Bacteria as a versatile tool for protein delivery
Cell biological research is often hampered by the lack of appropriate methods that allow the fast, synchronized and efficient delivery of proteins into eukaryotic cells. The most widely used technique is DNA transfection ultimately leading to expression of the desired protein. This results in a heterogeneous and unsynchronized cell population after an incubation time of 12-48 hours. Hence, the protein under study is often present for far longer than physiologically relevant or desirable. Therefore, standard DNA transfection methods are of “limited use because they are active for time periods that exceed the duration of the signaling process they control so that transient activities cannot be investigated” (from Inoue et al., Nature Methods 2005).
Our aim is to provide a novel technique matching the above mentioned needs and that is further suited for combination with -omics technologies and allows for biochemical experiments. Our method is based on a bacterial protein delivery nanomachine. Several Gram-negative bacteria encode a sophisticated nanomachine, called type 3 secretion system (T3SS), which has the function to deliver bacterial proteins into eukaryotic host cells. We have removed all bacterial proteins to be delivered and instead introduced proteins of choice to be translocated into the eukaryotic cells (Figure 1). This system offers distinct advantages: protein delivery is fast, synchronized, homogenous and efficient (Figure 2). Furthermore, our tool can be applied to almost all available cell lines with consistent performance.
Wide range of applications
The advantages of direct protein delivery into target cells make our technology attractive for various applications. In combination with proteomics one can for example study protein interactions or signaling dynamics. The convenient combination with mass spectrometry allows studying the impact of a protein at a systems level (e.g. by phosphoproteomics). The fast and synchronous delivery further enables researchers to introduce their protein of interest in a timed fashion. This allows studying short-lived signaling events and signaling kinetics (e.g. membrane dynamics or protein transport) or the impact of a protein during different stages of the cell cycle. Furthermore, the function of toxic proteins can be investigated.
Figure 1: Schematic representation of the type 3 secretion system delivering proteins (green) into a eukaryotic target cell.
Figure 2: Yersinia-based delivery of a myc-tagged protein into HeLa cells is homogeneous, efficient and tunable. The cells were incubated with a control strain or a strain secreting a myc-tagged protein. After 1 hour, cells were fixed and stained with an antibody for myc (green) and with Hoechst (nuclei, blue).
The ease of genetic manipulation of our system renders it a powerful tool to study protein function by screening point- and domain-mutations. In addition, the modest cost and simple protocols make the technology attractive for high-throughput screening assays. Finally, the system has already been successfully used to deliver proteins in zebrafish.
Interested in using our technology for your assays? We are actively looking for collaborations to validate our technology for different applications. Please feel free to contact us!