A streamlined cloning and toxicity evaluation strategy of toxin-antitoxin systems in insect cells.

Toxin-antitoxin (TA) systems, which are prevalent in bacteria and archaea, have significant potential applications in eukaryotic cells due to their characteristic ability to inhibit cell growth. In particular, type II TA systems, where both the toxin and antitoxin are proteins, can serve as lethal and rescue genes in genetic pest management. However, traditional methods of cloning these systems often encounter difficulties due to cryptic promoter driven expression of toxins during the cloning process. To address this issue, we have developed a novel cloning method that incorporates an additional prokaryotic promoter to express the antitoxin, thus preventing the deleterious effects of toxin expression in bacterial cells. This method allows for efficient and straightforward construction of toxin vectors and the rapid screening of effective TA systems in insect cells. Our study focused on evaluating the toxicity of various type II TA systems in Sf9 insect cells. We cloned and tested toxins and antitoxins from Escherichia coli, Streptococcus pneumoniae, and Rickettsia rickettsii. The toxicity of each system was assessed by the number of red fluorescent cells post-transfection. Our results demonstrated that toxins such as MazF (E. coli-2782), RelE (Spn-1223), and RelE (Spn-1104) exhibited high levels of toxicity in Sf9 cells, while other toxins showed varying degrees of effectiveness. The corresponding antitoxins were able to neutralize the toxins in a dose-dependent manner, although some antitoxins, like MnT (R. felis-0357) and MazE (E. coli-4224), were ineffective in insect cells. This study highlights the potential for applying type II TA systems in genetic pest management and other eukaryotic applications. Our novel cloning approach provides a robust platform for the rapid and efficient evaluation of TA systems, paving the way for future research and application in pest control and beyond.