The population variability of Yersinia pestis in soil samples from the natural focus of plague

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.     

Retracing the Evolutionary Path that Led to Flea-Borne Transmission of Yersinia pestis

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IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection

Pneumonic plague is one of the world's most deadly infectious diseases. The causative bacterium, Yersinia pestis, has the potential to be exploited as a biological weapon, and no vaccine is available. Vaccinating B cell-deficient mice with D27-pLpxL, a live attenuated Y. pestis strain, induces cell-mediated protection against lethal pulmonary Y. pestis challenge. In this article, we demonstrate that prime/boost vaccination with D27-pLpxL confers better protection than prime-only vaccination. The improved survival does not result from enhanced bacterial clearance but is associated with increased levels of IL-17 mRNA and protein in the lungs of challenged mice. The boost also increases pulmonary numbers of IL-17-producing CD4 T cells. Interestingly, most of these cells simultaneously produce canonical type 1 and type 17 cytokines; most produce IL-17 and TNF-α, and many produce IL-17, TNF-α, and IFN-γ. Neutralizing IL-17 counteracts the improved survival associated with prime/boost vaccination without significantly impacting bacterial burden. Thus, IL-17 appears to mediate the enhanced protection conferred by booster immunization. Although neutralizing IL-17 significantly reduces neutrophil recruitment to the lungs of mice challenged with Y. pestis, this impact is equally evident in mice that receive one or two immunizations with D27-pLpxL, suggesting it cannot suffice to account for the improved survival that results from booster immunization. We conclude that IL-17 plays a yet to be identified role in host defense that enhances protection against pulmonary Y. pestis challenge, and we suggest that pneumonic plague vaccines should aim to induce mixed type 1 and type 17 cellular responses.     

Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

Genetic basis of the variability of nitrate reduction trait in Yersinia pestis strains

The genetic basis of the varying ability to reduce nitrate in strains belonging to different biovars and subspecies of plague-causing microbe has been investigated and the inability to reduce nitrate observed in different intraspecies groups of Yersinia pestis has been shown to stem from mutations in different genes involved in the expression of this trait. The absence of denitrifying activity in strains of altaica and hissarica subspecies was not due to a mutation at position 613 of the periplasmic reductase napA observed in the strains of the biovar medievalis of the main subspecies, but rather was due to a mutation in the sequence encoding the nitrate-binding domain of the ABC transporter protein SsuA; a thymine insertion (+T) was detected at position 302 from the start of the ssuA gene. Five strains of biovar antiqua isolated at different times in Mongolia, China, and Africa were shown to lack the ability to reduce nitrate. A PCR test targeting two chromosomal regions containing deletions of 19 and 24 bp in size has been developed for the identification of strains of the biovar medievalis. This test can be combined with the test for the marker mutation in the napA gene for a more reliable detection of Y. pestis strains belonging to this biovar.     

Yersinia pestis infection and laboratory conditions alter flea-associated bacterial communities

We collected Oropsylla montana from rock squirrels, Spermophilus varigatus, and infected a subset of collected fleas with Yersinia pestis, the etiological agent of plague. We used bar-tagged DNA pyrosequencing to characterize bacterial communities of wild, uninfected controls and infected fleas. Bacterial communities within Y. pestis-infected fleas were substantially more similar to one another than communities within wild or control fleas, suggesting that infection alters the bacterial community in a directed manner such that specific bacterial lineages are severely reduced in abundance or entirely eliminated from the community. Laboratory conditions also significantly altered flea-associated bacterial communities relative to wild communities, but much less so than Y. pestis infection. The abundance of Firmicutes decreased considerably in infected fleas, and Bacteroidetes were almost completely eliminated from both the control and infected fleas. Bartonella and Wolbachia were unaffected or responded positively to Y. pestis infection.     

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.     

Neutrophils are important in early control of lung infection by Yersinia pestis

In this paper we evaluate the role of neutrophils in pneumonic plague. Splenic neutrophils from naïve BALB/c mice were found to reduce numbers of culturable Yersinia pestis strain GB in suspension. A murine, BALB/c, intranasal model of pneumonic plague was used in conjunction with in vivo neutrophil ablation, using the GR-1 antibody. This treatment reduced neutrophil numbers without affecting other leukocyte numbers. Neutrophil ablated mice exhibited increased bacterial colonisation of the lung 24 h post infection. Furthermore, exposure of Y. pestis to human neutrophils resulted in a 5-fold reduction in the number of viable bacterial cells, whereas, PBMCs had no effect.     

