Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli

Abstract

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/β domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

Keywords: Escherichia coli; effectors; pathogenesis; ubiquitination.

Fig. 1.

The NleG5-1 NMR and crystal structures reveal a conserved N-terminal fold. (A) Bundle of the 20 lowest-energy NMR models representing the solution structure of NleG5-1 for residues 1–100 (PDB ID code 6B3N). (B) The most conserved residues in the NleG5-1 N-terminal domain form the core of the fold. Depiction of the most conserved residues between NleGs made using the ConSurf server (40) and the multiple sequence alignment shown in SI Appendix, Fig. S1. (C) The crystal structure of full-length NleG5-1 (PDB ID code 5VGC) with the N-terminal domain colored in blue as in A and the U-box domain in purple. Due to slight secondary structure annotation differences the first β-strand in the NMR structure is annotated as β0.

Fig. 2.

Human protein targets of NleG5-1 and NleG2-3 are MED15 and HK2, respectively. AP-MS experiments with U937 cell lysate reveals the human protein target candidates for NleG5-1 (A) and NleG2-3 (B). AP-MS results are displayed as the average number of peptides identified for the indicated human proteins in at least four MS runs (two MS runs for each replicate AP experiment). To identify the direct interactors from the AP-MS candidate pools, Y2H experiments were performed with human protein candidates and NleG2-3 (C) or NleG5-1 (D), confirming interactions between MED15 and NleG5-1 and between HK2 and NleG2-3.

Fig. 3.

NleG5-1 and NleG2-3 bind and degrade host targets in human cells. (A) NleG2-3 and NleG5-1 bind and degrade their host targets following ectopic expression in HEK293T cells using the vector pcDNA3.1/nFLAG-DEST and their subsequent immunoprecipitation. (B) The indicated FLAG NleG2-3 and NleG5-1 constructs were expressed in EPEC O126:H7 ΔnleG and used to infect HeLa cells, which were then detected by anti-FLAG immunofluorescence (green) and costained for nucleus (DAPI, blue) and F-actin (phalloidin, red). (C) NleG2-3, NleG5-1, and their indicated mutants were expressed in EPEC O126:H7 ΔnleG and used to infect HeLa cells in culture along with wild-type and secretion-deficient (ΔescN) EPEC and probed for target degradation. Immunoblotting of the targets reveals specific degradation by both NleG2-3 and NleG5-1 during infection. GAPDH is a loading control in A and C.