Functional characterisation of Lp_2714, an EAL-domain protein from Lactobacillus plantarum

Bioinformatic analysis of lp_2714 from Lactobacillus plantarum WCFS1 demonstrates that it encodes an EAL-domain protein associated with a membrane targeting signal-sequence. Comparison of the predicted primary amino-acid sequence of Lp_2714 shows that it lacks critical catalytic residues and heterologous expression has determined that it does not encode a functional phosphodiesterase. We designate Lp_2714 as a class-3 EAL domain protein probably involved in regulating polysaccharide synthesis on the cell surface the cell.     

The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid A plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.     

The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis

Ec DOS is a heme-based gas sensor enzyme that catalyzes conversion from cyclic-di-GMP to linear-di-GMP in response to gas molecules, such as oxygen, CO and NO. Ec DOS contains an N-terminal heme-binding PAS domain and C-terminal phosphodiesterase domain. Based on crystal structures of the isolated heme-binding domain, it is suggested that the FG loop is involved in intra-molecular signal transduction to the catalytic domain. We generated nine full-length proteins mutated at ionic and non-ionic polar residues between positions 83 and 96 corresponding to the F-helix and FG loop, and examined the heme binding properties, autoxidation rates, and catalytic activities of mutant proteins. N84A and R85A mutant proteins displayed lower heme binding affinities, consistent with the finding that Asn84 interacts with propionate of protoporphyrin IX, and Arg85 with Asp40 on the heme proximal side. Autoxidation rates (0.058-0.54 min⁻¹) of R91A, S96A and K89A/R91A/E93A mutant proteins were significantly higher than that (0.0053 min⁻¹) of wild-type protein, suggesting that these residues in the FG loop form heme distal architecture conferring stability to the Fe(II)-O₂ complex. Catalytic activities of N84A and R85A mutant proteins with low heme affinity were significantly higher than those of wild-type protein in the absence of gas molecules. Accordingly, we propose that loss of heme binding enhances basal catalysis without the gas molecule, consistent with previous reports on heme inhibition of Ec DOS catalysis.     

鼠疫菌噬菌体效价噬菌斑测定法的建立

目的建立鼠疫菌噬菌体噬菌斑效价测定方法。方法通过分析细菌接种浓度、孵育吸附时间及培养温度等参数,建立鼠疫菌噬菌体效价测定方法,并分析其精密性;建立鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准。结果经优化后确定细菌接种浓度为7×108/mL,不需孵育吸附,培养温度为29℃,所建立的检测方法精密性较好,用于鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准应不低于1×106PFU/mL。结论建立了鼠疫菌噬菌体噬菌斑效价测定方法,为鼠疫菌噬菌体及疫苗质量控制奠定了基础。     

Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.     

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。     

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.     

鼠疫的流行病学概述

鼠疫是由鼠疫耶尔森菌(Yersinia pestis)引起严重危害人类健康的烈性传染病。本文介绍了鼠疫病原体——鼠疫耶尔森菌的一般特性及生物学特性, 并对国内、外鼠疫疫情现状进行总结。目前鼠疫在全球范围内的流行已进入新的活跃期,世界卫生组织将鼠疫列为近20年来重新流行的急性传染病之一。当前,全球疫区主要分布在非洲、亚洲和南美洲。我国人间鼠疫自20世纪80年代开始处于明显回升势态,近10年流行逐渐下降,但防控形势依然艰巨。     

鼠疫耶尔森氏菌LcrV基因的克隆及序列分析

为了研究鼠疫耶尔森氏菌（Y.pestis）保护性抗原V蛋白,从基因库中查得Y.pestis LcrV基因DNA序列,针对序列设计合成了一对PCR扩增引物,以本所保存的Y.pestis菌种为模板进行基因扩增,结果获得长约980bp的DNA片段。将扩增产物回收纯化,克隆至pGEM-T载体,构建重组载体pGEN-T/ypV,经过PCR,酶切鉴定,并对pGEM-T/ypV中的V基因片段进行测序,分析测序结果与己知序列相同,表明获得了LcrV基因。     

Colorado animal‐based plague surveillance systems: relationships between targeted animal species and prediction efficacy of areas at risk for humans

