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Sample records for pestis em roedores

  1. Yersinia pestis Lineages in Mongolia

    PubMed Central

    Kiefer, Daniel; Damdindorj, Tserennorov; Dashdavaa, Otgonbaatar; Khurelsukh, Tungalag; Zöller, Lothar; Wölfel, Roman; Le Flèche, Philippe; Scholz, Holger C.

    2012-01-01

    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

  2. Microgravity Effects on Yersinia Pestis Virulence

    NASA Astrophysics Data System (ADS)

    Lawal, A.; Abogunde, O.; Jejelowo, O.; Rosenzweig, J.-A.

    2010-04-01

    Microgravity effects on Yersinia pestis proliferation, cold growth, and type three secretion system function were evaluated in macrophage cell infections, HeLa cell infections, and cold growth plate assays.

  3. Yersinia pestis--etiologic agent of plague.

    PubMed Central

    Perry, R D; Fetherston, J D

    1997-01-01

    Plague is a widespread zoonotic disease that is caused by Yersinia pestis and has had devastating effects on the human population throughout history. Disappearance of the disease is unlikely due to the wide range of mammalian hosts and their attendant fleas. The flea/rodent life cycle of Y. pestis, a gram-negative obligate pathogen, exposes it to very different environmental conditions and has resulted in some novel traits facilitating transmission and infection. Studies characterizing virulence determinants of Y. pestis have identified novel mechanisms for overcoming host defenses. Regulatory systems controlling the expression of some of these virulence factors have proven quite complex. These areas of research have provide new insights into the host-parasite relationship. This review will update our present understanding of the history, etiology, epidemiology, clinical aspects, and public health issues of plague. PMID:8993858

  4. Early emergence of Yersinia pestis as a severe respiratory pathogen.

    PubMed

    Zimbler, Daniel L; Schroeder, Jay A; Eddy, Justin L; Lathem, Wyndham W

    2015-06-30

    Yersinia pestis causes the fatal respiratory disease pneumonic plague. Y. pestis recently evolved from the gastrointestinal pathogen Y. pseudotuberculosis; however, it is not known at what point Y. pestis gained the ability to induce a fulminant pneumonia. Here we show that the acquisition of a single gene encoding the protease Pla was sufficient for the most ancestral, deeply rooted strains of Y. pestis to cause pneumonic plague, indicating that Y. pestis was primed to infect the lungs at a very early stage in its evolution. As Y. pestis further evolved, modern strains acquired a single amino-acid modification within Pla that optimizes protease activity. While this modification is unnecessary to cause pneumonic plague, the substitution is instead needed to efficiently induce the invasive infection associated with bubonic plague. These findings indicate that Y. pestis was capable of causing pneumonic plague before it evolved to optimally cause invasive infections in mammals.

  5. Early emergence of Yersinia pestis as a severe respiratory pathogen

    PubMed Central

    Zimbler, Daniel L.; Schroeder, Jay A.; Eddy, Justin L.; Lathem, Wyndham W.

    2015-01-01

    Yersinia pestis causes the fatal respiratory disease pneumonic plague. Y. pestis recently evolved from the gastrointestinal pathogen Y. pseudotuberculosis; however, it is not known at what point Y. pestis gained the ability to induce a fulminant pneumonia. Here we show that the acquisition of a single gene encoding the protease Pla was sufficient for the most ancestral, deeply rooted strains of Y. pestis to cause pneumonic plague, indicating that Y. pestis was primed to infect the lungs at a very early stage in its evolution. As Y. pestis further evolved, modern strains acquired a single amino-acid modification within Pla that optimizes protease activity. While this modification is unnecessary to cause pneumonic plague, the substitution is instead needed to efficiently induce the invasive infection associated with bubonic plague. These findings indicate that Y. pestis was capable of causing pneumonic plague before it evolved to optimally cause invasive infections in mammals. PMID:26123398

  6. [Yersinia pestis and plague - an update].

    PubMed

    Stock, Ingo

    2014-12-01

    The plague of man is a severe, systemic bacterial infectious disease. Without antibacterial therapy, the disease is associated with a high case fatality rate, ranging from 40% (bubonic plague) to nearly 100% (septicemic and pneumonic plague). The disease is caused by Yersinia pestis, a non-motile, gram-negative, facultative anaerobic bacterium belonging to the family of Enterobacteriaceae. In nature, Y. pestis has been found in several rodent species and some other small animals such as shrews. Within its reservoir host, Y. pestis circulates via flea bites. Transmission of Y. pestis to humans occurs by the bite of rat fleas, other flea vectors or by non vectorial routes, e. g., handling infected animals or consumption of contaminated food. Human-to-human transmission of the pathogen occurs primarily through aerosol droplets. Compared to the days when plague was a pandemic scourge, the disease is now relatively rare and limited to some rural areas of Africa. During the last ten years, however, plague outbreaks have been registered repea- tedly in some African regions. For treatment of plague, streptomycin is still considered the drug of choice. Chloramphenicol, doxycycline, gentamicin and ciprofloxacin are also promising drugs. Recombinant vaccines against plague are in clinical development.

  7. Global Expression Studies of Yersinia Pestis Pathogenicity

    SciTech Connect

    Garcia, E; Motin, V; Brubaker, R; Fitch, P

    2002-10-15

    The aim of these studies continues to be the investigation into the molecular mechanisms that underlie the virulence process in Yersinia pestis. In particular, the focus of this work centers on the identification of novel genes and pathways responsible for the pathogenic properties of this organism. In spite of more than four decades of intense investigation in this field, the dilemma as to what makes Y. pestis such a virulent and lethal pathogen remains unanswered. The method being employed makes use microarray technology (DNA chip) that enables the examination of the global activities of the whole complement of genes in this pathogen. Two primary resources available to the investigators (one directly obtained from a separate CBNP-funded project) make these studies possible: (1) Whole genome comparisons of the genes in Y. pestis and its near neighbors with attenuated or non pathogenic characteristics, and (2) the ability to duplicate in vitro, conditions that mimic the infection process of this pathogen. This year we have extended our studies from the original work of characterizing the global transcriptional regulation in Y. pestis triggered during temperature transition from 26 C to 37 C (roughly conditions found in the flea vector and the mammalian host, respectively) to studies of regulation encountered during shift between growth from conditions of neutral pH to acidic pH (the latter conditions, those mimic the environment found inside macrophages, a likely environment found by these cells during infection.). For this work, DNA arrays containing some 5,000 genes (the entire genome of Y. pestis plus those genes found uniquely in the enteropathogen, and near neighbor, Y. pseudotuberculosis) are used to monitor the simultaneous expression levels of each gene of known and unknown function in Y. pestis. Those genes that are up-regulate under the experimental conditions represent genes potentially involved in the pathogenic process. The ultimate role in

  8. Nucleotide sequences specific to Yersinia pestis and methods for the detection of Yersinia pestis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Motin, Vladinir L.

    2009-02-24

    Nucleotide sequences specific to Yersinia pestis that serve as markers or signatures for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  9. Proteomic Characterization of Yersinia pestis Virulence

    SciTech Connect

    Chromy, B; Murphy, G; Gonzales, A; Fitch, J P; McCutchen-Maloney, S L

    2005-01-05

    Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditions were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.

  10. Pathogenicity of Yersinia pestis Synthesis of 1-Dephosphorylated Lipid A

    PubMed Central

    Sun, Wei; Six, David A.; Reynolds, C. Michael; Chung, Hak Suk; Raetz, Christian R. H.

    2013-01-01

    Synthesis of Escherichia coli LpxL, which transfers a secondary laurate chain to the 2′ position of lipid A, in Yersinia pestis produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::PlpxL lpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated Y. pestis vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from Francisella tularensis in Y. pestis yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of Y. pestis in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD50) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type Y. pestis KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to Salmonella enterica, expression of LpxE does not attenuate the virulence of Y. pestis. PMID:23357387

  11. Yersinia pestis Ail: multiple roles of a single protein

    PubMed Central

    Kolodziejek, Anna M.; Hovde, Carolyn J.; Minnich, Scott A.

    2012-01-01

    Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague—a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (OMPs) are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed (1) inhibition of the bactericidal properties of complement, (2) attachment and Yersinia outer proteins (Yop) delivery to host tissue, (3) prevention of PMNL recruitment to the lymph nodes, and (4) inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen. PMID:22919692

  12. Expression profiling of Yersinia pestis during mouse pulmonary infection.

    PubMed

    Lawson, Jonathan N; Lyons, C Rick; Johnston, Stephen Albert

    2006-11-01

    Yersinia pestis, the causative agent of plague, can be transmitted by infected flea bite or inhaled aerosol. Both routes of infection have a high mortality rate, and pneumonic infections of Y. pestis represent a significant concern as a tool of bioterrorism. Understanding the transcriptional program of this pathogen during pulmonary infection should be valuable in understanding plague pathogenesis, as well as potentially offering insights into new vaccines and therapeutics. Toward this goal we developed a long oligonucleotide microarray to the plague bacillus and evaluated the expression profiles of Y. pestis in vitro and in the mouse pulmonary infection model in vivo. The in vitro analysis compared expression patterns at 27 versus 37 degrees C, as a surrogate of the transition from the flea to the mammalian host. The in vivo analysis used intranasal challenge to the mouse lung. By amplifying the Y. pestis RNA from individual mouse lungs we were able to map the transcriptional profile of plague at postinfection days 1 to 3. Our data present a very different transcriptional profile between in vivo and in vitro expression, suggesting Y. pestis responds to a variety of host signals during infection. Of note was the number of genes found in genomic regions with altered %GC content that are upregulated within the mouse lung environment. These data suggest these regions may provide particularly promising targets for both vaccines and therapeutics. PMID:17132091

  13. Pneumonic Plague: The Darker Side of Yersinia pestis.

    PubMed

    Pechous, Roger D; Sivaraman, Vijay; Stasulli, Nikolas M; Goldman, William E

    2016-03-01

    Inhalation of the bacterium Yersinia pestis results in primary pneumonic plague. Pneumonic plague is the most severe manifestation of plague, with mortality rates approaching 100% in the absence of treatment. Its rapid disease progression, lethality, and ability to be transmitted via aerosol have compounded fears of the intentional release of Y. pestis as a biological weapon. Importantly, recent epidemics of plague have highlighted a significant role for pneumonic plague during outbreaks of Y. pestis infections. In this review we describe the characteristics of pneumonic plague, focusing on its disease progression and pathogenesis. The rapid time-course, severity, and difficulty of treating pneumonic plague highlight how differences in the route of disease transmission can enhance the lethality of an already deadly pathogen.

  14. Reannotation of Yersinia pestis Strain 91001 Based on Omics Data.

    PubMed

    Mao, Yiqing; Yang, Xianwei; Liu, Yang; Yan, Yanfeng; Du, Zongmin; Han, Yanping; Song, Yajun; Zhou, Lei; Cui, Yujun; Yang, Ruifu

    2016-09-01

    Yersinia pestis is among the most dangerous human pathogens, and systematic research of this pathogen is important in bacterial pathogenomics research. To fully interpret the biological functions, physiological characteristics, and pathogenesis of Y. pestis, a comprehensive annotation of its entire genome is necessary. The emergence of omics-based research has brought new opportunities to better annotate the genome of this pathogen. Here, the complete genome of Y. pestis strain 91001 was reannotated using genomics and proteogenomics data. One hundred and thirty-seven unreliable coding sequences were removed, and 41 homologous genes were relocated with their translational initiation sites, while the functions of seven pseudogenes and 392 hypothetical genes were revised. Moreover, annotations of noncoding RNAs, repeat sequences, and transposable elements have also been incorporated. The reannotated results are freely available at http://tody.bmi.ac.cn. PMID:27382076

  15. Investigating the ?Trojan Horse? Mechanism of Yersinia pestis Virulence

    SciTech Connect

    McCutchen-Maloney, S L; Fitch, J P

    2005-02-08

    Yersinia pestis, the etiological agent of plague, is a Gram-negative, highly communicable, enteric bacterium that has been responsible for three historic plague pandemics. Currently, several thousand cases of plague are reported worldwide annually, and Y. pestis remains a considerable threat from a biodefense perspective. Y. pestis infection can manifest in three forms: bubonic, septicemic, and pneumonic plague. Of these three forms, pneumonic plague has the highest fatality rate ({approx}100% if left untreated), the shortest intervention time ({approx}24 hours), and is highly contagious. Currently, there are no rapid, widely available vaccines for plague and though plague may be treated with antibiotics, the emergence of both naturally occurring and potentially engineered antibiotic resistant strains makes the search for more effective therapies and vaccines for plague of pressing concern. The virulence mechanism of this deadly bacterium involves induction of a Type III secretion system, a syringe-like apparatus that facilitates the injection of virulence factors, termed Yersinia outer membrane proteins (Yops), into the host cell. These virulence factors inhibit phagocytosis and cytokine secretion, and trigger apoptosis of the host cell. Y. pestis virulence factors and the Type III secretion system are induced thermally, when the bacterium enters the mammalian host from the flea vector, and through host cell contact (or conditions of low Ca{sup 2+} in vitro). Apart from the temperature increase from 26 C to 37 C and host cell contact (or low Ca{sup 2+} conditions), other molecular mechanisms that influence virulence induction in Y. pestis are largely uncharacterized. This project focused on characterizing two novel mechanisms that regulate virulence factor induction in Y. pestis, immunoglobulin G (IgG) binding and quorum sensing, using a real-time reporter system to monitor induction of virulence. Incorporating a better understanding of the mechanisms of virulence

  16. Detection of a Yersinia pestis gene homologue in rodent samples.

    PubMed

    Giles, Timothy A; Greenwood, Alex D; Tsangaras, Kyriakos; Giles, Tom C; Barrow, Paul A; Hannant, Duncan; Abu-Median, Abu-Bakr; Yon, Lisa

    2016-01-01

    A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus) and of mice (Mus musculus and Apodemus sylvaticus) using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool) and Canada (Vancouver). The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker. PMID:27602258

  17. Detection of a Yersinia pestis gene homologue in rodent samples

    PubMed Central

    Greenwood, Alex D.; Tsangaras, Kyriakos; Giles, Tom C.; Barrow, Paul A.; Hannant, Duncan; Abu-Median, Abu-Bakr; Yon, Lisa

    2016-01-01

    A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus) and of mice (Mus musculus and Apodemus sylvaticus) using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool) and Canada (Vancouver). The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker. PMID:27602258

  18. Detection of a Yersinia pestis gene homologue in rodent samples

    PubMed Central

    Greenwood, Alex D.; Tsangaras, Kyriakos; Giles, Tom C.; Barrow, Paul A.; Hannant, Duncan; Abu-Median, Abu-Bakr; Yon, Lisa

    2016-01-01

    A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus) and of mice (Mus musculus and Apodemus sylvaticus) using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool) and Canada (Vancouver). The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker.

  19. Genome sequence of Yersinia pestis, the causative agent of plague.

    PubMed

    Parkhill, J; Wren, B W; Thomson, N R; Titball, R W; Holden, M T; Prentice, M B; Sebaihia, M; James, K D; Churcher, C; Mungall, K L; Baker, S; Basham, D; Bentley, S D; Brooks, K; Cerdeño-Tárraga, A M; Chillingworth, T; Cronin, A; Davies, R M; Davis, P; Dougan, G; Feltwell, T; Hamlin, N; Holroyd, S; Jagels, K; Karlyshev, A V; Leather, S; Moule, S; Oyston, P C; Quail, M; Rutherford, K; Simmonds, M; Skelton, J; Stevens, K; Whitehead, S; Barrell, B G

    2001-10-01

    The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

  20. yadBC of Yersinia pestis, a New Virulence Determinant for Bubonic Plague▿ †

    PubMed Central

    Forman, Stanislav; Wulff, Christine R.; Myers-Morales, Tanya; Cowan, Clarissa; Perry, Robert D.; Straley, Susan C.

    2008-01-01

    In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice. PMID:18025093

  1. Cultural and morphological properties of the vaccine strain Yersinia pestis EV NIIEG bacteria after photodynamic inactivation

    NASA Astrophysics Data System (ADS)

    Ulianova, Onega V.; Lyapina, Anna M.; Khizhnyakova, Mariya A.; Laskavy, Vladislav N.; Feodorova, Valentina A.; Ulyanov, Sergey S.

    2015-03-01

    New method of photoinactivation of plague microbes (bacteria Yersinia pestis) has been suggested. Rate of growth of colonies of Y. pestis EV NIIEG at specific regimes of photo processing have been analyzed. Dependence of growth on exposure time and concentrations of photosensitizer (methylene blue) has been studied. Number of colony forming units of Y. pestis EV NIIEG bacteria as a function of intensity of light and concentration of methylene blue has been scrutinized.

  2. Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination

    PubMed Central

    Yang, Kun; Park, Chae G; Cheong, Cheolho; Bulgheresi, Silvia; Zhang, Shusheng; Zhang, Pei; He, Yingxia; Jiang, Lingyu; Huang, Hongping; Ding, Honghui; Wu, Yiping; Wang, Shaogang; Zhang, Lin; Li, Anyi; Xia, Lianxu; Bartra, Sara S; Plano, Gregory V; Skurnik, Mikael; Klena, John D; Chen, Tie

    2015-01-01

    Yersinia pestis is a Gram-negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen-presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium-dependent (C-type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin-expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin-expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin-expressing transfectants is inhibited by purified Langerin, a DC-SIGN (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin)-like molecule, an anti-CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin-mediated binding of Y. pestis to APCs may promote its dissemination and infection. PMID:25829141

  3. Proteomic Characterization of Host Response to Yersinia pestis

    SciTech Connect

    Chromy, B; Perkins, J; Heidbrink, J; Gonzales, A; Murhpy, G; Fitch, J P; McCutchen-Maloney, S

    2004-05-11

    Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Y. pseudotuberculosis and Y. enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.

  4. Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

    PubMed

    Shannon, Jeffrey G; Bosio, Christopher F; Hinnebusch, B Joseph

    2015-03-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

  5. Dermal Neutrophil, Macrophage and Dendritic Cell Responses to Yersinia pestis Transmitted by Fleas

    PubMed Central

    Shannon, Jeffrey G.; Bosio, Christopher F.; Hinnebusch, B. Joseph

    2015-01-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis. PMID:25781984

  6. Microevolution and history of the plague bacillus, Yersinia pestis

    PubMed Central

    Achtman, Mark; Morelli, Giovanna; Zhu, Peixuan; Wirth, Thierry; Diehl, Ines; Kusecek, Barica; Vogler, Amy J.; Wagner, David M.; Allender, Christopher J.; Easterday, W. Ryan; Chenal-Francisque, Viviane; Worsham, Patricia; Thomson, Nicholas R.; Parkhill, Julian; Lindler, Luther E.; Carniel, Elisabeth; Keim, Paul

    2004-01-01

    The association of historical plague pandemics with Yersinia pestis remains controversial, partly because the evolutionary history of this largely monomorphic bacterium was unknown. The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS100 insertion elements. Eight populations were recognized by the three methods, and we propose an evolutionary tree for these populations, rooted on Yersinia pseudotuberculosis. The tree invokes microevolution over millennia, during which enzootic pestoides isolates evolved. This initial phase was followed by a binary split 6,500 years ago, which led to populations that are more frequently associated with human disease. These populations do not correspond directly to classical biovars that are based on phenotypic properties. Thus, we recommend that henceforth groupings should be based on molecular signatures. The age of Y. pestis inferred here is compatible with the dates of historical pandemic plague. However, it is premature to infer an association between any modern molecular grouping and a particular pandemic wave that occurred before the 20th century. PMID:15598742

  7. Effect of fat in ground beef on the growth and virulence plasmid (pYV) stability in Yersinia pestis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of the behavior of Yersinia pestis in food may be useful in the event Y. pestis is used in a bioterrorism attack on the food supply. However, there are no reports on the growth of plasmid-bearing (pYV) virulent Y. pestis in food. The growth of a conditionally virulent pYV-bearing Yersini...

  8. Distribution of Yersinia pestis pIP1202-like Multidrug Resistance Plasmids Among Foodborne Pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibiotic resistance in Yersinia pestis is rare and constitutes a significant threat given that antibiotics are used for both plague treatment and for prevention of human-to-human transmission. For this reason, the discovery of a multiple antimicrobial resistant (MDR) isolate of Y. pestis (strain I...

  9. Inactivation of avirulent Yersinia pestis on food and food contact surfaces by ultraviolet light and freezing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia pestis, the causative agent of plague, can occasionally be contracted as a naso-pharangeal or gastrointestinal illness through consumption of contaminated meat. In this study, the use of 254 nm ultraviolet light (UV-C) to inactivate a multi-isolate cocktail of avirulent Y. pestis on food an...

  10. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis

    SciTech Connect

    Chain, Patrick S. G.; Carniel, E.; Larimer, Frank W; Lamerdin, Jane; Vergez, Lisa; Land, Miriam L; Motin, V. L.; Brubaker, R. R.; Fowler, J.; Hinnebusch, J.; Marceau, M.; Medigue, Claudine; Chenal-Francisque, V.; Souza, B.; Dacheux, D.; Elliott, J. M.; Derbise, A.; Hauser, Loren John; Garcia, Emilio

    2004-09-01

    Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.

  11. Insights into the genome evolution of Yersinia pestis through whole genome comparison with Yersinia pseudotuberculosis

    SciTech Connect

    Souza, B; Stoutland, P; Derbise, A; Georgescu, A; Elliott, J; Land, M; Marceau, M; Motin, V; Hinnebusch, J; Simonet, M; Medigue, C; Dacheux, D; Chenal-Francisque, V; Regala, W; Brubaker, R R; Carniel, E; Chain, P; Verguez, L; Fowler, J; Garcia, E; Lamerdin, J; Hauser, L; Larimer, F

    2004-01-24

    Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons to available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveals 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, represent the only new genetic material in Y. pestis acquired since the divergence from Y. pseudotuberculosis. In contrast, 149 new pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive IS-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of pre-existing gene expression pathways appear to be more important than acquisition of new genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.

  12. Inactivation of avirulent pgm+ and delta pgm Yersinia pestis by ultraviolet light (UV-C)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia pestis is the causative agent of bubonic plague. Though not considered a foodborne pathogen, Y. pestis can survive, and even grow, in some foods, and the foodborne route of transmission is not without precedent. As such, concerns exist over the possible intentional contamination of foods wi...

  13. Inactivation of avirulent Yersinia pestis in beef bologna by gamma irradiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia pestis, a psychrotrophic pathogen capable of growth at refrigeration temperatures, can cause pharyngeal and gastrointestinal plague in humans as a result of eating contaminated foods. Because Y. pestis is listed as a select agent for food safety and defense, evaluation of food safety interv...

  14. Identification and cloning of a fur regulatory gene in Yersinia pestis.

    PubMed Central

    Staggs, T M; Perry, R D

    1991-01-01

    Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization. Images PMID:1898928

  15. A bibliography of literature pertaining to plague (Yersinia pestis)

    USGS Publications Warehouse

    Ellison, Laura E.; Frank, Megan K. Eberhardt

    2011-01-01

    Plague is an acute and often fatal zoonotic disease caused by the bacterium Yersinia pestis. Y. pestis mainly cycles between small mammals and their fleas; however, it has the potential to infect humans and frequently causes fatalities if left untreated. It is often considered a disease of the past; however, since the late 1800s, plagueis geographic range has expanded greatly, posing new threats in previously unaffected regions of the world, including the Western United States. A literature search was conducted using Internet resources and databases. The keywords chosen for the searches included plague, Yersinia pestis, management, control, wildlife, prairie dogs, fleas, North America, and mammals. Keywords were used alone or in combination with the other terms. Although this search pertains mostly to North America, citations were included from the international research community, as well. Databases and search engines used included Google (http://www.google.com), Google Scholar (http://scholar.google.com), SciVerse Scopus (http://www.scopus.com), ISI Web of Knowledge (http://apps.isiknowledge.com), and the USGS Library's Digital Desktop (http://library.usgs.gov). The literature-cited sections of manuscripts obtained from keyword searches were cross-referenced to identify additional citations or gray literature that was missed by the Internet search engines. This Open-File Report, published as an Internet-accessible bibliography, is intended to be periodically updated with new citations or older references that may have been missed during this compilation. Hence, the authors would be grateful to receive notice of any new or old papers that the audience (users) think need to be included.

  16. Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis

    PubMed Central

    Pennington, Jarrod; Miller, Virginia L.

    2013-01-01

    SUMMARY Autotransporters, the largest family of secreted proteins in Gram negative bacteria, perform a variety of functions, including adherence, cytotoxicity, and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to bubonic infection of the mouse model. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologs but is not conserved in the Y. enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis-specific plasmid pPCP1. Together, these data show that post-translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence. PMID:23701256

  17. Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity.

    PubMed

    Bobrov, Alexander G; Perry, Robert D

    2006-02-01

    Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired. PMID:16436060

  18. Pestoides F, and Atypical Yersinia pestis Strain from the Former Soviet Union

    SciTech Connect

    Garcia, E; Worsham, P; Bearden, S; Malfatti, S; Lang, D; Larimer, F; Lindler, L; Chain, P

    2007-01-05

    Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lacking the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout a comprehensive genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla{sup -} strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains, reveals a series of differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single {approx}7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet

  19. Pestoides F, an atypical Yersinia pestis strain from the former Soviet Union.

    SciTech Connect

    Garcia, Emilio; Worsham, Patricia; Bearden, S.; Malfatti, Stephanie; Lang, D.; Larimer, Frank W; Lindler, L.; Chain, Patrick S. G.

    2007-01-01

    Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lack of the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout a comprehensive genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla(-) strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains reveals differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single approximately 7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet sequenced.

  20. Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages

    PubMed Central

    Ponnusamy, Duraisamy; Clinkenbeard, Kenneth D.

    2015-01-01

    Background Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections. Principal Findings In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype. Conclusions These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a

  1. Functional characterization of Yersinia pestis aerobic glycerol metabolism.

    PubMed

    Willias, Stephan P; Chauhan, Sadhana; Motin, Vladimir L

    2014-11-01

    Yersinia pestis biovar Orientalis isolates have lost the capacity to ferment glycerol. Herein we provide experimental validation that a 93 bp in-frame deletion within the glpD gene encoding the glycerol-3-phosphate dehydrogenase present in all biovar Orientalis strains is sufficient to disrupt aerobic glycerol fermentation. Furthermore, the inability to ferment glycerol is often insured by a variety of additional mutations within the glpFKX operon which prevents glycerol internalization and conversion to glycerol-3-phosphate. The physiological impact of functional glpFKX in the presence of dysfunctional glpD was assessed. Results demonstrate no change in growth kinetics at 26 °C and 37 °C. Mutants deficient in glpD displayed decreased intracellular accumulation of glycerol-3-phosphate, a characterized inhibitor of cAMP receptor protein (CRP) activation. Since CRP is rigorously involved in global regulation Y. pestis virulence, we tested a possible influence of a single glpD mutation on virulence. Nonetheless, subcutaneous and intranasal murine challenge was not impacted by glycerol metabolism. As quantified by crystal violet assay, biofilm formation of the glpD-deficient KIM6+ mutant was mildly repressed; whereas, chromosomal restoration of glpD in CO92 resulted in a significant increase in biofilm formation. PMID:25220241

  2. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    SciTech Connect

    Smith, Natasha; Kim, Sook-Kyung; Reddy, Prasad T.; Gallagher, D. Travis

    2006-03-01

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V{sub M} = 3.0 Å{sup 3} Da{sup −1}. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

  3. Whole genome multilocus sequence typing as an epidemiologic tool for Yersinia pestis.

    PubMed

    Kingry, Luke C; Rowe, Lori A; Respicio-Kingry, Laurel B; Beard, Charles B; Schriefer, Martin E; Petersen, Jeannine M

    2016-04-01

    Human plague is a severe and often fatal zoonotic disease caused by Yersinia pestis. For public health investigations of human cases, nonintensive whole genome molecular typing tools, capable of defining epidemiologic relationships, are advantageous. Whole genome multilocus sequence typing (wgMLST) is a recently developed methodology that simplifies genomic analyses by transforming millions of base pairs of sequence into character data for each gene. We sequenced 13 US Y. pestis isolates with known epidemiologic relationships. Sequences were assembled de novo, and multilocus sequence typing alleles were assigned by comparison against 3979 open reading frames from the reference strain CO92. Allele-based cluster analysis accurately grouped the 13 isolates, as well as 9 publicly available Y. pestis isolates, by their epidemiologic relationships. Our findings indicate wgMLST is a simplified, sensitive, and scalable tool for epidemiologic analysis of Y. pestis strains. PMID:26778487

  4. Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago.

    PubMed

    Rasmussen, Simon; Allentoft, Morten Erik; Nielsen, Kasper; Orlando, Ludovic; Sikora, Martin; Sjögren, Karl-Göran; Pedersen, Anders Gorm; Schubert, Mikkel; Van Dam, Alex; Kapel, Christian Moliin Outzen; Nielsen, Henrik Bjørn; Brunak, Søren; Avetisyan, Pavel; Epimakhov, Andrey; Khalyapin, Mikhail Viktorovich; Gnuni, Artak; Kriiska, Aivar; Lasak, Irena; Metspalu, Mait; Moiseyev, Vyacheslav; Gromov, Andrei; Pokutta, Dalia; Saag, Lehti; Varul, Liivi; Yepiskoposyan, Levon; Sicheritz-Pontén, Thomas; Foley, Robert A; Lahr, Marta Mirazón; Nielsen, Rasmus; Kristiansen, Kristian; Willerslev, Eske

    2015-10-22

    The bacteria Yersinia pestis is the etiological agent of plague and has caused human pandemics with millions of deaths in historic times. How and when it originated remains contentious. Here, we report the oldest direct evidence of Yersinia pestis identified by ancient DNA in human teeth from Asia and Europe dating from 2,800 to 5,000 years ago. By sequencing the genomes, we find that these ancient plague strains are basal to all known Yersinia pestis. We find the origins of the Yersinia pestis lineage to be at least two times older than previous estimates. We also identify a temporal sequence of genetic changes that lead to increased virulence and the emergence of the bubonic plague. Our results show that plague infection was endemic in the human populations of Eurasia at least 3,000 years before any historical recordings of pandemics.

  5. A strain of Yersinia pestis with a mutator phenotype from the Republic of Georgia.

    PubMed

    Rajanna, Chythanya; Ouellette, Gary; Rashid, Mohammed; Zemla, Adam; Karavis, Mark; Zhou, Carol; Revazishvili, Tamara; Redmond, Brady; McNew, Lauren; Bakanidze, Lela; Imnadze, Paata; Rivers, Bryan; Skowronski, Evan W; O'Connell, Kevin P; Sulakvelidze, Alexander; Gibbons, Henry S

    2013-06-01

    We describe here a strain of Yersinia pestis, G1670A, which exhibits a baseline mutation rate elevated 250-fold over wild-type Y. pestis. The responsible mutation, a C to T substitution in the mutS gene, results in the transition of a highly conserved leucine at position 689 to arginine (mutS(L689R)). When the MutSL 689R protein of G1670A was expressed in a ΔmutS derivative of Y. pestis strain EV76, mutation rates observed were equivalent to those observed in G1670A, consistent with a causal association between the mutS mutation and the mutator phenotype. The observation of a mutator allele in Yersinia pestis has potential implications for the study of evolution of this and other especially dangerous pathogens.

  6. Early Divergent Strains of Yersinia pestis in Eurasia 5,000 Years Ago

    PubMed Central

    Rasmussen, Simon; Allentoft, Morten Erik; Nielsen, Kasper; Orlando, Ludovic; Sikora, Martin; Sjögren, Karl-Göran; Pedersen, Anders Gorm; Schubert, Mikkel; Van Dam, Alex; Kapel, Christian Moliin Outzen; Nielsen, Henrik Bjørn; Brunak, Søren; Avetisyan, Pavel; Epimakhov, Andrey; Khalyapin, Mikhail Viktorovich; Gnuni, Artak; Kriiska, Aivar; Lasak, Irena; Metspalu, Mait; Moiseyev, Vyacheslav; Gromov, Andrei; Pokutta, Dalia; Saag, Lehti; Varul, Liivi; Yepiskoposyan, Levon; Sicheritz-Pontén, Thomas; Foley, Robert A.; Lahr, Marta Mirazón; Nielsen, Rasmus; Kristiansen, Kristian; Willerslev, Eske

    2015-01-01

    Summary The bacteria Yersinia pestis is the etiological agent of plague and has caused human pandemics with millions of deaths in historic times. How and when it originated remains contentious. Here, we report the oldest direct evidence of Yersinia pestis identified by ancient DNA in human teeth from Asia and Europe dating from 2,800 to 5,000 years ago. By sequencing the genomes, we find that these ancient plague strains are basal to all known Yersinia pestis. We find the origins of the Yersinia pestis lineage to be at least two times older than previous estimates. We also identify a temporal sequence of genetic changes that lead to increased virulence and the emergence of the bubonic plague. Our results show that plague infection was endemic in the human populations of Eurasia at least 3,000 years before any historical recordings of pandemics. PMID:26496604

  7. Fast and Simple Detection of Yersinia pestis Applicable to Field Investigation of Plague Foci

    PubMed Central

    Simon, Stéphanie; Demeure, Christian; Lamourette, Patricia; Filali, Sofia; Plaisance, Marc; Créminon, Christophe; Volland, Hervé; Carniel, Elisabeth

    2013-01-01

    Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples. PMID:23383008

  8. Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

    PubMed Central

    Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

    2011-01-01

    Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

  9. Temperature-dependence of yadBC phenotypes in Yersinia pestis.

    PubMed

    Uittenbogaard, Annette M; Myers-Morales, Tanya; Gorman, Amanda A; Welsh, Erin; Wulff, Christine; Hinnebusch, B Joseph; Korhonen, Timo K; Straley, Susan C

    2014-02-01

    YadB and YadC are putative trimeric autotransporters present only in the plague bacterium Yersinia pestis and its evolutionary predecessor, Yersinia pseudotuberculosis. Previously, yadBC was found to promote invasion of epithelioid cells by Y. pestis grown at 37 °C. In this study, we found that yadBC also promotes uptake of 37 °C-grown Y. pestis by mouse monocyte/macrophage cells. We tested whether yadBC might be required for lethality of the systemic stage of plague in which the bacteria would be pre-adapted to mammalian body temperature before colonizing internal organs and found no requirement for early colonization or growth over 3 days. We tested the hypothesis that YadB and YadC function on ambient temperature-grown Y. pestis in the flea vector or soon after infection of the dermis in bubonic plague. We found that yadBC did not promote uptake by monocyte/macrophage cells if the bacteria were grown at 28 °C, nor was there a role of yadBC in colonization of fleas by Y. pestis grown at 21 °C. However, the presence of yadBC did promote recoverability of the bacteria from infected skin for 28 °C-grown Y. pestis. Furthermore, the gene for the proinflammatory chemokine CXCL1 was upregulated in expression if the infecting Y. pestis lacked yadBC but not if yadBC was present. Also, yadBC was not required for recoverability if the bacteria were grown at 37 °C. These findings imply that thermally induced virulence properties dominate over effects of yadBC during plague but that yadBC has a unique function early after transmission of Y. pestis to skin.

  10. The Role of Early-Phase Transmission in the Spread of Yersinia pestis

    PubMed Central

    EISEN, REBECCA J.; DENNIS, DAVID T.; GAGE, KENNETH L.

    2015-01-01

    Early-phase transmission (EPT) of Yersinia pestis by unblocked fleas is a well-documented, replicable phenomenon with poorly defined mechanisms. We review evidence demonstrating EPT and current knowledge on its biological and biomechanical processes. We discuss the importance of EPT in the epizootic spread of Y. pestis and its role in the maintenance of plague bacteria in nature. We further address the role of EPT in the epidemiology of plague. PMID:26336267

  11. Adhesive Properties of YapV and Paralogous Autotransporter Proteins of Yersinia pestis

    PubMed Central

    Nair, Manoj K. M.; De Masi, Leon; Yue, Min; Galván, Estela M.; Chen, Huaiqing; Wang, Fang

    2015-01-01

    Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs. PMID:25690102

  12. Development of an improved selective agar medium for isolation of Yersinia pestis.

    PubMed

    Ber, Raphael; Mamroud, Emanuelle; Aftalion, Moshe; Tidhar, Avital; Gur, David; Flashner, Yehuda; Cohen, Sara

    2003-10-01

    Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

  13. Detections of Yersinia pestis East of the Known Distribution of Active Plague in the United States.

    PubMed

    Mize, Erica L; Britten, Hugh B

    2016-02-01

    We examined fleas collected from black-tailed prairie dog (Cynomys ludovicianus) burrows from 2009 through 2011 in five national park units east of the known distribution of active plague across the northern Great Plains for the presence of Yersinia pestis. Across all national park units, Oropsylla tuberculata and Oropsylla hirsuta were the most common fleas collected from prairie dog burrows, 42.4% and 56.9%, respectively, of the 3964 fleas collected from burrow swabbing. Using a nested PCR assay, we detected 200 Y. pestis-positive fleas from 3117 assays. In total, 6.4% of assayed fleas were Y. pestis positive and 13.9% of prairie dog burrows swabbed contained Y. pestis-positive fleas. Evidence of the presence of Y. pestis was observed at all national park units except Devils Tower National Monument in Wyoming. We detected the presence of Y. pestis without large die-offs, i.e., enzootic sylvatic plague, east of the known distribution of active plague and near the eastern edge of the present distribution of black-tailed prairie dogs. This study, in combination with previous work suggests that sylvatic plague likely occurs across the range of black-tailed prairie dogs and should now be treated as endemic across this range. PMID:26771845

  14. Detections of Yersinia pestis East of the Known Distribution of Active Plague in the United States.

    PubMed

    Mize, Erica L; Britten, Hugh B

    2016-02-01

    We examined fleas collected from black-tailed prairie dog (Cynomys ludovicianus) burrows from 2009 through 2011 in five national park units east of the known distribution of active plague across the northern Great Plains for the presence of Yersinia pestis. Across all national park units, Oropsylla tuberculata and Oropsylla hirsuta were the most common fleas collected from prairie dog burrows, 42.4% and 56.9%, respectively, of the 3964 fleas collected from burrow swabbing. Using a nested PCR assay, we detected 200 Y. pestis-positive fleas from 3117 assays. In total, 6.4% of assayed fleas were Y. pestis positive and 13.9% of prairie dog burrows swabbed contained Y. pestis-positive fleas. Evidence of the presence of Y. pestis was observed at all national park units except Devils Tower National Monument in Wyoming. We detected the presence of Y. pestis without large die-offs, i.e., enzootic sylvatic plague, east of the known distribution of active plague and near the eastern edge of the present distribution of black-tailed prairie dogs. This study, in combination with previous work suggests that sylvatic plague likely occurs across the range of black-tailed prairie dogs and should now be treated as endemic across this range.

  15. Regulation and expression of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis

    SciTech Connect

    Sample, A.K.

    1987-01-01

    It is shown in this thesis that cells of Lcr/sup +/, Pst/sup -/ Y. pestis KIM are able to express Yops at levels comparable to that of Lcr/sup +/ Yersinia pseudotuberculosis. Pulse-chase radiolabeling with /sup 35/S-methionine was used to demonstrate that Lcr/sup +/, Pst/sup +/ Y. pestis synthesized at least 11 distinct peptides during the low calcium response and that seven of the labeled peptides were rapidly degraded. These seven peptides were stably expressed in Lcr/sup +/, Pst/sup -/ Y. pestis and were of identical molecular weights as the Yops expressed by that strain. Radiolabeled fragments of low molecular weight accumulated in the extracellular medium of Pst/sup +/ cultures and were assumed to be stable degradation fragments derived from Yops. It was also shown that the set of stable peptides, including V antigen, were made during restriction by both Pst/sup +/ and Pst/sup -/ Y. pestis KIM and were located primarily within the cytoplasm. Those radiolabeled peptides which underwent proteolytic degradation in Pst/sup +/ Y. pestis were localized to the outer membrane and extracellular medium in the Pst/sup -/ strain. It is concluded that the failure of Lcr/sup +/, Pst/sup +/ Y. pestis to express Yops is the result of post-translational degradation and is not a block in the synthesis of Yops.

  16. Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis.

    PubMed

    Zimbler, Daniel L; Eddy, Justin L; Schroeder, Jay A; Lathem, Wyndham W

    2015-11-09

    Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague.

  17. Genome-scale reconstruction of the metabolic network in Yersinia pestis, strain 91001

    SciTech Connect

    Navid, A; Almaas, E

    2009-01-13

    The gram-negative bacterium Yersinia pestis, the aetiological agent of bubonic plague, is one the deadliest pathogens known to man. Despite its historical reputation, plague is a modern disease which annually afflicts thousands of people. Public safety considerations greatly limit clinical experimentation on this organism and thus development of theoretical tools to analyze the capabilities of this pathogen is of utmost importance. Here, we report the first genome-scale metabolic model of Yersinia pestis biovar Mediaevalis based both on its recently annotated genome, and physiological and biochemical data from literature. Our model demonstrates excellent agreement with Y. pestis known metabolic needs and capabilities. Since Y. pestis is a meiotrophic organism, we have developed CryptFind, a systematic approach to identify all candidate cryptic genes responsible for known and theoretical meiotrophic phenomena. In addition to uncovering every known cryptic gene for Y. pestis, our analysis of the rhamnose fermentation pathway suggests that betB is the responsible cryptic gene. Despite all of our medical advances, we still do not have a vaccine for bubonic plague. Recent discoveries of antibiotic resistant strains of Yersinia pestis coupled with the threat of plague being used as a bioterrorism weapon compel us to develop new tools for studying the physiology of this deadly pathogen. Using our theoretical model, we can study the cell's phenotypic behavior under different circumstances and identify metabolic weaknesses which may be harnessed for the development of therapeutics. Additionally, the automatic identification of cryptic genes expands the usage of genomic data for pharmaceutical purposes.

