Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System

Lingling Chen,Shreyas Joshi,Timothy L Yahr,Zhida Zheng,Guozhou Chen,Evan D Brutinel

doi:10.1074/jbc.m611664200

Lingling Chen, Shreyas Joshi + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m611664200

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Abstract

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

Highlights

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of pneumonia and urinary tract infections in intensive care units [1, 2]
Under Ca2ϩ replete conditions, ExsE is retained in the cytoplasm because of the lack of type III secretory activity and forms a complex with ExsC
The tighter interaction of the ExsCExsE (1 nM) complex when compared with the ExsC-ExsD (18 nM) complex is supportive of a model in which preferential binding of ExsC to ExsE results in inhibition of T3SS gene expression in the absence of type III secretory activity

Summary

The abbreviations used are

T3SS, type III secretion system; Tev, tobacco etch virus; AUC, analytical ultracentrifugation; ITC, isothermal titration calorimetry. In the present study the oligomeric states of ExsE, ExsC, ExsD, as well as the complexes of ExsC-ExsD and ExsC-ExsE were characterized by analytical ultracentrifugation By this technique purified ExsE exists as monomers, ExsC forms homodimers, and ExsD self-associates into homotrimers. The subunit stoichiometry of the ExsC-ExsD and ExsC-ExsE complexes was resolved to 2:2 and 2:1, respectively It appears that the trimeric ExsD dissociates in forming the heterotetrameric ExsC-ExsD complex, whereas the dimeric ExsC maintains during complex formation. The tighter interaction of the ExsCExsE (1 nM) complex when compared with the ExsC-ExsD (18 nM) complex is supportive of a model in which preferential binding of ExsC to ExsE results in inhibition of T3SS gene expression in the absence of type III secretory activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION