Lethal factor of Clostridium histolyticum kills cells by apoptosis

In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

Expression of the colH Gene Encoding Clostridium histolyticum Collagenase in Bacillus subtilis and Its Application to Enzyme Purification

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

Protease Inhibitors: Synthesis of L-Alanine Hydroxamate Sulfonylated Derivatives as Inhibitors of Clostridium Histolyticum Collagenase

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collage-nase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl-and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P₂, and P₃ sites, in order to achieve tight binding to the enzyme.     

Addition of polyadenylic acid to RNA by ATP: polynucleotidylexotransferase partially purified from HeLa cells

An enzyme that catalyzes the addition of polyadenylic acid to several RNAs was partially resolved by salt precipitation and DEAE-cellulose chromatography from the cytoplasmic component of HeLa cells. ATP and manganese are required for the sequential addition of AMP moieties to lengthen RNA primers. The presence of endogenous primer and endonucleolytic activity associated with the enzyme are indicated.     

Synergism among three purified cellulolytic components of Clostridium thermocellum

Abstract Three clostridial cellulases viz. a hydrophilic cellobiohydrolase (CBH3), a hydrophobic endoglucanase (EG1), and an aggregate-forming hydrophilic endoglucanase (EG5), all purified from recombinant strains of Escherichia coli , were used in different combinations to reconstitute the synergistic effect during cellulose hydrolysis. EG1 and EG5 were weakly active on crystalline cellulose, if added separately or together in the reaction mixture. However, when CBH3 was added to the reaction mixture, its hydrolytic activity was increased to 1.8-fold in the presence of EG1 and EG5. A further increase in the activity from 1.8 to 2.2-fold was observed when calcium and dithiothreitol were added to the reaction mixture containing all three enzymes and filter paper as substrate. The synergistic effect remained unaffected even when EG1 was replaced by its 33-amino acid C-terminal deleted variant BL35. BL35 was less active compared to EG1, but was equally hydrophobic as EG1. These results suggest that the hydrophobic interaction between cellulolytic components and/or with the crystalline substrate is important for positive synergistic effect.     

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]     

Hemagglutinating activity of trypsinized Clostridium perfringens enterotoxin

Abstract The hemagglutinating activity of Clostridium perfringens enterotoxin (CPE) was studied after trypsin treatment. Untreated CPE did not show any hemagglutinating activity to human type A, B, and O, sheep, chicken, horse, guinea-pig, or rabbit erythrocytes. Trypsinized CPE resulted in a more than 100-fold increase in hemagglutinating activity with rabbit erythrocytes only. Other erythrocytes and trypsinized rabbit erythrocytes were not agglutinated at all. The hemagglutinating activity of CPE was also found on treatment with a lysine-specific proteinase. On the other hand, trypsinized CPE did not significantly increase the cytotoxic and enterotoxic activities. The binding reaction between trypsinized and rabbit erythrocytes was not inhibited by any mono-, di-, or polysaccharides, glycoproteins or ganglioside mixtures. These results suggest that the hydrolysis of bonds involving lysine residues is mainly required for hemagglutinating activity, and that the receptor for trypsinized CPE on rabbit erythrocytes is probably the protein moiety.     

Inhibition of Clostridium histolyticum collagenases by phosphonamide peptide inhibitors

Several phosphonamide peptides having the general structure R-PO(OH)-Xaa-Yaa-Zaa were synthesized and tested for inhibition of Clostridium histolyticum collagenase. Inhibition was found to depend on the nature of R, Xaa, Yaa and Zaa such that the maximal affinity (Ki = 5 nM) was observed when R = p-nitrophenylethyl, Xaa = Gly, Yaa = Pro and Zaa = 2-aminohexanoic acid; this represents the tightest binding of inhibitor reported to date for any bacterial collagenase. Substitution of the p-nitrophenylethyl by a methyl group led to a 500-fold decrease of the potency, highlighting the existence of optimal interaction between the nitrophenylethyl side chain and one subsite of the enzyme. Replacement of the NH group in glycine residue (Xaa position) by -O- or -N-CH3 produces significantly less potent inhibitors, presumably due in part to the loss of a hydrogen bond between the inhibitor and collagenase active site. These phosphonamidates are thought to be acting as transition-state analogues of the peptide substrate.     

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb Cla I/ Sph I chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum , SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.     

