Identification of critical components for anti-tumor activity of attenuated Salmonella.

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VNP20009, an attenuated Salmonella bacterium, preferentially replicates to high levels in solid tumors, relative to normal tissues, and possesses significant anti-tumor activity in murine animal models. During the course of generating new bacterial vectors, we discovered that some of the strains lost the tumor growth inhibition property while maintaining the ability to replicate and accumulate to high levels in tumors in vivo. In order to develop a better understanding of the anti-tumor activity induced by TAPET vectors, we undertook proteomic and genomic approaches to identify the potential tumoricidal components of VNP20009 and other closely related strains. VNP69, a variant derived from VNP20009, lost its anti-tumor activity after transposon mutagenesis, suggesting that the inserted transposon disrupted a gene or operon that directly or indirectly affects bacterial anti-tumor activity. Thus, we used genomic walking technology to identify the location of the transposon insertion and 2-D gel electrophoresis to confirm the disappearance of the gene product in VNP69. When the disrupted gene, gshB (encoding glutathione synthetase), was cloned and re-introduced into VNP69, the anti-tumor activity could be restored in a B16 melanoma-transplanted animal model. Loss of the gene in VNP69 also caused significant changes in the protein expression profile (more than 20 proteins affected), based on 2-D gel analysis, of VNP69 as compared to VNP25, which has anti-tumor activity. These data suggest that the identified gene could be a critical component of or may regulate other factors critical to the anti-tumor activity of Salmonella.