  18. Inactivation of avirulent Yersinia pestis on food and food contact surfaces by ultraviolet light and freezing.

    PubMed

    Sommers, Christopher H; Sheen, Shiowshuh

    2015-09-01

    Yersinia pestis, the causative agent of plague, can occasionally be contracted as a naso-pharyngeal or gastrointestinal illness through consumption of contaminated meat. In this study, the use of 254 nm ultraviolet light (UV-C) to inactivate a multi-isolate cocktail of avirulent Y. pestis on food and food contact surfaces was investigated. When a commercial UV-C conveyor was used (5 mW/cm(2)/s) 0.5 J/cm(2) inactivated >7 log of the Y. pestis cocktail on agar plates. At 0.5 J/cm(2), UV-C inactivated ca. 4 log of Y. pestis in beef, chicken, and catfish, exudates inoculated onto high density polypropylene or polyethylene, and stainless steel coupons, and >6 log was eliminated at 1 J/cm(2). Approximately 1 log was inactivated on chicken breast, beef steak, and catfish fillet surfaces at a UV-C dose of 1 J/cm(2). UV-C treatment prior to freezing of the foods did not increase the inactivation of Y. pestis over freezing alone. These results indicate that routine use of UV-C during food processing would provide workers and consumers some protection against Y. pestis.

  19. Characterization of Residual Medium Peptides from Yersinia pestis Cultures

    SciTech Connect

    Clowers, Brian H.; Wunschel, David S.; Kreuzer, Helen W.; Engelmann, Heather E.; Valentine, Nancy B.; Wahl, Karen L.

    2013-04-03

    Using a range of common microbial medium formulations (TSB, BHI, LB, and G-media), two attenuated strains of Y. pestis (KIM D27 (pgm-) and KIMD1 lcr-) were cultivated in triplicate. These cellular suspensions were used to develop a method of extracting residual medium peptides from the final microbial preparation to assess their relative abundance and identity. Across the conditions examined, which included additional cellular washing and different forms of microbial inactivation, residual medium peptides were detected. Despite the range of growth medium sources used and the associated manufacturing processes used in their production, a high degree of peptide similarity was observed for a given medium recipe. These results demonstrate that residual medium peptides are retained using traditional microbial cultivation techniques and may be used to inform forensic investigations with respect to production deduction.

  20. Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

    SciTech Connect

    Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J.; Noinaj, Nicholas; Felek, Suleyman; Tsang, Tiffany M.; Krukonis, Eric S.; Hinnebusch, B. Joseph; Buchanan, Susan K.

    2012-01-30

    Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.

  1. Yersinia pestis and Yersinia pseudotuberculosis infection: a regulatory RNA perspective

    PubMed Central

    Martínez-Chavarría, Luary C.; Vadyvaloo, Viveka

    2015-01-01

    Yersinia pestis, responsible for causing fulminant plague, has evolved clonally from the enteric pathogen, Y. pseudotuberculosis, which in contrast, causes a relatively benign enteric illness. An ~97% nucleotide identity over 75% of their shared protein coding genes is maintained between these two pathogens, leaving much conjecture regarding the molecular determinants responsible for producing these vastly different disease etiologies, host preferences and transmission routes. One idea is that coordinated production of distinct factors required for host adaptation and virulence in response to specific environmental cues could contribute to the distinct pathogenicity distinguishing these two species. Small non-coding RNAs that direct posttranscriptional regulation have recently been identified as key molecules that may provide such timeous expression of appropriate disease enabling factors. Here the burgeoning field of small non-coding regulatory RNAs in Yersinia pathogenesis is reviewed from the viewpoint of adaptive colonization, virulence and divergent evolution of these pathogens. PMID:26441890

  2. Physiological basis of the low calcium response in Yersinia pestis.

    PubMed Central

    Fowler, J M; Brubaker, R R

    1994-01-01

    It is established that duplication in vitro of that amount of Ca2+ (2.5 mM) and Mg2+ (1.5 mM) present in blood permits vegetative growth of Yersinia pestis with repression of virulence factors encoded by the Lcr plasmid (Lcr+); similar simulation of intracellular fluid (no Ca2+ and 20 mM Mg2+) promotes bacteriostasis with induction of these virulence determinants. However, proliferation of yersiniae in mice occurs primarily within necrotic focal lesions (supplied by Ca(2+)-deficient host cell cytoplasm) within visceral organs rather than in Ca(2+)-sufficient blood. The present study addressed this enigma by defining conditions necessary for achieving vegetative growth of Lcr+ yersiniae at 37 degrees C in simulated intracellular fluid. Maximum optical densities were increased by substitution of K+ for Na+ and elimination of Cl-; the combination of Na+ plus L-glutamate was selectively toxic to Lcr+ cells. This phenomenon was attributed in part to the absence of aspartase in Y. pestis (a lesion known to facilitate massive accumulation of L-aspartate via transamination of the oxalacetate pool by L-glutamate). Replacement of L-glutamate by exogenous L-aspartate or alpha-ketoglutarate reversed this toxicity by favoring retention of oxalacetate. Proliferation of Lcr+ cells in a medium containing K+ and L-aspartate but lacking added Ca2+ and Na+ was markedly enhanced by increasing the concentration of fermentable carbohydrate. Accordingly, in the worst-case scenario (i.e., added Na+, Cl-, and L-glutamate), Lcr+ yersiniae underwent restriction of growth after one doubling, and in the best-case scenario (i.e., added K+ and L-aspartate), the organisms completed more than five doublings, thereby achieving full-scale growth. Both of these Ca(2+)-deficient media promoted maximum induction of Mg(2+)-induced V antigen, a virulence factor encoded by the Lcr plasmid. PMID:7960099

  3. A live attenuated strain of Yersinia pestis KIM as a vaccine against plague.

    PubMed

    Sun, Wei; Six, David; Kuang, Xiaoying; Roland, Kenneth L; Raetz, Christian R H; Curtiss, Roy

    2011-04-01

    Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P(BAD) promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P(lpxL)lpxL ΔP(crp21)::TT araC P(BAD)crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD(50) of the mutant in mice, when administered subcutaneously or intranasally was >10(7)-times and >10(4)-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10(4) live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.

  4. Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague.

    PubMed

    Vagima, Yaron; Zauberman, Ayelet; Levy, Yinon; Gur, David; Tidhar, Avital; Aftalion, Moshe; Shafferman, Avigdor; Mamroud, Emanuelle

    2015-05-01

    Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel

  5. A live attenuated strain of Yersinia pestis KIM as a vaccine against plague

    PubMed Central

    Sun, Wei; Six, David; Kuang, Xiaoying; Roland, Kenneth L; Raetz, Christian R.H.; Curtiss, Roy

    2011-01-01

    Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2′-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC PBAD promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::PlpxL lpxL ΔPcrp21::TT araC PBAD crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD50 of the mutant in mice, when administered subcutaneously or intranasally was >107-times and >104-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6 × 107 wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2 × 104 live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate. PMID:21320544

  6. Inactivation of avirulent Yersinia pestis in Butterfield's phosphate buffer and frankfurters by UVC (254 nm) and gamma radiation.

    PubMed

    Sommers, Christopher H; Cooke, Peter H

    2009-04-01

    Yersinia pestis is the causative agent of plague. Although rare, pharyngeal plague in humans has been associated with consumption or handling of meat prepared from infected animals. The risks of contracting plague from consumption of deliberately contaminated food are currently unknown. Gamma radiation is a penetrating form of electromagnetic radiation, and UVC radiation is used for decontamination of liquids or food surfaces. Gamma radiation D10-values (the radiation dose needed to inactivate 1 log unit pathogen) were 0.23 (+/-0.01) and 0.31 (+/-0.03) kGy for avirulent Y. pestis inoculated into Butterfield's phosphate buffer and onto frankfurter surfaces, respectively, at 0 degree C. A UVC radiation dose of 0.25 J/cm2 inactivated avirulent Y. pestis suspended in Butterfield's phosphate buffer. UVC radiation doses of 0.5 to 4.0 J/cm2 inactivated 0.97 to 1.20 log units of the Y. pestis surface inoculated onto frankfurters. A low gamma radiation dose of 1.6 kGy could provide a 5-log reduction and a UVC radiation dose of 1 to 4 J/cm2 would provide a 1-log reduction of Y. pestis surface inoculated onto frankfurters. Y. pestis was capable of growth on frankfurters during refrigerated storage (10 degrees C). Gamma radiation of frankfurters inhibited the growth of Y. pestis during refrigerated storage, and UVC radiation delayed the growth of Y. pestis.

  7. A procedure for monitoring the presence of the virulence plasmid (pYV) in Yersinia pestis under culture conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenicity of Yersinia pestis depends on the presence of a virulence plasmid (pYV). The unstable nature of pYV in Y. pestis leads to the eventual outgrowth of pYV less cells due its higher growth rate. Thus, it was necessary to develop procedures to monitor the presence of the plasmid durin...

  8. A procedure for maintenance of the virulence plasmid (pYV) in Yersinia pestis under culture conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenicity of Yersinia pestis depends on the presence of a virulence plasmid (pYV). The unstable nature of pYV in Y. pestis leads to the eventual outgrowth of pYV less cells due to its higher growth rate. Thus, it was necessary to develop procedures to monitor the presence of the plasmid du...

  9. An experimentally-supported genome-scale metabolic network reconstruction for Yersinia pestis CO92

    PubMed Central

    2011-01-01

    Background Yersinia pestis is a gram-negative bacterium that causes plague, a disease linked historically to the Black Death in Europe during the Middle Ages and to several outbreaks during the modern era. Metabolism in Y. pestis displays remarkable flexibility and robustness, allowing the bacterium to proliferate in both warm-blooded mammalian hosts and cold-blooded insect vectors such as fleas. Results Here we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92 and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins, 963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional requirements characteristic of this strain. Conclusions Y. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data and correctly reproduces known phenotypes, provides an in silico platform with which to investigate the metabolism of this important human pathogen. PMID:21995956

  10. Resistance to Innate Immunity Contributes to Colonization of the Insect Gut by Yersinia pestis

    PubMed Central

    Earl, Shaun C.; Rogers, Miles T.; Keen, Jennifer; Bland, David M.; Houppert, Andrew S.; Miller, Caitlynn; Temple, Ian; Anderson, Deborah M.; Marketon, Melanie M.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic and pneumonic plague, is typically a zoonotic vector-borne disease of wild rodents. Bacterial biofilm formation in the proventriculus of the flea contributes to chronic infection of fleas and facilitates efficient disease transmission. However prior to biofilm formation, ingested bacteria must survive within the flea midgut, and yet little is known about vector-pathogen interactions that are required for flea gut colonization. Here we establish a Drosophila melanogaster model system to gain insight into Y. pestis colonization of the insect vector. We show that Y. pestis establishes a stable infection in the anterior midgut of fly larvae, and we used this model system to study the roles of genes involved in biofilm production and/or resistance to gut immunity stressors. We find that PhoP and GmhA both contribute to colonization and resistance to antimicrobial peptides in flies, and furthermore, the data suggest biofilm formation may afford protection against antimicrobial peptides. Production of reactive oxygen species in the fly gut, as in fleas, also serves to limit bacterial infection, and OxyR mediates Y. pestis survival in both insect models. Overall, our data establish the fruit fly as an informative model to elucidate the relationship between Y. pestis and its flea vector. PMID:26177454

  11. Transcriptomic and innate immune responses to Yersinia pestis in the lymph node during bubonic plague.

    PubMed

    Comer, Jason E; Sturdevant, Daniel E; Carmody, Aaron B; Virtaneva, Kimmo; Gardner, Donald; Long, Dan; Rosenke, Rebecca; Porcella, Stephen F; Hinnebusch, B Joseph

    2010-12-01

    A delayed inflammatory response is a prominent feature of infection with Yersinia pestis, the agent of bubonic and pneumonic plague. Using a rat model of bubonic plague, we examined lymph node histopathology, transcriptome, and extracellular cytokine levels to broadly characterize the kinetics and extent of the host response to Y. pestis and how it is influenced by the Yersinia virulence plasmid (pYV). Remarkably, dissemination and multiplication of wild-type Y. pestis during the bubonic stage of disease did not induce any detectable gene expression or cytokine response by host lymph node cells in the developing bubo. Only after systemic spread had led to terminal septicemic plague was a transcriptomic response detected, which included upregulation of several cytokine, chemokine, and other immune response genes. Although an initial intracellular phase of Y. pestis infection has been postulated, a Th1-type cytokine response associated with classical activation of macrophages was not observed during the bubonic stage of disease. However, elevated levels of interleukin-17 (IL-17) were present in infected lymph nodes. In the absence of pYV, sustained recruitment to the lymph node of polymorphonuclear leukocytes (PMN, or neutrophils), the major IL-17 effector cells, correlated with clearance of infection. Thus, the ability to counteract a PMN response in the lymph node appears to be a major in vivo function of the Y. pestis virulence plasmid.

  12. Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

    PubMed Central

    Caulfield, Adam J.; Rangel, Stephanie M.; Lathem, Wyndham W.

    2014-01-01

    Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand. PMID:25198697

  13. An Experimentally-Supported Genome-Scale Metabolic Network Reconstruction for Yersinia pestis CO92

    SciTech Connect

    Charusanti, Pep; Chauhan, Sadhana; Mcateer, Kathleen; Lerman, Joshua A.; Hyduke, Daniel R.; Motin, Vladimir L.; Ansong, Charles; Adkins, Joshua N.; Palsson, Bernhard O.

    2011-10-13

    Yersinia pestis is a gram-negative bacterium that causes plague, a disease linked historically to the Black Death in Europe during the Middle Ages and to several outbreaks during the modern era. Metabolism in Y. pestis displays remarkable flexibility and robustness, allowing the bacterium to proliferate in both warm-blooded mammalian hosts and cold-blooded insect vectors such as fleas. Here we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92 and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins, 963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional requirements characteristic of this strain. Y. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data and correctly reproduces known phenotypes, thus provides an in silico platform with which to investigate the metabolism of this important human pathogen.

  14. Yersinia pestis Requires Host Rab1b for Survival in Macrophages

    PubMed Central

    Connor, Michael G.; Pulsifer, Amanda R.; Price, Christopher T.; Abu Kwaik, Yousef; Lawrenz, Matthew B.

    2015-01-01

    Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH. PMID:26495854

  15. A draft genome of Yersinia pestis from victims of the Black Death

    PubMed Central

    Bos, Kirsten I.; Schuenemann, Verena J.; Golding, G. Brian; Burbano, Hernán A.; Waglechner, Nicholas; Coombes, Brian K.; McPhee, Joseph B.; DeWitte, Sharon N.; Meyer, Matthias; Schmedes, Sarah; Wood, James; Earn, David J. D.; Herring, D. Ann; Bauer, Peter; Poinar, Hendrik N.; Krause, Johannes

    2013-01-01

    Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard1. This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348–1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347–1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y. pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y. pestis infections. PMID:21993626

  16. [Modeling of interaction between Yersinia pestis and Tetrahymena pyriformis in experimental ecosystems].

    PubMed

    Breneva, N V; Maramovich, A S

    2008-01-01

    Modeling of interaction Yersinia pestis-Tetrahymena pyriformis in artificial soil ecosystem (ASE) containing soil of burrows of main carrier from Gorno-Altayski natural plague reservoir, as well as in physiological solution (PS) and in Hottinger broth (HB). Optimal proportion of bacterial and protozoa cells was possible to obtain and depended from virulence of Y. pestis and environmental conditions. In ASE at 18-22 degrees C association was the most stable under the microbial burden of 100 microbial cells (m.c.) per infusorian. Resistance of plague agent to phagocytosis by T. pyriformis was determined by strain's virulence. Avirulent strain Y. pestis [cyrillic letter: see text]-2377 was rapidly eliminated by protozoan in HB, PS and in ASE under the burden of 10 m.c per infusorian. Y. pestis [cyrillic letter: see text]-3443 with selective virulence compared with [cyrillic letter: see text]-2377 preserved in association longer in any tested medium. Highly virulent Y. pestis [cyrillic letter: see text]-3448 was the most resistant to phagocytosis by T. pyriformis.

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

    PubMed

    Hinnebusch, B Joseph; Rosso, Marie-Laure; Schwan, Tom G; Carniel, Elisabeth

    2002-10-01

    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. PMID:12406213

  18. Eighteenth century Yersinia pestis genomes reveal the long-term persistence of an historical plague focus.

    PubMed

    Bos, Kirsten I; Herbig, Alexander; Sahl, Jason; Waglechner, Nicholas; Fourment, Mathieu; Forrest, Stephen A; Klunk, Jennifer; Schuenemann, Verena J; Poinar, Debi; Kuch, Melanie; Golding, G Brian; Dutour, Olivier; Keim, Paul; Wagner, David M; Holmes, Edward C; Krause, Johannes; Poinar, Hendrik N

    2016-01-01

    The 14th-18th century pandemic of Yersinia pestis caused devastating disease outbreaks in Europe for almost 400 years. The reasons for plague's persistence and abrupt disappearance in Europe are poorly understood, but could have been due to either the presence of now-extinct plague foci in Europe itself, or successive disease introductions from other locations. Here we present five Y. pestis genomes from one of the last European outbreaks of plague, from 1722 in Marseille, France. The lineage identified has not been found in any extant Y. pestis foci sampled to date, and has its ancestry in strains obtained from victims of the 14th century Black Death. These data suggest the existence of a previously uncharacterized historical plague focus that persisted for at least three centuries. We propose that this disease source may have been responsible for the many resurgences of plague in Europe following the Black Death. PMID:26795402

  19. Comparative transcriptome analysis of Yersinia pestis in response to hyperosmotic and high-salinity stress.

    PubMed

    Han, Yanping; Zhou, Dongsheng; Pang, Xin; Zhang, Ling; Song, Yajun; Tong, Zongzhong; Bao, Jingyue; Dai, Erhei; Wang, Jin; Guo, Zhaobiao; Zhai, Junhui; Du, Zongmin; Wang, Xiaoyi; Wang, Jian; Huang, Peitang; Yang, Ruifu

    2005-04-01

    DNA microarray was used as a tool to investigate genome-wide transcriptional responses of Yersinia pestis to hyperosmotic and high-salinity stress. Hyperosmotic stress specifically upregulated genes responsible for ABC-type transport and the cytoplasmic accumulation of certain polysaccharides, while high-salinity stress induced the transcription of genes encoding partition proteins and several global transcriptional regulators. Genes whose transcription was enhanced by both kinds of stress comprised those encoding osmoprotectant transport systems and a set of virulence determinants. The number of genes downregulated by the two kinds of stress was much lower than that of upregulated genes, suggesting that neither kind of stress severely depresses cellular processes in general. Many differentially regulated genes still exist whose functions remain unknown. Y. pestis recognized high-salinity and hyperosmotic stress as different kinds of environmental stimuli, and different mechanisms enabled acclimation to these two kinds of stress, although Y. pestis still executed common mechanisms to accommodate both types of stress.

  20. Transcriptome analysis of acyl-homoserine lactone-based quorum sensing regulation in Yersinia pestis [corrected].

    PubMed

    LaRock, Christopher N; Yu, Jing; Horswill, Alexander R; Parsek, Matthew R; Minion, F Chris

    2013-01-01

    The etiologic agent of bubonic plague, Yersinia pestis, senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.

  1. In Vitro Antibiotic Susceptibilities of Yersinia pestis Determined by Broth Microdilution following CLSI Methods

    PubMed Central

    Hershfield, Jeremy; Marchand, Charles; Miller, Lynda; Halasohoris, Stephanie; Purcell, Bret K.; Worsham, Patricia L.

    2015-01-01

    In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, β-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance. PMID:25583720

  2. Regulation of Yersina pestis Virulence by AI-2 Mediated Quorum Sensing

    SciTech Connect

    Segelke, B; Hok, S; Lao, V; Corzett, M; Garcia, E

    2010-03-29

    The proposed research was motivated by an interest in understanding Y. pestis virulence mechanisms and bacteria cell-cell communication. It is expected that a greater understanding of virulence mechanisms will ultimately lead to biothreat countermeasures and novel therapeutics. Y. pestis is the etiological agent of plague, the most devastating disease in human history. Y. pestis infection has a high mortality rate and a short incubation before mortality. There is no widely available and effective vaccine for Y. pestis and multi-drug resistant strains are emerging. Y. pestis is a recognized biothreat agent based on the wide distribution of the bacteria in research laboratories around the world and on the knowledge that methods exist to produce and aerosolize large amounts of bacteria. We hypothesized that cell-cell communication via signaling molecules, or quorum sensing, by Y. pestis is important for the regulation of virulence factor gene expression during host invasion, though a causative link had never been established. Quorum sensing is a mode of intercellular communication which enables orchestration of gene expression for many bacteria as a function of population density and available evidence suggests there may be a link between quorum sensing and regulation of Y. pesits virulence. Several pathogenic bacteria have been shown to regulate expression of virulence factor genes, including genes encoding type III secretion, via quorum sensing. The Y. pestis genome encodes several cell-cell signaling pathways and the interaction of at least three of these are thought to be involved in one or more modes of host invasion. Furthermore, Y. pestis gene expression array studies carried out at LLNL have established a correlation between expression of known virulence factors and genes involved in processing of the AI-2 quorum sensing signal. This was a basic research project that was intended to provide new insights into bacterial intercellular communication and how it is

  3. Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India.

    PubMed

    Khushiramani, Rekha; Tuteja, Urmil; Shukla, Jyoti; Batra, Harsh Vardhan

    2004-05-01

    The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins

  4. Yersinia pestis with regulated delayed attenuation as a vaccine candidate to induce protective immunity against plague.

    PubMed

    Sun, Wei; Roland, Kenneth L; Kuang, Xiaoying; Branger, Christine G; Curtiss, Roy

    2010-03-01

    Two mutant strains of Yersinia pestis KIM5+, a Deltacrp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P(BAD) crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD(50)s) of the Deltacrp and araC P(BAD) crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5+, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 x 10(7) CFU of the Deltacrp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 x 10(4) CFU of the araC P(BAD) crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.

  5. Intrinsic plasmids influence MicF-mediated translational repression of ompF in Yersinia pestis

    PubMed Central

    Liu, Zizhong; Wang, Haili; Wang, Hongduo; Wang, Jing; Bi, Yujing; Wang, Xiaoyi; Yang, Ruifu; Han, Yanping

    2015-01-01

    Yersinia pestis, which is the causative agent of plague, has acquired exceptional pathogenicity potential during its evolution from Y. pseudotuberculosis. Two laterally acquired plasmids, namely, pMT1 and pPCP1, are specific to Y. pestis and are critical for pathogenesis and flea transmission. Small regulatory RNAs (sRNAs) commonly function as regulators of gene expression in bacteria. MicF, is a paradigmatic sRNA that acts as a post-transcriptional repressor through imperfect base pairing with the 5′-UTR of its target mRNA, ompF, in Escherichia coli. The high sequence conservation and minor variation in the RNA duplex of MicF-ompF has been reported in Yersinia. In this study, we utilized super-folder GFP reporter gene fusion to validate the post-transcriptional MicF-mediated regulation of target mRNA ompF in Y. pestis. Unexpectedly, upon MicF overexpression, the slightly upregulated expression of OmpF were found in the wild-type strain, which contradicted the previously established model. Interestingly, the translational repression of ompF target fusions was restored in the intrinsic plasmids-cured Y. pestis strain, suggesting intrinsic plasmids influence the MicF-mediated translational repression of ompF in Y. pestis. Further examination showed that plasmid pPCP1 is likely the main contributor to the abolishment of MicF-mediated translational repression of endogenous or plasmid-borne ompF. It represents that the possible roles of intrinsic plasmids should be considered upon investigating sRNA-mediated gene regulation, at least in Y. pestis, even if the exact mechanism is not fully understood. PMID:26347736

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

    PubMed

    Nham, Toan; Filali, Sofia; Danne, Camille; Derbise, Anne; Carniel, Elisabeth

    2012-01-01

    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.

  7. Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

    PubMed Central

    Zimbler, Daniel L.; Eddy, Justin L.; Schroeder, Jay A.

    2015-01-01

    Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. PMID:26553463

  8. [Plague in Algeria: about five strains of Yersinia pestis isolated during the outbreak of June 2003].

    PubMed

    Lounici, M; Lazri, M; Rahal, K

    2005-02-01

    In this study, we isolated and identified five strains of Yersinia pestis during an epidemic occurred in west of Algeria in June 2003. The bacteriological identification was confirmed by bacteriophage susceptibility. All these strains belonged to the biovar Orientalis (they did not ferment glycerol but did reduce nitrate to nitrite) which caused the current pandemic. The in vitro activities of antimicrobial agents used to treat plague and recommended for prophylaxis, showed that they are active against all strains. The comparison of these strains by plasmid profile analyse demonstrated that all isolates had three plasmids: 110, 70 and 9.5 kb, which are present in Y. pestis strains.

  9. Rapid identification of Yersinia pestis and Brucella melitensis by chip-based continuous flow PCR

    NASA Astrophysics Data System (ADS)

    Dietzsch, Michael; Hlawatsch, Nadine; Melzer, Falk; Tomaso, Herbert; Gärtner, Claudia; Neubauer, Heinrich

    2012-06-01

    To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

  10. Yersinia pestis DNA from Skeletal Remains from the 6th Century AD Reveals Insights into Justinianic Plague

    PubMed Central

    Harbeck, Michaela; Seifert, Lisa; Hänsch, Stephanie; Wagner, David M.; Birdsell, Dawn; Parise, Katy L.; Wiechmann, Ingrid; Grupe, Gisela; Thomas, Astrid; Keim, Paul; Zöller, Lothar; Bramanti, Barbara; Riehm, Julia M.; Scholz, Holger C.

    2013-01-01

    Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19th and 20th centuries, during which plague was spread around the world, and the second pandemic of the 14th–17th centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6th–8th centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics. PMID:23658525

  11. Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.

    PubMed

    Harbeck, Michaela; Seifert, Lisa; Hänsch, Stephanie; Wagner, David M; Birdsell, Dawn; Parise, Katy L; Wiechmann, Ingrid; Grupe, Gisela; Thomas, Astrid; Keim, Paul; Zöller, Lothar; Bramanti, Barbara; Riehm, Julia M; Scholz, Holger C

    2013-01-01

    Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.

  12. Origins of Yersinia pestis Sensitivity to the Arylomycin Antibiotics and the Inhibition of Type I Signal Peptidase

    PubMed Central

    Steed, Danielle B.; Liu, Jian; Wasbrough, Elizabeth; Miller, Lynda; Halasohoris, Stephanie; Miller, Jeremy; Somerville, Brandon; Hershfield, Jeremy R.

    2015-01-01

    Yersinia pestis is the etiologic agent of the plague. Reports of Y. pestis strains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported that Y. pestis strain KIM6+, unlike most Enterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range of Y. pestis strains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of the Y. pestis SPase for the antibiotic and that alterations to each component of the Y. pestis lipopolysaccharide—O antigen, core, and lipid A—make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity against Y. pestis and potentially other Gram-negative pathogens. PMID:25896690

  13. Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

    PubMed Central

    Zauberman, Ayelet; Tidhar, Avital; Levy, Yinon; Bar-Haim, Erez; Halperin, Gideon; Flashner, Yehuda; Cohen, Sara; Shafferman, Avigdor; Mamroud, Emanuelle

    2009-01-01

    An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 107-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD50 of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the

  14. Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    PubMed Central

    Sergueev, Kirill V.; He, Yunxiu; Borschel, Richard H.; Nikolich, Mikeljon P.; Filippov, Andrey A.

    2010-01-01

    Background Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. Methodology/Principal Findings The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Conclusions/Significance Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria. PMID:20596528

  15. Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century.

    PubMed

    Seifert, Lisa; Wiechmann, Ingrid; Harbeck, Michaela; Thomas, Astrid; Grupe, Gisela; Projahn, Michaela; Scholz, Holger C; Riehm, Julia M

    2016-01-01

    Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered. PMID:26760973

  16. Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century.

    PubMed

    Seifert, Lisa; Wiechmann, Ingrid; Harbeck, Michaela; Thomas, Astrid; Grupe, Gisela; Projahn, Michaela; Scholz, Holger C; Riehm, Julia M

    2016-01-01

    Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.

  17. Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century

    PubMed Central

    Seifert, Lisa; Wiechmann, Ingrid; Harbeck, Michaela; Thomas, Astrid; Grupe, Gisela; Projahn, Michaela; Scholz, Holger C.; Riehm, Julia M.

    2016-01-01

    Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered. PMID:26760973

  18. Yersinia pseudotuberculosis ST42 (O:1) Strain Misidentified as Yersinia pestis by Mass Spectrometry Analysis.

    PubMed

    Gérôme, Patrick; Le Flèche, Philippe; Blouin, Yann; Scholz, Holger C; Thibault, François M; Raynaud, Françoise; Vergnaud, Gilles; Pourcel, Christine

    2014-06-12

    We report here the draft sequence of strain CEB14_0017, alias HIAD_DUP, recovered from a human patient and initially identified as Yersinia pestis by mass spectrometry analysis. Genotyping based on tandem repeat polymorphism assigned the strain to Yersinia pseudotuberculosis sequence type 42 (ST42). The total assembly length is 4,894,739 bp.

  19. Yersinia pseudotuberculosis ST42 (O:1) Strain Misidentified as Yersinia pestis by Mass Spectrometry Analysis

    PubMed Central

    Gérôme, Patrick; Le Flèche, Philippe; Blouin, Yann; Scholz, Holger C.; Thibault, François M.; Raynaud, Françoise; Vergnaud, Gilles

    2014-01-01

    We report here the draft sequence of strain CEB14_0017, alias HIAD_DUP, recovered from a human patient and initially identified as Yersinia pestis by mass spectrometry analysis. Genotyping based on tandem repeat polymorphism assigned the strain to Yersinia pseudotuberculosis sequence type 42 (ST42). The total assembly length is 4,894,739 bp. PMID:24926044

  20. Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties

    PubMed Central

    Knirel, Y.A.; Anisimov, A.P.

    2012-01-01

    The present review summarizes data pertaining to the composition and structure of the carbohydrate moiety (core oligosaccharide) and lipid component (lipid A) of the various forms of lipopolysaccharide (LPS), one of the major pathogenicity factors ofYersinia pestis, the cause of plague. The review addresses the functions and the biological significance of genes for the biosynthesis of LPS, as well as the biological properties of LPS in strains from various intraspecies groups ofY. pestis and their mutants, including the contribution of LPS to the resistance of bacteria to factors of the innate immunity of both insect-vectors and mammal-hosts. Special attention is paid to temperature-dependent variations in the LPS structure, their genetic control and roles in the pathogenesis of plague. The evolutionary aspect is considered based on a comparison of the structure and genetics of the LPS ofY. pestis and other enteric bacteria, including otherYersinia species. The prospects of development of live plague vaccines created on the basis ofY. pestis strains with the genetically modified LPS are discussed. PMID:23150803

  1. Genome assemblies for 11 Yersinia pestis strains isolated in the Caucasus region

    SciTech Connect

    Zhgenti, Ekaterine; Johnson, Shannon L.; Davenport, Karen W.; Chanturia, Gvantsa; Daligault, Hajnalka E.; Chain, Patrick S.; Nikolich, Mikeljon P.

    2015-09-17

    Yersinia pestis, the causative agent of plague, is endemic to the Caucasus region but few reference strain genome sequences from that region are available. We present the improved draft or finished assembled genomes from 11 strains isolated in the nation of Georgia and surrounding countries.

  2. Genome assemblies for 11 Yersinia pestis strains isolated in the Caucasus region

    DOE PAGESBeta

    Zhgenti, Ekaterine; Johnson, Shannon L.; Davenport, Karen W.; Chanturia, Gvantsa; Daligault, Hajnalka E.; Chain, Patrick S.; Nikolich, Mikeljon P.

    2015-09-17

    Yersinia pestis, the causative agent of plague, is endemic to the Caucasus region but few reference strain genome sequences from that region are available. We present the improved draft or finished assembled genomes from 11 strains isolated in the nation of Georgia and surrounding countries.

  3. Analysis of temperature-dependent changes in the metabolism of Yersinia pestis.

    NASA Astrophysics Data System (ADS)

    Navid, Ali; Almaas, Eivind

    2008-03-01

    The gram-negative bacterium Yersinia pestis is the aetiological agent of bubonic plague, a zoonotic infection that occurs through the bite of a flea. It has long been known that Y. pestis has different metabolic needs upon transition from the flea gut environment (26 C) to that of a mammalian host (37 C). To study this and other outstanding questions about metabolic function of Y. pestis, we used the available genomic, biochemical and physiological data to develop a constraint-based flux balance model of metabolism in the avirulent 91001 strain (biovar Mediaevalis) of this organism. Utilizing two sets of whole-genome DNA microarray expression data, we examined the system level changes that occur when Y. pestis acclimatizes to temperature shifts. Our results point to fundamental changes in its oxidative metabolism of sugars and use of amino acids, in particular that of arginine. This behavior is indicative of an inefficient metabolism that could be caused by adaptation to life in a nutrient rich environment.

  4. Comparative Proteomic Studies of Yersinia pestis Strains Isolated from Natural Foci in the Republic of Georgia

    PubMed Central

    Nozadze, Maia; Zhgenti, Ekaterine; Meparishvili, Maia; Tsverava, Lia; Kiguradze, Tamar; Chanturia, Gvantsa; Babuadze, Giorgi; Kekelidze, Merab; Bakanidze, Lela; Shutkova, Tatiana; Imnadze, Paata; Francesconi, Stephen C.; Obiso, Richard; Solomonia, Revaz

    2015-01-01

    Yersinia pestis, the causative agent of plague, is a highly virulent bacterium responsible for millions of human deaths throughout history. In the last decade, two natural plague foci have been described in the Republic of Georgia from which dozens of Y. pestis strains have been isolated. Analyses indicate that there are genetic differences between these strains, but it is not known if these differences are also reflected in protein expression. We chose four strains of Y. pestis (1390, 1853, 2944, and 8787) from the National Center for Disease Control and Public Health collection for proteomic studies based on neighbor-joining tree genetic analysis and geographical loci of strain origin. Proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. Select Y. pestis strains were grown under different physiological conditions and their proteomes were compared: (1) 28°C without calcium; (2) 28°C with calcium; (3) 37°C without calcium; and (4) 37°C with calcium. Candidate proteins were identified and the differences in expression of F1 antigen, tellurium-resistance protein, and outer membrane protein C, porin were validated by Western blotting. The in vitro cytotoxicity activity of these strains was also compared. The results indicate that protein expression and cytotoxic activities differ significantly among the studied strains; these differences could contribute to variations in essential physiological functions in these strains. PMID:26528469

  5. Posttranscriptional Regulation of the Yersinia pestis Cyclic AMP Receptor Protein Crp and Impact on Virulence

    PubMed Central

    Lathem, Wyndham W.; Schroeder, Jay A.; Bellows, Lauren E.; Ritzert, Jeremy T.; Koo, Jovanka T.; Price, Paul A.; Caulfield, Adam J.; Goldman, William E.

    2014-01-01

    ABSTRACT The cyclic AMP receptor protein (Crp) is a transcriptional regulator that controls the expression of numerous bacterial genes, usually in response to environmental conditions and particularly by sensing the availability of carbon. In the plague pathogen Yersinia pestis, Crp regulates the expression of multiple virulence factors, including components of the type III secretion system and the plasminogen activator protease Pla. The regulation of Crp itself, however, is distinctly different from that found in the well-studied Escherichia coli system. Here, we show that at physiological temperatures, the synthesis of Crp in Y. pestis is positively regulated at the posttranscriptional level. The loss of the small RNA chaperone Hfq results in decreased Crp protein levels but not in steady-state Crp transcript levels, and this regulatory effect occurs within the 5′ untranslated region (UTR) of the Crp mRNA. The posttranscriptional activation of Crp synthesis is required for the expression of pla, and decoupling crp from Hfq through the use of an exogenously controlled promoter and 5′ UTR increases Pla protein levels as well as partially rescues the growth defect associated with the loss of Hfq. Finally, we show that both Hfq and the posttranscriptional regulation of Crp contribute to the virulence of Y. pestis during pneumonic plague. The Hfq-dependent, posttranscriptional regulation of Crp may be specific to Yersinia species, and thus our data help explain the dramatic growth and virulence defects associated with the loss of Hfq in Y. pestis. PMID:24520064

  6. Novel Yersinia Pestis Toxin that Resembles Bacillus Anthracis Edema Factor: Study of Activity and Structural Modeling

    SciTech Connect

    Motin, V; Garcia, E; Barsky, D; Zemla, A

    2003-02-05

    The goal of this project was to begin both experimental and computational studies of the novel plague toxin to establish its biological properties and create its 3D-model. The project was divided into two parts. (1) Experimental--This part was devoted to determine distribution of the genes encoding novel plague toxin among different isolates of Y.pestis. If the EF-like activity is important for Y.pestis pathogenicity, it is anticipated that all highly virulent strains will contain the toxin genes. Also, they proposed to initiate research to investigate the functionality of the novel Y.pestis toxin that they hypothesize is likely to significantly contribute to the virulence of this dangerous microbe. this research design consisted of amplification, cloning and expression in E.coli the toxin genes followed by affinity purification of the recombinant protein that can be further used for testing of enzymatic activity. (2) Computational--The structural modeling of the putative EF of Y.pestis was based on multiple sequence alignments, secondary structure predictions, and comparison with 3D models of the EF of B. anthracis. The x-ray structure of the last has been recently published [Nature. 2002. 415(Jan):396-402]. The final model was selected after detailed analysis to determine if the structure is consistent with the biological function.

  7. Mutations, mutation rates, and evolution at the hypervariable VNTR loci of Yersinia pestis.

    PubMed

    Vogler, Amy J; Keys, Christine E; Allender, Christopher; Bailey, Ira; Girard, Jessica; Pearson, Talima; Smith, Kimothy L; Wagner, David M; Keim, Paul

    2007-03-01

    VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogens, including Yersinia pestis the etiologic agent of plague, because of their great diversity. Diversity is driven largely by mutation but little is known about VNTR mutation rates, factors affecting mutation rates, or the mutational mechanisms. The molecular epidemiological utility of VNTRs will be greatly enhanced when this foundational knowledge is available. Here, we measure mutation rates for 43 VNTR loci in Y. pestis using an in vitro generated population encompassing approximately 96,000 generations. We estimate the combined 43-locus rate and individual rates for 14 loci. A comparison of Y. pestis and Escherichia coli O157:H7 VNTR mutation rates and products revealed a similar relationship between diversity and mutation rate in these two species. Likewise, the relationship between repeat copy number and mutation rate is nearly identical between these species, suggesting a generalized relationship that may be applicable to other species. The single- versus multiple-repeat mutation ratios and the insertion versus deletion mutation ratios were also similar, providing support for a general model for the mutations associated with VNTRs. Finally, we use two small sets of Y. pestis isolates to show how this general model and our estimated mutation rates can be used to compare alternate phylogenies, and to evaluate the significance of genotype matches, near-matches, and mismatches found in empirical comparisons with a reference database. PMID:17161849

  8. Rapid diagnostic test that uses isocitrate lyase activity for identification of Yersinia pestis.

    PubMed

    Hillier, S L; Charnetzky, W T

    1981-04-01

    The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.

  9. Inactivation of avirulent Yersinia pestis in beef bologna by gamma irradiation.

    PubMed

    Sommers, Christopher H; Niemira, Brendan A

    2011-04-01

    Yersinia pestis, a psychrotrophic pathogen capable of growth at refrigeration temperatures, can cause pharyngeal and gastrointestinal plague in humans that consume contaminated foods. Because Y. pestis is listed as a select agent for food safety and defense, evaluation of food safety intervention technologies for inactivation of this pathogen is needed. Ionizing (gamma) radiation is a safe and effective intervention technology that can inactivate pathogens in raw and processed meats, produce, and seafood. In this study, we investigated the effect of temperature on the ability of ionizing radiation to inactivate avirulent Y. pestis in beef bologna. The mean (±standard error of the mean) radiation D(10)-values (the radiation dose needed to inactivate 1 log unit or 90% of the population of a microorganism) for avirulent Y. pestis suspended in beef bologna samples were 0.20 (±0.01), 0.22 (±0.01), 0.25 (±0.02), 0.31 (±0.01), 0.35 (±0.01), and 0.37 (±0.01) kGy at temperatures of 5, 0, -5, -10, -15, and -20°C, respectively. When incorporated into a three-dimensional mesh, the predictive model followed a parabolic fit (R(2) = 0.84), where the log reduction = -0.264 - (0.039 × temp) - (3.833 × dose) - (0.0013 × temp(2)) - (0.728 × dose(2)). These results indicate that ionizing radiation would be an effective technology for control of Y. pestis in ready-to-eat fine emulsion sausage products. PMID:21477478

  10. Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans).

    PubMed

    Vernati, G; Edwards, W H; Rocke, T E; Little, S F; Andrews, G P

    2011-01-01

    Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.

