Destabilization of phospholipid model membranes by YplA, a phospholipase A2 secreted by Yersinia enterocolitica

Yersinia enterocolitica produces a virulence-associated phospholipase A(2) (YplA) that is secreted via its flagellar type-III secretion apparatus. When the N-terminal 59 amino acids of YplA are removed (giving YplA(S)), it retains phospholipase activity; however, it is altered with respect to the apparent kinetics of hydrolysis using fluorescent phospholipid substrates in micellar form. To explore the physical properties of YplA more carefully, Langmuir phospholipid monolayers were used to study the association of YplA with biological membranes. YPlA and YplA(S) both associate with Langmuir monolayers, but YplA(S) appears to interact better at low initial lipid densities while YplA interacts better at higher densities. This may indicate that the N-terminus of YplA has a role in mediating its initial interaction with compact cellular membranes, which is consistent with spectroscopic observations that fluorescein-labeled YplA may interact more readily with the nonpolar region of liposomes than does YplA(S).     

鼠疫减毒活疫苗研究现状与展望

鼠疫(plague)是由鼠疫耶尔森氏菌(Yersinia pesits)引起的烈性传染病,在人类历史上曾造成约2亿人的死亡,在我国被列为甲类传染病。由于鼠疫菌具有高度致病性、传染性,被列为最具潜力的生物战剂和生物恐怖剂。在面临鼠疫威胁时,疫苗是最为有力的武器。鼠疫疫苗研究中,减毒活疫苗是重要的研究方向,现就鼠疫减毒活疫苗的研究现状进行综述,为新疫苗的研制提供参考。     

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y. enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y. enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y. frederiksenii hybridization group 2 (unnamed genomospecies 2). This finding was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified.     

The impact of abiotic factors (temperature and glucose) on physicochemical properties of lipids from Yersinia pseudotuberculosis

The impact of the availability of glucose in nutrition medium and growth temperature on the composition and thermotropic behavior of lipids from Yersinia pseudotuberculosis (Enterobacteriaceae) was studied. Y. pseudotuberculosis was grown in nutrition broth (NB) with/without glucose at 8 and 37 degrees C, corresponding to the temperatures of saprophytic and parasitic phases of this bacterium life. The decrease of phosphatidylethanolamine, phosphatidylglycerol and unsaturated fatty acids and the parallel increase of lysophosphatidylethanolamine and diphosphatidylglycerol and saturated and cyclopropane acids were the most significant changes with temperature in bacterial phospholipid (PL) classes and fatty acids, respectively. Glucose did not effect the direction of temperature-induced changes in the contents of PLs, fatty acids, however it enhanced (for PLs) or diminished (for fatty acids) intensity of these changes. The thermally induced transitions of lipids were studied by differential scanning calorimetry (DSC). It was revealed that the addition of glucose to NB induced a sharp shift of DSC thermograms to lower temperatures in the "warm" variants of bacteria. The peak maximum temperature (Tmax) of thermal transitions dropped from 50 to 26 degrees C that is the optimal growth temperature of Y. pseudotuberculosis. Tmax of total lipids of the cells grown at 8 degrees C without glucose in NB was equal to growth temperature that corresponded to the classical mechanism of homeoviscous adaptation of bacteria. An addition of glucose to NB at this growth temperature caused the subsequent reduction of Tmax to -8 degrees C, while the temperature ranges of thermograms were not substantially changed. So, not only the temperature growth of bacteria, but also the presence of glucose in NB can modify the physical state of lipids from Y. pseudotuberculosis. In this case, both factors affect additively. It is suggested that glucose influences some membrane-associated proteins and then the fluidity of lipid matrix through temperature-inducible genes.     

Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system

Interactions among the Yersinia secretion (Ysc) proteins of Yersinia pestis were explored using the yeast two-hybrid system. Various pairwise combinations of the yscEFGHIKLN and Q genes fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast, and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of yscN and yscL, yscL and yscQ, and yscQ and yscK resulted in high levels of reporter gene activation. These results suggest that YscL interacts with both YscN and YscQ, and that YscQ interacts with both YscL and YscK. Three-hybrid analyses using plasmid pDELA to target a third hybrid protein to the yeast nucleus was used to detect the formation of ternary protein complexes. Using the three-hybrid system, YscQ expressed from plasmid pDELA was able to bring together the YscK and YscL fusion proteins. In a similar manner, YscL expressed from plasmid pDELA was able to bring together the YscN and YscQ fusion proteins. Together, these results suggest that a complex composed of YscN, YscQ, YscK and YscL is involved in the assembly and/or function of the Y. pestis type III secretion apparatus.     

