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.     

Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion

Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.     

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 structural and functional analysis of type III periplasmic and substrate binding proteins: their role in bacterial siderophore and heme transport

In Escherichia coli the Fhu, Fep and Fec transport systems are involved in the uptake of chelated ferric iron-siderophore complexes, whereas in pathogenic strains heme can also be used as an iron source. An essential step in these pathways is the movement of the ferric-siderophore complex or heme from the outer membrane transporter across the periplasm to the cognate cytoplasmic membrane ATP-dependent transporter. This is accomplished in each case by a dedicated periplasmic binding protein (PBP). Ferric-siderophore binding PBPs belong to the PBP protein superfamily and adopt a bilobal type III structural fold in which the two independently folded amino and carboxy terminal domains are linked together by a single long α-helix of approximately 20 amino acids. Recent structural studies reveal how the PBPs of the Fhu, Fep, Fec and Chu systems are able to bind their corresponding ligands. These complex structures will be discussed and placed in the context of our current understanding of the entire type III family of Gram-negative periplasmic binding proteins and related Gram-positive substrate binding proteins.     

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.     

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.     

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.     

Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel beta-sheet flanked by three alpha-helices, SycT lacks the dimerization alpha-helix and has an additional beta-strand capable of undergoing a conformational change. The dimer interface consists of two beta-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group.     

致病菌III型分泌系统分子伴侣序列保守性分析及新伴侣的预测

很多革兰氏阴性致病菌使用Ⅲ型分泌系统(type Ⅲ secretion system,TTSS)将毒力因子直接注射到宿主细胞质中,其中某些效应蛋白(即毒素)需要专一的Ⅲ型分泌系统分子伴侣才能有效分泌。尽管2000年Cheng等提出这些分子伴侣中应该存在保守的氨基酸序列或保守的分泌信号,但是以往的研究并没有发现保守的氨基酸序列或分泌信号。文章作者通过生物信息学模体搜索对45个Ⅲ型分泌系统分子伴侣的氨基酸序列进行分析,找出5个与Ⅲ型分泌系统分子伴侣功能有关的保守模体和模体组合。其中一个为已知的,一个比已知的34氨基酸多肽重复(tetratdco peptide repeat,TPR)模体更具有Ⅲ型分泌系统分子伴侣SycD家族特征,其他3个为新的模体。每个Ⅲ型分泌系统分子伴侣家族包含一个或多个模体,这揭示了同类分子伴侣序列中确实存在保守的位点,暗示着同类分子伴侣可能存在相同的空间结构和功能,进一步支持了Birtanlan有关相同的空间结构具有普遍TTSS三维分泌信号的假说,并基于此分析和同源分析预测出27个新的可能的Ⅲ型分泌系统分子伴侣。     

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.     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP

Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion‐competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well‐characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two‐hybrid, co‐precipitation, cross‐linking and size exclusion chromatography we show that Slc1 (SycE‐like chaperone 1; CT043) specifically interacts with a 200‐amino‐acid residue N‐terminal region of TARP (TARP^1–200). Slc1 formed homodimers in vitro, as shown in cross‐linking and gel filtration experiments. Biochemical analysis of an isolated Slc1–TARP^1–200 complex was consistent with a characteristic 2:1 chaperone–effector stoichiometry. Furthermore, Slc1 was co‐immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.     

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.     

Crystal structure of the heteromolecular chaperone, AscE-AscG, from the type III secretion system in Aeromonas hydrophila

Background

The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62–116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion.

Methodology/Principal Findings

Here, we report the crystal structure of the ordered AscG_1–61 region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG_1–61 complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4–12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG_1–61 comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG_1–61 complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex.

Conclusion/Significance

The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.     

Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli

The locus of enterocyte effacement of enteropathogenic Escherichia coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein-protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two-hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching-effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. coli secretion of T ir.     

HpaC controls substrate specificity of the Xanthomonas type III secretion system

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein-protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition.     

Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.