  11. Immunological and clinical response of coyotes (Canis latrans) to experimental inoculation with Yersinia pestis.

    PubMed

    Baeten, Laurie A; Pappert, Ryan; Young, John; Schriefer, Martin E; Gidlewski, Thomas; Kohler, Dennis; Bowen, Richard A

    2013-10-01

    Multiple publications have reported the use of coyotes (Canis latrans) in animal-based surveillance efforts for the detection of Yersinia pestis. Coyotes are likely exposed via flea bite or oral routes and are presumed to be resistant to the development of clinical disease. These historic data have only been useful for the evaluation of the geographic distribution of Y. pestis in the landscape. Because the canid immunologic response to Y. pestis has not been thoroughly characterized, we conducted experimental inoculation of captive-reared, juvenile coyotes (n = 8) with Y. pestis CO92 via oral or intradermal routes. We measured the humoral response to Y. pestis fraction 1 capsular protein (anti-F1) and found a significant difference between inoculation groups in magnitude and duration of antibody production. The anti-F1 titers in animals exposed intradermally peaked at day 10 postinoculation (PI; range = 1∶32 to 1∶128) with titers remaining stable at 1∶32 through week 12. In contrast, orally inoculated animals developed higher titers (range = 1∶256 to 1∶1,024) that remained stable at 1∶256 to 1∶512 through week 6. No clinical signs of disease were observed, and minimal changes were noted in body temperature, white blood cell counts, and acute phase proteins during the 7 days PI. Gross pathology was unremarkable, and minimal changes were noted in histopathology at days 3 and 7 PI. Rechallenge at 14 wk PI via similar dosage and routes resulted in marked differences in antibody response between groups. Animals in the orally inoculated group produced a striking increase in anti-F1 titers (up to 1∶4,096) within 3 days, whereas there was minimal to no increase in antibody response in the intradermal group. Information gathered from this experimental trial may provide additional insight into the spatial and temporal evaluation of coyote plague serology.

  12. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

    PubMed

    Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L; Wormald, Michael M; Fast, Randy L; Worsham, Patricia L; Cote, Christopher K; Amemiya, Kei; Dimitrov, Dimiter S

    2010-10-13

    Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  13. Antigenic profiling of Yersinia pestis infection in the Wyoming coyote (Canis latrans)

    USGS Publications Warehouse

    Vernati, G.; Edwards, W.H.; Rocke, T.E.; Little, S.F.; Andrews, G.P.

    2011-01-01

    Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.

  14. Amino acid and structural variability of Yersinia pestis LcrV protein

    SciTech Connect

    Anisimov, A P; Dentovskaya, S V; Panfertsev, E A; Svetoch, T E; Kopylov, P K; Segelke, B W; Zemla, A; Telepnev, M V; Motin, V L

    2009-11-09

    The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium which is generally conserved between the epidemic strains of Yersinia pestis. They investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from ten Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed them to classify the LcrV alleles into five sequence types (A-E). They observed that the strains of different Y. pestis subspecies can have the same typ of LcrV, and different types of LcrV can exist within the same natural plague focus. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in oligomerization of LcrV. The importance of the latter property in emergence of epidemic strains of Y. pestis during evolution of this pathogen will need to be further investigated.

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

    PubMed Central

    Bland, David M.; Hinnebusch, B. Joseph

    2016-01-01

    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

  16. The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background.

    PubMed

    Weening, Eric H; Cathelyn, Jason S; Kaufman, Greer; Lawrenz, Matthew B; Price, Paul; Goldman, William E; Miller, Virginia L

    2011-02-01

    Yersinia pestis is a highly pathogenic Gram-negative organism and the causative agent of bubonic and pneumonic plague. Y. pestis is capable of causing major epidemics; thus, there is a need for vaccine targets and a greater understanding of the role of these targets in pathogenesis. Two prime Y. pestis vaccine candidates are the usher-chaperone fimbriae Psa and Caf. Herein we report that Y. pestis requires, in a nonredundant manner, both PsaA and Caf1 to achieve its full pathogenic ability in both pneumonic and bubonic plague in C57BL/6J mice. Deletion of psaA leads to a decrease in the organ bacterial burden and to a significant increase in the 50% lethal dose (LD₅₀) after subcutaneous infection. Deletion of caf1 also leads to a significant decrease in the organ bacterial burden but more importantly leads to a significantly greater increase in the LD₅₀ than was observed for the ΔpsaA mutant strain after subcutaneous infection of C57BL/6J mice. Furthermore, the degree of attenuation of the Δcaf1 mutant strain is mouse background dependent, as the Δcaf1 mutant strain was attenuated to a lesser degree in BALB/cJ mice by the subcutaneous route than in C57BL/6J mice. This observation that the degree of requirement for Caf1 is dependent on the mouse background indicates that the virulence of Y. pestis is dependent on the genetic makeup of its host and provides further support for the hypothesis that PsaA and Caf1 have different targets. PMID:21115720

  17. Two Distinct Yersinia pestis Populations Causing Plague among Humans in the West Nile Region of Uganda

    PubMed Central

    Respicio-Kingry, Laurel B.; Yockey, Brook M.; Acayo, Sarah; Kaggwa, John; Apangu, Titus; Kugeler, Kiersten J.; Eisen, Rebecca J.; Griffith, Kevin S.; Mead, Paul S.; Schriefer, Martin E.; Petersen, Jeannine M.

    2016-01-01

    Background Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Since the 1990s, Africa has accounted for the majority of reported human cases. In Uganda, plague cases occur in the West Nile region, near the border with Democratic Republic of Congo. Despite the ongoing risk of contracting plague in this region, little is known about Y. pestis genotypes causing human disease. Methodology/Principal Findings During January 2004–December 2012, 1,092 suspect human plague cases were recorded in the West Nile region of Uganda. Sixty-one cases were culture-confirmed. Recovered Y. pestis isolates were analyzed using three typing methods, single nucleotide polymorphisms (SNPs), pulsed field gel electrophoresis (PFGE), and multiple variable number of tandem repeat analysis (MLVA) and subpopulations analyzed in the context of associated geographic, temporal, and clinical data for source patients. All three methods separated the 61 isolates into two distinct 1.ANT lineages, which persisted throughout the 9 year period and were associated with differences in elevation and geographic distribution. Conclusions/Significance We demonstrate that human cases of plague in the West Nile region of Uganda are caused by two distinct 1.ANT genetic subpopulations. Notably, all three typing methods used, SNPs, PFGE, and MLVA, identified the two genetic subpopulations, despite recognizing different mutation types in the Y. pestis genome. The geographic and elevation differences between the two subpopulations is suggestive of their maintenance in highly localized enzootic cycles, potentially with differing vector-host community composition. This improved understanding of Y. pestis subpopulations in the West Nile region will be useful for identifying ecologic and environmental factors associated with elevated plague risk. PMID:26866815

  18. Role of the Yersinia pestis yersiniabactin iron acquisition system in the incidence of flea-borne plague.

    PubMed

    Sebbane, Florent; Jarrett, Clayton; Gardner, Donald; Long, Daniel; Hinnebusch, B Joseph

    2010-12-17

    Plague is a flea-borne zoonosis caused by the bacterium Yersinia pestis. Y. pestis mutants lacking the yersiniabactin (Ybt) siderophore-based iron transport system are avirulent when inoculated intradermally but fully virulent when inoculated intravenously in mice. Presumably, Ybt is required to provide sufficient iron at the peripheral injection site, suggesting that Ybt would be an essential virulence factor for flea-borne plague. Here, using a flea-to-mouse transmission model, we show that a Y. pestis strain lacking the Ybt system causes fatal plague at low incidence when transmitted by fleas. Bacteriology and histology analyses revealed that a Ybt-negative strain caused only primary septicemic plague and atypical bubonic plague instead of the typical bubonic form of disease. The results provide new evidence that primary septicemic plague is a distinct clinical entity and suggest that unusual forms of plague may be caused by atypical Y. pestis strains.

  19. Yersinia pestis activates both IL-1β and IL-1 receptor antagonist to modulate lung inflammation during pneumonic plague.

    PubMed

    Sivaraman, Vijay; Pechous, Roger D; Stasulli, Nikolas M; Eichelberger, Kara R; Miao, Edward A; Goldman, William E

    2015-03-01

    Pneumonic plague is the most rapid and lethal form of Yersinia pestis infection. Increasing evidence suggests that Y. pestis employs multiple levels of innate immune evasion and/or suppression to produce an early "pre-inflammatory" phase of pulmonary infection, after which the disease is highly inflammatory in the lung and 100% fatal. In this study, we show that IL-1β/IL-18 cytokine activation occurs early after bacteria enter the lung, and this activation eventually contributes to pulmonary inflammation and pathology during the later stages of infection. However, the inflammatory effects of IL-1β/IL-1-receptor ligation are not observed during this first stage of pneumonic plague. We show that Y. pestis also activates the induction of IL-1 receptor antagonist (IL-1RA), and this activation likely contributes to the ability of Y. pestis to establish the initial pre-inflammatory phase of disease.

  20. 78 FR 23207 - Availability of an Environmental Assessment for Field Testing of a Yersinia Pestis Vaccine, Live...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-18

    ... Animal and Plant Health Inspection Service Availability of an Environmental Assessment for Field Testing of a Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector AGENCY: Animal and Plant Health Inspection... Plant Health Inspection Service has prepared an environmental assessment concerning authorization...

  1. Differential plague-transmission dynamics determine Yersinia pestis population genetic structure on local, regional, and global scales

    PubMed Central

    Girard, Jessica M.; Wagner, David M.; Vogler, Amy J.; Keys, Christine; Allender, Christopher J.; Drickamer, Lee C.; Keim, Paul

    2004-01-01

    Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly mutable marker loci allow determination of Y. pestis population genetic structure and tracking of transmission patterns at two spatial scales within a single focus. In addition, we found that in vitro mutation rates for these loci are similar to those observed in vivo, which allowed us to develop a mutation-rate-based model to examine transmission mechanisms. Our model suggests there are two primary components of plague ecology: a rapid expansion phase for population growth and dispersal followed by a slower persistence phase. This pattern seems consistent across local, regional, and even global scales. PMID:15173603

  2. YfbA, a Yersinia pestis Regulator Required for Colonization and Biofilm Formation in the Gut of Cat Fleas

    PubMed Central

    Tam, Christina; Demke, Owen; Hermanas, Timothy; Mitchell, Anthony; Hendrickx, Antoni P. A.

    2014-01-01

    For transmission to new hosts, Yersinia pestis, the causative agent of plague, replicates as biofilm in the foregut of fleas that feed on plague-infected animals or humans. Y. pestis biofilm formation has been studied in the rat flea; however, little is known about the cat flea, a species that may bridge zoonotic and anthroponotic plague cycles. Here, we show that Y. pestis infects and replicates as a biofilm in the foregut of cat fleas in a manner requiring hmsFR, two determinants for extracellular biofilm matrix. Examining a library of transposon insertion mutants, we identified the LysR-type transcriptional regulator YfbA, which is essential for Y. pestis colonization and biofilm formation in cat fleas. PMID:24391055

  3. Roles of chaperone/usher pathways of Yersinia pestis in a murine model of plague and adhesion to host cells.

    PubMed

    Hatkoff, Matthew; Runco, Lisa M; Pujol, Celine; Jayatilaka, Indralatha; Furie, Martha B; Bliska, James B; Thanassi, David G

    2012-10-01

    Yersinia pestis and many other Gram-negative pathogenic bacteria use the chaperone/usher (CU) pathway to assemble virulence-associated surface fibers termed pili or fimbriae. Y. pestis has two well-characterized CU pathways: the caf genes coding for the F1 capsule and the psa genes coding for the pH 6 antigen. The Y. pestis genome contains additional CU pathways that are capable of assembling pilus fibers, but the roles of these pathways in the pathogenesis of plague are not understood. We constructed deletion mutations in the usher genes for six of the additional Y. pestis CU pathways. The wild-type (WT) and usher deletion strains were compared in the murine bubonic (subcutaneous) and pneumonic (intranasal) plague infection models. Y. pestis strains containing deletions in CU pathways y0348-0352, y1858-1862, and y1869-1873 were attenuated for virulence compared to the WT strain by the intranasal, but not subcutaneous, routes of infection, suggesting specific roles for these pathways during pneumonic plague. We examined binding of the Y. pestis WT and usher deletion strains to A549 human lung epithelial cells, HEp-2 human cervical epithelial cells, and primary human and murine macrophages. Y. pestis CU pathways y0348-0352 and y1858-1862 were found to contribute to adhesion to all host cells tested, whereas pathway y1869-1873 was specific for binding to macrophages. The correlation between the virulence attenuation and host cell binding phenotypes of the usher deletion mutants identifies three of the additional CU pathways of Y. pestis as mediating interactions with host cells that are important for the pathogenesis of plague.

  4. Duration of plague (Yersinia pestis) outbreaks in black-tailed prairie dog (Cynomys ludovicianus) colonies of northern Colorado.

    PubMed

    St Romain, Krista; Tripp, Daniel W; Salkeld, Daniel J; Antolin, Michael F

    2013-09-01

    Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs. PMID:24057801

  5. CRP-Mediated Carbon Catabolite Regulation of Yersinia pestis Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA

    PubMed Central

    Willias, Stephan P.; Chauhan, Sadhana; Lo, Chien-Chi; Chain, Patrick S. G.; Motin, Vladimir L.

    2015-01-01

    The natural transmission of Yersinia pestis is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate Y. pestis biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon Y. pestis biofilm production. Our findings demonstrate that Y. pestis biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of crp in both CO92 and KIM6+ Y. pestis strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter Y. pestis biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized hms biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote Y. pestis biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon Y. pestis biofilm production. Contrary to what has been described for E. coli, Y. pestis biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of csrA significantly impaired Y. pestis biofilm production. CsrA was found to promote Y. pestis biofilm production independent of glycogen regulation. Loss of csrA did not significantly alter relative hmsH, hmsP, or hmsT mRNA abundance. However, deletion of hmsP in the csrA-deficient mutant enabled excessive biofilm production, suggesting Csr

  6. Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge.

    PubMed

    Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Senina, Svetlana; Perlin, David; Crystal, Ronald G; Boyer, Julie L

    2009-04-01

    Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

  7. A New Generation Microarray for the Simultaneous Detection and Identification of Yersinia pestis and Bacillus anthracis in Food

    PubMed Central

    Goji, Noriko; MacMillan, Trevor; Amoako, Kingsley Kwaku

    2012-01-01

    The use of microarrays as a multiple analytic system has generated increased interest and provided a powerful analytical tool for the simultaneous detection of pathogens in a single experiment. A wide array of applications for this technology has been reported. A low density oligonucleotide microarray was generated from the genetic sequences of Y. pestis and B. anthracis and used to fabricate a microarray chip. The new generation chip, consisting of 2,240 spots in 4 quadrants with the capability of stripping/rehybridization, was designated as “Y-PESTIS/B-ANTHRACIS 4x2K Array.” The chip was tested for specificity using DNA from a panel of bacteria that may be potentially present in food. In all, 37 unique Y. pestis-specific and 83 B. anthracis-specific probes were identified. The microarray assay distinguished Y. pestis and B. anthracis from the other bacterial species tested and correctly identified the Y. pestis-specific oligonucleotide probes using DNA extracted from experimentally inoculated milk samples. Using a whole genome amplification method, the assay was able to detect as low as 1 ng genomic DNA as the start sample. The results suggest that oligonucleotide microarray can specifically detect and identify Y. pestis and B. anthracis and may be a potentially useful diagnostic tool for detecting and confirming the organisms in food during a bioterrorism event. PMID:23125935

  8. Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

    SciTech Connect

    Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.; Wunschel, David S.; Bruckner-Lea, Cindy J.; Hutchison, Janine R.

    2015-12-23

    ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection

  9. Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague.

    PubMed

    Hinnebusch, B Joseph; Jarrett, Clayton O; Callison, Julie A; Gardner, Donald; Buchanan, Susan K; Plano, Gregory V

    2011-12-01

    The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.

  10. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    PubMed Central

    Shou, Yulin; Graves, Steven W.; Bradbury, Andrew R. M.

    2011-01-01

    Background Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection. Methods Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA. Results Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. Conclusions Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of sc

  11. Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis)

    USGS Publications Warehouse

    Rocke, T.E.; Iams, K.P.; Dawe, S.; Smith, S.R.; Williamson, J.L.; Heisey, D.M.; Osorio, J.E.

    2009-01-01

    In previous studies, we demonstrated protection against plague in mice and prairie dogs using a raccoon pox (RCN) virus-vectored vaccine that expressed the F1 capsular antigen of Yersinia pestis. In order to improve vaccine efficacy, we have now constructed additional RCN-plague vaccines containing two different forms of the lcrV (V) gene, including full-length (Vfull) and a truncated form (V307). Mouse challenge studies with Y. pestis strain CO92 showed that vaccination with a combination of RCN-F1 and the truncated V construct (RCN-V307) provided the greatest improvement (P = 0.01) in protection against plague over vaccination with RCN-F1 alone. This effect was mediated primarily by anti-F1 and anti-V antibodies and both contributed independently to increased survival of vaccinated mice.

  12. Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague.

    PubMed Central

    Leary, S E; Williamson, E D; Griffin, K F; Russell, P; Eley, S M; Titball, R W

    1995-01-01

    The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague. PMID:7622205

  13. Crystal Structure of the Protease-Resistant Core Domain of Yersinia Pestis Virulence Factor Yopr

    SciTech Connect

    Schubot,F.; Cherry, S.; Austin, B.; Tropea, J.; Waugh, D.

    2005-01-01

    Yersinia pestis, the causative agent of the plague, employs a type III secretion system (T3SS) to secrete and translocate virulence factors into the cytoplasm of mammalian host cells. One of the secreted virulence factors is YopR. Little is known about the function of YopR other than that it is secreted into the extracellular milieu during the early stages of infection and that it contributes to virulence. Hoping to gain some insight into the function of YopR, we determined the crystal structure of its protease-resistant core domain, which consists of residues 38--149 out of 165 amino acids. The core domain is composed of five {alpha}-helices that display unexpected structural similarity with one domain of YopN, a central regulator of type III secretion in Y. pestis. This finding raises the possibility that YopR may play a role in the regulation of type III secretion.

  14. Effects of land use on plague (Yersinia pestis) activity in rodents in Tanzania.

    PubMed

    McCauley, Douglas J; Salkeld, Daniel J; Young, Hillary S; Makundi, Rhodes; Dirzo, Rodolfo; Eckerlin, Ralph P; Lambin, Eric F; Gaffikin, Lynne; Barry, Michele; Helgen, Kristofer M

    2015-04-01

    Understanding the effects of land-use change on zoonotic disease risk is a pressing global health concern. Here, we compare prevalence of Yersinia pestis, the etiologic agent of plague, in rodents across two land-use types-agricultural and conserved-in northern Tanzania. Estimated abundance of seropositive rodents nearly doubled in agricultural sites compared with conserved sites. This relationship between land-use type and abundance of seropositive rodents is likely mediated by changes in rodent and flea community composition, particularly via an increase in the abundance of the commensal species, Mastomys natalensis, in agricultural habitats. There was mixed support for rodent species diversity negatively impacting Y. pestis seroprevalence. Together, these results suggest that land-use change could affect the risk of local transmission of plague, and raise critical questions about transmission dynamics at the interface of conserved and agricultural habitats. These findings emphasize the importance of understanding disease ecology in the context of rapidly proceeding landscape change.

  15. Proteomic characterization of host response to Yersinia pestis and near neighbors.

    PubMed

    Chromy, Brett A; Perkins, Julie; Heidbrink, Jenny L; Gonzales, Arlene D; Murphy, Gloria A; Fitch, J Patrick; McCutchen-Maloney, Sandra L

    2004-07-23

    Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Yersinia pseudotuberculosis and Yersinia enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.

  16. Susceptibility of the Siberian polecat to subcutaneous and oral Yersinia pestis exposure.

    PubMed

    Castle, K T; Biggins, D; Carter, L G; Chu, M; Innes, K; Wimsatt, J

    2001-10-01

    To determine if the Siberian polecat (Mustela eversmannii) represents a suitable model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela nigripes), polecats were exposed to 10(3), 10(7), or 10(10) Yersinia pestis organisms by subcutaneous injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse. Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 10(3) group, 100% mortality in the 10(7) and 10(10) groups, and 83% mortality in the mouse-fed group). Within the challenged group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly longer than those receiving higher parenteral doses. Within challenged animals, mean survival time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal output; time to onset of lethargy and other signs was also related to risk of dying and/or plague dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while two seropositive animals later died. This study confirms that the Siberian polecat is susceptible to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets in future plague studies and related vaccine trials.

  17. Investigation of Yersinia pestis Laboratory Adaptation through a Combined Genomics and Proteomics Approach

    PubMed Central

    Clowers, Brian H.; Deatherage Kaiser, Brooke L.; Lin, Andy; Hutchison, Janine R.; Melville, Angela M.; Wagner, David M.; Keim, Paul S.; Foster, Jeffrey T.; Kreuzer, Helen W.

    2015-01-01

    The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement. PMID:26599979

  18. [PCR-derived technology in gene identification and typing of Yersinia pestis].

