The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function

Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.     

Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells

Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.     

Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis

Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa , on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia , YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa . Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants ( yopB / yopD ) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.     

YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation

Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155ΔD_278–292) was constructed that is devoid of this region. Phenotypically, this small in frame ΔyopD_278–292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame ΔyopD_278–292 deletion and ΔyopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.     

YopK regulates the Yersinia pestis type III secretion system from within host cells

The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.     

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.     

The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity.

During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.     

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.     

Yersinia controls type III effector delivery into host cells by modulating Rho activity

Yersinia pseudotuberculosis binds to beta1 integrin receptors, and uses the type III secretion proteins YopB and YopD to introduce pores and to translocate Yop effectors directly into host cells. Y. pseudotuberculosis lacking effectors that inhibit Rho GTPases, YopE and YopT, have high pore forming activity. Here, we present evidence that Y. pseudotuberculosis selectively modulates Rho activity to induce cellular changes that control pore formation and effector translocation. Inhibition of actin polymerization decreased pore formation and YopE translocation in HeLa cells infected with Y. pseudotuberculosis. Inactivation of Rho, Rac, and Cdc42 by treatment with Clostridium difficile toxin B inhibited pore formation and YopE translocation in infected HeLa cells. Expression of a dominant negative form of Rac did not reduce the uptake of membrane impermeable dyes in HeLa cells infected with a pore forming strain YopEHJT(-). Similarly, the Rac inhibitor NSC23766 did not decrease pore formation or translocation, although it efficiently hindered Rac-dependent bacterial uptake. In contrast, C. botulinum C3 potently reduced pore formation and translocation, implicating Rho A, B, and/or C in the control of the Yop delivery. An invasin mutant (Y. pseudotuberculosis invD911E) that binds to beta1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the beta1 integrin signaling pathway, by inhibiting Src kinase activity, negatively affected YopE translocation. Additionally, Y. pseudotuberculosis infection activated Rho by a mechanism that was dependent on YopB and on high affinity bacteria interaction with beta1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of beta1 integrin receptors, stimulates actin polymerization and activates the translocation process, and that once the Yops are translocated, the action of YopE or YopT terminate delivery of Yops and prevents pore formation.     

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.     

TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expression and is required for polarized translocation of Yop effectors

Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.     

Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu

Gram-negative bacteria use type III machines to inject toxic proteins into the cytosol of eukaryotic cells. Pathogenic Yersinia species export 14 Yop proteins by the type III pathway and some of these, named effector Yops, are targeted into macrophages, thereby preventing phagocytosis and allowing bacterial replication within lymphoid tissues. Hitherto, YopB/YopD were thought to insert into the plasma membrane of macrophages and to promote the import of effector Yops into the eukaryotic cytosol. We show here that the type III machines of yersiniae secrete three proteins into the extracellular milieu (YopB, YopD and YopR). Although intrabacterial YopD is required for the injection of toxins into eukaryotic cells, secreted YopB, YopD and YopR are dispensable for this process. Nevertheless, YopB, YopD and YopR are essential for the establishment of Yersinia infections in a mouse model system, suggesting that type III secretion machines function to deliver virulence factors into the extracellular milieu also.     

Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae.

Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE-containing S. typhimurium expressed a YopE-mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.     

The V Antigen of Yersinia pestis Regulates Yop Vectorial Targeting as Well as Yop Secretion through Effects on YopB and LcrG

Yersinia pestis expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca²⁺ concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca²⁺ as part of the low-Ca²⁺ response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of lcrV and yop operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed lcrG deletion strain, a ΔlcrG2 mutant, of Y. pestis that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca²⁺ and to dramatically decrease Yop expression and secretion in growth medium lacking Ca²⁺. Overexpression of both LcrG and LcrV in the ΔlcrG2 strain restored wild-type levels of Yop expression and Ca²⁺ control of Yop secretion. Surprisingly, when HeLa cells were infected with the ΔlcrG2 strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV’s regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.     

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.     

Type III secretion translocon assemblies that attenuate Yersinia virulence

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti‐host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α‐helix motif at the C‐terminus. Mutants YopD_I262P and YopD_K267P poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host‐membrane insertion of the YopD–YopB translocon. Mutants YopD_A263P and YopD_A270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.     

The V-antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation

Type III-mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic Yersinia. LcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti-LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV-induced protective immunity.     

The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.     

TyeA, a protein involved in control of Yop release and in translocation of Yersinia Yop effectors.

Extracellular Yersinia spp. disarm the immune system by injecting the effector Yersinia outer proteins (Yops) into the target cell. Yop secretion is triggered by contact with eukaryotic cells or by Ca2+ chelation. Two proteins, YopN and LcrG, are known to be involved in Yop-secretion control. Here we describe TyeA, a third protein involved in the control of Yop release. Like YopN, TyeA is localized at the bacterial surface. A tyeA knock-out mutant secreted Yops in the presence of Ca2+ and in the absence of eukaryotic cells. Unlike a yopN null mutant, the tyeA mutant was defective for translocation of YopE and YopH, but not YopM, YopO and YopP, into eukaryotic cells. This is the first observation suggesting that Yop effectors can be divided into two sets for delivery into eukaryotic cells. TyeA was found to interact with the translocator YopD and with residues 242-293 of YopN. In contrast with a yopN null mutant, a yopNDelta248-272 mutant was also unable to translocate YopE and YopH. Our results suggest that TyeA forms part of the translocation-control apparatus together with YopD and YopN, and that the interaction of these proteins is required for selective translocation of Yops inside eukaryotic cells.     

Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells

Pathogenic bacteria of the genus Yersinia release in vitro a set of antihost proteins called Yops. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis transfers YopE across the host cell plasma membrane. To facilitate the study of this translocation process, we constructed a recombinant Yersinia enterocolitica strain producing YopE fused to a reporter enzyme. As a reporter, we selected the calmodulin-dependent adenylate cyclase of Borde-tella pertussis and we monitored the accumulation of cyclic AMP (cAMP). Since bacteria do not produce calmodulin, cyclase activity marks the presence of hybrid enzyme in the cytoplasmic compartment of the eukaryotic cell. Infection of a monolayer of HeLa cells by the recombinant Y. enterocolitica strain led to a significant increase of cAMP. This phenomenon was dependent not only on the integrity of the Yop secretion pathway but also on the presence of YopB and/or YopD. It also required the presence of the adhesin YadA at the bacterial surface. In contrast, the phenomenon was not affected by cytochalasin D, indicating that internalization of the bacteria themselves was not required for the translocation process. Our results demonstrate that Y. enterocolitica is able to transfer hybrid proteins into eukaryotic cells. This system can be used not only to study the mechanism of YopE translocation but also the fate of the other Yops or even of proteins secreted by other bacterial pathogens.