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.     

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.     

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.     

Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB-dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5-1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore-forming streptolysin O from Streptococcus pyogenes. Upon infection, cells released the low-molecular-weight marker BCECF (623 Da) but not the high-molecular-weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red-X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG.     

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.     

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127-195 of LcrV and to amino acids 106-173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.     

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.     

Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST–YopB hybrid proteins and SycD in Escherichia coli , we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST–YopB in E. coli . By analysis of a series of truncated GST–YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia . In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB–YopD and LcrV.     

Coordinate involvement of invasin and Yop proteins in a Yersinia pseudotuberculosis-specific class I-restricted cytotoxic T cell-mediated response

Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.     

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.     

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.     

Translocators YopB and YopD from Yersinia enterocolitica form a multimeric integral membrane complex in eukaryotic cell membranes

The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in Yersinia enterocolitica) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.     

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.     

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.     

A study of the YopD-lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.     

Yersinia pestis YopD 150-287 fragment is partially unfolded in the native state

Yersinia pestis, a human and animal pathogen, uses the type III secretion system (T3SS) for delivering virulence factors and effectors into the host cells. The system is conserved in animal pathogens and is hypothesized to deliver the virulence factors directly from bacterial to mammalian cells through a pore composed of YopB and YopD translocation proteins. The YopB and YopD translocator proteins must be delivered first to form a functional pore in the mammalian cell. The criteria by which Yersinia selects the two proteins for initial delivery are not known and we hypothesized that the extensive binding by the chaperone and partial unfolding of the unbound region may be the criteria for selection. The YopB and YopD translocator proteins, unlike other effectors, have a common chaperone SycD, which binds through multiple regions. Due to the small size of the pore, we hypothesized that many of the transported virulence factors, translocators YopB and YopD included, are delivered in a partially unfolded state stabilized by binding to specific chaperones. The YopD protein binds the chaperone through amino acid (a.a.) 53-149 and a.a. 278-292 regions but biophysical characterization of YopD has not been possible due to the lack of an expression system for soluble, large fragments of the protein. In our present work, we demonstrated that the YopD 150-287 peptide fragment, almost the full soluble C-terminal part, including the non-interacting peptide fragment YopD 150-277, was partially unfolded in its native state by a combination of biophysical methods: circular dichroism, quasi-elastic light scattering, chemical unfolding and 8-anilino-1-naphthalene sulfonate (ANS) binding. The secondary structure of the peptide converted easily between alpha-helical and random coil states at neutral pH, and the alpha-helical state was almost fully recovered by lowering the temperature to 263 K. The current results suggest that YopD 150-287 peptide may have the postulated transport-competent state in its native form.     

Expression and Association of the Yersinia pestis Translocon Proteins,YopB and YopD,Are Facilitated by Nanolipoprotein Particles

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.     

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.     

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.