The Toxicokinetics And Tissue Distribution Of Shiga Toxin Type 2 (Stx2) In Rats

Shiga toxin (Stx), a major virulence factor of Shiga toxin-producing Escherichia coli (STEC), is a potent exotoxin with neurotoxic, cytotoxic, and enterotoxic activities. Stx has been shown to be responsible for hemorrhagic colitis and hemolytic uremic syndrome (HUS) clinically, which remains to be the leading cause of acute pediatric renal failure. In recent years, Stx has been listed as one of prohibited biological toxins, and it has served as a potential toxin agent for use in biological warfare and bioterrorism. Approximately 15% of HUS cases involve children. To date, the comprehensive biodistribution profile of Stx2 has been poorly described. In this study, iodine-125 (125I) was used as an indicator to describe the in vivo Stx2 biodistribution profile in rats. Besides, we also try to establish new ways with the confocal laser scanning microscopy (CLSM), isotopic tracer method and the in vivo imagine technology to study Stx2 in vivo and vitro.Part 1. The preparation and purification of Stx2The preparation of Stx2 is our first problem to be solved. There are several papers reporting cloning and expression of separate subunits, but few papers about expression of the holotoxins. It is difficult to get high yield of recombinant Shiga toxins with biological activities. First, two subunits have to be expressed and then assembled into the whole complete structure of AB5. Second, Shiga toxins have to be purified with a high-purify from the various proteins. We have developed an easy way to build recombinant plasmid pET32a-stx2 and adopted the Studier auto-induction method to get high yield of rStx2 in our previously work. Auto-induction systems express even highly toxic target proteins at high levels. The molecular weight of A subunit was approximately 32kDa, B subunit was about 7.7kDa and holotoxin rStx2 was approximately 70kDa. rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The purity of the protein obtained was about 96.6% by one step purification with optimum condition, which was analyzed by Bandscan5.0 software. The final obtained rStx2 possessed HeLa cell CD50 value about 500pg and LD50 value approximately 2.3μg/kg. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening and so on.Part 2. Labeling of Stx2 with iodine-125(125I)Stx2, a protein with the molecule weight of 70kDa, has a complicated metabolism course in vivo. There has not yet a standard for evaluating Stx2 in vivo, and the method and technique of protein polypeptide drug was always used as a reference. Iodine-125(125I) has been used to label Stx in many researches in vitro. In this research, we used 125I which has a 60-days half life as the radioactive tracer to label the protein of Stx2.5×105 cpm/μg of the specific activities for iodinated samples in this study were determined. Greater than 93% of recovered radioactive material was protein-bound as determined by an Automatic Liquid Chromatography Separation Chromatography. The protein concentration of 125I-Stx2 was 0.6mg/ml that determined by the bicinchoninic acid (BCA) protein assay kit. To verify that radiolabeling of the toxins did not affect activity, a hela cell cytotoxicity assay was performed to compare labeled and unlabeled toxin, and revealed that the 50% cytolethal doses of the reassembled 125I-Stx2 preparations were equal to the unlabeled Stx2. Besides, the toxicity activity of 125I-Stx2 and Stx2 were also compared in BALB/C mice, the results showed no difference between them.Part 3. The toxicokinetics and tissue distribution of Shiga toxin type 2(Stx2) in ratsShiga toxin type 2, a major virulence factor produced by the Shiga toxin-producing Escherichia coli, is a potential toxin agent of bioterrorism. Understand the tissue targeting and the toxicokinetics of Stx2 may help to establish a new flat for evaluating the treatment on HUS. Despite this, the systemic fate of Stx2 in vivo has not been clearly characterized. In this study, iodine-125 (125I) was used as an indicator to describe the in vivo Stx2 biodistribution profile. The rats were injected intravenously (i.v.) with 125I-Stx2 at three doses of 5.1-127.5μg/kg body weight. Stx2 had a short distribution half-life (t1/2α, less than 6 min) and a long elimination half-life in rat. The toxicokinetics of Stx2 in rats was dose dependent and non-linear. Stx2 concentrations in various tissues were detected at 5 min,0.5 h, and 72 h post-injection. High radioactivity was found in the lungs, kidneys, nasal turbinates, and sometimes in the eyes, which has never been reported in previous studies. In addition, serious lesions were found mainly in the kidneys and thymus.Part 4. The initial establishment of detections with the confocal laser scanning microscope (CLSM) and the in vivo imagine technology on Stx2With the development of science and technology, bio-detection methods in the field constantly updated. Based on the needs of post-test work, we have to establish some new detection methods which are more advanced and accurate. Through these methods we will complement the previous gaps or inadequate means of detection, and close to the international scientific researches. In this paper, we described the dynamic process of Stx2 in HeLa cells with CLSM. Stx2 mainly distributed in the cell membrane at Omin after Stx2 incubated with HeLa cells, while mainly around the nucleus at 60min. Stx2 partly remain on the cell membrane and partly around the nucleus at 30min. The result is consistent with some reports. Besides, we have also observed the migration of Stx2 with the in vivo imagine technology in mouse. We found the fluorescence in liver increased within the first 30min after i.v., while the fluorescence in bladder increased from 30min, and reach a high level at 50min. The results suggest that the liver might be a main target organ and the bladder might be a main excretory organ in the BALB/c-nu mouse.