Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif

Area of Science:

• Molecular biology
• Cellular signaling
• Bacterial pathogenesis

Background:

• 14-3-3 proteins are key regulators in signal transduction and checkpoint control.
• They typically bind phosphoserine-phosphorylated ligands, but also interact with some unphosphorylated proteins like bacterial toxin ExoS.
• The mechanism and significance of the ExoS-14-3-3 interaction are not fully understood.

Purpose of the Study:

• To identify the specific amino acid sequence on ExoS responsible for 14-3-3 binding.
• To investigate the role of this interaction in ExoS-mediated ADP-ribosylation of Ras.
• To determine the functional consequences of disrupting the ExoS-14-3-3 interaction on ExoS activity.

Main Methods:

• In vitro and in vivo assays to map the 14-3-3 binding site on ExoS.
• Analysis of Ras ADP-ribosylation by wild-type and mutant ExoS.
• Cell-based assays to assess ExoS-induced changes in cell morphology and viability.

Main Results:

• The specific amino acid sequence on ExoS responsible for 14-3-3 interaction was identified.
• This interaction is critical for ExoS to ADP-ribosylate the endogenous target Ras, both in vitro and in vivo.
• Disruption of the 14-3-3 binding site on ExoS impairs Ras inactivation, reduces cell morphology changes, and decreases bacterial killing activity.

Conclusions:

• A specific ExoS sequence mediates interaction with 14-3-3 proteins.
• This interaction is essential for ExoS virulence, particularly for Ras ADP-ribosylation and subsequent cellular effects.
• Targeting the ExoS-14-3-3 interaction could be a strategy to inhibit bacterial virulence.