    PubMed

    Wang, Mei; Tang, Xinyuan; Wang, Zuyun

    2015-01-01

    Application of the PCR-derived technology in gene identification and genotypes of different ecotype Yersinia pestis to make the high-throughput experimental results can reflect the epidemic history and compare the diversity in genome, pathogenicity, so that results from these experiments provide an important basis for clinical diagnosis, treatment and origin. But the experiment should be considered typing ability, practicality, budget and other experimental factors or conditions, because each PCR-derivative technology has advantages and disadvantages.

  19. Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

    PubMed Central

    Zhou, Lei; Guo, Zhaobiao; Zhou, Dongsheng; Zhai, Junhui; Yang, Ruifu

    2010-01-01

    Background Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C. Methods/Principal Findings TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥102 copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here. Conclusions/Significance The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance. PMID:20231881

  20. Investigation of Yersinia pestis Laboratory Adaptation through a Combined Genomics and Proteomics Approach.

    PubMed

    Leiser, Owen P; Merkley, Eric D; Clowers, Brian H; Deatherage Kaiser, Brooke L; Lin, Andy; Hutchison, Janine R; Melville, Angela M; Wagner, David M; Keim, Paul S; Foster, Jeffrey T; Kreuzer, Helen W

    2015-01-01

    The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement. PMID:26599979

  1. Molecular Survey of Bartonella Species and Yersinia pestis in Rodent Fleas (Siphonaptera) From Chihuahua, Mexico.

    PubMed

    Fernández-González, Adriana M; Kosoy, Michael Y; Rubio, André V; Graham, Christine B; Montenieri, John A; Osikowicz, Lynn M; Bai, Ying; Acosta-Gutiérrez, Roxana; Ávila-Flores, Rafael; Gage, Kenneth L; Suzán, Gerardo

    2016-01-01

    Rodent fleas from northwestern Chihuahua, Mexico, were analyzed for the presence of Bartonella and Yersinia pestis. In total, 760 fleas belonging to 10 species were tested with multiplex polymerase chain reaction analysis targeting the gltA (338-bp) and pla genes (478-bp) of Bartonella and Y. pestis, respectively. Although none was positive for Y. pestis, 307 fleas were infected with Bartonella spp., resulting in an overall prevalence of 40.4%. A logistic regression analysis indicated that the presence of Bartonella is more likely to occur in some flea species. From a subset of Bartonella-positive fleas, phylogenetic analyses of gltA gene sequences revealed 13 genetic variants clustering in five phylogroups (I–V), two of which were matched with known pathogenic Bartonella species (Bartonella vinsonii subsp. arupensis and Bartonella washoensis) and two that were not related with any previously described species or subspecies of Bartonella. Variants in phylogroup V, which were mainly obtained from Meringis spp. fleas, were identical to those reported recently in their specific rodent hosts (Dipodomys spp.) in the same region, suggesting that kangaroo rats and their fleas harbor other Bartonella species not reported previously. Considering the Bartonella prevalence and the flea genotypes associated with known pathogenic Bartonella species, we suggest that analysis of rodent and flea communities in the region should continue for their potential implications for human health. Given that nearby locations in the United States have reported Y. pestis in wild animals and their fleas, we suggest conducting larger-scale studies to increase our knowledge of this bacterium.

  2. Investigation of Yersinia pestis Laboratory Adaptation through a Combined Genomics and Proteomics Approach.

    PubMed

    Leiser, Owen P; Merkley, Eric D; Clowers, Brian H; Deatherage Kaiser, Brooke L; Lin, Andy; Hutchison, Janine R; Melville, Angela M; Wagner, David M; Keim, Paul S; Foster, Jeffrey T; Kreuzer, Helen W

    2015-01-01

    The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement.

  3. Investigation of Yersinia pestis laboratory adaptation through a combined genomics and proteomics approach

    DOE PAGESBeta

    Leiser, Owen P.; Merkley, Eric D.; Clowers, Brian H.; Kaiser, Brooke L. Deatherage; Lin, Andy; Hutchison, Janine R.; Melville, Angela M.; Wagner, David M.; Keim, Paul S.; Foster, Jeff; et al

    2015-11-24

    Here, the bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a Parallel Serial Passage Experimentmore » (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS-based proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism, envelope biogenesis, iron storage and acquisition, and a type VI secretion system. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement.« less

  4. Molecular Survey of Bartonella Species and Yersinia pestis in Rodent Fleas (Siphonaptera) From Chihuahua, Mexico.

    PubMed

    Fernández-González, Adriana M; Kosoy, Michael Y; Rubio, André V; Graham, Christine B; Montenieri, John A; Osikowicz, Lynn M; Bai, Ying; Acosta-Gutiérrez, Roxana; Ávila-Flores, Rafael; Gage, Kenneth L; Suzán, Gerardo

    2016-01-01

    Rodent fleas from northwestern Chihuahua, Mexico, were analyzed for the presence of Bartonella and Yersinia pestis. In total, 760 fleas belonging to 10 species were tested with multiplex polymerase chain reaction analysis targeting the gltA (338-bp) and pla genes (478-bp) of Bartonella and Y. pestis, respectively. Although none was positive for Y. pestis, 307 fleas were infected with Bartonella spp., resulting in an overall prevalence of 40.4%. A logistic regression analysis indicated that the presence of Bartonella is more likely to occur in some flea species. From a subset of Bartonella-positive fleas, phylogenetic analyses of gltA gene sequences revealed 13 genetic variants clustering in five phylogroups (I–V), two of which were matched with known pathogenic Bartonella species (Bartonella vinsonii subsp. arupensis and Bartonella washoensis) and two that were not related with any previously described species or subspecies of Bartonella. Variants in phylogroup V, which were mainly obtained from Meringis spp. fleas, were identical to those reported recently in their specific rodent hosts (Dipodomys spp.) in the same region, suggesting that kangaroo rats and their fleas harbor other Bartonella species not reported previously. Considering the Bartonella prevalence and the flea genotypes associated with known pathogenic Bartonella species, we suggest that analysis of rodent and flea communities in the region should continue for their potential implications for human health. Given that nearby locations in the United States have reported Y. pestis in wild animals and their fleas, we suggest conducting larger-scale studies to increase our knowledge of this bacterium. PMID:26576933

  5. Cethromycin-mediated protection against the plague pathogen Yersinia pestis in a rat model of infection and comparison with levofloxacin.

    PubMed

    Rosenzweig, Jason A; Brackman, Sheri M; Kirtley, Michelle L; Sha, Jian; Erova, Tatiana E; Yeager, Linsey A; Peterson, Johnny W; Xu, Ze-Qi; Chopra, Ashok K

    2011-11-01

    The Gram-negative plague bacterium, Yersinia pestis, has historically been regarded as one of the deadliest pathogens known to mankind, having caused three major pandemics. After being transmitted by the bite of an infected flea arthropod vector, Y. pestis can cause three forms of human plague: bubonic, septicemic, and pneumonic, with the latter two having very high mortality rates. With increased threats of bioterrorism, it is likely that a multidrug-resistant Y. pestis strain would be employed, and, as such, conventional antibiotics typically used to treat Y. pestis (e.g., streptomycin, tetracycline, and gentamicin) would be ineffective. In this study, cethromycin (a ketolide antibiotic which inhibits bacterial protein synthesis and is currently in clinical trials for respiratory tract infections) was evaluated for antiplague activity in a rat model of pneumonic infection and compared with levofloxacin, which operates via inhibition of bacterial topoisomerase and DNA gyrase. Following a respiratory challenge of 24 to 30 times the 50% lethal dose of the highly virulent Y. pestis CO92 strain, 70 mg of cethromycin per kg of body weight (orally administered twice daily 24 h postinfection for a period of 7 days) provided complete protection to animals against mortality without any toxic effects. Further, no detectable plague bacilli were cultured from infected animals' blood and spleens following cethromycin treatment. The antibiotic was most effective when administered to rats 24 h postinfection, as the animals succumbed to infection if treatment was further delayed. All cethromycin-treated survivors tolerated 2 subsequent exposures to even higher lethal Y. pestis doses without further antibiotic treatment, which was related, in part, to the development of specific antibodies to the capsular and low-calcium-response V antigens of Y. pestis. These data demonstrate that cethromycin is a potent antiplague drug that can be used to treat pneumonic plague.

  6. The effect of growth temperature on the nanoscale biochemical surface properties of Yersinia pestis.

    PubMed

    Wang, Congzhou; Stanciu, Cristina E; Ehrhardt, Christopher J; Yadavalli, Vamsi K

    2016-08-01

    Yersinia pestis, the causative agent of plague, has been responsible for several recurrent, lethal pandemics in history. Currently, it is an important pathogen to study owing to its virulence, adaptation to different environments during transmission, and potential use in bioterrorism. Here, we report on the changes to Y. pestis surfaces in different external microenvironments, specifically culture temperatures (6, 25, and 37 °C). Using nanoscale imaging coupled with functional mapping, we illustrate that changes in the surfaces of the bacterium from a morphological and biochemical standpoint can be analyzed simultaneously using atomic force microscopy. The results from functional mapping, obtained at a single cell level, show that the density of lipopolysaccharide (measured via terminal N-acetylglucosamine) on Y. pestis grown at 37 °C is only slightly higher than cells grown at 25 °C, but nearly three times higher than cells maintained at 6 °C for an extended period of time, thereby demonstrating that adaptations to different environments can be effectively captured using this technique. This nanoscale evaluation provides a new microscopic approach to study nanoscale properties of bacterial pathogens and investigate adaptations to different external environments. PMID:27259520

  7. Protein markers for identification of Yersinia pestis and their variation related to culture

    SciTech Connect

    Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.; Clowers, Brian H.; Sorensen, Christina M.; Valentine, Nancy B.; Mahoney Fahey, Christine M.; Wietsma, Thomas W.; Wahl, Karen L.

    2013-12-11

    The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

  8. Eighteenth century Yersinia pestis genomes reveal the long-term persistence of an historical plague focus

    PubMed Central

    Bos, Kirsten I; Herbig, Alexander; Sahl, Jason; Waglechner, Nicholas; Fourment, Mathieu; Forrest, Stephen A; Klunk, Jennifer; Schuenemann, Verena J; Poinar, Debi; Kuch, Melanie; Golding, G Brian; Dutour, Olivier; Keim, Paul; Wagner, David M; Holmes, Edward C; Krause, Johannes; Poinar, Hendrik N

    2016-01-01

    The 14th–18th century pandemic of Yersinia pestis caused devastating disease outbreaks in Europe for almost 400 years. The reasons for plague’s persistence and abrupt disappearance in Europe are poorly understood, but could have been due to either the presence of now-extinct plague foci in Europe itself, or successive disease introductions from other locations. Here we present five Y. pestis genomes from one of the last European outbreaks of plague, from 1722 in Marseille, France. The lineage identified has not been found in any extant Y. pestis foci sampled to date, and has its ancestry in strains obtained from victims of the 14th century Black Death. These data suggest the existence of a previously uncharacterized historical plague focus that persisted for at least three centuries. We propose that this disease source may have been responsible for the many resurgences of plague in Europe following the Black Death. DOI: http://dx.doi.org/10.7554/eLife.12994.001 PMID:26795402

  9. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    SciTech Connect

    Hong-geller, Elizabeth; Valdez, Yolanda E; Shou, Yulin; Yoshida, Thomas M; Marrone, Babetta L; Dunbar, John

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  10. New insights into how Yersinia pestis adapts to its mammalian host during bubonic plague.

    PubMed

    Pradel, Elizabeth; Lemaître, Nadine; Merchez, Maud; Ricard, Isabelle; Reboul, Angéline; Dewitte, Amélie; Sebbane, Florent

    2014-03-01

    Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, "per-pool" screening method that we have developed. Our data showed that in addition to genes involved in physiological adaptation and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site--probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.

  11. A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim

    PubMed Central

    Feldman, Michal; Harbeck, Michaela; Keller, Marcel; Spyrou, Maria A.; Rott, Andreas; Trautmann, Bernd; Scholz, Holger C.; Päffgen, Bernd; Peters, Joris; McCormick, Michael; Bos, Kirsten; Herbig, Alexander; Krause, Johannes

    2016-01-01

    The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany. PMID:27578768

  12. Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus.

    PubMed

    Zhou, Dongsheng; Tong, Zongzhong; Song, Yajun; Han, Yanping; Pei, Decui; Pang, Xin; Zhai, Junhui; Li, Min; Cui, Baizhong; Qi, Zhizhen; Jin, Lixia; Dai, Ruixia; Du, Zongmin; Wang, Jin; Guo, Zhaobiao; Wang, Jian; Huang, Peitang; Yang, Ruifu

    2004-08-01

    Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from Microtus-related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y. pestis and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.

  13. New Insights into How Yersinia pestis Adapts to Its Mammalian Host during Bubonic Plague

    PubMed Central

    Pradel, Elizabeth; Lemaître, Nadine; Merchez, Maud; Ricard, Isabelle; Reboul, Angéline; Dewitte, Amélie; Sebbane, Florent

    2014-01-01

    Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, “per-pool” screening method that we have developed. Our data showed that in addition to genes involved in physiological adaption and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site – probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability. PMID:24675805

  14. Evaluation of swabs and transport media for the recovery of Yersinia pestis.

    PubMed

    Gilbert, Sarah E; Rose, Laura J; Howard, Michele; Bradley, Meranda D; Shah, Sanjiv; Silvestri, Erin; Schaefer, Frank W; Noble-Wang, Judith

    2014-01-01

    The Government Accountability Office report investigating the surface sampling methods used during the 2001 mail contamination with Bacillus anthracis brought to light certain knowledge gaps that existed regarding environmental sampling with biothreat agents. Should a contamination event occur that involves non-spore forming biological select agents, such as Yersinia pestis, surface sample collection and processing protocols specific for these organisms will be needed. Two Y. pestis strains (virulent and avirulent), four swab types (polyester, macrofoam, rayon, and cotton), two pre-moistening solutions, six transport media, three temperatures, two levels of organic load, and four processing methods (vortexing, sonicating, combined sonicating and vortexing, no agitation) were evaluated to determine the conditions that would yield the highest percent of cultivable Y. pestis cells after storage. The optimum pre-moistening agent/transport media combination varied with the Y. pestis strain and swab type. Directly inoculated macrofoam swabs released the highest percent of cells into solution (93.9% recovered by culture) and rayon swabs were considered the second best swab option (77.0% recovered by culture). Storage at 4°C was found to be optimum for all storage times and transport media. In a worst case scenario, where the Y. pestis strain is not known and sample processing and analyses could not occur until 72h after sampling, macrofoam swabs pre-moistened with PBS supplemented with 0.05% Triton X-100 (PBSTX), stored at 4°C in neutralizing buffer (NB) as a transport medium (PBSTX/NB) or pre-moistened with NB and stored in PBSTX as a transport medium (NB/PBSTX), then vortexed 3min in the transport medium, performed significantly better than all other conditions for macrofoam swabs, regardless of strain tested (mean 12 - 72h recovery of 85.9-105.1%, p<0.001). In the same scenario, two combinations of pre-moistening medium/transport medium were found to be optimal for

  15. VALIDATION OF COOKING TIMES AND TEMPERATURES FOR THERMAL INACTIVATION OF YERSINIA PESTIS STRAINS KIM5 AND CDC-A1112 IN GROUND BEEF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The thermal stability of Yersinia pestis inoculated into retail ground beef (25 per cent fat) and heated in a temperature-controlled water bath or cooked on commercial grills was evaluated. Irradiated ground beef (3-g portions) was inoculated with ca. 6.7 log10 CFU/g of Y. pestis strain KIM5 and hea...

  16. Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

    NASA Astrophysics Data System (ADS)

    Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

    2004-08-01

    Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

  17. Entry of Yersinia pestis into the viable but nonculturable state in a low-temperature tap water microcosm.

    PubMed

    Pawlowski, David R; Metzger, Daniel J; Raslawsky, Amy; Howlett, Amy; Siebert, Gretchen; Karalus, Richard J; Garrett, Stephanie; Whitehouse, Chris A

    2011-01-01

    Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.

  18. Historical Y. pestis Genomes Reveal the European Black Death as the Source of Ancient and Modern Plague Pandemics.

    PubMed

    Spyrou, Maria A; Tukhbatova, Rezeda I; Feldman, Michal; Drath, Joanna; Kacki, Sacha; Beltrán de Heredia, Julia; Arnold, Susanne; Sitdikov, Airat G; Castex, Dominique; Wahl, Joachim; Gazimzyanov, Ilgizar R; Nurgaliev, Danis K; Herbig, Alexander; Bos, Kirsten I; Krause, Johannes

    2016-06-01

    Ancient DNA analysis has revealed an involvement of the bacterial pathogen Yersinia pestis in several historical pandemics, including the second plague pandemic (Europe, mid-14(th) century Black Death until the mid-18(th) century AD). Here we present reconstructed Y. pestis genomes from plague victims of the Black Death and two subsequent historical outbreaks spanning Europe and its vicinity, namely Barcelona, Spain (1300-1420 cal AD), Bolgar City, Russia (1362-1400 AD), and Ellwangen, Germany (1485-1627 cal AD). Our results provide support for (1) a single entry of Y. pestis in Europe during the Black Death, (2) a wave of plague that traveled toward Asia to later become the source population for contemporary worldwide epidemics, and (3) the presence of an historical European plague focus involved in post-Black Death outbreaks that is now likely extinct. PMID:27281573

  19. Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.

    PubMed

    Chen, Yuhuang; Duan, Ran; Li, Xu; Li, Kewei; Liang, Junrong; Liu, Chang; Qiu, Haiyan; Xiao, Yuchun; Jing, Huaiqi; Wang, Xin

    2015-12-01

    The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia.

  20. Historical Y. pestis Genomes Reveal the European Black Death as the Source of Ancient and Modern Plague Pandemics.

    PubMed

    Spyrou, Maria A; Tukhbatova, Rezeda I; Feldman, Michal; Drath, Joanna; Kacki, Sacha; Beltrán de Heredia, Julia; Arnold, Susanne; Sitdikov, Airat G; Castex, Dominique; Wahl, Joachim; Gazimzyanov, Ilgizar R; Nurgaliev, Danis K; Herbig, Alexander; Bos, Kirsten I; Krause, Johannes

    2016-06-01

    Ancient DNA analysis has revealed an involvement of the bacterial pathogen Yersinia pestis in several historical pandemics, including the second plague pandemic (Europe, mid-14(th) century Black Death until the mid-18(th) century AD). Here we present reconstructed Y. pestis genomes from plague victims of the Black Death and two subsequent historical outbreaks spanning Europe and its vicinity, namely Barcelona, Spain (1300-1420 cal AD), Bolgar City, Russia (1362-1400 AD), and Ellwangen, Germany (1485-1627 cal AD). Our results provide support for (1) a single entry of Y. pestis in Europe during the Black Death, (2) a wave of plague that traveled toward Asia to later become the source population for contemporary worldwide epidemics, and (3) the presence of an historical European plague focus involved in post-Black Death outbreaks that is now likely extinct.

  1. Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P.

    PubMed

    Ko, Jae-hyeong; Izadjoo, Mina; Altman, Sidney

    2008-08-01

    External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

  2. Molecualr Cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid

    PubMed Central

    Jahanian-Najafabadi, A.; Soleimani, M.; Azadmanesh, K.; Mostafavi, E.; Majidzadeh-A, K.

