Influence of cultivation conditions on the expression of Yersinia pestis YopE

With the use three types of nutrient media made it possible to study the specific features of the biosynthesis of YopE, one of the main effector proteins, coded by Yersinia pestis virulence plasmid. This protein was proved to be produced practically at all stages of Y. pestis parasitism in the host body. The above-mentioned antigen was found capable of being synthesized, depending on the conditions of Y. pestis cultivation, in the form of membrane-linked (extracellularly and under phagosomal conditions) or secreted substance, mainly in phagolysosome. In the latter case the maximum level of its expression was registered. The experimental confirmation of YopE localization in the form of superficially localized antigen/receptor at the period of the extracellular growth of bacteria is presented, which suggests its important role in the realization of the virulent properties of Y. pestis and, together with the known data on the protective properties of the antigen, indicates the prospects of its use as the basis for the creation of new chemical antiplague vaccine.     

Use of protein microarray to identify gene expression changes of Yersinia pestis at different temperatures

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 °C, and mammalian hosts, which have a body temperature of 37 °C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague serum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 °C, or adsorbed by Y. pestis cultivated at 26 and 37 °C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 °C but not at 26 °C, or expressed at significantly higher levels at 37 °C than at 26 °C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 °C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.     

鼠疫耶尔森菌毒力蛋白YopE新功能的初步研究

鼠疫耶尔森菌外部蛋白E（YopE）是鼠疫耶尔森菌的6种分泌蛋白之一,主要通过其144位的”精氨酸手指”结构与细胞膜耦联蛋白RhoGTP酶相互作用发挥功能.本文构建YopE及其144位突变体YopE(R144A)的可诱导表达系统,并优化了诱导条件. 用该系统结合流式细胞技术检测YopE和YopE(R144A)对细胞凋亡、细胞周期和细胞活性氧(ROS)水平的影响.结果显示：YopE(R144A)促进HeLa细胞凋亡|使G0/G1期细胞比例上升,G2/M期细胞比例下降;随着YopE(R144A)表达量增加,p21蛋白的表达量也增加| YopE(R144A)也能抑制细胞ROS的产生.研究结果提示,YopE在细胞内可能存在新的致病靶点.     

鼠疫耶尔森氏菌基因表达的环境调节

鼠疫耶尔森氏菌是烈性传染病鼠疫的病原菌,该菌在媒介(跳蚤)和宿主(哺乳动物)之间的循环过程中,基因表达适应环境谱的变化。本介绍鼠疫耶尔森氏菌适应环境信号如不同温度、离子浓度、pH等条件下的基因表达调控研究现状。     

New plasmidovars of Yersinia pestis isolated in Mongolia]

The plasmid spectres of 122 strains of Yersinia pestis isolated in Mongolia from patients, wild mammals and arthropods were studied. The populations of three plasmidovars of Yersinia pestis were found to be circulating in the natural foci of plague in Mongolia. The first plasmidovar harbours three plasmids with mol masses 6, 47, 65 Md. The second and third plasmidovars contain the plasmids with mol masses 6, 16, 47, 65 Md and 8, 47, 75-80 Md.     

Plasmid heterogeneity in populations of Yersinia pestis strains]

Yersinia pestis strains with the typical plasmid patterns were shown to have the heterogenic populations. Heterogeneity is increased by cultivation passages in artificial nutrient media and is manifested in plasmid elimination within several clones, plasmid integration into the chromosome, appearance of auxiliary plasmids or the ones with increased molecular masses. Passages of strains in experimental animals result in populations homogeneity with the typical plasmid patterns within all clones tested. The clones having changed the plasmid content and selected from heterogenic populations pertain their properties when cultivated in nutrient media and passaged in experimental animals.     