Identification of Yersinia pestis as the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting

Eighteen isolates of bacteria obtained from the sputum of pneumonic plague patients and from the liver and spleen of rodents from the plague-affected areas of India during 1994-1995 when analyzed by 16S rDNA analysis clearly demonstrated that all 18 isolates exhibit an average similarity of 98.5% with the genus Yersinia and 99.1% with Yersinia pestis, thus identifying the isolates as Y. pestis. The isolates from the human plague patients were found to be genetically more homogeneous compared to the isolates from the rodents which were more heterogeneous. An epidemiological linkage among the rodents and human patients is also indicated by 16S rDNA analysis, which suggests that only a sub-population of the rodents was probably the source of the infectious pathogen to the humans initiating the outbreak of the epidemic. The results of the randomly amplified DNA polymorphisms (RAPD)-based DNA fingerprinting are in agreement with the above conclusions.     

The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis

In Yersinia pestis, biofilm formation is stimulated by HmsT, a GGDEF-domain containing protein that synthesizes cyclic-di-GMP (c-di-GMP), and inhibited by HmsP, an EAL-domain protein. Only the EAL-domain portion of HmsP is required to inhibit biofilm formation. The EAL domain of HmsP was purified as a 6XHis-tag fusion protein and demonstrated to have phosphodiesterase activity using bis(p-nitrophenyl) phosphate (bis-pNPP) as a substrate. This enzymatic activity was strictly manganese dependent. A critical residue (E506) of HmsP within the EAL domain, that is required for inhibition of biofilm formation, is also essential for this phosphodiesterase activity. While the proposed function of EAL-domain proteins is to linearize c-di-GMP, this is a direct demonstration of the required phosphodiesterase activity of a purified EAL-domain protein.     

Glucose as a growth medium factor regulating lipid composition of Yersinia pseudotuberculosis

Effects of glucose and growth temperature on Yersinia pseudotuberculosis O:1b serovar lipid composition have been studied. These growth parameters were shown to have drastic effects on biosynthetic processes in the pseudotuberculosis bacteria. The temperature effect is the most universal, extending to cell growth and to free lipid and lipopolysaccharide content and composition; it is most conspicuous in the bacteria cultivated on glucose-containing nutrient broth. The effect of glucose is selective, affecting only free lipids and depending on temperature (glucose favors phospholipid (PL) synthesis in the cold and inhibits it at 37°C); the effect of glucose is more evident in the cold. Determination of the contents of individual PL in percent dry bacterial weight indicates that the most obvious effect of glucose and/or growth temperature is on phosphatidylethanolamine (PE) content: on both media and at both temperatures an overall decrease in PL content stems from the inhibition of PE synthesis and is attended by decreasing ratio of neutral to acidic lipids.     

Retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters

AIMS: To investigate the retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters and effects on the microbial community composition. METHODS AND RESULTS: Sand filter columns were loaded (70 mm day(-1)) with fish farm wastewater and a suspension (10(8) CFU ml(-1)) of Y. ruckeri. Bacterial numbers and protozoan numbers were determined by plate counts and epifluorescence microscopy, respectively, and microbial biomass and community composition were assessed by phospholipid fatty acids (PLFA) analysis. Concentrations of Y. ruckeri in the filter effluent decreased from 10(8) to 10(3)-10(5) CFU ml(-1) during the experiment. Numbers of Y. ruckeri in the sand decreased from 10(6) CFU g(-1) dry weight (DW) sand to 10(4) CFU g(-1) DW sand. In contrast, microbial biomass determined with plate counts and total PLFA increased during the whole experiment. Principal component analysis (PCA) revealed a change in microbial community composition with time, with the most pronounced change in surface layers and towards the end of the experiment. Protozoan numbers increased from ca 0-600 cells g(-1) DW sand, indicating the establishment of a moderate population of bacterial grazers. CONCLUSIONS: The removal of Y. ruckeri improved during the experiment. Introduction of Y. ruckeri to the sand filter columns stimulated growth of other micro-organisms, which in turn caused a shift in the microbial community composition in the sand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the understanding of the dynamics of sand filters subjected to a high loading of a pathogenic bacterium and can therefore be used in future work were the overall aim is to provide a more reliable and efficient removal of pathogenic bacteria in biological sand filter systems.     

Analysis of Genomic DNA from Medieval Plague Victims Suggests Long-Term Effect of Yersinia pestis on Human Immunity Genes

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes, we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggest that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe.