YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB-dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5-1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore-forming streptolysin O from Streptococcus pyogenes. Upon infection, cells released the low-molecular-weight marker BCECF (623 Da) but not the high-molecular-weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red-X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG.     

Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST–YopB hybrid proteins and SycD in Escherichia coli , we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST–YopB in E. coli . By analysis of a series of truncated GST–YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia . In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB–YopD and LcrV.     

DsbA Is Required for Stable Expression of Outer Membrane Protein YscC and for Efficient Yop Secretion in Yersinia pestis

The role of the periplasmic disulfide oxidoreductase DsbA in Yop secretion was investigated in Yersinia pestis. A Y. pestis dsbA mutant secreted reduced amounts of the V antigen and Yops and expressed reduced amounts of the full-sized YscC protein. Site-directed mutagenesis of the four cysteine residues present in the YscC protein resulted in defects similar to those found in the dsbA mutant. These results suggest that YscC contains at least one disulfide bond that is essential for the function of this protein in Yop secretion.     

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.     

Heparin interferes with translocation of Yop proteins into HeLa cells and binds to LcrG, a regulatory component of the Yersinia Yop apparatus

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin-agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin-binding motifs. In vitro production and secretion by Y . enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE–Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.     

Effect of preparations of protein Yop encoded by Yersinia pestis calcium-dependence plasmid on mice

The impact of the preparations of Y. pestis secreted proteins Yop (YopH-M, YopB, YopD-N, YopE) on mice immunized with 3 s.c. injections was studied. Though these proteins failed to protect the animals from plague, they stimulated the immunobiological transformation in the immunized animals. YopB and YopD-N were found to have the highest immunobiological activity with respect to mice. The preparation of YopB induced the production of the highest titers of specific antibodies and stimulated cell-mediated immune response. The injection of YopD-N to mice led to a considerable decrease in the proliferative capacity of splenocytes in vitro in response to stimulation with nonspecific mitogen ConA, as well as to pathological changes in the kidneys.     

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.     

Roles of LcrG and LcrV during Type III Targeting of Effector Yops by Yersinia enterocolitica

Yersinia enterocolitica target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of lcrG caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations in lcrV reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused DeltalcrGV yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in lcrG alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.     

鼠疫菌V抗原基因在大肠杆菌中的克隆及表达

从鼠疫杆菌野毒株总DNA中利用PCR方法扩增出V抗原编码基因,将此基因克隆到大肠杆菌的表达质粒pET42b(+)中,经IPTG诱导后,在大肠杆菌BL21(DE3)细胞中成功地表达出鼠疫菌V抗原蛋白。目的蛋白表达量占菌体总蛋白的32.8%。     

Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface

Background

Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results

In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion

Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.     

Generation and Characterization of Murine Monoclonal Antibodies to Recombinant YopM, YopB and LcrV Proteins of Yersinia pestis

Summary YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains.     

Interactions of the type III secretion pathway proteins LcrV and LcrG from Yersinia pestis are mediated by coiled-coil domains

The type III secretion system is used by pathogenic Yersinia to translocate virulence factors into the host cell. A key component is the multifunctional LcrV protein, which is present on the bacterial surface prior to host cell contact and up-regulates translocation by blocking the repressive action of the LcrG protein on the cytosolic side of the secretion apparatus. The functions of LcrV are proposed to involve self-interactions (multimerization) and interactions with other proteins including LcrG. Coiled-coil motifs predicted to be present are thought to play a role in mediating these protein-protein interactions. We have purified recombinant LcrV, LcrG, and site-directed mutants of LcrV and demonstrated the structural integrity of these proteins using circular dichroism spectroscopy. We show that LcrV interacts both with itself and with LcrG and have obtained micromolar and nanomolar affinities for these interactions, respectively. The effects of LcrV mutations upon LcrG binding suggest that coiled-coil interactions indeed play a significant role in complex formation. In addition, comparisons of secretion patterns of effector proteins in Yersinia, arising from wild type and mutants of LcrV, support the proposed role of LcrG in titration of LcrV in vivo but also suggest that other factors may be involved.     

Expression and Association of the Yersinia pestis Translocon Proteins,YopB and YopD,Are Facilitated by Nanolipoprotein Particles

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.     

Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU_CC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca²⁺ conditions) and calcium-depleted medium (−Ca²⁺ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca²⁺-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca²⁺ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca²⁺ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab''s group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhB_TM. The hydrophilic C-terminal domain (FlhB_C) is predicted to protrude into the cytosol. In addition, FlhB_C can be further divided into two subdomains, FlhB_CN (amino acids 211 to 269) and FlhB_CC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhB_C is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscU_CC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscU_C) can be divided into the YscU_CN and YscU_CC subdomains (Fig. ​(Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscU_CC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.Open in a separate windowFIG. 1.Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed at the site http://www.cbs.dtu.dk/services/TMHMM. IM, inner membrane. (B) E. coli expressing C-terminally His-tagged YscU_C was induced with IPTG, which was followed by sonication and solubilization and denaturation of the protein in binding buffer (8 M urea and 10 mM imidazole). The lysate (lane L) was flushed over the Ni column, and the flowthrough (lane FT) was collected. The column was washed five times with binding buffer, and the wash fractions (lanes W1 to W5) were collected. Elution buffer (8 M urea and 300 mM imidazole) was flushed over the column to release proteins bound to the column, resulting in the eluate (lane E). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain a urea concentration of 0.2 M and incubated at 21°C overnight. The resulting overnight eluate fraction (lane E/ON) was TCA precipitated and taken up in binding buffer. Samples were analyzed by 15% Tris-Tricine SDS-PAGE. The cleavage of YscU_C-His₆ to YscU_CC-His₆ and YscU_CN was verified by N-terminal sequencing. All fractions were volume corrected. Lane ST contained a protein standard.     

鼠疫耶尔森氏菌rV270抗原的克隆、表达及其免疫保护作用评价

目的：为研制鼠疫亚单位疫苗,克隆、表达并纯化去除产生免疫抑制作用序列后的鼠疫耶尔森氏菌LcrV抗原（rV270）。方法：依据已知的LcrV的核苷酸序列,避开其产生免疫抑制作用的区段设计引物,扩增rV270基因并克隆到pET24a载体中,在大肠杆菌BL21中表达His-rV270融合蛋白：表达产物先后经Co^2＋亲和层析和Sephacryl S-200HR凝胶柱纯化,并在纯化过程中应用凝血酶切除His标塔;氢氧化铝佐剂吸附重组抗原后免疫BALB／c小鼠,初次免疫后第21天加强免疫1次,第5周使用104CFU鼠疫菌141强毒株攻毒,测定其免疫保护作用。结果：rV270以可溶性方式表达;应用Co^2＋亲和层析柱和Sephacryl S-200HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度的重组蛋白;攻毒实验中实验组小鼠全部存活,而对照组全部死亡。结论：获得了具有良好免疫保护作用的rV270蛋白,可用于鼠疫亚单位疫苗的研究。     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.