鼠疫耶尔森氏菌VcrV基因在大肠杆菌中的高效表达

将测序后的鼠疫耶尔森氏菌（Yersinia pestis)LcrV基因重组质粒pGEM－T／ypV酶切,克隆于原核表达载体pBV220,构建成pBV/ypV表达质粒,转化大肠杆菌DH5α,进行PCR及酶切鉴定,筛选阳性克隆,进行温控诱导表达,SDS－PAGE检测表达产物,在相对分子质量38000处有－表达条带,经薄层扫描分析目的蛋白带占全菌蛋白的38．4％以上,主要以可溶形式存在。     

Characterization of Yersinia pestis fibrinolysin using monoclonal antibody]

Immunochemical identity of Y. pestis fibrinolysin and coagulase is demonstrated using monoclonal antibodies. These substances are proven to exist as complex proteins with two independent activities. Possible causes of this phenomenon are discussed. Coagulase antigenic determinants are involved in specific fluorescence of Y. pestis cells grown at 28 degrees C. A new original method for screening the hybridomas producing monoclonal antibodies is proposed, based on inhibition of the functional activity of antigen.     

建立基于RNA免疫共沉淀技术的鼠疫耶尔森菌Hfq蛋白相关sRNA的体内验证方法

目的：建立RNA免疫共沉淀方法,为鼠疫耶尔森菌Hfq蛋白相关非编码小RNA（sRNA）提供体内验证方法。方法：首先在RNA结合蛋白Hfq下游加入Flag标签,用Flag标签抗体进行免疫共沉淀,获得蛋白-RNA复合物,然后从沉淀的蛋白-RNA复合物中分离得到纯化的RNA;通过Western印迹检测各步骤Hfq蛋白的表达,再利用Northern印迹检测目的sRNA--RyhB1和RyhB2。结果：构建了带有Flag标签的RNA结合蛋白Hfq的载体,此载体转导入hfq缺失株后与鼠疫菌野生株的生长曲线无明显差异;通过RNA-蛋白免疫共沉淀技术鉴定出已知与鼠疫菌Hfq蛋白结合的2个sRNA--RyhB1和RyhB2。结论：建立了利用RNA-蛋白免疫共沉淀鉴定与鼠疫菌Hfq蛋白结合的sRNA的技术,为细菌sRNA的验证、功能研究和体内蛋白质与RNA相互作用研究提供了有利工具。     

Antigen biosynthesis during the limitation of Yersinia pestis growth

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.     

Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.     

Characterization of the Antigenic Subunits of the Envelope Protein of Yersinia pestis

Chemical, physical, and immunological properties of the envelope antigen of Yersinia pestis strains have been investigated. The antigen consists of two components with isoelectric points (pI) of 4.6 and 4.8. One component (pI 4.6) is a protein bound to a small carbohydrate moiety identified as an oligomeric galactan; the other component (pI 4.8) is a simple protein. These two components are antigenically identical. In buffered solution, the antigen exists as aggregates of molecular weights larger than 300,000. The aggregates dissociate into a variety of smaller molecular weight forms depending on the nature of the treatment for dissociation. Each aggregate can be further dissociated into a single antigenic subunit fraction containing protein and glycoprotein species with molecular weights in the range from 15,000 to 17,000. The subunits can be obtained by a dissociation treatment with 0.1% mercaptoethanol in 0.25% sodium dodecyl sulfate at 95 C for 5 min. The subunits will readily reaggregate into a variety of larger molecular weight forms on the removal of dodecyl sulfate.     

Plasmid composition of Yersinia pestis strains from different natural foci]

The study of the plasmid composition of 246 Y. pestis strains from different natural foci in the USSR and other countries revealed that 173 strains (70%) carried three known plasmids with a molecular weight of about 6, 45-50 and 60 megadaltons (MD) respectively. In 20 strains (8%) obtained from different sources additional cryptic plasmids were detected. In some cases the absence of one or two typical plasmids was observed. Replicon pPst was shown to have quite constant molecular weight (6 MD), whereas plasmids pCad and especially pFra exhibited certain variations of their molecular weight (45-49 MD and 60-149 MD respectively) in strains of different origin.