    2015-01-01

    Yersinia pestis which is the causative agent of pneumonic plague and distributed in all continents has led to many deaths during the history. Because of its high mortality rate, it must be diagnosed and treated at the earliest time post infection and therefore, rapid diagnostic tests are required. In the present study, we cloned the coding sequence of F1 capsular antigen of the bacteria in the pBAD/gIII plasmid for later expression and purification of the protein to produce poly and monoclonal antibodies against this antigen, and subsequently to develop rapid and efficient diagnostics tools for Y. pestis infections. PMID:26430461

  3. Protective efficacy of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and nonencapsulated Yersinia pestis.

    PubMed

    Andrews, G P; Strachan, S T; Benner, G E; Sample, A K; Anderson, G W; Adamovicz, J J; Welkos, S L; Pullen, J K; Friedlander, A M

    1999-03-01

    To evaluate the role of Yersinia outer proteins (Yops) in conferring protective immunity against plague, six yop loci from Yersinia pestis were individually amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant proteins were purified and injected into mice. Most Yop-vaccinated animals succumbed to infection with either wild-type encapsulated Y. pestis or a virulent, nonencapsulated isogenic variant. Vaccination with YpkA significantly prolonged mean survival time but did not increase overall survival of mice infected with the nonencapsulated strain. The only significant protection against death was observed in YopD-vaccinated mice challenged with the nonencapsulated strain. PMID:10024607

  4. Polymorphism of the Cysteine Protease YopT from Yersinia pestis.

    PubMed

    Platonov, Mikhail E; Svetoch, Tat'yana E; Evseeva, Vera V; Knyazeva, Anastasiya I; Dentovskaya, Svetlana V; Motin, Vladimir L; Uversky, Vladimir N; Anisimov, Andrey P

    2016-01-01

    Antibiotic therapy of plague is hampered by the recent isolation of Yersinia pestis strain resistant to all of antibiotics recommended for cure. This has constrained a quest for new antimicrobials taking aim at alternative targets. Recently Y. pestis cysteine protease YopT has been explored as a potential drug target. Targets conserved in the pathogen populations should be more efficacious; therefore, we evaluated intraspecies variability in yopT genes and their products. 114 Y. pestis isolates were screened. Only two YopT full-size isoforms were found among them. The endemic allele (N149) was present in biovar caucasica from Dagestan-highland natural plague focus # 39. The biovar caucasica strains from Transcaucasian highland (# 4-6) and Pre-Araks (# 7) plague foci also contained the N149 allele. These strains from foci # 4 7 possessed a truncated version of YopT that was a consequence of a frame-shift due to the deletion of a single nucleotide at position 71 bp. Computational analyses showed that although the SNP at the position 149 has a very minimal effect of the intrinsic disorder propensity of YopT proteins, whereas the N-terminal truncations of the YopT detected in bv. caucasica strains Pestoides F_YopT1 and F_YopT2, and Pestoides G generated isoforms with the significantly modified intrinsic disorder propensities and with reduced capability to interact with lost ability to utilize their N-terminal tail for the disorder-based interactions with biological partners. Considering that representatives of biovar caucasica were reported to be the reason of sporadic cases of human plague, this study supports the necessity of additional testing of globally disseminated YopT (S149) isoform as a potential target for treatment of plague caused by the strains producing different YopT isoforms. PMID:26845766

  5. Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide

    PubMed Central

    Chung, Hak Suk; Raetz, Christian R. H.

    2011-01-01

    Several Gram-negative pathogens, including Yersinia pestis, Burkholderia cepacia, and Acinetobacter haemolyticus, synthesize an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), known as d-glycero-d-talo-oct-2-ulosonic acid (Ko), in which the axial hydrogen atom at the Kdo 3-position is replaced with OH. Here we report a unique Kdo 3-hydroxylase (KdoO) from Burkholderia ambifaria and Yersinia pestis, encoded by the bamb_0774 (BakdoO) and the y1812 (YpkdoO) genes, respectively. When expressed in heptosyl transferase-deficient Escherichia coli, these genes result in conversion of the outer Kdo unit of Kdo2-lipid A to Ko in an O2-dependent manner. KdoO contains the putative iron-binding motif, HXDXn>40H. Reconstitution of KdoO activity in vitro with Kdo2-lipid A as the substrate required addition of Fe2+, α-ketoglutarate, and ascorbic acid, confirming that KdoO is a Fe2+/α-ketoglutarate/O2-dependent dioxygenase. Conversion of Kdo to Ko in Kdo2-lipid A conferred reduced susceptibility to mild acid hydrolysis. Although two enzymes that catalyze Fe2+/α-ketoglutarate/O2-dependent hydroxylation of deoxyuridine in fungal extracts have been reported previously, kdoO is the first example of a gene encoding a deoxy-sugar hydroxylase. Homologues of KdoO are found exclusively in Gram-negative bacteria, including the human pathogens Burkholderia mallei, Yersinia pestis, Klebsiella pneumoniae, Legionella longbeachae, and Coxiella burnetii, as well as the plant pathogen Ralstonia solanacearum. PMID:21178073

  6. Bacillus anthracis, Francisella tularensis and Yersinia pestis. The most important bacterial warfare agents - review.

    PubMed

    Pohanka, M; Skládal, P

    2009-01-01

    There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.

  7. Detection of Rickettsia felis, Rickettsia typhi, Bartonella Species and Yersinia pestis in Fleas (Siphonaptera) from Africa

    PubMed Central

    Leulmi, Hamza; Socolovschi, Cristina; Laudisoit, Anne; Houemenou, Gualbert; Davoust, Bernard; Bitam, Idir; Raoult, Didier; Parola, Philippe

    2014-01-01

    Little is known about the presence/absence and prevalence of Rickettsia spp, Bartonella spp. and Yersinia pestis in domestic and urban flea populations in tropical and subtropical African countries. Methodology/Principal findings Fleas collected in Benin, the United Republic of Tanzania and the Democratic Republic of the Congo were investigated for the presence and identity of Rickettsia spp., Bartonella spp. and Yersinia pestis using two qPCR systems or qPCR and standard PCR. In Xenopsylla cheopis fleas collected from Cotonou (Benin), Rickettsia typhi was detected in 1% (2/199), and an uncultured Bartonella sp. was detected in 34.7% (69/199). In the Lushoto district (United Republic of Tanzania), R. typhi DNA was detected in 10% (2/20) of Xenopsylla brasiliensis, and Rickettsia felis was detected in 65% (13/20) of Ctenocephalides felis strongylus, 71.4% (5/7) of Ctenocephalides canis and 25% (5/20) of Ctenophthalmus calceatus calceatus. In the Democratic Republic of the Congo, R. felis was detected in 56.5% (13/23) of Ct. f. felis from Kinshasa, in 26.3% (10/38) of Ct. f. felis and 9% (1/11) of Leptopsylla aethiopica aethiopica from Ituri district and in 19.2% (5/26) of Ct. f. strongylus and 4.7% (1/21) of Echidnophaga gallinacea. Bartonella sp. was also detected in 36.3% (4/11) of L. a. aethiopica. Finally, in Ituri, Y. pestis DNA was detected in 3.8% (1/26) of Ct. f. strongylus and 10% (3/30) of Pulex irritans from the villages of Wanyale and Zaa. Conclusion Most flea-borne infections are neglected diseases which should be monitored systematically in domestic rural and urban human populations to assess their epidemiological and clinical relevance. Finally, the presence of Y. pestis DNA in fleas captured in households was unexpected and raises a series of questions regarding the role of free fleas in the transmission of plague in rural Africa, especially in remote areas where the flea density in houses is high. PMID:25299702

  8. Yersiniae other than Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis: the ignored species.

    PubMed

    Sulakvelidze, A

    2000-04-01

    The genus Yersinia is composed of 11 species, of which three (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) have been exhaustively characterized. The remaining eight species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) have not been studied extensively and, because of the absence of classical Yersinia virulence markers, are generally considered to be nonpathogenic. However, recent data suggest that some of these eight species may cause disease by virtue of their having virulence factors distinct from those of Y. enterocolitica. These data raise intriguing questions about the mechanisms by which these species interact with their host cells and elicit human disease.

  9. Genome-scale reconstruction of the metabolic network in Yersinia pestis CO92

    NASA Astrophysics Data System (ADS)

    Navid, Ali; Almaas, Eivind

    2007-03-01

    The gram-negative bacterium Yersinia pestis is the causative agent of bubonic plague. Using publicly available genomic, biochemical and physiological data, we have developed a constraint-based flux balance model of metabolism in the CO92 strain (biovar Orientalis) of this organism. The metabolic reactions were appropriately compartmentalized, and the model accounts for the exchange of metabolites, as well as the import of nutrients and export of waste products. We have characterized the metabolic capabilities and phenotypes of this organism, after comparing the model predictions with available experimental observations to evaluate accuracy and completeness. We have also begun preliminary studies into how cellular metabolism affects virulence.

  10. Differential Regulation of c-di-GMP Metabolic Enzymes by Environmental Signals Modulates Biofilm Formation in Yersinia pestis.

    PubMed

    Ren, Gai-Xian; Fan, Sai; Guo, Xiao-Peng; Chen, Shiyun; Sun, Yi-Cheng

    2016-01-01

    Cyclic diguanylate (c-di-GMP) is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs), HmsT and HmsD and one phosphodiesterase (PDE), HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD, and HmsP in Y. pestis. Biofilm formation was higher in the presence of non-lethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulate activity of DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments. PMID:27375563

  11. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

    PubMed Central

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  12. Growth of a plasmid-bearing (pYV) Yersinia pestis KIM5 in retail raw ground pork

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia pestis can cause oro-pharyngeal plague as a result of consumption or handling of meat from infected animals. Thus, food naturally or intentionally contaminated can have a role in the dissemination of human plague. The growth of a conditionally virulent plasmid (pYV)-bearing rifampicin-res...

  13. Differential Regulation of c-di-GMP Metabolic Enzymes by Environmental Signals Modulates Biofilm Formation in Yersinia pestis.

    PubMed

    Ren, Gai-Xian; Fan, Sai; Guo, Xiao-Peng; Chen, Shiyun; Sun, Yi-Cheng

    2016-01-01

    Cyclic diguanylate (c-di-GMP) is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs), HmsT and HmsD and one phosphodiesterase (PDE), HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD, and HmsP in Y. pestis. Biofilm formation was higher in the presence of non-lethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulate activity of DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

  14. Inactivation of avirulent Yersinia pestis in butterfield's phosphate buffer and frankfurters by UVC (254 nm) and gamma irradiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia pestis is the causative agent of plague. While rare, pharyngeal plague in humans has been associated with consumption or handling of meat prepared from infected animals. The risks of contracting plague from consumption of deliberately contaminated meat are currently unknown. Gamma radiat...

  15. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes.

    PubMed

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  16. Differential Regulation of c-di-GMP Metabolic Enzymes by Environmental Signals Modulates Biofilm Formation in Yersinia pestis

    PubMed Central

    Ren, Gai-Xian; Fan, Sai; Guo, Xiao-Peng; Chen, Shiyun; Sun, Yi-Cheng

    2016-01-01

    Cyclic diguanylate (c-di-GMP) is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs), HmsT and HmsD and one phosphodiesterase (PDE), HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD, and HmsP in Y. pestis. Biofilm formation was higher in the presence of non-lethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulate activity of DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments. PMID:27375563

  17. [Yersinia pestis factors, assuring circulation and maintenance of the plague pathogen in natural foci ecosystems. Report 1].

    PubMed

    Anisimov, A P

    2002-01-01

    Everlasting reproduction of Yersinia pestis, plague bacillus in natural pestholes needs virulent causative agent to invade into the host entity, be potent to overcome protection powers of the rodent organism and to pullulate to entail bacteriemia for subsequent conveyance the plague bacillus to the new host by fleas. All of legs of life cyclic patterns of Yersinia pestis are maintained by a number of plague bacillus factors acting jointly or separately, participating at the different stages of infectious process or conveyance. However these factors provide the perpetuation of the plague bacillus in the ecosystems of natural pestholes only acting in conjunction independently on their distinct contributions. Not only biomolecules, organellas and bacteria systems ensured the pursuance of virulent properties, but other factors, essential for survival of Yersinia pestis and the relationship between separate virulence factors and expression of the different genes of housekeeping and virulence of plague bacillus are considered in this review. The report I covers the problems concerned with adaptational plasticity of Yersinia pestis, it represents the classification of plague causative factors, securing its perpetuation in the environmental space, and discussion the factors promoting plague bacillus survival in the host entity. Not only wellknown publications, but papers in out-of-the-way or hard-to-reach, especially for English-reading experts, editions, also were used to compile this communication. The English version of this review may be requested from Alerton Press. PMID:12243063

  18. Effects of Land Use on Plague (Yersinia pestis) Activity in Rodents in Tanzania

    PubMed Central

    McCauley, Douglas J.; Salkeld, Daniel J.; Young, Hillary S.; Makundi, Rhodes; Dirzo, Rodolfo; Eckerlin, Ralph P.; Lambin, Eric F.; Gaffikin, Lynne; Barry, Michele; Helgen, Kristofer M.

    2015-01-01

    Understanding the effects of land-use change on zoonotic disease risk is a pressing global health concern. Here, we compare prevalence of Yersinia pestis, the etiologic agent of plague, in rodents across two land-use types—agricultural and conserved—in northern Tanzania. Estimated abundance of seropositive rodents nearly doubled in agricultural sites compared with conserved sites. This relationship between land-use type and abundance of seropositive rodents is likely mediated by changes in rodent and flea community composition, particularly via an increase in the abundance of the commensal species, Mastomys natalensis, in agricultural habitats. There was mixed support for rodent species diversity negatively impacting Y. pestis seroprevalence. Together, these results suggest that land-use change could affect the risk of local transmission of plague, and raise critical questions about transmission dynamics at the interface of conserved and agricultural habitats. These findings emphasize the importance of understanding disease ecology in the context of rapidly proceeding landscape change. PMID:25711606

  19. In Vivo Transcriptional Profiling of Yersinia pestis Reveals a Novel Bacterial Mediator of Pulmonary Inflammation

    PubMed Central

    Pechous, Roger D.; Broberg, Christopher A.; Stasulli, Nikolas M.; Miller, Virginia L.

    2015-01-01

    ABSTRACT Inhalation of Yersinia pestis results in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed the in vivo transcription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes, ybtX, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion of ybtX in another lethal respiratory pathogen, Klebsiella pneumoniae, also resulted in diminished host inflammation during infection. Thus, our in vivo transcriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia. PMID:25691593

  20. Forensic Signature Detection of Yersinia Pestis Culturing Practices Across Institutions Using a Bayesian Network

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Corley, Courtney D.; McCue, Lee Ann; Clowers, Brian H.; Dowling, Chase P.; Wahl, Karen L.; Wunschel, David S.; Kreuzer, Helen W.

    2014-03-21

    The field of bioforensics is focused on the analysis of evidence from a biocrime. Existing laboratory analyses can identify the specific strain of an organism in the evidence, as well signatures of the specific culture batch of organisms, such as low-frequency contaminants or indicators of growth and processing methods. To link these disparate types of physical data to potential suspects, investigators may need to identify institutions or individuals whose access to strains and culturing practices match those identified from the evidence. In this work we present a Bayesian statistical network to fuse different types of analytical measurements that predict the production environment of a Yersinia pestis sample under investigation with automated test processing of scientific publications to identify institutions with a history of growing Y. pestis under similar conditions. Furthermore, the textual and experimental signatures were evaluated recursively to determine the overall sensitivity of the network across all levels of false positives. We illustrate that institutions associated with several specific culturing practices can be accurately selected based on the experimental signature from only a few analytical measurements. These findings demonstrate that similar Bayesian networks can be generated generically for many organisms of interest and their deployment is not prohibitive due to either computational or experimental factors.

  1. Biomarker Candidate Identification in Yersinia Pestis Using Organism-Wide Semiquantitative Proteomics

    SciTech Connect

    Hixson, Kim K.; Adkins, Joshua N.; Baker, Scott E.; Moore, Ronald J.; Smith, Richard D.; McCutchen-Maloney, Sandra L.; Lipton, Mary S.

    2006-11-03

    Yersinia pestis, the causative agent of plague, is listed by the CDC as a level A select pathogen. To better enable detection, intervention and treatment of Y. pestis infections, it is necessary to understand its protein expression under conditions that promote or inhibit virulence. To this end, we have utilized a novel combination of the accurate mass and time tag methodology of mass spectrometry and clustering analysis using OmniViz™ to compare the protein abundance changes of 992 identified proteins under four growth conditions. Temperature and Ca2+ concentration were used to trigger virulence associated protein expression fundamental to the low calcium response. High-resolution liquid chromatography and electrospray ionization mass spectrometry were utilized to determine protein identity and abundance on the genome-wide level. The cluster analyses revealed, in a rapid visual platform, the reproducibility of the current method as well as relevant protein abundance changes of expected and novel proteins relating to a specific growth condition and sub-cellular location. Using this method, 89 proteins were identified as having a similar abundance change profile to 29 known virulence associated proteins, providing additional biomarker candidates for future detection and vaccine development strategies.

  2. Genome-Wide Mutant Fitness Profiling Identifies Nutritional Requirements for Optimal Growth of Yersinia pestis in Deep Tissue

    PubMed Central

    Palace, Samantha G.; Proulx, Megan K.; Lu, Shan; Baker, Richard E.

    2014-01-01

    ABSTRACT Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes—encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)—both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes. PMID:25139902

  3. The Yfe and Feo transporters are involved in microaerobic growth and virulence of Yersinia pestis in bubonic plague.

    PubMed

    Fetherston, Jacqueline D; Mier, Ildefonso; Truszczynska, Helena; Perry, Robert D

    2012-11-01

    The Yfe/Sit and Feo transport systems are important for the growth of a variety of bacteria. In Yersinia pestis, single mutations in either yfe or feo result in reduced growth under static (limited aeration), iron-chelated conditions, while a yfe feo double mutant has a more severe growth defect. These growth defects were not observed when bacteria were grown under aerobic conditions or in strains capable of producing the siderophore yersiniabactin (Ybt) and the putative ferrous transporter FetMP. Both fetP and a downstream locus (flp for fet linked phenotype) were required for growth of a yfe feo ybt mutant under static, iron-limiting conditions. An feoB mutation alone had no effect on the virulence of Y. pestis in either bubonic or pneumonic plague models. An feo yfe double mutant was still fully virulent in a pneumonic plague model but had an ∼90-fold increase in the 50% lethal dose (LD(50)) relative to the Yfe(+) Feo(+) parent strain in a bubonic plague model. Thus, Yfe and Feo, in addition to Ybt, play an important role in the progression of bubonic plague. Finally, we examined the factors affecting the expression of the feo operon in Y. pestis. Under static growth conditions, the Y. pestis feo::lacZ fusion was repressed by iron in a Fur-dependent manner but not in cells grown aerobically. Mutations in feoC, fnr, arcA, oxyR, or rstAB had no significant effect on transcription of the Y. pestis feo promoter. Thus, the factor(s) that prevents repression by Fur under aerobic growth conditions remains to be identified.

  4. Complete Genome Sequence of Yersinis pestis Strains Antiqua and Nepa1516: Evidence of Gene Reduction in an Emerging Pathogen

    SciTech Connect

    Chain, Patrick S; Hu, Ping; Malfatti, Stephanie; Radnedge, Lyndsay; Larimer, Frank W; Vergez, Lisa; Worsham, Patricia; Chu, May C; Anderson, Gary L

    2006-01-01

    Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the ''classical'' antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.

  5. Complete Genome Sequence of Yersinia pestis Strains Antiqua and Nepal516: Evidence of Gene Reduction in an Emerging Pathogen†

    PubMed Central

    Chain, Patrick S. G.; Hu, Ping; Malfatti, Stephanie A.; Radnedge, Lyndsay; Larimer, Frank; Vergez, Lisa M.; Worsham, Patricia; Chu, May C.; Andersen, Gary L.

    2006-01-01

    Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the “classical” antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans. PMID:16740952

  6. Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

    SciTech Connect

    Zhang, C G; Gonzales, A D; Choi, M W; Chromy, B A; Fitch, J P; McCutchen-Maloney, S L

    2004-05-20

    Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in human monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different

  7. Real-Time Characterization of Virulence Factor Expression in Yersinia pestis Using a Green Fluorescent Protein Reporter System

    SciTech Connect

    Forde, C; Rocco, J; Fitch, J P; McCutchen-Maloney, S

    2004-06-09

    A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE/yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time. Basal expression levels observed for the Y. pestis promoters, expressed as percentages of the positive control with GFP under the control of the lac promoter, were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%) and yscN (0.8%). The yopE reporter showed the strongest gene induction following temperature transition from 26 C to 37 C. The induction levels of the other virulence factors, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11 fold), yscN (7 fold), yopK (6 fold), lcrE (3 fold), yopT (2 fold), and sycE (2 fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells.

  8. Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages.

    PubMed

    Noel, Betty L; Lilo, Sarit; Capurso, Daniel; Hill, Jim; Bliska, James B

    2009-10-01

    Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-gamma)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-gamma and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-gamma impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-gamma. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-gamma-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-gamma to survive and induce apoptosis in murine macrophages.

  9. Yersinia pestis Can Bypass Protective Antibodies to LcrV and Activation with Gamma Interferon To Survive and Induce Apoptosis in Murine Macrophages ▿

    PubMed Central

    Noel, Betty L.; Lilo, Sarit; Capurso, Daniel; Hill, Jim; Bliska, James B.

    2009-01-01

    Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-γ)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-γ and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-γ impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-γ. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-γ-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-γ to survive and induce apoptosis in murine macrophages. PMID:19710295

  10. Application of the flow cytometry for determination of the amount of DNA in Yersinia pestis cells under the influence of serotonin (5-hydroxytryptamine)

    NASA Astrophysics Data System (ADS)

    Korsukov, Vladimir N.; Shchukovskaya, Tatyana N.; Kravtsov, Alexander L.; Popov, Youri A.

    2002-07-01

    Using flow cytometry a low DNA content in inoculated Yersinia pestis EV cells have been shown at the beginning of culture in Hottinger broth pH 7.2. The dependence serotonin action of its concentration on DNA content have been demonstrated. Serotonin accelerated Yersinia pestis culture growth during cultivation in Hottinger broth pH 7.2 both at 28 degrees C and 37 degrees C at concentration of 10-5 M.

