Transgenic Biosynthesis of Trypanothione Protects Escherichia coli from Radiation-Induced Toxicity

Abstract

Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H 2 DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by 137 Cs γ radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent.

Resveratrol induced inhibition of Escherichia coli proceeds via membrane oxidation and independent of diffusible reactive oxygen species generation

“…This in conjunction with external supplementation of antioxidants to scavenge the ROS provides valuable insight in to the events leading to macromolecular damage and cellular cytotoxicity. Fluorescence augmentation due to oxidation of DCFDA by ROS has been employed previously in E. coli to elucidate the role of ROS [22] . ROS generated by antibiotics when scavenged by externally supplemented antioxidants result in loss of their efficacy against the target bacteria [8] .…”

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

“…T. cruzi is naturally exposed to ROS produced by itself or by the host response to infection [47] , and this parasite has a full system based on trypanothione-thiol metabolism to deal with oxidative stress [48] . It was observed that E. coli cells expressing the T. cruzi trypanothione showed an increased protection against genomic DNA damage (4.6-fold) and resistance to gamma rays (4.3-fold), as well as a decrease in intracellular levels of ROS [49] . Contrary to the expected finding, we found in this study that gamma radiation did not induce a massive expression of key antioxidant genes.…”

Escherichia coliglutathionylspermidine synthetase/amidase: phylogeny and effect on regulation of gene expression

“…These post‐translational modifications protect proteins from oxidative stress and can regulate their functions (Fairlamb & Cerami, ), at least in part due to presence of trypanothione (Krieger et al ., ). Thus, when the genes for trypanothione synthetase and reductase from Trypanosoma cruzi were introduced into E. coli , the cells were protected from radiation‐induced DNA damage (Fitzgerald et al ., ).…”

“…Despite these changes in gene expression, we have not found any difference in the two strains (gss + vs. gss À ) in their growth rate, their sensitivity to oxygen, the toxicity of copper sulfate or cadmium sulfate, or survival after long-time storage (data not shown). As one of the older speculations suggested a function in protecting DNA (Krieger et al, 2000;Fitzgerald et al, 2010), we also tested their sensitivity to UV radiation, but found no significant difference in either survival or development of fluorouracil-resistant mutations (data not shown). Although we could not find any difference in oxygen sensitivity, Chiang et al (2010) have reported slight increase in the sensitivity of a combined grxD and gssD double mutant to hydrogen peroxide, but no difference between gss + and gssDcells.…”

“…These post-translational modifications protect proteins from oxidative stress and can regulate their functions (Fairlamb & Cerami, 1992), at least in part due to presence of trypanothione (Krieger et al, 2000). Thus, when the genes for trypanothione synthetase and reductase from Trypanosoma cruzi were introduced into E. coli, the cells were protected from radiation-induced DNA damage (Fitzgerald et al, 2010). Although the high homology for the Gss sequences in the Enterobacteria suggests an important physiologic function for glutathionylspermidine in these organisms, no specific function has been described for this system in bacteria.…”