Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut

The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar.     

Vulnerabilities in Yersinia pestis caf operon are unveiled by a Salmonella vector

During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperature-dependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf during interaction with mammalian host.     

Adhesion pili in Yersinia pestis

Y. pestis cells cultivated at 37 degrees C are capable of agglutinating red blood cells of some animals, which is due to the appearance of pili. The adhesion pili consist of protein subunits with a molecular weight of the order of 12000 daltons, their isoionic point being at pH 4.7. The reaction of hemagglutination was inhibited by the mixture of ganglyosides, while the preliminary treatment of red blood cells with neuraminidases increased its effectiveness. The pili are supposed to take part in the expression of virulence.     

Yersinia pestis lineages in Mongolia

Background

Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia.

Methodology/Principal Findings

Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches.

Conclusions/Significance

We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Lectin of Yersinia pestis vaccine strain EV and its immunological properties

As the result of the chromatographic separation of Y. pestis EV membrane proteins, a protein fraction with hemagglutinating activity was obtained. The isolated preparation was glycoprotein with a molecular weight of 22 kD, contained 16% of carbohydrates and exhibited thermolabile properties. The determination of the carbohydrate specificity of this glycoprotein revealed that it belonged to the class of lectins. Changes in the content of 11 corticosteroids and the population composition of lymphocytes, as well as the detection of specific antibodies in the blood serum of guinea pigs immunized with lectin, were indicative of the fact that the preparation was sufficiently immunogenic and induced the activation of the processes of proliferation and activation of lymphocytes during immunogenesis. The lectin isolated from Y. pestis EV outer membrane may be regarded as an additional factor ensuring the contact of the pathogen with the cells of the body and as a promising component of combined plague vaccine.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

The formation of proventriculus block, alimentary activity and mortality of flea Amphipsylla primaris primaris infected with Yersinia pestis

The results of experiments held in 1982-1983 in Tuva plague natural focus with flea Amphipsylla primaris primaris (Jordan et Rothschild, 1915) from natural populations, whish were inflected and fed on specific host--flat-headed vole (Alticola strelzovi), are analyzed. The initial infectivity of the insects in autumn was higher than in spring: 90 and 50 % respectively. Accumulation of the agent in aggregated form in the organism of A. p. primaris, estimated by the quantity of fleas with and partial blocks, was more active in imago of both sexes in autumn than in spring, while sucking flea were observed in spring more often than in autumn. Irrespective of season, the part of males with visible accumulations of Y. pestis was more, and their alimentary activity was higher than that of females. Fleas died much more quickly in spring. Part of the males with proventriculus block exceeded that of females in spring experiment. Females with alimentary canal obstruction prevailed in autumn. Thus, sex of the insect and season of the experiment conducting influenced on all studied indices. Besides that, Y pestis ability for the proventriculus block formation in fleas during different seasons can change by the opposite way depending on sex of the ectoparasites.     

影响耶尔森氏鼠疫杆菌基因组密码子使用的因素分析

基因组密码子使用的影响因素分析有助于发现影响密码子使用的进化动力学 ,对发现和预测进化的方向和模式有重要的作用。同时 ,分析完整的基因组可以发现特定基因组中密码子的使用模式 ,从而重新设计高效的PCR引物和外源导入基因 ,促进外源基因在特定生物体中的高效率表达。导致瘟疫等外源性感染疾病的耶尔森氏鼠疫杆菌完整基因组序列已经测序公布。为了对鼠疫杆菌的同义密码子使用的进化模式有更加深入的了解 ,详细的研究分析鼠疫杆菌的基因组密码子的使用模式和影响密码子使用的因素。结果发现 ,尽管鼠疫杆菌基因组序列中“G” “C”含量相对较低 (4 7.6 4 % ) ,高水平表达基因的密码子第三位碱基使用胞嘧啶 (C)的频率比表达水平低的基因使用胞嘧啶 (C)有显著的提高 ,表达水平较低的基因在密码子的第三位碱基更趋向使用鸟嘌呤 (G)。在表达水平高低的两组基因中 ,对密码子的第三位碱基使用腺嘌呤 (A)和胸腺嘧啶 (T)总体上趋于随机使用。基因的表达水平与对应分析的第一条向量轴呈高度相关 (R =0 .6 3,P <0 .0 0 0 1)。通过分析比较表达水平高低两组基因的密码子使用模式发现 ,基因的表达水平对于密码子使用有显著的影响。GC skew分析结果显示 ,复制转录阶段的选择对密码子使用有一定的影响。在不同长度     

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。     

Comparative and evolutionary genomics of Yersinia pestis

Yersinia pestis is the causative agent of plague, causing three human plague pandemics in history. Comparative and evolutionary genomics of Y. pestis are extensively discussed in this review. Understanding the genomic variability and the adaptive evolution of Y. pestis from the genomic point of view will contribute greatly to plague detection, identification, control and prevention.     

The response regulator PhoP negatively regulates Yersinia pseudotuberculosis and Yersinia pestis biofilms

A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans , but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP , which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans , deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis , a phoP mutant made more extensive biofilms in vitro than did the wild type. Expression of HmsT, a diguanylate cyclase that positively regulates biofilms, is diminished in Y. pseudotuberculosis strains with functional PhoP.