Ultrasound Imaging Properties of Heterologously Synthesized Gas Vesicles from Halophilic Archaeon

Biosynthetic gas vesicles (GVs), as novel nanoscale ultrasound contrast agents, exhibit unique potential in biomedical ultrasound imaging. For example, they are expected to have better tissue penetration through the tumor vasculature for detecting tumor cells by the design of GV-based acoustic probes. Of all these GVs, GVs from <i>Halobacterium</i> sp. NRC-1 possess the largest size (over 200 nm) and are nearly spherical in shape, endowing them with stronger acoustic signals and better tumor penetration. However, their genetic manipulation is relatively difficult due to the requirement of a high-salt cytoplasmic environment for their expression and assembly, limiting the application of biosynthetic technology for modulating their structural features in heterologous host cells. In this study, we cloned the gene cluster encoding GVs from <i>Halobacterium</i> sp. NRC-1 and transformed it into <i>Haloferax volcanii</i>, an archaeal species naturally incapable of producing GVs. The genetically engineered <i>Haloferax volcanii</i> successfully synthesized functional GVs (GV_vol) with a similar size and shape to naturally synthesized GVs from <i>Halobacterium</i> sp. NRC-1 (GV_halo). The ultrasound imaging properties of GV_vol heterologously synthesized in <i>Haloferax volcanii</i> were compared with naturally synthesized GV_halo in vitro and in vivo, showing that GV_vol could achieve a mean signal intensity of 113.6 ± 0.9 a.u. in vitro and a peak intensity of 121.5 ± 0.8 a.u. in vivo in the kidney, compared with 115.7 ± 0.5 a.u. and 119.0 ± 0.5 a.u. for GV_halo, respectively. These findings confirm the functional integrity of heterologously synthesized GV_vol and its potential for biomedical applications. Our study provides a solid experimental foundation for genetically tailoring <i>Halobacterium</i> GV properties to optimize biomedical imaging performance.