R01AI172097

Characterization of Assembly and Activation of the Shigella Type III Secretion Injectisome - Project Summary

Shigella causes bacillary dysentery with high worldwide morbidity and childhood mortality. Complications include cognitive and developmental impairment in children suffering multiple diarrheal episodes each year. Shigella's main virulence factor is its Type III Secretion System (T3SS), which delivers effector proteins into host cells to promote pathogen entry.

T3SSs are shared by many gram-negative pathogens. The injectisome comprises an external needle and tip complex for delivering translocators and effectors, a basal body that spans the bacterial envelope, and a cytoplasmic sorting platform (SP) that energizes and controls secretion.

We pioneered visualizing the SP by cryo-electron tomography (cryo-ET). Since then, we and others have identified the components of the SP. We now propose studies to explore structural differences between the "on" and "off" secretion states for the in situ injectisome. We will also perform parallel biochemical analysis of the SP sub-assemblies and visualize the Shigella injectisome-host membrane interface in situ.

Using cryo-ET methods, we will view the SP pods at a 1-nm or better resolution. We will then use biochemical and molecular methods to develop models of assembly and function. In our investigation of the SP, we will explore the movement of protein domains at the SP interface with the inner membrane ring.

In parallel, we will examine another important cytoplasmic component of the injectisome - the export gate nonamer formed by MxiA. This component undergoes structural rearrangements in the absence of the SP. We will also trap different secretion substrates within the in situ injectisome to determine how the overall structure compares for three different states: the "on" injectisome (ipad null strain) and two forms of "off" injectisomes (MxiH null strain and effector-blocked strains). These comparisons will provide functional insight into SP and export gate communication and association.

We propose three complementary aims:
1) Define the makeup, intermediate states, and structural requirements at the SP/inner-membrane ring (IR) interface that allow SP assembly and guide type III secretion.
2) Correlate export gate structural features with secretion status using complementary cryo-ET and molecular methods.
3) Identify the structural changes associated with trapping substrates within the in situ injectisome and begin generating the first high-resolution picture of the injectisome-host membrane interface in situ using cryo-ET.

Improved cryo-ET methods provide an unprecedented view of substructures within the Shigella injectisome in situ, revealing elements that cannot be studied using purified needle complexes. This is reflected in the preliminary data presented here. We can now visualize these sub-structures, target them for molecular analysis, and purify them for in vitro biochemical and biophysical analysis.

The T3SS is an essential virulence determinant for many pathogens, but we still lack the structural understanding needed to determine the mechanisms underlying type III secretion.

University Of Missouri System

NOT APPLICABLE

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

Columbia, Missouri 652114405 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 391% from $630,893 to $3,097,969.