Development of ultrasound-visualized tumor-targeting engineered bacteria for precise tumor therapy

<i>In situ</i> imaging diagnosis and precise treatment of deep tumor tissues are hotspots in life sciences and medical research. In recent years, using focused ultrasound to remotely control engineered bacteria for drug release has become one of the methods for precise <i>in vivo</i> drug delivery. However, non-visualized engineered bacteria pose challenges for precise control within the body. Therefore, there is an urgent need for an engineered bacterial vector capable of deep tissue imaging to precisely locate bacteria <i>in vivo</i>. Acoustic reporter genes (ARGs) are biological elements used for deep tissue imaging, with gene clusters over 8 kb. However, ARGs are often tested on plasmids, which hinders stable expression <i>in vivo</i> and limits the space for inserting components that regulate drug release. Therefore, we used the attenuated <i>Salmonella typhimurium</i> VNP20009, known for its tumor-targeting ability, as the chassis bacteria. By using CRISPR-Cas9 technology, we inserted ARGs into the genome and optimized the promoter strength and copy number for ARG expression, constructing ultrasound-visible engineered bacteria expressing gas vesicles on the genome. Additionally, by knocking out the stress protein gene <i>htrA</i> in VNP20009, we increased the maximum injection dose by tenfold and the tumor specificity by a hundredfold. The constructed ultrasound-visible engineered bacteria can stably synthesize gas vesicles and output ultrasound signals while directly carrying drug plasmids for tumor therapy. Our research provides an effective vector for diagnosis and precise treatment.