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.