Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered in Clostridium botulinum Type C

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.Clostridium botulinum is a gram-positive, spore-forming, anaerobic bacterium. Cultures of this species produce poisonous botulinum neurotoxins (BoNTXs) that are lethal to humans and animals and are classified into seven types, types A to G, based on the antigenicity of the BoNTXs produced (actually, the cultures producing type G toxin were recently classified in a new species, Clostridium argentinens [40]).C. botulinum type C and D cultures produce a binary C2 toxin in addition to C (or C1) and D BoNTXs; this additional toxin consists of two nonlinked proteins, C2I and C2II (28), that occur independently in the culture supernatant and are not chemically joined to each other. The C. botulinum C2 toxin used here is a representative of the family of binary actin-ADP-ribosylating toxins, which includes, in addition to C2 toxin, the Clostridium perfringens iota toxin, Clostridium difficile toxin, Clostridium spiroforme toxin, and the vegetative insecticidal proteins from Bacillus cereus (4).The enzyme component of C2 toxin (C2I) ADP ribosylates G-actin at arginine 177 (1). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding-translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (5). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. Subsequently, C2I translocates across the membrane into the cytosol through these C2IIa pores.The production of C1 and D BoNTXs is governed by bacteriophages (12, 13, 18, 19, 26), and both toxin genes have been cloned from the corresponding phage DNAs (16, 21). Recently, we determined the whole-genome sequence of a type C toxin-converting phage (c-st) genome (31). Eklund et al. reported that C2 toxin toxigenicity (mouse lethality) became clear when strains were cultured in fortified egg-meat medium and the culture supernatants were treated with trypsin (12). They also reported that C2 toxin production was not related to the BoNTX-converting phages; some non-BoNTX-producing cells produced C2 toxin. Fujii et al. (15) and Kimura et al. (22) determined the whole nucleotide sequences of the c2I and c2II genes and speculated that these genes might be located on the bacterial chromosome (15, 22).In this study, we determined that these genes are present not on the cell chromosome but on a large plasmid; we first speculated that this was this was the case based on the results of both pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis and then confirmed it by determining the complete nucleotide sequence of the plasmid. Since the plasmid was extremely unstable, we could not purify the complete plasmid DNA; therefore, we determined the whole-genome sequence by using the inverse PCR method, which enables rapid determination of the flanking regions of unknown sequences and determination of unidentified sequences in the genome, and the primer-walking method. This is the first case in which an entire DNA sequence of a large plasmid was determined using only these two procedures. The process used to determine the whole-plasmid DNA sequence and several interesting features of the plasmid are described below.     

Nucleotide sequence analysis of the enterotoxigenic Escherichia coli Ent plasmid

We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (LT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The LT and STIa region was located 13.5 kb apart and was surrounded by three IS1s and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.     

