Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing Escherichia coli.
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Shiga toxins (Stx) produced by Escherichia coli cause systemic vascular damage that manifests as edema disease in swine and hemolytic uremic syndrome in humans. In vitro, Stx inhibit protein synthesis and, depending on circumstances, induce necrosis, apoptosis, or both. The mechanism of in vivo Stx-mediated vascular damage is not known. The ability of Stx to cause apoptosis of vasculature in vivo was studied in pigs with edema disease that was produced by oral inoculation with Stx-producing E. coli. Arterioles of ileum and brain were evaluated by terminal dUTP nick-end labeling (TUNEL) assay for DNA fragmentation in myocytes (10 infected pigs, 5 control pigs) and by transmission electron microscopy for ultrastructural changes characteristic of apoptosis (17 infected pigs, 8 control pigs). In comparison with controls, increased numbers of TUNEL-positive arterioles were detected in 6/10 (60%) subclinically affected pigs 14-15 days after inoculation. Ultrastructurally, lesions in myocytes consisted of lysis (necrosis), with cytoplasmic debris and nuclear fragments contained between intact basement membranes. Endothelial cell changes ranged from acute swelling to necrosis and detachment from basement membrane. Subclinically affected pigs (n = 14) tended to have changes predominantly in myocytes, whereas pigs with clinical illness (n = 3) more commonly had changes in endothelial cells. The arteriolar lesions and clinical signs of edema disease are attributed to the effects of Stx on vasculature. Therefore, our findings suggest that the Stx-induced arteriolar lesions seen in this study were primarily necrotic, not apoptotic. We suspect that necrosis was the principal cause of the DNA fragmentation detected. (+info)
Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.
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Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome-based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease. (+info)
Prevalence of non-O157:H7 shiga toxin-producing Escherichia coli in diarrheal stool samples from Nebraska.
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We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT- SMAC) culture, enterohemorrhagic E. coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR). Fourteen (4.2%) of 335 specimens were positive by at least one method (CT-SMAC culture [6 of 14], EHEC enzyme immunoassay [13 of 14], stx1,2 PCR [14 of 14]). Six contained serogroup O157, while non-O157 were as prevalent as O157 serogroups. (+info)
Detection and characterization of Shiga toxin-producing Escherichia coli from seagulls.
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Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from a seagull in Japan were examined. A total of 50 faecal samples was collected on a harbour bank in Hokkaido, Japan, in July 1998. Two different STEC strains, whose serotypes were O136:H16 and O153:H-, were isolated from the same individual by PCR screening; both of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing active Stx2 and Stx1, respectively. They harboured large plasmids, but did not carry the haemolysin or eaeA genes of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, pulsed-field gel electrophoresis analysis (PFGE), and the stx genes sequences, the isolates were different. Phylogenic analysis of the deduced Stx amino acid sequences demonstrated that the Stx toxins of seagull-origin STEC were closely associated with those of the human-origin, but not those of other animal-origin STEC. In addition, Stx2phi-K7 phage purified from O136 STEC resembled Stx2phi-II from human-origin O157:H7, and was able to convert non-toxigenic E. coli to STEC. These results suggest that birds may be one of the important carriers in terms of the distribution of STEC. (+info)
Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives.
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A large number of plant and bacterial toxins with enzymatic activity on intracellular targets are now known. These toxins enter cells by first binding to cell surface receptors, then they are endocytosed and finally they become translocated into the cytosol from an intracellular compartment. In the case of the plant toxin ricin and the bacterial toxin Shiga toxin, this happens after retrograde transport through the Golgi apparatus and to the endoplasmic reticulum. The toxins are powerful tools to reveal new pathways in intracellular transport. Furthermore, knowledge about their action on cells can be used to combat infectious diseases where such toxins are involved, and a whole new field of research takes advantage of their ability to enter the cytosol for therapeutic purposes in connection with a variety of diseases. This review deals with the mechanisms of entry of ricin and Shiga toxin, and the attempts to use such toxins in medicine are discussed. (+info)
Shiga toxin activates p38 MAP kinase through cellular Ca(2+) increase in Vero cells.
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We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Shiga toxin (Stx)-induced Vero cell injury. Consonant with cell injury, Stx caused a transient extracellular signal-regulated kinase1/2 (ERK1/2) and a sustained p38 MAPK phosphorylation. p38 MAPK inhibitors (SB 203580 and PD 169316), but not an ERK1/2 kinase inhibitor (PD 98059), partially inhibited the Stx-induced cell death. BAPTA-AM, a Ca(2+) chelator, reduced both cell injury and p38 MAPK phosphorylation. Antioxidants reduced Stx1-induced p38 MAPK phosphorylation. These data indicate that Stx activates p38 MAPK through an increase in intracellular Ca(2+) and reactive oxygen species, and this signaling is involved in Stx-induced cell death. (+info)
Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network.
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Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway. (+info)
Molecular and phenotypic characterization of potentially new Shigella dysenteriae serotype.
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From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96-204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96-204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes. (+info)