A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis

Human pathogenic Yersinia resist host defences, in part through the expression and delivery of a set of plasmid-encoded virulence proteins termed Yops. A number of these Yops are exported from the bacteria directly into the cytoplasm of their eukaryotic host's cells upon contact with these cells. The secreted YopN protein (also known as LcrE) is required to block Yop secretion in the presence of calcium in vitro or before contact with a eukaryotic cell in vivo. In this study, we characterize the role of the tyeA, sycN and yscB gene products in the regulation of Yop secretion in Yersinia pestis. Mutants specifically defective in the expression of TyeA, SycN or YscB were no longer able to block Yop secretion in the presence of calcium. In addition, the secretion of YopN was specifically reduced in both the sycN and the yscB deletion mutants. Protein cross-linking and immunoprecipitation studies in conjunction with yeast two-hybrid analyses showed that SycN and YscB interact with one another to form a SycN/YscB complex. Yeast three-hybrid analyses demonstrated that the SycN/YscB complex, but not SycN or YscB alone, specifically associates with YopN. SycN and YscB share amino acid sequence similarity and structural similarities with the specific Yop chaperones SycE and SycH. Together, these results indicate that a complex composed of SycN and YscB functions as a specific chaperone for YopN in Y. pestis.     

Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN,tyeA, sycN,yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies

Yersinia pestis, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase-dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk-tagged YopE (YopE129-Elk) and YopN (YopN293-Elk) into HeLa cells. Y. pestis yopN, tyeA, sycN and yscB deletion mutants showed reduced levels of YopE129-Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN293-Elk is translocated into HeLa cells and that this process is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN293-Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN293-Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.     

Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica

During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.     

Expression of a functional secreted YopN-TyeA hybrid protein in Yersinia pestis is the result of a +1 translational frameshift event

YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis, but not by Yersinia enterocolitica. DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.     

YscB of Yersinia pestis Functions as a Specific Chaperone for YopN

Following contact with a eucaryotic cell, Yersinia species pathogenic for humans (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37°C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or “contact-dependent,” secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the yscB gene product in the regulation of Yop secretion in Y. pestis. A yscB deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of yscB in trans completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in yopN eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in Y. pestis.     

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.     

Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis

Yersinia pestis, the causative agent of plague, utilizes a type III secretion system (T3SS) to inject effector proteins directly into the cytosol of mammalian cells where they interfere with signal transduction pathways that regulate actin cytoskeleton dynamics and inflammation, thereby enabling the bacterium to avoid engulfment and destruction by macrophages. Type III secretion normally does not occur in the absence of close contact with eukaryotic cells. Negative regulation is mediated in part by a multiprotein complex that has been proposed to act as a physical impediment to type III secretion by blocking the entrance to the secretion apparatus prior to contact with mammalian cells. This complex is composed of YopN, its heterodimeric secretion chaperone SycN-YscB, and TyeA. Here, we report two crystal structures of YopN in complex with its heterodimeric secretion chaperone SycN-YscB and the co-regulatory protein TyeA, respectively. By merging these two overlapping structures, it was possible to construct a credible theoretical model of the YopN-SycN-YscB-TyeA complex. The modeled assembly features the secretion signaling elements of YopN at one end of an elongated structure and the secretion regulating TyeA binding site at the other. A patch of highly conserved residues on the surface of the C-terminal alpha-helix of TyeA may mediate its interaction with structural components of the secretion apparatus. Conserved arginine residues that reside inside a prominent cavity at the dimer interface of SycN-YscB were mutated in order to investigate whether they play a role in targeting the YopN-chaperone complex to the type III secretion apparatus. One of the mutants exhibited a phenotype that is consistent with this hypothesis.     

Yersinia enterocolitica TyeA, an intracellular regulator of the type III machinery, is required for specific targeting of YopE, YopH, YopM, and YopN into the cytosol of eukaryotic cells

Pathogenic Yersinia species employ type III machines to target effector Yops into the cytosol of eukaryotic cells. Yersinia tyeA mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907-1918, 1998). We have examined the injection of Yop proteins by tyeA mutant yersiniae with the digitonin fractionation technique. We find that tyeA mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.     

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.     

Genetically Engineered Frameshifted YopN-TyeA Chimeras Influence Type III Secretion System Function in Yersinia pseudotuberculosis

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.     

Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae

The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.     

Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1–100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.     

The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion

Activation of bacterial virulence-associated type III secretion systems (T3SSs) requires direct contact between a bacterium and a eukaryotic cell. In Yersinia pestis, the cytosolic LcrG protein and a cytosolic YopN-TyeA complex function to block T3S in the presence of extracellular calcium and prior to contact with a eukaryotic cell. The mechanism by which the bacterium senses extracellular calcium and/or cell contact and transmits these signals to the cytosolic compartment is unknown. We report here that YscF, a small protein that polymerizes to form the external needle of the T3SS, is essential for the calcium-dependent regulation of T3S. Alanine-scanning mutagenesis was used to identify YscF mutants that secrete virulence proteins in the presence and absence of calcium and prior to contact with a eukaryotic cell. Interestingly, one of the YscF mutants that exhibited constitutive T3S was unable to translocate secreted proteins across the eukaryotic plasma membrane. These data indicate that the YscF needle is a multifunctional structure that participates in virulence protein secretion, in translocation of virulence proteins across eukaryotic membranes and in the cell contact- and calcium-dependent regulation of T3S.     

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.     

YscP and YscU regulate substrate specificity of the Yersinia type III secretion system

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.     

Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y.enterocolitica is internalized inside the cytosol of PU5-1.8 macrophages by the YopB, D, N delivery apparatus.

The Yersinia Yop virulon is an anti-host system made up of four elements: (i) a type III secretion system called Ysc; (ii) a system designed to deliver bacterial proteins into eukaryotic target cells (YopB, YopD); (iii) a control element (YopN); and (iv) a set of intracellularly delivered proteins designed to disarm these cells or disrupt their communications (YopE, YopH and possibly others). YopM, another Yop protein, binds thrombin and is thus presumed to act as an extracellular effector. Here, we analyzed YopM from Y.enterocolitica and we wondered whether it could also be delivered inside eukaryotic cells. To answer this question we applied the Yop-Cya reporter strategy. Hybrids made of 141 or 100 N-terminal residues of YopM fused to Cya were delivered inside PU5-1.8 macrophages by recombinant Y.enterocolitica strains. YopB and YopD were required as translocators. Leakage of the reporters into the macrophage culture supernatant during the bacterial infection increased strongly when YopN was missing, showing that YopN is involved in the control of delivery of YopM inside eukaryotic cells. YopN itself was not delivered into the macrophages. In conclusion, YopM is translocated inside the eukaryotic cells and its physiopathological role should be revised or completed.     

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.