艰难梭菌A毒素的细胞致死活性研究

本文报道艰难梭菌Ａ毒素对４种培养细胞的细胞致死活性的探讨。４种培养细胞为Ｖｅｒｏ（非洲绿猴肾细胞）细胞、ＴＰＣ─１（人甲状腺肿瘤细胞）细胞、ＮＩＨ３Ｔ３细胞（小鼠成纤维细胞）及将ｒａｓ癌基因转基因于ＮＩＨ３Ｔ３细胞的ＮＩＨ３Ｔ３ｒａｓ细胞。应用台酚蓝排除能试验、噻唑蓝（ＭＴＳ）比色、细胞膜损害测定试验、荧光显微术观察细胞核的形态变化等测定Ａ毒素细胞致死活性。实验表明：４种培养细胞系对Ａ毒素细胞圆缩化作用的敏感性依次为ＮＩＨ３Ｔ３ｒａｓ,ＴＰＣ─１,Ｖｅｒｏ,ＮＩＨ３Ｔ３细胞。而对Ａ毒素细胞致死活性的敏感性依次为ＴＰＣ─１,ＮＩＨ３Ｔ３,Ｖｅｒｏ,ＮＩＨ３Ｔ３ｒａｓ细胞。从而得知Ａ毒素的细胞致死活性不但依细胞种类不同而不同,而且也不一定与Ａ毒素的细胞圆缩化作用有关。肿瘤细胞ＴＰＣ─１细胞对Ａ毒素致死活性有特殊敏感性。以上结果对探索抗癌新药的研制具有重要意义。     

Extracellular protectants produced by Clostridium perfringens cells at elevated temperatures

Aim:  The mechanisms of adaptation of Clostridium perfringens to high temperatures are not well understood. In this work, the involvement of extracellular compounds in protection to heat was determined.
Methods and Results:  Cells were grown in fluid thioglycollate medium or chicken broth. When mid-log phase was reached, they were heat-shocked at 50°C for 30 min. Then cultures were centrifuged and supernatants were transferred to nonshocked cells. Heat tolerance of these cells was performed at 55°C. Viable cells were determined. In some cases, supernatants were heated at 65°C or 100°C or treated with trypsin. Supernatants were fractionated and PAGE was made of fractions showing heat-protective activity. When C. perfringens was exposed to a heat shock at 50°C, extracellular factors were found in the culture supernatant that provided protection to cells not exposed to a heat shock. The extracellular factors were sensitive to heat and trypsin treatment suggesting a protein component. SDS-PAGE analysis of supernatant fractions from heat-treated cells revealed two induced proteins (56 and 125 kDa) that could be involved in heat tolerance.
Conclusion:  In this work, the presence and thermoprotective activity of extracellular factors produced by C. perfringens under a heat shock was demonstrated.
Significance and Impact of the Study:  The detection of thermoprotective extracellular factors of C . perfringens will aid in our understanding of the physiology of survival of C. perfringens in foods.     

神经毒素原性酪酸梭菌与E型肉毒梭菌毒素的精制及特性比较

对首次自Ｅ型肉毒中毒食品中分离到的一株神经毒素原性酪酸梭菌（ＬＣＬ１５５）所产生的神经毒素,同Ｅ型肉毒梭菌（Ｅ１５３）所产生的神经毒素进行了精制及特性比较,发现（１）两菌神经毒素的分子量,Ｎａｔｉｖｅ－ＰＡＧＥ测试均为３２０ｋＤａ;ＳＤＳ－ＰＡＧＥ测试则均为１４７ｋＤａ,非毒性非血凝素部分均为１２８ｋＤａ;用胰蛋白酶激活神经毒素后发现两菌神经毒素均由分子量为１０３ｋＤａ的Ｈ链和４８ｋＤａ的Ｌ链组成。（２）两菌神经毒素柱层析图像基本一致,但在菌体毒素提取效果及精制效果诸方面,分离的酪酸梭菌却都较差。（３）胰蛋白酶激活试验表明：两菌神经毒素达到最大毒力所需激活时间不等。在相同温度下,分离的酪酸梭菌毒素只需５ｍｉｎ,而Ｅ型肉毒梭菌毒素却需３０ｍｉｎ,提示两菌神经毒素激活动力学上存在差异。（４）琼脂双扩散试验结果表明两菌神经毒素的抗原性是一致的,没有发现沉淀线呈交叉或部分交叉现象。     

Nutritional aspects of cytotoxin production by Clostridium difficile.

Arginine was the only amino acid used by Clostridium difficile that permitted cytotoxin synthesis in a peptone-based medium. Synthesis of cytotoxin was delayed when glucose was used as the substrate. Addition of rifampin or puromycin to cultures prior to release of cytotoxin inhibited the release of cytotoxin, suggesting that a protein essential for cytotoxin release is synthesized after cytotoxin is synthesized.