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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Citation
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.