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.     

Modulation of the host innate immune and inflammatory response by translocated bacterial proteins

Bacterial secretion systems play a central role in interfering with host inflammatory responses to promote replication in tissue sites. Many intracellular bacteria utilize secretion systems to promote their uptake and survival within host cells. An intracellular niche can help bacteria avoid killing by phagocytic cells, and may limit host sensing of bacterial components. Secretion systems can also play an important role in limiting host sensing of bacteria by translocating proteins that disrupt host immune signalling pathways. Extracellular bacteria, on the other hand, utilize secretion systems to prevent uptake by host cells and maintain an extracellular niche. Secretion systems, in this case, limit sensing and inflammatory signalling which can occur as bacteria replicate and release bacterial products in the extracellular space. In this review, we will cover the common mechanisms used by intracellular and extracellular bacteria to modulate innate immune and inflammatory signalling pathways, with a focus on translocated proteins of the type III and type IV secretion systems.     

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

Modulation of Rho GTPases by type III secretion system translocated effectors of Yersinia

Pathogenic species of the bacterial genus Yersinia subdue the immune system to proliferate and spread within the host organism. For this purpose yersiniae employ a type III secretion apparatus which governs injection of six effector proteins (Yersinia outer proteins; Yops) into host cells. Yops control various regulatory and signalling proteins in a unique and highly specific manner. YopE, YopT, and YpkA/YopO modulate the activity of Rho GTP-binding proteins, whereas YopH dephosphorylates phospho-tyrosine residues in focal adhesion proteins. Furthermore, YopP/YopJ and YopM affect cell survival/apoptosis and cell proliferation, respectively. In this review the focus will be on the biochemistry and cellular effects of YopT, YopE, YopO/YpkA, and YopH.     

Manipulation of host signalling pathways by anthrax toxins

Infectious microbes face an unwelcoming environment in their mammalian hosts, which have evolved elaborate multicelluar systems for recognition and elimination of invading pathogens. A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes. Here I review recent progress in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.     

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.     

The tranquilizing injection of Yersinia proteins: a pathogen's strategy to resist host defense.

Pathogenic bacteria of the genus Yersinia possess a type III secretion apparatus by which they can inject up to six effector proteins into host cells. These so-called effector Yops (Yersinia outer proteins) disrupt cellular immune defense functions such as TNF-alpha release, O2-production or phagocytosis and thereby allow Yersinia to grow extracellularly. Recent findings indicate that the effector Yops are highly active proteins that engage in crucial eukaryotic signaling mechanisms. For instance, the translocated tyrosine phosphatase YopH dephosphorylates the focal adhesion proteins paxillin and p130Cas within target cells. Furthermore, the Yersinia effector YopP is able to induce apoptosis in macrophages presumably by blocking MAP kinase and NFKB mediated signaling events. Here we discuss recent advances concerning the intracellular targets and biochemical signaling mechanisms regulated by the translocated Yersinia effectors.     

Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosis

Type III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild-type or Yop-Y. pseudotuberculosis strains were assayed for interleukin (IL)-8 production. HeLa cells infected with a YopEHJ- triple mutant released significantly more IL-8 than HeLa cells infected with isogenic wild-type, YopE-, YopH- or YopJ- bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL-8 production. IL-8 production required YopB, but did not require YopD, pore formation or invasin-mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen-activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ- mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells.     

A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes

The bacterial pathogen Yersinia pseudotuberculosis uses type III secretion machinery to translocate Yop effector proteins through host cell plasma membranes. A current model suggests that a type III translocation channel is inserted into the plasma membrane, and if Yops are not present to fill the channel, the channel will form a pore. We examined the possibility that Yops act within the host cell to prevent pore formation. Yop- mutants of Y.pseudotuberculosis were assayed for pore-forming activity in HeLa cells. A YopE- mutant exhibited high levels of pore-forming activity. The GTPase-downregulating function of YopE was required to prevent pore formation. YopE+ bacteria had increased pore-forming activity when HeLa cells expressed activated Rho GTPases. Pore formation by YopE- bacteria required actin polymerization. F-actin was concentrated at sites of contact between HeLa cells and YopE- bacteria. The data suggest that localized actin polymerization, triggered by the type III machinery, results in pore formation in cells infected with YopE- bacteria. Thus, translocated YopE inhibits actin polymerization to prevent membane damage to cells infected with wild-type bacteria.     

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.     

YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5 kDa). Its secretion required an intact Ysc apparatus and SycT (15.0 kDa, pI 4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia , producing a hybrid YopT–adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (ΔHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells.     

