Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori

Bacterial-mediated cancer therapy has shown great promise in in vivo tumour models with increased survival rates post-bacterial treatment. Improving efficiency of bacterial-mediated tumour regression has focused on controlling and exacerbating bacterial cytotoxicity towards tumours. One mechanism that has been used to carry this out is the process of bactofection where post-invasion, bacteria deliver plasmid-borne mammalian genes into target cells for expression. Here we utilised the cancer-targeting Salmonella Typhimurium strain, SL7207, to carry out bactofection into triple negative breast cancer MDA-MB-231 cells. However, we noted that post-transformation with the commonly used mammalian expression vector pEGFP, S. Typhimurium became filamentous, attenuated and unable to invade target cells efficiently. Filamentation did not occur in Escherichia coli-transformed with the same plasmid. Further investigation identified the region inducing S. Typhimurium filamentation as being the f1 origin of replication (f1 ori), an artefact of historic use of mammalian plasmids for single stranded DNA production. Other f1 ori-containing plasmids also induced the attenuated phenotype, while removal of the f1 ori from pEGFP restored S. Typhimurium virulence and increased the bactofection capacity. This work has implications for interpretation of prior bactofection studies employing f1 ori-containing plasmids in S. Typhimurium, while also indicating that future use of S. Typhimurium in targeting tumours should avoid the use of these plasmids.