The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence

A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.     

GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure

The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.     

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

The proteasome pathway destabilizes Yersinia outer protein E and represses its antihost cell activities

Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

The Legionella pneumophila Effector VipA Is an Actin Nucleator That Alters Host Cell Organelle Trafficking

Legionella pneumophila, the causative agent of Legionnaires'' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.     

Exploiting pathogenic Escherichia coli to model transmembrane receptor signalling

Many microbial pathogens manipulate the actin cytoskeleton of eukaryotic target cells to promote their internalization, intracellular motility and dissemination. Enteropathogenic and enterohaemorrhagic Escherichia coli, which both cause severe diarrhoeal disease, can adhere to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. As pedestal assembly is triggered by E. coli virulence factors that mimic several host cell-signalling components, such as transmembrane receptors, their cognate ligands and cytoplasmic adaptor proteins, it can serve as a powerful model system to study eukaryotic transmembrane signalling. Here, we consider the impact of recent data on our understanding of both E. coli pathogenesis and cell biology, and the rich prospects for exploiting these bacterial factors as versatile tools to probe cellular signalling pathways.     

Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton

A family of nine Salmonella typhimurium type III secretion effectors with a conserved amino-terminus have been defined. Three family members (SifA, SifB and SseJ) have previously been demonstrated to localize to the Salmonella-containing vacuole and to Salmonella-induced filaments. In contrast, we demonstrate that two other family members, SspH2 and SseI, co-localized with the polymerizing actin cytoskeleton. These proteins also interacted with the mammalian actin cross-linking protein filamin in the yeast two-hybrid assay through their highly conserved amino-terminal domains. This amino-terminus was sufficient to direct localization to the polymerizing actin cytoskeleton, suggesting that the interaction with filamin is important for this subcellular localization. In addition, SspH2 co-localized with vacuole-associated actin polymerizations (VAP) induced by intracellular bacteria through the Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS). SspH2 interacted with the actin-binding protein profilin in the yeast two-hybrid assay and by affinity chromatography. This interaction was highly specific to SspH2 and was mediated by its carboxy-terminus. Furthermore, SspH2 inhibited the rate of actin polymerization in vitro, suggesting that it functions to reduce or remodel VAP. Strains with mutations in sspH2 and sseI retained the ability to form VAP. However, a third intracellular virulence factor, spvB, which ADP-ribosylates actin, strongly inhibited VAP formation in HeLa cells, suggesting a more subtle effect for SspH2 and SseI on the actin cytoskeleton.     

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments

Exoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa . The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia . Recently, YopE was found to be translocated into the host cell by a bacteria–cell contact-dependent mechanism involving the ysc -encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia , including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa , by analogy with Yersinia , targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.     

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia , the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al ., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK -mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK -mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.     

Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes.

Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and, furthermore, that YerA is not required to target YopE-Cya proteins to the secretion complex. Taken together, our results suggest that traversing the bacterial and eukaryotic membranes occurs by separate processes that recognize distinct domains of YopE and that these processes are not dependent on YerA activity.     

A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton.     

Direct modulation of the host cell cytoskeleton by Salmonella actin-binding proteins

Invasive Salmonella trigger their own uptake into non-phagocytic eukaryotic cells by delivering virulence proteins that stimulate signaling pathways and remodel the actin cytoskeleton. It has recently emerged that Salmonella encodes two actin-binding proteins, SipC and SipA, which together efficiently nucleate actin polymerization and stabilize the resulting supramolecular filament architecture. Therefore, Salmonella might directly initiate actin polymerization independently of the cellular Arp2/3 complex early in the cell entry process. This is an unprecedented example of a direct intervention strategy to facilitate entry of a pathogen into a target cell. Here, we discuss the Salmonella actin-binding proteins and how they might function in combination with entry effectors that stimulate Rho GTPases. We propose that membrane-targeted bacterial effector proteins might trigger actin polymerization through diverse mechanisms during cell entry by bacterial pathogens.     

Rho1 directs formin-mediated actin ring assembly during budding yeast cytokinesis

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring.     

EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42

Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 ( Shigella ), SifA, SifB ( Salmonella ) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism.     

Activity modulation of the bacterial Rho GAP YopE: an inspiration for the investigation of mammalian Rho GAPs

The Yersinia enterocolitica Rho GTPase Activating Protein (Rho GAP) YopE belongs to a group of bacterial virulence factors that is translocated into infected target cells by a type three secretion system. Structurally and biochemically YopE resembles eukaryotic Rho GAPs which control various cellular functions by modulating the activity of Rho GTP binding proteins. Here we summarise the published information on cellular effects, Rho protein substrates, compartmentalisation and turnover of YopE. A fascinating picture evolves of how this virulence factor integrates in host cellular regulatory mechanisms to fine tune bacterial pathogenicity.     

Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.

Pathogenic bacteria of the species Yersinia, including Yersinia pestis, block phagocytosis by macrophages. This process involves the YopE protein, which induces disruption of the host cell actin microfilament structure. Here, we show that the contact between the pathogen and the mammalian cell induces expression and then polarized transfer of YopE into the eukaryotic cell. While the bacteria remain at the surface of the target cell, the YopE cytotoxin is transferred through the host cell plasma membrane and YopE is only recovered within the cytosol of the target cell. The results suggest that the pathogen senses cell structures and focuses the transfer of YopE to occur solely at the interaction zone between the bacterium and the eukaryotic cell. The regulation of this process is shown to involve surface-located YopN sensor protein of the bacterium.     

The mammalian endocytic cytoskeleton

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and invaginates to form a vesicle that buds off—along with its contents—into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.