Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion

Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.     

The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

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.     

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.     

Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.     

YopD of Yersinia pestis Plays a Role in Negative Regulation of the Low-Calcium Response in Addition to Its Role in Translocation of Yops

Yersinia pestis produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37°C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a yopD mutation in both wild-type and secretion-defective (ysc) Y. pestis to determine if the lcrH phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single yopD mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the yopD mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.     

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.     

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.     

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 cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes

Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type III secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N-terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc-binding domain in YopE (residues 15–50) and YopH (residues 20–70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes.