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 2.94 Å 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.     

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.     

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.     

Yersinia type III secretion: send in the effectors

Pathogenic Yersinia spp (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) have evolved an exquisite method for delivering powerful effectors into cells of the host immune system where they inhibit signaling cascades and block the cells' response to infection. Understanding the molecular mechanisms of this system has provided insight into the processes of phagocytosis and inflammation.     

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU^C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU^C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU^C indicates that the global structure of the protein is retained in cleaved YscU^C. The structure of YscU^C variant N263D reveals that wild type YscU^C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.     

Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as “chaperones” to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.     

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.     

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.     

Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN

Type III secretion is a transport mechanism by which bacteria secrete proteins across their cell envelope. This protein export pathway is used by two different bacterial nanomachines: the flagellum and the injectisome. An indispensable component of these secretion systems is an ATPase similar to the F₁-ATPase β subunit. Here we characterize EscN, an enteropathogenic Escherichia coli type III ATPase. A recombinant version of EscN, which was fully functional in complementation tests, was purified to homogeneity. Our results demonstrate that EscN is a Mg²⁺-dependent ATPase (k_cat 0.35 s⁻¹). We also define optimal conditions for the hydrolysis reaction. EscN displays protein concentration-dependent activity, suggesting that the specific activity changes with the oligomeric state of the protein. The presence of active oligomers was revealed by size exclusion chromatography and native gel electrophoresis.     

Genetic characterization of pAsa6, a new plasmid from Aeromonas salmonicida subsp. salmonicida that encodes a type III effector protein AopH homolog

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536 bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.     

Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon

Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca(2+)-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca(2+) levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca(2+), and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca(2+) levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.     

Anti-microbial susceptibility of Yersinia pseudotuberculosis and Yersinia enterocolitica under different cultural conditions

Generally, Yersinia pseudotuberculosis and Y. enterocolitica grown at 37°C had increased susceptibility to antibiotics than when grown at 25°C. The susceptibility to kanamycin, cephalothin, tetracycline and chloramphenicol of Yersinia was also influenced by the growth medium and gas composition.N. Markova, T. Radoucheva, L. Ilieva and D. Veljanov are with the Institute of Microbiology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria.     

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

鸭疫里默氏杆菌九型分泌系统(T9SS)分泌蛋白B739_0093的功能鉴定

【目的】鸭疫里默氏杆菌是引起鸭浆膜炎的一种革兰氏阴性病原菌,该菌编码的九型分泌系统(type IX secretion system, T9SS)参与滑动、致病等过程。前期研究结果表明,鸭疫里默氏杆菌CH-1株B739_0093基因在限铁培养条件明显上调。序列分析表明,B739_0093编码蛋白含有一个T9SS分泌蛋白保守的C端结构域,然而其具体功能未知。本研究旨在鉴定该基因编码蛋白是否被T9SS分泌,以及在该菌致病中的作用。【方法】用荧光定量PCR检测B739_0093是否被铁离子和铁转运调节子(ferric uptake regulator, Fur)调控;用大肠杆菌表达重组B739_0093截短蛋白制备多克隆抗体,通过Western blotting检测是否由T9SS分泌;构建B739_0093基因缺失株,通过毒力和定殖试验鉴定B739_0093在鸭疫里默氏杆菌致病中的功能。【结果】荧光定量PCR结果表明,B739_0093基因在铁离子限制性培养基明显上调,此调控是由调控蛋白Fur介导的;Western blotting结果显示,B739_0093基因编码蛋白在亲本株RA CH-1主要定位在分泌物中,而在T9SS缺失株定位在菌体且不能在分泌物被检测到;与亲本株RA CH-1相比,RA CH-1ΔB739_0093对雏鸭的致病力减弱,在雏鸭各组织器官的定殖能力明显降低。【结论】B739_0093基因编码蛋白是由鸭疫里默氏杆菌T9SS分泌的,其表达受铁离子及Fur调控,并且参与了该菌的致病。     

Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.     

铜绿假单胞菌T6SS的组装、分泌、功能和调控

作为一种对抗真核细胞和原核细胞的强有力细菌武器,Ⅵ型分泌系统(type Ⅵ secretion system,T6SS)广泛存在于革兰氏阴性菌中.铜绿假单胞菌是一种对多种抗生素具有耐药性并能够在人体引发急性和慢性感染的条件致病菌,它编码3套独立的T6SS,分别为H1-、H2-和H3-T6SS.T6SS通过介导细菌间竞争...     

副溶血性弧菌分泌系统在致病力中作用的研究进展

致病菌借助分泌系统将特异蛋白直接注入宿主细胞内,破坏宿主细胞内的多种信号通路,是导致细菌定殖和感染的有效途径。作为一种重要的食源性致病菌,副溶血性弧菌(Vibrio parahaemolyticus)的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)和Ⅵ型分泌系统(Type Ⅵ secretion system,T6SS)是其对宿主细胞产生致病性的重要因素。本文综述了副溶血性弧菌T3SS和T6SS效应物在致病力中的具体作用,以及相关调控机理,为进一步了解由副溶血性弧菌导致的病症,研究其致病机理以及寻找致病性靶标提供参考。     

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.