Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.     

The mitochondrial pathway in yeast apoptosis

Mitochondria are not only important for the energetic status of the cell, but are also the fatal organelles deciding about cellular life and death. Complex mitochondrial features decisive for cell death execution in mammals are present and functional in yeast: AIF and cytochrome c release to the cytosol, mitochondrial fragmentation as well as mitochondrial hyperpolarisation followed by an oxidative burst, and breakdown of mitochondrial membrane potential. The easy accessibility of mitochondrial manipulations such as repression of respiration by growing yeast on glucose or deletion of mitochondrial DNA (rho⁰) on the one hand and the unique ability of yeast cells to grow on non-fermentable carbon sources by switching on mitochondrial respiration on the other hand have made yeast an excellent tool to delineate the necessity for mitochondria in cell death execution. Yeast research indicates that the connection between mitochondria and apoptosis is intricate, as abrogation of mitochondrial function can be either deleterious or beneficial for the cell depending on the specific context of the death scenario. Surprisingly, mitochondrion dependent yeast apoptosis currently helps to understand the aetiology (or the complex biology) of lethal cytoskeletal alterations, ageing and neurodegeneration. For example, mutation of mitochondrial superoxide dismutase or CDC48/VCP mutations, both implicated in several neurodegenerative disorders, are associated with mitochondrial impairment and apoptosis in yeast.     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Sugar-induced apoptosis in yeast cells

Sugars induce death of Saccharomyces cerevisiae within a few hours in the absence of additional nutrients to support growth; by contrast, cells incubated in water or in the presence of other nutrients without sugar remain viable for weeks. Here we show that this sugar-induced cell death (SICD) is characterized by rapid production of reactive oxygen species (ROS), RNA and DNA degradation, membrane damage, nucleus fragmentation and cell shrinkage. Addition of ascorbic acid to sugar-incubated cells prevents SICD, indicating that SICD is initiated by ROS. The lack of a protection mechanism against SICD suggests that sugars use to be the limiting nutrients for yeast and are probably depleted before all other nutrients. Being the limiting nutrient, sugars became the growth-stimulating agent, signaling the presence of sufficient nutrients for growth, but in the absence of the complementing nutrients they induce apoptotic death.     

志贺毒素及其预防治疗剂的研究进展

志贺毒素（Shigatoxin,Stx）主要由肠出血性大肠杆菌（EHEC）产生,是其主要的致病毒力因子,可通过引起急性肾衰竭导致死亡。迄今为止尚没有可推荐的治疗方案能够有效地预防或治疗Stx引起的疾病。目前,对于Stx的研究热点主要包括：Stx尚未清楚的致病机理研究,Stx与HUS的相关性研究,以及预防、治疗由Stx引起的疾病的研究。本文就以上几方面对国内外的相关研究进行总结及讨论。     

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157:H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P>0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg x ml(-1), but not at lower concentrations.     

Mechanostasis in apoptosis and medicine

Mechanostasis describes a complex and dynamic process where cells maintain equilibrium in response to mechanical forces. Normal physiological loading modes and magnitudes contribute to cell proliferation, tissue growth, differentiation and development. However, cell responses to abnormal forces include compensatory apoptotic mechanisms that may contribute to the development of tissue disease and pathological conditions. Mechanotransduction mechanisms tightly regulate the cell response through discrete signaling pathways. Here, we provide an overview of links between pro- and anti-apoptotic signaling and mechanotransduction signaling pathways, and identify potential clinical applications for treatments of disease by exploiting mechanically-linked apoptotic pathways.     

Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages

The nucleotide sequence of an 11,142-bp region including the stx ₂ operon in the genome of the temperate bacteriophage 933W in the EDL933 strain of Escherichia coli O157 was determined and compared to the respective regions derived from other lambdoid bacteriophages. In phage 933W, a region of ORFs interlinked by overlapping start-stop codons (ATGA) was detected preceding the toxin gene. These ORFs show a high degree of sequence identity to genes of the nin region of phage λ. Immediately downstream of these nin genes we identified an ORF that may code for an antiterminator similar to the λ Q protein. It is concluded that toxin expression is directly associated with the initiation of cell lysis. Downstream of the stx ₂ operon we identified an ORF that is homologous to the holin gene S of bacteriophage PA-2. PCR primers were designed, which, based on a comparison of the phage sequences, appeared to be common to both stx ₁ - and stx ₂ -harbouring phages. However, only seven of the 22 STEC strains investigated from serogroups O157, O26, O103 and O111 yielded the expected PCR amplification product. The data reported here may be useful in developing new strategies for inhibiting the expression of Stx and for developing universal diagnostic primers for use in tracking the origin and evolution of Shiga toxins and the phages that carry them. Received: 24 February 1999 / Accepted: 7 September 1999     

