Search

Membrane-bound organelles serve as platforms for the assembly of multi-protein complexes that function as hubs of signal transduction in eukaryotic cells. Microbial pathogens have evolved virulence factors that reprogram these host signaling responses, but the underlying molecular mechanisms are poorly understood. Here we test the ability of ~200 type III and type IV effector proteins from six Gram-negative bacterial species to interact with the eukaryotic plasma membrane and intracellular organelles. We show that over 30% of the effectors localize to yeast and mammalian cell membranes, including a subset of previously uncharacterized Legionella effectors that appear to be able to regulate yeast vacuolar fusion. A combined genetic, cellular, and biochemical approach supports that some of the tested bacterial effectors can bind to membrane phospholipids and may regulate membrane trafficking. Finally, we show that the type III effector IpgB1 from Shigella flexneri may bind to acidic phospholipids and regulate actin filament dynamics., Microbial pathogens secrete effector proteins into host cells to affect cellular functions. Here, the authors use a yeast-based screen to study around 200 effectors from six bacterial species, showing that over 30% of them interact with the eukaryotic plasma membrane or intracellular organelles.

Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages., Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms. IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

Summary Bidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.

An irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a Shigella flexneri type III effector protein with cysteine protease activity, is described. Nearly one per cent of eukaryotic proteins are modified with N-myristoyl groups that facilitate dynamic protein–protein and protein–membrane interactions. This means that N-myristoylation is important for cellular signaling but also makes it an inviting target for pathogens seeking to modulate a host cell's signalling landscape. Neal Alto and colleagues describe a previously unrecognized pathogenic mechanism involving irreversible protein demyristoylation catalysed by IpaJ, a Shigella flexneri type III effector protein with cysteine protease activity. IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. 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.

Pathogenic strains of Escherichia coli translocate many proteins into the host cell to promote virulence. The structure of one of these proteins, EspG from E. coli O157:H7, has been determined in a complex with two host enzymes and its mechanism dissected. These structures reveal how EspG disrupts endomembrane trafficking pathways by specifically recognizing the GTP-bound active state of the host's ARF6 enzyme during the vesicle budding reaction at membrane organelles. EspG directly activates PAK kinase by trapping an unfolded transition state in the kinase activation cascade. Pathogenic Escherichia coli translocate many proteins into the host cell to promote virulence. It is now shown that one of these proteins, EspG, which is present in enterohaemorrhagic E. coli, interferes with the host signalling network. 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 components3,4,5,6. 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 A 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 A 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.