AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling

Area of Science:

• Microbiology
• Cell Biology
• Biochemistry

Background:

• Vibrio parahaemolyticus is a pathogen that utilizes type III secretion systems to deliver effector proteins into host cells.
• The effector VopS is known to disrupt the actin cytoskeleton, leading to cell rounding.
• Rho GTPases are key regulators of the actin cytoskeleton.

Purpose of the Study:

• To elucidate the molecular mechanism by which VopS inhibits Rho GTPases.
• To identify the specific modification VopS imparts on Rho GTPases.
• To investigate the consequences of this modification on actin dynamics and explore broader implications for posttranslational modifications.

Main Methods:

• In vitro biochemical assays to test VopS activity on Rho GTPases.
• Mass spectrometry to identify modified residues and the modifying group.
• Cell-based assays to assess actin cytoskeleton integrity and Rho GTPase effector interactions.

Main Results:

• VopS covalently modifies Rho, Rac, and Cdc42 GTPases by attaching adenosine 5'-monophosphate (AMP) to a conserved threonine residue.
• This AMPYLATION event, a novel posttranslational modification, prevents Rho GTPases from interacting with their downstream effectors.
• The inhibition of Rho GTPase signaling leads to the collapse of the actin cytoskeleton in infected cells.
• Evidence suggests VopS can also AMPYLATE eukaryotic proteins, indicating a potentially broader role in cellular signaling.

Conclusions:

• VopS employs a unique AMPYLATION mechanism to inactivate Rho GTPases, thereby subverting host cell actin dynamics.
• The discovery of VopS-mediated AMPYLATION expands the known repertoire of posttranslational modifications and their roles in molecular signaling.
• This finding provides insights into bacterial pathogenesis and opens new avenues for understanding cellular regulation.