The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G₁ maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1^GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1^GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1^GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1^GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit complex that functions as an E3 ubiquitin ligase for various cell cycle proteins (19, 46). Proteins ubiquitinated by APC/C are recognized and degraded by the 26S proteasome to ensure proper cell cycle progression. APC/C activity is strictly dependent on coactivator proteins that interact with APC/C during specific phases of the cell cycle. Cdh1 (also known as Fzr, Hct1, or Srw) is one of the coactivators that maintain APC/C activity from anaphase of mitosis until the end of the G₁ phase of the cell cycle (43, 53).The role of Cdh1 (APC/C^Cdh1) on cell-cycle progression has been well studied; however, several studies have shed light into another aspect of Cdh1''s function. For example, expression of Cdh1 is not restricted to cycling cells; APC/C^Cdh1 is also present and active in quiescent cultured cells (9). Furthermore, immunohistochemical analysis has shown that Cdh1 is expressed in a wide variety of tissues that are predominantly composed of postmitotic cells, such as neurons, where APC/C^Cdh1 has a high cyclin B ubiquitination activity (1, 16). It has been reported that APC/C^Cdh1 promotes axonal growth and patterning (20) and is required for neuronal survival (1). These results highlight the importance of the APC/C activator Cdh1 in neurons. However, Cdh1 has also been shown to participate in the differentiation of tissues such as the muscle (25). Given that Cdh1 is ubiquitously expressed in organs containing quiescent cells, there might be additional roles for Cdh1.Rho GTPase proteins play a central role in the regulation of cell shape, polarity, and locomotion via their effects on actin polymerization, actomyosin contractility, cell adhesion, and microtubule dynamics (13). Small G proteins, which include Rho, act as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. The latter form of Rho proteins interacts with and activates downstream effector proteins. The activity of Rho GTPases is controlled by three class of key regulators: (i) guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP to GTP for their activation (41); (ii) GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activity for their inactivation (8); and (iii) guanine nucleotide dissociation inhibitors (GDIs), which interact with GDP-bound Rho GTPases and sequester them in the cytoplasm to inhibit the exchange of GDP to GTP (33). In addition to these canonical regulations, recent studies indicate that the ubiquitination pathway is also involved in the modulation of Rho GTPase activity. Smurf1, which is a HECT domain E3 ubiquitin ligase, controls the local levels of RhoA at the cell periphery by targeting it for degradation (40, 55). Therefore, the regulatory mechanisms of Rho GTPase activity seem to be more complex than previously thought. It thus remains to be clarified whether other ubiquitin ligases also play a role in Rho signaling by targeting its components directly or indirectly.In this study, we found that the APC/C activator Cdh1 modulated actin organization. Mouse embryonic fibroblasts (MEFs) derived from a homozygous Cdh1 gene-trapped ([GT] Cdh1^GT/GT) mouse model displayed decreased numbers of stress fibers and focal adhesions (FAs). Consistent with these phenotypes, Rho activity was apparently reduced in Cdh1-deficient cells. Cdh1 regulated Rho activity via the targeting of p190 for degradation. We also found that Cdh1 knockdown cells showed decreased motility, which was rescued by codepletion of p190. Furthermore, phenotypic similarities between Cdh1^GT/GT embryos and ROCK (also known as Rho-kinase, which is the important Rho downstream effector of actin cytoskeleton formation) knockout (KO) mice (44, 49) support our notion that Cdh1 plays a role in the Rho/ROCK signaling axis. Collectively, our findings suggest an alternative role for Cdh1 other than cell cycle regulation and reveal Cdh1 as a new regulator of Rho.     

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITC-PTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.     

Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model

Recently, T cell cytokines such as IL-17 and IFN-γ have been shown to play important roles in the progression of brain injury induced by ischemia. We have shown that IL-23 from infiltrated macrophages activates γδT cells, thereby inducing IL-17 from these cells. However, deletion of the IL-23 gene in mice showed a more dramatic protective effect against brain ischemia reperfusion (I/R) model than γδT cell depletion did, suggesting that IL-23 plays some other pivotal role in brain injury in addition to its role in IL-17 induction. To develop therapeutic methods based on these findings, we examined the effect of the JAK kinase inhibitor CP-690550 and an anti-IL12/23 monoclonal antibody on an I/R model. CP-690550 efficiently inhibited IL-17 production from memory T cells in vitro and partly suppressed infarct volume increase after I/R. Anti-p40 antibody, which blocks both IL-12 and IL-23, efficiently suppressed I/R injury and improved recovery of neurological deficits. The number of IL-17-producing cells was decreased by anti-p40 antibody treatment. Thus the JAK inhibitor and anti-p40 antibody, both of which have already been under trial for the treatment of several human inflammatory diseases, appear to be promising therapeutic agents for the amelioration of stroke.     

Identification of a β-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.)

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing β-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an ～4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS–PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K_m and V_max values of TAG were 31.7 μM and 0.25 μkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific β-glucosidase hydrolyzing TAG, which releases the physiologically active TA.     

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1-β1 and PFDα2-β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1-β1; CPNα and PFDα2-β2; CPNβ and PFDα1-β1; and CPNβ and PFDα2-β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNβ and PFDα2-β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2-β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1-β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2-β2 is specific for CPNβ.     

Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases

Background  

Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrP^Sc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrP^Sc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood.