Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. The assembly and function of the Yersinia T3S apparatus is dependent upon 21 essential Yersinia secretion (Ysc) proteins. Following assembly of a functional base structure, the T3S apparatus secretes only early substrates (YscF, YscI and YscP) that function to assemble the rod (YscI) and needle (YscF) structures;however, little is known about the unique features of YscF and other early substrates that enable their selective recognition and secretion. In this proposal, we will characterize the unique molecular features that identify YscF as an early T3S substrate;as well as the components of the T3S apparatus that recognize these features. Importantly, cytoplasmic YscF is found exclusively in a 1:1:1 complex with its heterodimeric YscE/YscG chaperone, an interaction that is essential to prevent proteolytic degradation of YscF. We also provide experimental evidence that the YscE/YscG chaperone is essential for YscF secretion independent of its role in stabilizing YscF. Initially, we will investigate the role of the YscF N-terminal secretion signal in the hierarchical secretion of YscF. In addition, YscE and YscG point mutants that bind and stabilize YscF, but do not secrete YscF will be identified. Together, these studies will define the unique molecular features of YscF and the YscE/YscG chaperone that are required for recognition of the YscEFG complex and selective secretion of YscF. Finally, we will use characterize the role of a YscKYscQYscL sorting platform and YscN ATPase in the recognition of native and mutant YscEFG complexes. These studies will define the recognition events that allow the T3S apparatus to differentiate early and late T3S substrates, a process that is essential for establishing a secretion hierarchy.

Public Health Relevance

The overall goal of this project is to understand how pathogenic bacteria assemble specialized protein secretion systems that allow them to inject toxins directly into human cells. Drugs that interfere with these assembly events would be expected to disarm these pathogens and allow natural defense mechanisms to eliminate the bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI101823-02
Application #
8501366
Study Section
Special Emphasis Panel (ZRG1-IDM-A (80))
Program Officer
Mukhopadhyay, Suman
Project Start
2012-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$215,730
Indirect Cost
$74,730
Name
University of Miami School of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Mueller, K E; Plano, G V; Fields, K A (2014) New frontiers in type III secretion biology: the Chlamydia perspective. Infect Immun 82:2-9