phase movements of the contacting atoms at the interface. The self‐coupled motions at the interface indicated loss of conformational entropy due to binding. Therefore, such motions disfavor the formation of the bound state. We observed that the ligand‐coupled motions are embedded in the motions of unbound Ubiquitin. In conclusion, multispecificity of Ubiquitin can be characterized by an intricate balance of the ligand‐ and self‐coupled motions, both of which are embedded in the motions of the unbound form.     

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.     

Laboratory-based evaluation of TOX A/B QUIK CHEK "NISSUI" to detect toxins A and B of clostridium difficile]

The TOX A/B QUIK CHEK "NISSUI" which detects both toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile in stool specimens through immunochromatography was first approved to be released in Japan, and we evaluated its accuracy. In the evaluation, the TOX A/B QUIK CHEK "NISSUI" could correctly detect TcdA and TcdB in solution and in stool specimens spiked with culture broth of TcdA and/or TcdB-producing isolates of C. difficile. The minimum detectable concentrations for TcdA and TcdB were determined to be < or =0.32 ng/ml and < or =0.63 ng/ml, respectively. The TOX A/B QUIK CHEK "NISSUI" gave the consistent results with the colon-endoscopic diagnosis, that is, all the 10 stool specimens from the patients with pseudomembranous colitis were read as being positive, but negative for five patients without any C. difficile-associated disease (CDAD). Of 10 positive stool specimens, one was read as being negative by the commercially available test reagents that can detect only TcdA. In clinical evaluation, a total of 240 stool specimens were tested. Of these, the TOX A/B QUIK CHEK "NISSUI" gave 19 positive results, and TcdA and/or TcdB-producing strains of C. difficile were successfully isolated from all the positive stool specimens, except one. Whereas, of 221 negative stool specimens, 28 isolates of C. difficile were recovered and 11 isolates were identified as TcdA and/or TcdB-producing strains. With these results, it can be concluded that the TOX A/B QUIK CHEK "NISSUI" can correctly detect both TcdA and TcdB of C. difficile, and should be promptly applied to clinical microbiology laboratory to make a definite diagnosis of CDAD, particularly for the CDAD caused by the TcdA-negative but TcdB-positive mutant strains.     

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.     

The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA(1-1874)) and found that this mutant was still cytopathic. However, TcdA(1-1874) possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP-mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA(1-1874) to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA(1-1874) exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

A survey of metronidazole and vancomycin resistance in strains of Clostridium difficile isolated in Warsaw, Poland

The drug of choice used to treat Clostridium difficile-associated diarroea (CDAD) are metronidazole and vancomycin. Information about emergence of antimicrobial resistance among C. difficile strains to metronidazole and intermediate resistance to vancomycin in some countries are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 193 C. difficile strains isolated in our diagnostic laboratory between year 1998 and 2003 from patients adults and children suffering from CDAD. Among these strains, 142 produced toxin A and B (TcdA(+)TcdB(+)), 43 only B (TcdA(-)TcdB(+)) and 8 were nontoxigenic. We have not observed any differences in susceptibility to metronidazole and vancomycin between all C. difficile strains under investigation (toxinogenic and non-toxinogenic). Resistance to metronidazole and vancomycin was not observed.     

ProMode: a database of normal mode analyses on protein molecules with a full-atom model

