DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses a type III secretion system (T3SS) to deliver virulence proteins into host cells to promote pathogen entry into these cells. The type III secretion apparatus (T3SA) consists of a basal body that spans both bacterial membranes and an external needle that provides the conduit for unidirectional delivery of translocator and effector proteins. From its position at the T3SA needle tip, IpaD controls the critical first step of type III secretion -recruitment of the fist translocator protein, IpaB, to the needle tip. From here, IpaB interacts with host cell membrane components to mobilize the final translocator protein, IpaC, to the needle tip, resulting in the onset of full secretion induction. Shigella provides a unique system for exploring the distinct steps of T3SA needle tip complex assembly and the environmental factors that control this process. We have solved the structures of the Shigella needle, needle tip proteins and a significant portion of IpaB. We have also extensively examined the biochemical properties of these proteins and used electron microscopy (EM) methods to reconstruct the nascent T3SA needle tip complex. In this investigation, we will build upon this substantial foundation to provid a significant step forward in what we know about IpaB with application for understanding T3SSs in general. We hypothesize that IpaB assumes a distinct structural context at the T3SA needle tip where it adopts an IpaD-associated oligomeric state that is essential for its function (e.g. membrane penetration) as part of the Shigella T3SA needle tip complex. To test this hypothesis, the specific aims of this investigation are: 1) Determine the in situ structural features of the primed T3SA needle tip using electron microscopy-based reconstruction; 2) Determine the crystal structure of IpaB in its monomeric state along with its general oligomeric structure; 3) Determine the role of IpaB oligomerization in lipid-interaction functions and determine regions involved in the protein-protein interactions involved in tip complex maturation. IpaB forms a stable structure when bound by its chaperone (IpgC) in the cytoplasm and this structure queues it for secretion. Upon recruitment to the T3SA needle tip, it interacts with itself and IpaD to assume the role of sensor of host cell contact. These regulated intermediary states would be similar to those in other systems at comparable steps and they thus represent a fundamental shared mechanistic feature of type III secretion systems. We know that IpaB is the central component that links T3SA assembly and function and our team is applying an integrated set of diverse methodologies to help understand the complex role of IpaB in the onset of type III secretion. This study is anticipated to reveal mechanistic aspects of T3SS may be targeted by anti-infective agents.

Shigella flexneri is a diarrheal pathogen that is a global public health problem. Its virulence requires a type III secretion system (T3SS) that allows it to invade human intestinal cells. We have developed a method for examining the individual steps of type III secretion induction in S. flexneri and we now propose to determine the structure-function basis of the second step T3SS activation. This will ultimately allow us to target the S. flexneri T3SS to prevent disease caused by this pathogen.