Human plague risks (Yersinia pestis infection) are greatest when epizootics cause high mortality among this bacterium's natural rodent hosts. Therefore, health departments in plague‐endemic areas commonly establish animal‐based surveillance programs to monitor Y. pestis infection among plague hosts and vectors. The primary objectives of our study were to determine whether passive animal‐based plague surveillance samples collected in Colorado from 1991 to 2005 were sampled from high human plague risk areas and whether these samples provided information useful for predicting human plague case locations. By comparing locations of plague‐positive animal samples with a previously constructed GIS‐based plague risk model, we determined that the majority of plague‐positive Gunnison's prairie dogs (100%) and non‐prairie dog sciurids (85.82%), and moderately high percentages of sigmodontine rodents (71.4%), domestic cats (69.3%), coyotes (62.9%), and domestic dogs (62.5%) were recovered within 1 km of the nearest area posing high peridomestic risk to humans. In contrast, the majority of white‐tailed prairie dog (66.7%), leporid (cottontailed and jack rabbits) (71.4%), and black‐tailed prairie dog (93.0%) samples originated more than 1 km from the nearest human risk habitat. Plague‐positive animals or their fleas were rarely (one of 19 cases) collected within 2 km of a case exposure site during the 24 months preceding the dates of illness onset for these cases. Low spatial accuracy for identifying epizootic activity prior to human plague cases suggested that other mammalian species or their fleas are likely more important sources of human infection in high plague risk areas. To address this issue, epidemiological observations and multi‐locus variable number tandem repeat analyses (MLVA) were used to preliminarily identify chipmunks as an under‐sampled, but potentially important, species for human plague risk in Colorado.     

Retrospective study of a plague outbreak by multiplex-PCR

AIMS: To determine the effectiveness of multiplex-PCR in Yersinia pestis identification in samples preserved in Cary & Blair medium and to evaluate if this technique would uncover Y. pestis-positives among culture-negative samples. METHODS AND RESULTS: Multiplex-PCR was used to detect Y. pestis in Cary & Blair preserved bubo aspirates from experimentally infected guinea pigs and to re-analyze samples from a plague outbreak after prolonged storage in Cary & Blair. Variation in the target genes amplification was observed over time. CONCLUSIONS: Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.     

Structural modification of a base disaccharide alters antiadhesion properties towards Yersinia pestis

Incubation in the presence of structurally modified disaccharides altered the in vitro attachment of Yersinia pestis GB to three human respiratory epithelial cell lines. Each disaccharide resulted in decreased attachment to the alveolar epithelial (A549) cell line. The best inhibitor of attachment for each cell line was the benzylated derivative of Galbeta1-4GalNAc. Highly negatively charged saccharides were efficient inhibitors, particularly for the bronchial epithelial (BEAS2-B) cell line. The data indicate that targeted modification of receptor ligands could offer a novel therapeutic preventing Y. pestis attachment to host cells.     

Modelling of steric structure of a periplasmic molecular chaperone Caf1M of Yersinia pestis, a prototype member of a subfamily with characteristic structural and functional features

Abstract Steric structure of Caf1M, a periplasmic molecular chaperone of Yersinia pestis , was reconstructed by computer modelling based on a statistically significant primary structure homology between Caf1M and PapD protein from Escherichia coli , and using the known atomic coordinates obtained by the X-ray crystallography for PapD. In the three-dimensional model of Caf1M an accessory sequence between F1 and G1 β-strands (as compared to PapD) can form a strain-specific part of the binding pocket of surface organell subunits. This accessory sequence decreases the depth of the binding pocket. The characteristic structural feature of the subfamily of periplasmic molecular chaperones with the accessory sequence (Caf1M subfamily) is the existence of exposed to a solvent Cys residues in F1 and G1 β-strands which can form disulfide bond in the putative binding pocket. The characteristic functional feature of Caf1M subfamily is the chaperoning of more simple compositions of virulence-associated surface organells (in the case of Y. pestis a capsule consists of only F1 protein). Highly conserved R₈₂ and D₉₃, located at the domain surface remote from the putative subunit binding pocket, can participate in direct contacts with the conserved portion of molecular usher proteins.     

pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor

Abstract It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.     

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。