Dr. E. coli: The smallest injector in the world

Structure of Type III secretion apparatus

   Type III secretion apparatus have a syringe like structure. It can be visible under electro microscope.(2) Its length is about 80nm and the diameter of its needle channel is about 20Å.(3) The length is about 1/10 and the diameter is about 1/400 of an E.coli cell’s minor axis. Thus this is the smallest injector in the world. (Fig.1 a~c)

How does it function?

   When the needle tip attaches to the host cell membrane a translocator complex that is also secreted by the T3SS is assembled on the host cell membrane and mediates the passage of the effector proteins through the target cell membrane. On the other hand an effector protein, which have a unique T3SS secretion signal domain on its N-terminal, is recognized by the specific chaperone and form an effector-chaperone complex. The secretion machinery, including a T3-secretion-associated ATPase, recognizes the complex. Then, the ATPase stripes the chaperone from the complex, which remains within the bacterial cell, and mediates the unfolding and threading of the effector protein through the central channel of the needle complex. Finally, the translocated effectors re-fold within the host cell to carry out their function. (Fig.2)

Motivation

   It is valuable to develop a system that can modulate cells’ behavior impersistently by injecting a desired “protein” directly into cells using a non-pathogenic strain. This system can be applied for many ways. For example, – Inject p53 to kill cancer cells selectively – Inject Yamanaka factors to induce iPS cells And fortunately It was reported in 2007 that T3SS encoded in Salmonella Pathogenesity Island 2(SPI2) is functional in vitro on the E.coli(K-12) strain.(9) However, the T3SS encoded in SPI-2 naturally function inside of the phagosome of the target cell.(8) So, there was no report about whether the SPI-2 T3SS, that is cloned on E.coli(K-12), can inject a heterologous protein from outside of the target cell or not. That is why we have decided to put this challenging project into practice.