In vitro and in vivo antitumor activity of Laz-expressing Salmonella Typhimurium YB1 via optimized secretion and regulation.

Abstract

Salmonella enterica serovar Typhimurium strain YB1 is a genetically engineered obligate anaerobe with strong tumor-targeting potential. In this study, we evaluated YB1 as a delivery platform for Laz, a lipidated azurin-derived protein, using three secretion strategies: a native lipoprotein signal peptide (SPase II; YDL), a Sec-dependent secretory signal peptide (SPase I; YDN), and a Type III secretion signal (YDP). Laz expression was controlled by the aTc-inducible Pxyl/tet promoter on a pET29a backbone. To eliminate the confounding effects of metabolic burden, we constructed a TetR-matched control plasmid (named YC*) to be used as a comparator. Compared with YC*, Laz-expressing YB1 strains significantly reduced the viability of MCF-7 cells: YDL (50.69 ± 5.51%, p < 0.001), YDN (52.05 ± 4.69%, p < 0.001), and YDP (51.03 ± 3.92%, p < 0.001). This effect was dependent on induction conditions, with stronger responses observed at higher aTc concentrations. In vivo evaluation using the Ehrlich carcinoma mouse model revealed increased tumor necrosis and reduced mitotic activity in bacterially-treated groups. Among the constructs, YDL exhibited higher Laz expression and export, which was associated with more pronounced effects in vitro and consistent trends in vivo. Collectively, these findings support the feasibility of YB1-mediated Laz delivery as a bacterial delivery platform and highlight the importance of matched regulatory architecture in the design of engineered bacterial therapeutics. This study provides a functional evaluation of Laz-associated cytotoxic effects in the YB1 system while indicating areas for further investigation.