Functional analysis of the Shigella flexneri type III secretion system distal needle tip complex

Many disease-causing bacteria attack the cells our bodies by using a complex machine designed to inject bacterial toxins directly into the attacked cell. This machine directly contacts the targeted cell by using a tiny hollow needle that projects from the surface of the bacterium. Contact between the cell and the needle switches on the machine within the bacterium so that bacterial toxins travel through the needle and are injected into the cell. We want to study the detailed structure of this needle, and in particular of its external tip, to help us understand how the bacterium knows that the needle has touched a suitable host cell. We then want to understand how the needle tip alters itself to insert into the cell and allow passage of the relatively large bacterial toxins through the narrow central channel of the needle. We hope that a good understanding of this system will suggest new ways to interfere with the process. Such knowledge may lead to the eventual design of novel antimicrobials and vaccines at a time when antibiotic resistance is increasing ever faster.

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens of animals and plants. We study the T3SS of Shigella flexneri, an agent of bacillary dysentery in humans, as a model to understand key functional mechanisms of T3SS. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow needle protruding from the bacterial surface. Physical contact with eukaryotic host cells initiates secretion and leads to formation of a pore, formed by the bacterial proteins IpaB and IpaC, in the host-cell membrane. Through this pore, further bacterial proteins, termed effectors and facilitating host cell invasion, are translocated. Understanding the molecular details of this protein transport process, the core function of all T3SSs, is the focus of our laboratory. We showed that the needle is implicated in host cell sensing and secretion regulation and that its distal tip contains components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we revealed tip complexes of differing compositions and functional states, that appear to represent the molecular events surrounding host cell sensing and pore formation. The interaction between IpaD and IpaB at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle and its secretion channel and hence direct effector translocation.
Our present goal is to investigate crucial aspects of our new working model. This can be subdivided into:
1) Understanding the molecular mechanism of mature tip complex assembly prior to host cell contact.
2) Determining the physiological mechanism of tip complex activation for secretion.
3) Investigating the development of any conformational changes within the tip complex during artificial or physiological activation and translocon insertion.
This work is key to understand all T3SSs because the needle, the translocon and the IpaD-type adaptor are highly conserved components. IpaD homologues in other T3SSs are so far the only T3SS components to which disease-protecting antibodies can be raised. Understanding the molecular mechanism of T3SS activation also offers the possibility of rational drug design, an appealing option given ever increasing antiboitic resistance in bacteria.