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A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

Date

2013-02

Authors

Ramu, Thangadurai
Prasad, Madhulika Esther
Connors, Erica
Mishra, Amit
Thomassin, Jenny-Lee
Leblanc, Jason
Rainey, Jan K.
Thomas, Nikhil A.

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Abstract

The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.

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Ramu, Thangadurai, Madhulika Esther Prasad, Erica Connors, Amit Mishra, et al. 2013. "A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion." Journal of Bacteriology 195(4): 740-756.

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