Thursday, April 3, 2025
4/3/2025
Bacterial Targets of T3SS Effector Proteases - Project Summary. Many Gram-negative bacterial pathogens interact with mammalian cells by using secretion systems to inject virulence proteins directly into infected host cells. Some of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of mammalian proteins by catalyzing the addition of unusual post- translational modifications. Type III secretion system (T3SS) effectors play essential roles in virulence and their mechanisms have provided great insight into the functions and components of the innate immune system. T3SS effectors are believed to be inactive until they are injected into host cells, where they then fold into their active conformations. However, recent work with the NleB and SseK glycosyltransferases from E. coli, Citrobacter rodentium, and Salmonella enterica has challenged that dogma. NleB glycosylates and activates the bacterial glutathione synthetase (GshB) enzyme, resulting in enhanced glutathione production and improved C. rodentium survival in oxidative stress conditions. SseK1 is active within Salmonella enterica, where it glycosylates and enhances the activity of several enzymes (GloA, GloB, GloC, and YajL) that are critical to the ability of Salmonella to resist methylglyoxal stress. The studies proposed here seek to extend previous findings and determine the extent to which other T3SS effectors with defined enzymatic activities are active within the bacterium. To do this, the E. coli T3SS effector proteases NleC, NleD, and EspL will be characterized for their intra-bacterial activities. The natural bacterial substrates of these proteases will be identified and the impact of proteolytic activities on pathogen protein abundance will be quantified. Recombinant systems will be developed to monitor the activity of NleC, NleD, and EspL in C. rodentium. The endogenous bacterial substrates of these effector proteases will be identified by using an unbiased, state-of- the-art proteomic approach named ‘terminal amine isotopic labeling of substrates (TAILS)’. The proposed studies represent the first comprehensive analysis of the activities of T3SS effector proteins within the bacterial cell, and as such, are likely to have a lasting, transformative impact on the field by demonstrating that effector functions are not simply limited to their well-known activities in modifying host cell proteins. Such concepts can readily be extended to other pathogens, other enzyme activities, and other secretion systems.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).