Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycT

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.     

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.     

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 type III secretion chaperone SycE promotes a localized disorder-to-order transition in the natively unfolded effector YopE

Many virulence-related, bacterial effector proteins are translocated directly into the cytosol of host cells by the type III secretion (TTS) system. Translocation of most TTS effectors requires binding by specific chaperones in the bacterial cytosol, although how chaperones promote translocation is unclear. To provide insight into the action of such chaperones, we studied the consequences of binding by the Yersinia chaperone SycE to the effector YopE by NMR. These studies examined the intact form of the effector, whereas prior studies have been limited to well ordered fragments. We found that YopE had the characteristics of a natively unfolded protein, with its N-terminal 100 residues, including its chaperone-binding (Cb) region, flexible and disordered in the absence of SycE. SycE binding caused a pronounced disorder-to-order transition in the Cb region of YopE. The effect of SycE was strictly localized to the Cb region, with other portions of YopE being unperturbed. These results provide stringent limits on models of chaperone action and are consistent with the chaperone promoting formation of a three-dimensional targeting signal in the Cb region of the effector. The target of this putative signal is unknown but appears to be a bacterial component other than the TTS ATPase YscN.     

Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 A crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals.     

Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins (Yersinia outer proteins (Yops)), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell. YopT is a cysteine protease which cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to their inactivation. Besides a biochemical characterisation of the molecular mechanism and substrate specificity also delivery into host cells with chaperone binding and guidance to the injection apparatus and the patho-physiological role of YopT have been studied and are summarised in this review.     

Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri

The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.     

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.     

Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a specialized type III secretion (TTS) system into the host cell cytosol. The efficient secretion of many effector proteins depends on the global TTS chaperone HpaB. Here, we identified a novel export control protein, HpaC, which significantly contributes to bacterial pathogenicity. Deletion of hpaC leads to a severe reduction in secretion of effector proteins and the putative type III translocon proteins HrpF and XopA. By contrast, secretion of the TTS pilus protein HrpE is not affected. We provide experimental evidence that HpaC differentiates between two classes of effector proteins. Using an in vivo reporter assay, we found that HpaC specifically promotes the translocation of the effector proteins XopJ and XopF1 into the plant cell, whereas AvrBs3 and XopC are efficiently translocated even in the absence of HpaC. Similar findings were obtained for HpaB. Inhibition of protein synthesis suggests that HpaB is involved in the secretion of stored effector proteins. Furthermore, protein-protein interaction studies revealed that HpaB and HpaC form an oligomeric protein complex and that they interact with members of both effector protein classes and the conserved TTS system component HrcV. Taken together, our data indicate that HpaB and HpaC play a central role in recruiting TTS substrates to the secretion apparatus.     

Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two-hybrid screen in yeast and a co-purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non-homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.     

Hierarchical delivery of an essential host colonization factor in enteropathogenic Escherichia coli

Many significant bacterial pathogens use a type III secretion system to inject effector proteins into host cells to disrupt specific cellular functions, enabling disease progression. The injection of these effectors into host cells is often dependent on dedicated chaperones within the bacterial cell. In this report, we demonstrate that the enteropathogenic Escherichia coli (EPEC) chaperone CesT interacts with a variety of known and putative type III effector proteins. Using pull-down and secretion assays, a degenerate CesT binding domain was identified within multiple type III effectors. Domain exchange experiments between selected type III effector proteins revealed a modular nature for the CesT binding domain, as demonstrated by secretion, chaperone binding, and infection assays. The CesT-interacting type III effector Tir, which is crucial for in vivo intestinal colonization, had to be expressed and secreted for efficient secretion of other type III effectors. In contrast, the absence of other CesT-interacting type III effectors did not abrogate effector secretion, indicating an unexpected hierarchy with respect to Tir for type III effector delivery. Coordinating the expression of other type III effectors with cesT in the absence of tir partially restored total type III effector secretion, thereby implicating CesT in secretion events. Collectively, the results suggest a coordinated mechanism involving both Tir and CesT for type III effector injection into host cells.     

Structure of the Yersinia type III secretory system chaperone SycE.

In the type III secretory system of bacterial pathogens, a large number of sequence-divergent but characteristically small (approximately 14-19 kDa), acidic (pI approximately 4-5) chaperone proteins have been identified. We present the 1.74 A resolution crystal structure of the Yersinia pseudotuberculosis chaperone SycE, whose action in promoting translocation of YopE into host macrophages is essential to Yersinia pathogenesis. SycE, a compact, globular dimer with a novel fold, has two large hydrophobic surface patches that may form binding sites for YopE or other type III components. These patches are formed by structurally key residues that are conserved among many chaperones, suggesting shared structural and functional relationships. A negative electrostatic potential covers almost the entire surface of SycE and is likely conserved in character, but not in detail, among chaperones. The structure provides the first structural insights into possible modes of action of SycE and type III chaperones in general.     

Yersinia type III effectors perturb host innate immune responses

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion

The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.     

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1

Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.     

Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD

Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 Å resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely α-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.