In recent times tyrosine phosphorylation has been shown to play a vital role in bacterial virulence, from regulation of capsule in Streptococcus pneumoniae, to modulation of T3SS function in Shigella flexneri. Furthermore, we and others have shown that tyrosine phosphorylation is more prevalent in bacteria than originally thought, with levels above that seen in eukaryotes. Included amongst proteins tyrosine phosphorylated in S. flexneri and E. coli are a range of DNA binding transcription factors including the response regulator OmpR and the global repressor H-NS, which are both known regulators of bacterial virulence. We were thus interested to see if tyrosine phosphorylation of these proteins modulated their regulatory ability, and thus their ability to control bacterial virulence.
In order to investigate this, we constructed phosphomimetic and phosphoablative substitutions in E. coli and S. flexneri on all sites of tyrosine phosphorylation in both OmpR and H-NS. We then investigated whether these substitutions modulated OmpR and H-NS activity, by analysing the level of regulated proteins OmpC and OmpF, as well as bacterial growth. This work suggested that tyrosine phosphorylation of both OmpR and H-NS modulated their regulatory abilities, with dysregulated OmpC/OmpF expression and aberrant growth similar to that seen with a complete deletion of the respective regulator itself. For OmpR, electrophoretic mobility shift assays showed that tyrosine phosphomimetic substitution resulted in a deficiency in the ability of OmpR to bind DNA, illustrating a mechanism for dysfunctional gene regulation.
This work unlocks another layer of complexity behind how bacterial pathogens regulate gene expression, and provides further evidence of the key role that tyrosine phosphorylation plays in bacterial virulence.