Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis

Abstract

BackgroundThe CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.ResultsCpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the λcI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.ConclusionsThis interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

The Legionella pneumophila CpxRA two‐component regulatory system: new insights into CpxR's function as a dual regulator and its connection to the effectors regulatory network

“…A). However, when the same two‐hybrid system was previously used, it was shown that the Y. pseudotuberculosis CpxR regulator self‐interacts only when one or both of the interactors contained only the CpxR receiver domain (Thanikkal et al ., ). Examination of the interaction between a full‐length L. pneumophila CpxR and the CpxR receiver domain as well between two CpxR receiver domains resulted in strong interaction (Fig.…”

“…A). Interestingly, when the Y. pseudotuberculosis CpxR regulator was examined using the same two‐hybrid system, no self‐interaction was observed using an identical mutation (Thanikkal et al ., ).…”

“…In Vibrio cholera, the CpxRA TCS was shown to be involved in envelope stress response and adaptation to low iron, and was recently shown to control the expression of virulence genes (Acosta et al, 2015a,b). In Yersinia pseudotuberculosis the CpxRA TCS has been shown to influence the contact of Yersinia with eukaryotic cells (Carlsson et al, 2007;Liu et al, 2012) and like in E. coli the conserved aspartic acid in the Yersinia CpxR receiver domain was shown to be critical for its function and self-interaction (Ronnebaumer et al, 2009;Thanikkal et al, 2012).…”

YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

“…WT: virulence of mutant bacteria was not statistically different from the parent; ND, not determined . g Determined from conventional yeast two-hybrid assay (YTH; Figure 5 ; Francis et al, 2000 ) and bacterial adenylate cyclase two hybrid (BACTH; electronic Supplementary Material, Figure S3 ; Thanikkal et al, 2012 ). WT: robust interaction between YopN and TyeA; Null: no detectable binding between YopN and TyeA; WT-like: a modest interaction between YopN and TyeA.…”

Elongation Factor P and Modifying Enzyme PoxA Are Necessary for Virulence of Shigella flexneri

“…Aspartate 51 of CpxR is phosphorylated by CpxA, leading to active CpxR, which can then stimulate transcription. Changing the aspartate residue to glutamate, which mimics aspartyl-phosphate, can result in constitutive activation (37,38). The S. flexneri efp mutant producing CpxR D51E was tested for invasion and plaque formation to determine whether CpxA-independent activation of CpxR would suppress the virulence defect of the efp mutant.…”