The WXXXE Effector Map Functions as a Potent and Specific Guanine Nucleotide Exchange Factor for CDC42

Many gram negative bacterial pathogens utilize a type three secretion system (TTSS) in order to infect eukaryotic cells. A TTSS looks and acts like a molecular syringe, allowing bacteria to inject effector proteins directly into the host cell cytoplasm. These effectors commandeer and manipulate host signaling pathways to support the lifecycle of the particular pathogen. Common targets for these effectors are small GTPases, particularly those controlling the actin cytoskeletal architecture. GTPases are molecular switches found within a diverse consortium of eukaryotic signaling pathways. As their name denotes, the primary enzymatic activity of GTPases is the conversion of bound guanine triphosphate (GTP) to guanine diphosphate (GDP) via the hydrolysis of the gamma phosphate. The oscillation between the "off" or GDP-bound form and the "on" or GTP-bound form is facilitated by the action of GTPase activating factors (GAPs) and Guanine Nucleotide Exchange Factors (GEFs). GAPs accelerate the intrinsic catalytic activity of the GTPase to physiologically relevant levels and lead to the rapid inactivation of a GTPase. GEFs conformationally eject bound GDP from inactivated GTPases allowing the binding of GTP and the activation GTPases and downstream pathways. Lipid modifications also allow GTPase activity to be localized within a eukaryotic cell. Their switch-like function, preeminence in signaling, and localized activity make GTPases great targets for bacterial effectors. Several members of a multispecies group of effectors known as the WxxxE family appear to mimic certain Rho GTPases. The WxxxE family is defined by the motif of an invariant tryptophan and glutamic acid separated by three variable residues. Given the frequent incidence of horizontal gene transfer in bacterial evolution, it is not surprising that such a family might exist between TTSS pathogens, but this family does not contain a high level of homology besides the titular motif. Upon expression in eukaryotic cells, some WxxxE effectors have been shown to produce phenotypes identical to those seen when over-expressing certain GTPases. For example, the Shigella effectors IpgB1 and IpgB2 produce Rac1-characteristic lamellipodia and RhoA-characteristic actin stress fibers. The enteropathogenic and enterohaemorrhagic E. coli effector Map produces filipodia similar to Cdc42. This study demonstrates that the enzymatic activity of EHEC O157:H7 Map as a specific GEF for cellular Cdc42. Structure/function studies were used to identify key residues and protein regions of Shigella IpgB2, E. coli Map, and Salmonella SifA that suggests that the WxxxE family functions through a SopE-like GEF biochemical activity. We propose that the WxxxE family members physically couple GTPase activation to downstream signaling pathways via a highly evolved pathogenic mechanism. These findings also suggest the possibility of GAP or GAP-like bacterial effectors counter to the WxxxEs over the course of pathogenesis, which is briefly explored in the EHEC effector EspH.