TMEM166, a novel transmembrane protein,regulates cell autophagy and apoptosis

Programmed cell death can be divided into apoptosis and autophagic cell death. We describe the biological activities of TMEM166 (transmembrane protein 166, also known as FLJ13391), which is a novel lysosome and endoplasmic reticulum-associated membrane protein containing a putative TM domain. Overexpression of TMEM166 markedly inhibited colony formation in HeLa cells. Simultaneously, typical morphological characteristics consistent with autophagy were observed by transmission electron microscopy, including extensive autophagic vacuolization and enclosure of cell organelles by double-membrane structures. Further experiments confirmed that the overexpression of TMEM166 increased the punctate distribution of MDC staining and GFP-LC3 in HeLa cells, as well as the LC3-II/LC3-I proportion. On the other hand, TMEM166-transfected HeLa and 293T cells succumbed to cell death with hallmarks of apoptosis including phosphatidylserine externalization, loss of mitochondrial transmembrane potential, caspase activation and chromatin condensation. Kinetic analysis revealed that the appearance of autophagy-related biochemical parameters preceded the nuclear changes typical of apoptosis in TMEM166-transfected HeLa cells. Suppression of TMEM166 expression by small interference RNA inhibited starvation-induced autophagy in HeLa cells. These findings show for the first time that TMEM166 is a novel regulator involved in both autophagy and apoptosis.     

志贺毒素及其预防治疗剂的研究进展

志贺毒素(Shigatoxin,Stx)主要由肠出血性大肠杆菌(EHEC)产生,是其主要的致病毒力因子,可通过引起急性肾衰竭导致死亡。迄今为止尚没有可推荐的治疗方案能够有效地预防或治疗Stx引起的疾病。目前,对于Stx的研究热点主要包括:Stx尚未清楚的致病机理研究,Stx与HUS的相关性研究,以及预防、治疗由Stx引起的疾病的研究。本文就以上几方面对国内外的相关研究进行总结及讨论。     

60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

猪水肿病毒素Stx2e的致Vero细胞凋亡作用

摘要：【目的】研究猪水肿病的致病因子志贺毒素2e（Shiga toxin 2e, Stx2e）的致病机理。【方法】以AO/EB荧光染色法、琼脂糖凝胶电泳法和Western blot等方法研究Stx2e对Vero细胞的致凋亡作用及其信号途径。【结果】从细胞形态学和染色质水平证明,Stx2e 能诱导Vero细胞凋亡,并表现出时间和浓度依赖性;同时引起caspase-3表达量明显上调,Bax、caspase-9的表达量没有明显变化。【结论】Stx2e对Vero细胞的致凋亡作用主要通过膜受体通路引起,线粒体信号通路所起的作用较小。     

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A₁, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.     

c-Jun contributes to amyloid beta-induced neuronal apoptosis but is not necessary for amyloid beta-induced c-jun induction

The role of gene expression in neuronal apoptosis may be cell- and apoptotic stimulus-specific. Previously, we and others showed that amyloid beta (Abeta)-induced neuronal apoptosis is accompanied by c-jun induction. Moreover, c-Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c-Jun in Abeta toxicity, we compared Abeta-induced apoptosis in neurons from murine fetal littermates that were deficient or wild-type with respect to c-Jun. We report that neurons deficient for c-jun are relatively resistant to Abeta toxicity, suggesting that c-Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Abeta treatment surprisingly led to an apparent activation of the c-jun promoter in both the c-jun-deficient and wild-type neurons, suggesting that c-Jun is not necessary for activation of the c-jun promoter. Indeed, several genes induced by Abeta in wild-type neurons were also induced in c-jun-deficient neurons, including c-fos, fosB, ngfi-B, and ikappaB. In summary, these results indicate that c-Jun contributes to Abeta-induced neuronal death but that c-Jun is not necessary for c-jun induction.     

Structural requirements for furin-induced cleavage and activation of Shiga toxin

Shiga toxin has a protease-sensitive site in the disulfide loop region of the A-chain. Cleavage of this site by furin is essential for rapid intoxication of cells by Shiga toxin. We have here investigated whether in addition to the Arg-X-X-Arg sequence, there are other structural requirements in the disulfide loop region for furin cleavage. A toxin mutant (Shiga-2D toxin) still containing the consensus motif for cleavage by furin, but lacking ten amino acids in the disulfide loop, was generated. Trypsin was able to cleave Shiga-2D toxin in vitro, demonstrating that the protease-sensitive region is intact. However, Shiga-2D toxin was not efficiently cleaved by furin either in vitro or in vivo. Furthermore, unless it was precleaved with trypsin, Shiga-2D toxin was much less toxic than wild type Shiga toxin in LoVo cells expressing functional furin. In contrast, LoVo/neo cells lacking functional furin were unable to activate both wild type Shiga toxin and Shiga-2D toxin. In conclusion, an extended loop structure is required for furin-induced cleavage of Shiga toxin.     

Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K⁺ (25 m M ) and then switched to physiological concentrations of K⁺ (5 m M ) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of G_s with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K⁺, whereas stimulation of G_o/G_i with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K⁺. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.