The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3–6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome1,2. The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems3. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus4. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen5,6. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

Toxins produced by Shigella bacteria undergo endosome-to-Golgi retrograde trafficking to evade degradation in the lysosome and reach the cytosol. Selyunin et al. performed a genome-wide siRNA screen and identify host factors required for the transport and toxicity of Shiga toxins.

Shiga toxin-producing E. coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared to STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (β4-β5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding β4-β5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.

Shiga toxin 1 (STx1) and 2 (STx2), produced by Shiga toxin–producing Escherichia coli, cause lethal untreatable disease. The toxins invade cells via retrograde trafficking. Direct early endosome-to-Golgi transport allows the toxins to evade degradative late endosomes. Blocking toxin trafficking, particularly at the early endosome-to-Golgi step, is appealing, but transport mechanisms of the more disease-relevant STx2 are unclear. Using data from a genome-wide siRNA screen, we discovered that disruption of the fusion of late endosomes, but not autophagosomes, with lysosomes blocked the early endosome-to-Golgi transport of STx2. A subsequent screen of clinically approved lysosome-targeting drugs identified tamoxifen (TAM) to be a potent inhibitor of the trafficking and toxicity of STx1 and STx2 in cells. The protective effect was independent of estrogen receptors but dependent on the weak base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal dynamics. Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or STx2. Thus, it may be possible to repurpose TAM for treating Shiga toxin–producing E. coli infections.