High catalytic efficiency and resistance to denaturing in bacterial Rho GTPase-activating proteins

Abstract

Several major bacterial pathogens use the type III secretion system (TTSS) to deliver virulence factors into host cells. Bacterial Rho GTPase activating proteins (RhoGAPs) comprise a remarkable family of type III secreted toxins that modulate cytoskeletal dynamics and manipulate cellular signaling pathways. We show that the RhoGAP activity of Salmonella SptP and Pseudomonas ExoS toxins is resistant to variations in the concentration of NaCl or MgCl(2), unlike the known salt dependant nature of the activity of some eukaryotic GAPs such as p190, RanGAP and p120GAP. Furthermore, SptP-GAP and ExoS-GAP display full activity after treatment at 80°C or with 6 m urea, which suggests that these protein domains are capable of spontaneous folding into an active state following denaturing such as what might occur upon transit through the TTSS needle. We determined the catalytic activity of bacterial GAPs for Rac1, CDC42 and RhoA GTPases and found that ExoS, in addition to Yersinia YopE and Aeromonas AexT toxins, display higher catalytic efficiencies for Rac1 and CDC42 than the known eukaryotic GAPs, making them the most catalytically efficient RhoGAPs known. This study expands our knowledge of the mechanism of action of GAPs and of the ways bacteria mimic host activities and promote catalysis of eukaryotic signaling proteins.

Regulation of Small GTPases by GEFs, GAPs, and GDIs

“…Domains homologous to the RhoGAP domain of Sptp are found in Pseudomonas ExoS and ExoT where they are associated with an ADP-ribosyltransferase domain, in Yersinia YopE, and in AexT from gastroenteritis-causing Aeromonas. The GAP domains of ExoS, SopE, and AexT have very strong GAP activities in vitro, suggesting that they are more efficient than their host counterparts, possibly due to their distinct fold(170). The ADP-ribosyl transferase domain in ExoS allows the bacteria to establish niches in the plasma membrane blebs of epithelial cells, independently of the RhoGAP activity(8).It took some time for another family of bacterial effectors carrying a conserved WxxxE motif to be grouped together (4) and to be eventually recognized as another family of RhoGEFs structurally related to the SopE family (reviewed in Ref.…”