MOTIVATION: Although information from protein dynamics simulation is important to understand principles of architecture of a protein structure and its function, simulations such as molecular dynamics and Monte Carlo are very CPU-intensive. Although the ability of normal mode analysis (NMA) is limited because of the need for a harmonic approximation on which NMA is based, NMA is adequate to carry out routine analyses on many proteins to compute aspects of the collective motions essential to protein dynamics and function. Furthermore, it is hoped that realistic animations of the protein dynamics can be observed easily without expensive software and hardware, and that the dynamic properties for various proteins can be compared with each other. RESULTS: ProMode, a database collecting NMA results on protein molecules, was constructed. The NMA calculations are performed with a full-atom model, by using dihedral angles as independent variables, faster and more efficiently than the calculations using Cartesian coordinates. In ProMode, an animation of the normal mode vibration is played with a free plug-in, Chime (MDL Information Systems, Inc.). With the full-atom model, the realistic three-dimensional motions at an atomic level are displayed with Chime. The dynamic domains and their mutual screw motions defined from the NMA results are also displayed. Properties for each normal mode vibration and their time averages, e.g. fluctuations of atom positions, fluctuations of dihedral angles and correlations between the atomic motions, are also presented graphically for characterizing the collective motions in more detail. AVAILABILITY: http://promode.socs.waseda.ac.jp     

The structure of Clostridium difficile toxin A glucosyltransferase domain bound to Mn(2+) and UDP provides insights into glucosyltransferase activity and product release

Clostridium?difficile toxin?A (TcdA) is a member of the large clostridial toxin family, and is responsible, together with C.?difficile toxin?B (TcdB), for many clinical symptoms during human infections. Like other large clostridial toxins, TcdA catalyzes the glucosylation of GTPases, and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA glucosyltransferase domain (TcdA-GT) in the apo form and in the presence of Mn(2+) and hydrolyzed UDP-glucose. These structures, together with the recently reported crystal structure of TcdA-GT bound to UDP-glucose, provide a detailed understanding of the conformational changes of TcdA that occur during the catalytic cycle. Indeed, we present a new intermediate conformation of a so-called 'lid' loop (residues?510-522 in TcdA), concomitant with the absence of glucose in the catalytic domain. The recombinant TcdA was expressed in Brevibacillus in the inactive apo form. High thermal stability of wild-type TcdA was observed only after the addition of both Mn(2+) and UDP-glucose. The glucosylhydrolase activity, which is readily restored after reconstitution with both these cofactors, was similar to that reported for TcdB. Interestingly, we found that ammonium, like K(+) , is able to activate the UDP-glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain the first crystal structure of TcdA-GT bound to the hydrolysis product UDP. Database ??Coordinates of apo-TcdA-GT and Mn(2+) -UDP-TcdA-GT are available in the Protein Data Bank under the accession numbers 4DMV and 4DMW, respectively.     

A coarse-grained normal mode approach for macromolecules: an efficient implementation and application to Ca(2+)-ATPase

A block normal mode (BNM) algorithm, originally proposed by Tama et al., (Proteins Struct. Func. Genet. 41:1-7, 2000) was implemented into the simulation program CHARMM. The BNM approach projects the hessian matrix into local translation/rotation basis vectors and, therefore, dramatically reduces the size of the matrix involved in diagonalization. In the current work, by constructing the atomic hessian elements required in the projection operation on the fly, the memory requirement for the BNM approach has been significantly reduced from that of standard normal mode analysis and previous implementation of BNM. As a result, low frequency modes, which are of interest in large-scale conformational changes of large proteins or protein-nucleic acid complexes, can be readily obtained. Comparison of the BNM results with standard normal mode analysis for a number of small proteins and nucleic acids indicates that many properties dominated by low frequency motions are well reproduced by BNM; these include atomic fluctuations, the displacement covariance matrix, vibrational entropies, and involvement coefficients for conformational transitions. Preliminary application to a fairly large system, Ca(2+)-ATPase (994 residues), is described as an example. The structural flexibility of the cytoplasmic domains (especially domain N), correlated motions among residues on domain interfaces and displacement patterns for the transmembrane helices observed in the BNM results are discussed in relation to the function of Ca(2+)-ATPase. The current implementation of the BNM approach has paved the way for developing efficient sampling algorithms with molecular dynamics or Monte Carlo for studying long-time scale dynamics of macromolecules.     

Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1_401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal receptors. This structural study of TcdB in complex with frizzled-2 and chondroitin sulfate proteoglycan 4 reveals how TcdB binds to human receptors and primes itself for host entry.