Bacterial guanine nucleotide exchange factors SopE-like and WxxxE effectors

Abstract

Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce "the trigger mechanism of cell entry." Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

FIG. 1.

SopE-like and WxxxE effectors. Multiple sequence alignment, with hierarchical clustering of IpgB1 and IpgB2 from Shigella flexneri 2a, EspM2 from EHEC O157:H7, EspT from C. rodentium, Map from EPEC E2348/69, the C-terminal, WxxxE-containing motif of SifA and SifB, SopE and SopE2 from S. Typhimurium, and BopE from B. pseudomallei. Similar and identical residues are highlighted in yellow. The WxxxE motif and the catalytic loops are boxed.

FIG. 2.

SopE and WxxxE effectors subvert actin dynamics. Serum-starved Swiss 3T3 cells were mock transfected or transfected with the mammalian expression vector pRK5 encoding myc-tagged IpgB2, EspM2 (not shown), Map, IpgB1, EspT, or the catalytic domain of SopE (residues 78 to 240) for 16 h. Ectopic expression of IpgB2 and EspM2 induced formation of stress fibers, Map induced filopodium formation, IpgB1 induced formation of membrane ruffles, while EspT and SopE induced membrane ruffles and lamellipodia. Actin was stained with Oregon Green phalloidin, and the myc tag was detected with monoclonal antibody.

FIG. 3.

Structures of SopE and the WxxxE effectors. (A) Overlay of the crystal structure of Map (yellow; Protein Data Bank [PDB] ID: 3gcg) (36), the crystal structure of the WxxxE-containing domain of SifA (green; PDB ID: 3cxb) (51), the model of EspM2 (purple) (4), and the crystal structure of the GEF domain of SopE (cyan; PDB ID: 1gzs) (14). Although these structures generally superimpose well, there are differences between the helical arrangements within the corresponding WxxxE region of SopE (red box). Moreover, differences in the orientation of the catalytic loop between SopE and Map and SifA and EspM2 could be attributed to the fact that the crystal structures of SopE and Map are solved in complex with Cdc42, while the SifA structure is that of an Rho GTPase-free effector. EspM2 was modeled on the SifA structure. (B) Within the WxxxE motif of Map, E78 makes two hydrogen bonds via the carboxyl oxygen to the backbone nitrogen of S130 (in helix 4), while the ring nitrogen of W74 makes a single hydrogen bond to the hydroxyl group of S130. The side chain of W74 is also buried away from the solvent in a hydrophobic pocket containing L58 and V62. The combination of these interactions orientates the catalytic loop (N terminal to helix 4) correctly, with respect to forming a complex with Cdc42. In SopE, Y106 and T110 are in positions equivalent to W74 and E78 in Map; however, they are not involved in interhelical contacts.

FIG. 4.

SopE-like and WxxxE bacterial effectors subvert host-cell pathways. Using the T3SS, Shigella (A), EPEC/EHEC (B), and Salmonella (C) inject the effectors IpgB1/IpgB2, EspT/Map/EspM, and SopE/SopE2/SifA/SifB, respectively. Except for SifA and SifB, these effectors activate a cascade of signal transduction pathways, starting with activation of Rho GTPases (either Rac1, RhoA, or Cdc42), which leads to actin polymerization. Membrane ruffles induced by IpgB1, EspT, SopE, and SopE2 allow bacterial engulfment and subsequent internalization into a bacterium-containing vacuole (BCV). Invasive EPEC and Salmonella remain in the BCV and induce the formation of intracellular actin comets and Salmonella-induced filaments (Sifs), respectively, while Shigella escapes to the cytosol, where it forms actin tails. In parallel to subversion of actin dynamics, IpgB1, IpgB2, SopE, and SopE2 induce inflammatory response, EspM, Map, SopE, and SopE2 induce tight junction alteration, and Map induces mitochondrial dysfunction. SopE and SifA, which is translocated across the SCV via the SPI-2 T3SS, play a role in maintaining the SCV. The activity of SifB remains unknown.