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.     

Defective innate cell response and lymph node infiltration specify Yersinia pestis infection

Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen.     

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     

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.     

The multi-locus VNTR-analysis in studies of the population structure of Yersinia pestis in natural foci

A collection of Yersinia pestis strains was investigated by the multi-locus VNTR analysis. All 9 used locuses were diverse, although they differed between themselves by the quantity of genotypes displaying 4 to 13 variations in the sample. The diversity index (DI) ranged from 0.18 (ms21) to 0.86 (ms46); 8 locuses had DI > 0.5. The statistical processing showed 55 individual genotypes in a group of 81 examined strains, which denoted a high discriminative potentiality of the typing system (DP = 0.98). On the basis of the cluster analysis, the genotypes were shared between 11 main groups. The strains belonging to one genotype group were found to originate, as a rule, from one natural focus. The suggested scheme of typing and of creating the databases of genotypes of plaque agent can be used to establish, with a high probability degree, the source of strains.     

鼠疫溶菌疫苗免疫小鼠的体液免疫应答

为选择以F1抗原为主要有效成分的鼠疫溶菌疫苗(Whole cell lysate of Yersinia pestis vaccine,WCLY)的免疫程序,设计了这组试验。在37℃培养鼠疫EV菌,通过超声波裂解法制备鼠疫溶菌疫苗。设计(0,2周)、(0,4周)、(0,2,4周)三种免疫程序,以每剂总蛋白量7.9μg、31.5μg和126.0μg三个剂量皮下接种NIH小鼠。分别在第一针免疫后2、4、8、12周采集血清,通过间接ELISA检测抗鼠疫菌F1抗原和总抗原抗体。结果显示:免疫后血清抗体上升很快,2周内即可测出;无论哪种免疫程序,至12周时抗体滴度仍保持高水平;加强免疫后,抗体水平在4周或8周达到较高,可与活疫苗免疫者相比;溶菌疫苗的接种剂量为7.9μg时,动物只出现轻度不良反应。提示鼠疫溶菌疫苗需要两剂免疫,最短可间隔2周,接种剂量应不超过7.9μg,疫苗中应富含F1抗原。     

Feeding Behavior Modulates Biofilm-Mediated Transmission of Yersinia pestis by the Cat Flea,Ctenocephalides felis

Background

The cat flea, Ctenocephalides felis, is prevalent worldwide, will parasitize animal reservoirs of plague, and is associated with human habitations in known plague foci. Despite its pervasiveness, limited information is available about the cat flea’s competence as a vector for Yersinia pestis. It is generally considered to be a poor vector, based on studies examining early-phase transmission during the first week after infection, but transmission potential by the biofilm-dependent proventricular-blocking mechanism has never been systematically evaluated. In this study, we assessed the vector competence of cat fleas by both mechanisms. Because the feeding behavior of cat fleas differs markedly from important rat flea vectors, we also examined the influence of feeding behavior on transmission dynamics.

Methodology/Principal Findings

Groups of cat fleas were infected with Y. pestis and subsequently provided access to sterile blood meals twice-weekly, 5 times per week, or daily for 4 weeks and monitored for infection, the development of proventricular biofilm and blockage, mortality, and the ability to transmit. In cat fleas allowed prolonged, daily access to blood meals, mimicking their natural feeding behavior, Y. pestis did not efficiently colonize the digestive tract and could only be transmitted during the first week after infection. In contrast, cat fleas that were fed intermittently, mimicking the feeding behavior of the efficient vector Xenopsylla cheopis, could become blocked and regularly transmitted Y. pestis for 3–4 weeks by the biofilm-mediated mechanism, but early-phase transmission was not detected.

Conclusions

The normal feeding behavior of C. felis, more than an intrinsic resistance to infection or blockage by Y. pestis, limits its vector competence. Rapid turnover of midgut contents results in bacterial clearance and disruption of biofilm accumulation in the proventriculus. Anatomical features of the cat flea foregut may also restrict transmission by both early-phase and proventricular biofilm-dependent mechanisms.