Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface

Jyl S Matson,Matthew L Nilles

doi:10.1186/1471-2180-2-16

Abstract

BackgroundSecretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.ResultsIn this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.ConclusionOur results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

Highlights

Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive
Truncations of LcrG were constructed and fused to the GAL4 activation domain of pACT2. These plasmids were co-transformed into yeast strain Y187 with full-length LcrV fused to the GAL4 DNA binding domain of plasmid pAS2-1 to determine if the LcrG truncations could continue to participate in an interaction with LcrV
Deletion of five additional amino acids from the N-terminus of the 7–40 construct or seven amino acids from the C-terminus of the 7–40 construct eliminated our ability to detect an interaction using this system (Fig. 1). This approximate region of LcrG is predicted to form a coiled-coil domain when LcrG is analyzed using the COILS algorithm [26]. These results suggest that the LcrG-LcrV interaction requires the N-terminus of LcrG and may involve a coiled-coil domain

Summary

Introduction

Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The causative agent of bubonic plague, harbors a large virulence plasmid that is essential for pathogenicity. This plasmid, called pCD1 [1] in Y. pestis, encodes for a type III secretion apparatus necessary for the translocation of effector proteins (Yops) into eukaryotic target cells [2]. The LCR is believed to mimic the response of Y. pestis in contact with eukaryotic cells when calcium ions are removed from the growth medium at 37°C. The result of this in vitro induction of viru-

Methods

Results

Conclusion