Phosphotyrosine-Mediated Regulation of Enterohemorrhagic
Escherichia coli
Virulence
Authors: Robertson Colin D. , Hazen Tracy H. , Kaper James B. , Rasko David A. , Hansen Anne-Marie 
Published: February 2018

Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic
Escherichia coli
(EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of
E. coli
identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of
ler
, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.

IMPORTANCE
Enterohemorrhagic
Escherichia coli
(EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.