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Characterization of assembly and activation of the Shigella type III secretion injectisome

Award Number: R01AI172097
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2027

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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 with complications that 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 is used to deliver effector proteins into host cells to promote pathogen entry. T3SSs are shared by many Gram negative pathogens with the injectisome comprising a(n): 1) external needle and tip complex for delivering translocators and effectors; 2) basal body that spans the bacterial envelope; and 3) cytoplasmic sorting platform (SP) that energizes and controls secretion. We pioneered visualizing the SP by cryo-electron tomography (cryo-ET) and 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 with parallel biochemical analysis of the SP sub-assemblies and the first visualization of the Shigella injectisome-host membrane interface in situ. We will use cryo-ET methods to view the SP pods at a 1-nm or better resolution and then use biochemical and molecular methods as we 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 extend our study to examine another important cytoplasmic component of the injectisome - the export gate nonamer formed by MxiA, which undergoes structural rearrangements in the absence of the SP. We will also exploit a system we’ve generated for trapping different secretion substrates within the in situ injectisome so that we can determine how the overall structure compares for three different states. These are the “on” injectisome (ipaD null strain) and two forms of “off” injectisomes (mxiH null strain and effector-blocked strains), which 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; and 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 to reveal elements that cannot be studied using purified needle complexes and 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 that underlie type III secretion.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $616,769 )
2026	2026	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Allergy and Infectious Diseases Research	000	5	6/26/2026	NON-COMPETING CONTINUATION	$616,769
														Subtotal = $616,769

Issue Date FY: 2025 ( Subtotal = $616,769 )
2025	2025	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Allergy and Infectious Diseases Research	000	4	8/6/2025	NON-COMPETING CONTINUATION	$616,769
														Subtotal = $616,769

Issue Date FY: 2024 ( Subtotal = $616,769 )
2024	2024	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Allergy and Infectious Diseases Research	000	3	7/2/2024	NON-COMPETING CONTINUATION	$616,769
														Subtotal = $616,769

Issue Date FY: 2023 ( Subtotal = $616,769 )
2023	2023	UNIVERSITY OF MISSOURI SYSTEM	121 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Allergy and Infectious Diseases Research	000	2	7/12/2023	NON-COMPETING CONTINUATION	$616,769
														Subtotal = $616,769

Issue Date FY: 2022 ( Subtotal = $630,893 )
2022	2022	UNIVERSITY OF MISSOURI SYSTEM	316 UNIVERSITY HALL	COLUMBIA	MO	65211	BOONE	USA	Allergy and Infectious Diseases Research	000	1	7/28/2022	NEW	$630,893
														Subtotal = $630,893

Grand Total All Awards = $3,097,969

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Characterization of assembly and activation of the Shigella type III secretion injectisome

Award Number: R01AI172097

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/01/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2027

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