Sensing of bacteria: NOD a lonely job

Recognition of bacteria by the vertebrate innate immune system relies on detection of invariant molecules by specialized receptors. The view is emerging that activation of both Toll-like receptors (TLRs) and Nod-like receptors (NLRs) by different bacterial agonists is important in order to mount an inflammatory response in the host. Priming of cells with peptidoglycan and products that are sensed by cytosolic-localized members of the NLR family have a synergistic effect on TLR signalling and vice versa. Currently, the underlying molecular mechanisms of this cross-talk between NLR and TLR signalling are beginning to emerge. These reveal that the two sensing-systems are non-redundant in bacterial recognition and that their cross-talk plays an important role in immunological homeostasis.     

Yersinia outer protein P inhibits CD8 T cell priming in the mouse infection model

Pathogenic yersiniae translocate a mixture of effector proteins called Yersinia outer proteins (Yops) into the cytosol of eukaryotic cells by their type III secretion system. YopP is one of the best characterized of these effector proteins and known to inhibit the proinflammatory response of the host by interfering with NF-kappaB signal transduction and inducing apoptosis of macrophages. The effects of YopP on the immune response were studied by a Yersinia Ag-independent approach using bacteria that translocate the well-characterized model Ag listeriolysin O of Listeria monocytogenes via their type III secretion system. In this study we demonstrate a novel function for YopP in vivo. It is shown for the first time that YopP not only counteracts the innate immune defense but also inhibits the adaptive immune system by suppressing the development of an effective CD8 T cell response in a mouse model. A possible mechanism for this could be the inhibition of Ag presentation by dendritic cells (DC). In vitro this is shown to be due to the rapid induction of programmed DC death and to inhibition of DC maturation. Using this approach we could further show that the listeriolysin O-specific CD8 T cells generated in vivo by the yopP mutant are functional and are able to protect mice against a lethal challenge with wild type Listeria.     

The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Type III secretion systems deliver effector proteins from Gram‐negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.     

Mammalian NLR proteins; discriminating foe from friend

Eukaryotic organisms of the plant and animal kingdoms have developed evolutionarily conserved systems of defence against microbial pathogens. These systems depend on the specific recognition of microbial products or structures by molecules of the host innate immune system. The first mammalian molecules shown to be involved in innate immune recognition of, and defence against, microbial pathogens were the Toll-like receptors (TLRs). These proteins are predominantly but not exclusively located in the transmembrane region of host cells. Interestingly, mammalian hosts were subsequently found to also harbour cytosolic proteins with analogous structures and functions to plant defence molecules. The members of this protein family exhibit a tripartite domain structure and are characterized by a central nucleotide-binding oligomerization domain (NOD). Moreover, in common with TLRs, most NOD proteins possess a C-terminal leucine-rich repeat (LRR) domain, which is required for the sensing of microbial products and structures. Recently, the name 'nucleotide-binding domain and LRR' (NLR) was coined to describe this family of proteins. It is now clear that NLR proteins play key roles in the cytoplasmic recognition of whole bacteria or their products. Moreover, it has been demonstrated in animal studies that NLRs are important for host defence against bacterial infection. This review will particularly focus on two subfamilies of NLR proteins, the NODs and 'NALPs', which specifically recognize bacterial products, including cell wall peptidoglycan and flagellin. We will discuss the downstream signalling events and host cell responses to NLR recognition of such products, as well as the strategies that bacterial pathogens employ to trigger NLR signalling in host cells. Cytosolic recognition of microbial factors by NLR proteins appears to be one mechanism whereby the innate immune system is able to discriminate between pathogenic bacteria ('foe') and commensal ('friendly') members of the host microflora.     

Bidirectional signaling betweenYersinia and its target cell

Preventing the early host immune defense allows pathogenicYersinia to proliferate in lymphatic tissue. This ability depends on signaling that occurs between the bacteria and the host cells. Following intimate contact with the target cell a signal is generated within the bacterium that results in increased expression of virulence-associated proteins that are subsequently delivered into the infected cell. These proteins, designated Yops, interfere with the host-cell signaling pathways that are normally activated to eliminate infectious agents. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Yersinia protein kinase YopO is activated by a novel G-actin binding process

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.     

Strategies used by Yersinia enterocolitica to evade killing by the host: thinking beyond Yops

Yersinia enterocolitica is an important gastrointestinal pathogen. Its pathogenicity has been attributed primarily to the plasmid encoded Yersinia outer proteins or Yops that are known to subvert the immune system. This review, however, highlights the role of Yop-independent mechanisms that help Y. enterocolitica evade immune system and contribute significantly to its survival in the host.     

Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

Background

Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.

Methodology/Principal Findings

In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.

Conclusions/Significance

Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.