Abstract
ABSTRACTEnteric 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.
Highlights
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
Of the 512 phosphotyrosine sites on 342 proteins combined in E. coli K-12 and Escherichia coli (EHEC) O157:H7 that we identified by a mass spectrometry-based phosphoproteomic approach, most relate to fundamental cell functions and virulence, indicating a central regulatory role of tyrosine phosphorylation in E. coli [27]
To assess whether phosphotyrosinemediated regulation by Cra occurs under glycolytic conditions where Cra Y47 was identified as phosphorylated [27], we compared the abilities of plasmid-encoded wild-type Cra and nonphosphorylatable Cra Y47F to complement T3SS expression in a
Summary
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. 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. We identified phosphotyrosine modifications on nine global transcriptional regulators associated with LEE expression [27], suggesting that phosphotyrosine signaling could play an important role in the control of EHEC virulence potential. We previously demonstrated that tyrosine phosphorylation of the regulator SspA positively affects the production and secretion of LEE-encoded T3SS proteins and is required for optimal A/E lesion formation by EHEC [27].
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