Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

Rebecca Solomon,Grace Mccrann,James B Bliska,Gloria I Viboud,Weibing Zhang,Dipshikha Chakravortty

doi:10.1371/journal.pone.0120471

Rebecca Solomon, Grace Mccrann + Show 4 more

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https://doi.org/10.1371/journal.pone.0120471

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Journal: PloS one	Publication Date: Mar 25, 2015
Citations: 5	License type: CC BY 4.0

Affiliation: Stony Brook University

Abstract

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.

Highlights

Pathogenic Yersinia species require the function of a type III secretion system (T3SS) to successfully establish infection
Because the readout for both assays is LDH release, we confirmed that under the infection conditions used in the macrophage cell death assay pore formation did not occur, i.e, infection with a pore forming strain that lacked YopJ did not cause LDH release from macrophages
Growth type was determined at 37°C with and without calcium, “TS” refers to temperature sensitive, and “CD” to 37°C calcium-dependent growth; YopD production and secretion were determined in a bacterial culture at low calcium conditions in the pellet and supernatants, respectively; pore formation was tested in HeLa cells by LDH release and uptake of impermeant dyes; signal activation of mitogen-activated protein kinases (MAPKs) and NFκB was assessed in HeLa cells. “interm” refers to intermediate phenotype

Summary

Introduction

Pathogenic Yersinia species require the function of a type III secretion system (T3SS) to successfully establish infection. This specialized secretion machinery delivers a series of effector proteins (YopE, H, J, K, M, O, T) into the host cell that target different cell signaling molecules to interfere with the host immune system [1, 2]. In vitro, Yop secretion can be stimulated by culturing bacteria at 37°C at low calcium concentration. This low calcium condition results in growth arrest. Deletion of any of those YopD regions results in a TS growth and deregulation of Yop synthesis [17]

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