  11. Single-Nucleotide Polymorphisms Reveal Spatial Diversity Among Clones of Yersinia pestis During Plague Outbreaks in Colorado and the Western United States

    PubMed Central

    Antolin, Michael F.; Andersen, Gary L.; Hu, Ping; Stokowski, Renee P.; Gage, Kenneth L.

    2015-01-01

    Abstract Background: In western North America, plague epizootics caused by Yersinia pestis appear to sweep across landscapes, primarily infecting and killing rodents, especially ground squirrels and prairie dogs. During these epizootics, the risk of Y. pestis transmission to humans is highest. While empirical models that include climatic conditions and densities of rodent hosts and fleas can predict when epizootics are triggered, bacterial transmission patterns across landscapes, and the scale at which Y. pestis is maintained in nature during inter-epizootic periods, are poorly defined. Elucidating the spatial extent of Y. pestis clones during epizootics can determine whether bacteria are propagated across landscapes or arise independently from local inter-epizootic maintenance reservoirs. Material and Methods: We used DNA microarray technology to identify single-nucleotide polymorphisms (SNPs) in 34 Y. pestis isolates collected in the western United States from 1980 to 2006, 21 of which were collected during plague epizootics in Colorado. Phylogenetic comparisons were used to elucidate the hypothesized spread of Y. pestis between the mountainous Front Range and the eastern plains of northern Colorado during epizootics. Isolates collected from across the western United States were included for regional comparisons. Results: By identifying SNPs that mark individual clones, our results strongly suggest that Y. pestis is maintained locally and that widespread epizootic activity is caused by multiple clones arising independently at small geographic scales. This is in contrast to propagation of individual clones being transported widely across landscapes. Regionally, our data are consistent with the notion that Y. pestis diversifies at relatively local scales following long-range translocation events. We recommend that surveillance and prediction by public health and wildlife management professionals focus more on models of local or regional weather patterns and

  12. Extraction of Aerosol-Deposited Yersinia pestis from Indoor Surfaces To Determine Bacterial Environmental Decay

    PubMed Central

    Bartlett, Ryan A.; Yeager, John J.; Leroux, Brian; Ratnesar-Shumate, Shanna; Dabisch, Paul

    2016-01-01

    ABSTRACT Public health and decontamination decisions following an event that causes indoor contamination with a biological agent require knowledge of the environmental persistence of the agent. The goals of this study were to develop methods for experimentally depositing bacteria onto indoor surfaces via aerosol, evaluate methods for sampling and enumerating the agent on surfaces, and use these methods to determine bacterial surface decay. A specialized aerosol deposition chamber was constructed, and methods were established for reproducible and uniform aerosol deposition of bacteria onto four coupon types. The deposition chamber facilitated the control of relative humidity (RH; 10 to 70%) following particle deposition to mimic the conditions of indoor environments, as RH is not controlled by standard heating, ventilation, and air conditioning (HVAC) systems. Extraction and culture-based enumeration methods to quantify the viable bacteria on coupons were shown to be highly sensitive and reproducible. To demonstrate the usefulness of the system for decay studies, Yersinia pestis persistence as a function of surface type at 21°C and 40% RH was determined to be >40%/min for all surfaces. Based upon these results, at typical indoor temperature and RH, a 6-log reduction in titer would expected to be achieved within 1 h as the result of environmental decay on surfaces without active decontamination. The developed approach will facilitate future persistence and decontamination studies with a broad range of biological agents and surfaces, providing agent decay data to inform both assessments of risk to personnel entering a contaminated site and decontamination decisions following biological contamination of an indoor environment. IMPORTANCE Public health and decontamination decisions following contamination of an indoor environment with a biological agent require knowledge of the environmental persistence of the agent. Previous studies on Y. pestis persistence have

  13. Toward a molecular pathogenic pathway for Yersinia pestis YopM

    PubMed Central

    Uittenbogaard, Annette M.; Chelvarajan, R. Lakshman; Myers-Morales, Tanya; Gorman, Amanda A.; Brickey, W. June; Ye, Zhan; Kaplan, Alan M.; Cohen, Donald A.; Ting, Jenny P.-Y.; Straley, Susan C.

    2012-01-01

    YopM is one of the six “effector Yops” of the human-pathogenic Yersinia, but its mechanism has not been defined. After delivery to J774A.1 monocyte-like cells, YopM can rapidly bind and activate the serine/threonine kinases RSK1 and PRK2. However, in infected mice, effects of Y. pestis YopM have been seen only after 24–48 h post-infection (p.i.). To identify potential direct effects of YopM in-vivo we tested for effects of YopM at 1 h and 16–18 h p.i. in mice infected systemically with 106 bacteria. At 16 h p.i., there was a robust host response to both parent and ΔyopM-1 Y. pestis KIM5. Compared to cells from non-infected mice, CD11b+ cells from spleens of infected mice produced more than 100-fold greater IFNγ. In the corresponding sera there were more than 100-fold greater amounts of IFNγ, G-CSF, and CXCL9, as well as more than 10-fold greater amounts of IL-6, CXCL10, and CXCL1. The only YopM-related differences were slightly lower CXCL10 and IL-6 in sera from mice infected 16 h with parent compared to ΔyopM-1 Y. pestis. Microarray analysis of the CD11b+ cells did not identify consistent transcriptional differences of ≥4-fold at 18 h p.i. However, at 1 h p.i. mRNA for early growth response transcription factor 1 (Egr1) was decreased when YopM was present. Bone marrow-derived macrophages infected for 1 h also expressed lower Egr1 message when YopM was present. Infected J774A.1 cells showed greater expression of Egr1 at 1 h p.i. when YopM was present, but this pattern reversed at 3 h. At 6 h p.i., Cxcl10 mRNA was lower in parent-strain infected cells. We conclude that decreased Egr1 expression is a very early transcriptional effect of YopM and speculate that a pathway may exist from RSK1 through Egr1. These studies revealed novel early transcriptional effects of YopM but point to a time after 18 h of infection when critical transitional events lead to later major effects on cytokine gene transcription. PMID:23248776

  14. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD.

    PubMed

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3',5'-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  15. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  16. [Change in the habitat of Yersinia pestis in the Gorno-Altaisk natural focus of plague].

    PubMed

    Korzun, V M; Chipanin, E V; Balakhonov, S V; Denisov, A V; Rozhdestvenskiĭ, E N; Mihaĭlov, E P; Iarygina, M B; Kosilko, S A

    2014-01-01

    The paper analyzes the change that occurred in the habitat of the causative agent of plague in its Gorno-Altaisk natural focus in 1961 to 2012. Since 1961 when the plague microbe was found to come from the southern slopes of the Saylyugem mountain range, which are located in Mongolia, to the northern slopes situated in Russia, a gradual expansion of the habitat of Yersenia pestis subsp. altaica had commenced in South-Eastern Altai. During the considered period, the area where epizootic manifestations were registered showed an 11-fold increase. In most cases, the spread of the plague pathogen within the focus was natural and occurred in the successive and closely related settlements of Mongolian pikas (Ochotona pallasi). By now, the plague microbe has been widely distributed in three populations of this small animal, which inhabit the territory of South-Eastern Altai.

  17. Seroprevalence rates and transmission of plague (Yersinia pestis) in mammalian carnivores.

    PubMed

    Salkeld, D J; Stapp, P

    2006-01-01

    Exposure to plague (Yersinia pestis) by flea-bites or consumption of infected rodents is common in mammalian carnivores in North America. Most carnivore species exhibit seroprevalence rates ranging from 3% to 100% in areas where plague occurs. Seroprevalence is highest in mustelids, intermediate in ursids, felids, and canids, and lowest in procyonids, probably reflecting variation in exposure rates as a function of dietary habits. Although conventional wisdom suggests that carnivores may only be important in plague ecology as vectors of infective fleas, animal-to-human (zoonotic) transmission suggests that mammalian carnivores can act as infectious hosts. Furthermore, a review of clinical investigations reveals that plague can be harvested from canid and felid hosts, and suggests the possibility of plague transmission between carnivores. Further study of plague transmission by carnivores in both wild and laboratory conditions is needed to understand the possible role of carnivores as wildlife reservoirs of plague. PMID:16989561

  18. Seroprevalence rates and transmission of plague (Yersinia pestis) in mammalian carnivores.

    PubMed

    Salkeld, D J; Stapp, P

    2006-01-01

    Exposure to plague (Yersinia pestis) by flea-bites or consumption of infected rodents is common in mammalian carnivores in North America. Most carnivore species exhibit seroprevalence rates ranging from 3% to 100% in areas where plague occurs. Seroprevalence is highest in mustelids, intermediate in ursids, felids, and canids, and lowest in procyonids, probably reflecting variation in exposure rates as a function of dietary habits. Although conventional wisdom suggests that carnivores may only be important in plague ecology as vectors of infective fleas, animal-to-human (zoonotic) transmission suggests that mammalian carnivores can act as infectious hosts. Furthermore, a review of clinical investigations reveals that plague can be harvested from canid and felid hosts, and suggests the possibility of plague transmission between carnivores. Further study of plague transmission by carnivores in both wild and laboratory conditions is needed to understand the possible role of carnivores as wildlife reservoirs of plague.

  19. Temperature regulation of the hemin storage (Hms+) phenotype of Yersinia pestis is posttranscriptional.

    PubMed

    Perry, Robert D; Bobrov, Alexander G; Kirillina, Olga; Jones, Heather A; Pedersen, Lisa; Abney, Jennifer; Fetherston, Jacqueline D

    2004-03-01

    In Yersinia pestis, the Congo red (and hemin) binding that is characteristic of the Hms+ phenotype occurs at temperatures up to 34 degrees C but not at higher temperatures. Manifestation of the Hms+ phenotype requires at least five proteins (HmsH, -F, -R, -S, and -T) that are organized into two separate operons: hmsHFRS and hmsT. HmsH and HmsF are outer membrane proteins, while HmsR, HmsS, and HmsT are predicted to be inner membrane proteins. We have used transcriptional reporter constructs, RNA dot blots, and Western blots to examine the expression of hms operons and proteins. Our studies indicate that transcription from the hmsHFRS and hmsT promoters is not regulated by the iron status of the cells, growth temperature, or any of the Hms proteins. In addition, the level of mRNA for both operons is not significantly affected by growth temperature. However, protein levels of HmsH, HmsR, and HmsT in cells grown at 37 degrees C are very low compared to those in cells grown at 26 degrees C, while the amounts of HmsF and HmsS show only a moderate reduction at the higher growth temperature. Neither the Pla protease nor a putative endopeptidase (Y2360) encoded upstream of hmsH is essential for temperature regulation of the Hms+ phenotype. However, HmsT at 37 degrees C is sensitive to degradation by Lon and/or ClpPX. Thus, the stability of HmsH, HmsR, and HmsT proteins likely plays a role in temperature regulation of the Hms+ phenotype of Y. pestis. PMID:14996794

  20. Evaluation of whole cell fixation methods for the analysis of nanoscale surface features of Yersinia pestis KIM.

    PubMed

    Wang, C; Stanciu, C E; Ehrhardt, C J; Yadavalli, V K

    2016-09-01

    Manipulation of viable Yersinia pestis (etiologic agent of plague) in the laboratory usually necessitates elevated biosafety and biocontainment procedures, even with avirulent or vaccine strains. To facilitate downstream biochemical or physical analyses in a Biosafety Level 1 laboratory environment, effective inactivation without affecting its intrinsic properties is critical. Here, we report on the morphological and biochemical changes to Y. pestis surfaces following four different fixation methods that render the cells nonviable. The results, obtained at the single cell level, demonstrate that methanol inactivation is best able to preserve bacterial morphology and bioactivity, enabling subsequent analysis. This nanoscale evaluation of the effects of inactivation on cell morphology and surface bioactivity may provide a crucial preparatory approach to study virulent pathogens in the lab setting using high-resolution microscopic techniques such as atomic force microscopy. PMID:27527609

  1. Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

    PubMed

    Sanapala, Shilpa; Rahav, Hannah; Patel, Hetal; Sun, Wei; Curtiss, Roy

    2016-05-01

    Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine. PMID:27060051

  2. Local Antibody Production Against the Murine Toxin of Yersinia pestis in a Golf Ball-Induced Granuloma

    PubMed Central

    Hillam, Robyn P.; Tengerdy, Robert P.; Brown, George L.

    1974-01-01

    The method of Garra and Baygorria, for localized antibody production, has been adapted for obtaining high-titered and monospecific antibodies against the murine toxin of Yersinia pestis. A hollow, perforated plastic golf ball surgically implanted under the skin of rabbits induced the formation of a granuloma. When the murine toxin of Y. pestis was injected directly into the granulomatous cavity, an increased amount of antibody was found in the granuloma fluid as compared with serum or with serum antibody obtained by conventional immunization. The granuloma antibody consisted mainly of immunoglobulin G, probably produced locally by lymphoid cells of the granuloma. The immune granuloma fluid and the granuloma tissue were rich in plasma cells and lymphocytes. The chemical composition of the granuloma fluid indicated that it was a transudate. Images PMID:4214770

  3. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization.

    PubMed Central

    Hill, J; Leary, S E; Griffin, K F; Williamson, E D; Titball, R W

    1997-01-01

    V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen. PMID:9353022

  4. Investigation of Yersinia pestis laboratory adaptation through a combined genomics and proteomics approach

    SciTech Connect

    Leiser, Owen P.; Merkley, Eric D.; Clowers, Brian H.; Kaiser, Brooke L. Deatherage; Lin, Andy; Hutchison, Janine R.; Melville, Angela M.; Wagner, David M.; Keim, Paul S.; Foster, Jeff; Kreuzer, Helen W.

    2015-11-24

    Here, the bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a Parallel Serial Passage Experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS-based proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism, envelope biogenesis, iron storage and acquisition, and a type VI secretion system. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement.

  5. Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India.

    PubMed

    Mahale, Kiran N; Paranjape, Pradyumna S; Marathe, Nachiket P; Dhotre, Dhiraj P; Chowdhury, Somak; Shetty, Sudarshan A; Sharma, Avinash; Sharma, Kaushal; Tuteja, Urmil; Batra, Harsh V; Shouche, Yogesh S

    2014-12-01

    We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139-147 contigs. Average sequencing depth for all four genomes was 21x. PMID:25320451

  6. Acquisition of maternal antibodies both from the placenta and by lactation protects mouse offspring from Yersinia pestis challenge.

    PubMed

    Qi, Zhizhen; Zhao, Haihong; Zhang, Qingwen; Bi, Yujing; Ren, Lingling; Zhang, Xuecan; Yang, Hanqing; Yang, Xiaoyan; Wang, Qiong; Li, Cunxiang; Zhou, Jiyuan; Xin, Youquan; Yang, Yonghai; Yang, Huiying; Du, Zongmin; Tan, Yafang; Han, Yanping; Song, Yajun; Zhou, Lei; Zhang, Pingping; Cui, Yujun; Yan, Yanfeng; Zhou, Dongsheng; Yang, Ruifu; Wang, Xiaoyi

    2012-11-01

    Artificially passive immunization has been demonstrated to be effective against Yersinia pestis infection in animals. However, maternal antibodies' protective efficacy against plague has not yet been demonstrated. Here, we evaluated the kinetics, protective efficacy, and transmission modes of maternal antibodies, using mice immunized with plague subunit vaccine SV1 (20 μg of F1 and 10 μg of rV270). The results showed that the rV270- and F1-specific antibodies could be detected in the sera of newborn mice (NM) until 10 and 14 weeks of age, respectively. There was no antibody titer difference between the parturient mice immunized with SV1 (PM-S) and the caesarean-section newborns (CSN) from the PM-S or between the lactating mice immunized by SV1 (LM-S) and the cross-fostered mice (CFM) during 3 weeks of lactation. The NM had a 72% protection against 4,800 CFU Y. pestis strain 141 challenge at 6 weeks of age, whereas at 14 weeks of age, NM all succumbed to 5,700 CFU of Y. pestis challenge. After 7 weeks of age, CFM had an 84% protection against 5,000 CFU of Y. pestis challenge. These results indicated that maternal antibodies induced by the plague subunit vaccine in mother mice can be transferred to NM by both placenta and lactation. Passive antibodies from the immunized mothers could persist for 3 months and provide early protection for NM. The degree of early protection is dependent on levels of the passively acquired antibody. The results indicate that passive immunization should be an effective countermeasure against plague during its epidemics. PMID:22933398

  7. The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1▿

    PubMed Central

    Haiko, Johanna; Laakkonen, Liisa; Juuti, Katri; Kalkkinen, Nisse; Korhonen, Timo K.

    2010-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis. PMID:20639337

  8. Recombinant murine toxin from Yersinia pestis shows high toxicity and β-adrenergic blocking activity in mice.

    PubMed

    Fan, Yanxiao; Zhou, Yazhou; Feng, Na; Wang, Qiong; Tian, Guang; Wu, Xiaohong; Liu, Zizhong; Bi, Yujing; Yang, Ruifu; Wang, Xiaoyi

    2016-05-01

    Yersinia pestis murine toxin (Ymt) encoded on pMT1 is a 61-kDa protein, a member of the phospholipase D superfamily, which is found in all the domains of life. It is considered to be an intracellular protein required for the survival of Y. pestis in the midgut of the flea, but the exact role of Ymt in the pathogenesis of Y. pestis has not been clarified. Purified Ymt is highly toxic to mice and rats, but the exact mechanism of the animals' death is unclear. Here, we prepared a recombinant Ymt in Escherichia coli BL21 cells, and determined its toxicity and activity. We demonstrated that recombinant Ymt was as toxic to mice as the native protein when administered via the intraperitoneal or intravenous route, and inhibited the elevation of blood sugar caused by adrenaline. We also demonstrated that recombinant Ymt was highly toxic to mice when administered via the muscular or subcutaneous route. We also show that the multiple organ congestion or hemorrhage caused by Ymt poisoning may explain the death of the mice. PMID:26774329

  9. Reciprocal regulation of Yersinia pestis biofilm formation and virulence by RovM and RovA

    PubMed Central

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    RovA is known to enhance Yersinia pestis virulence by directly upregulating the psa loci. This work presents a complex gene regulatory paradigm involving the reciprocal regulatory action of RovM and RovA on the expression of biofilm and virulence genes as well as on their own genes. RovM and RovA enhance and inhibit Y. pestis biofilm production, respectively, whereas RovM represses virulence in mice. RovM directly stimulates the transcription of hmsT, hmsCDE and rovM, while indirectly enhancing hmsHFRS transcription. It also indirectly represses hmsP transcription. By contrast, RovA directly represses hmsT transcription and indirectly inhibits waaAE-coaD transcription, while RovM inhibits psaABC and psaEF transcription by directly repressing rovA transcription. rovM expression is significantly upregulated at 26°C (the temperature of the flea gut) relative to 37°C (the warm-blooded host temperature). We speculate that upregulation of rovM together with downregulation of rovA in the flea gut would promote Y. pestis biofilm formation while inhibiting virulence gene expression, leading to a more transmissible infection of this pathogen in fleas. Once the bacterium shifts to a lifestyle in the warm-blooded hosts, inhibited RovM production accompanied by recovered RovA synthesis would encourage virulence factor production and inhibit biofilm gene expression. PMID:26984293

  10. Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague.

    PubMed

    Riehm, Julia M; Rahalison, Lila; Scholz, Holger C; Thoma, Bryan; Pfeffer, Martin; Razanakoto, Léa Mamiharisoa; Al Dahouk, Sascha; Neubauer, Heinrich; Tomaso, Herbert

    2011-02-01

    Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5'-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 (pla gene), and pMT1 (caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5'-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5'-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.

  11. Genetic variations of live attenuated plague vaccine strains (Yersinia pestis EV76 lineage) during laboratory passages in different countries.

    PubMed

    Cui, Yujun; Yang, Xianwei; Xiao, Xiao; Anisimov, Andrey P; Li, Dongfang; Yan, Yanfeng; Zhou, Dongsheng; Rajerison, Minoarisoa; Carniel, Elisabeth; Achtman, Mark; Yang, Ruifu; Song, Yajun

    2014-08-01

    Plague, one of the most devastating infectious diseases in human history, is caused by the bacterial species Yersinia pestis. A live attenuated Y. pestis strain (EV76) has been widely used as a plague vaccine in various countries around the world. Here we compared the whole genome sequence of an EV76 strain used in China (EV76-CN) with the genomes of Y. pestis wild isolates to identify genetic variations specific to the EV76 lineage. We identified 6 SNPs and 6 Indels (insertions and deletions) differentiating EV76-CN from its counterparts. Then, we screened these polymorphic sites in 28 other strains of EV76 lineage that were stored in different countries. Based on the profiles of SNPs and Indels, we reconstructed the parsimonious dissemination history of EV76 lineage. This analysis revealed that there have been at least three independent imports of EV76 strains into China. Additionally, we observed that the pyrE gene is a mutation hotspot in EV76 lineages. The fine comparison results based on whole genome sequence in this study provide better understanding of the effects of laboratory passages on the accumulation of genetic polymorphisms in plague vaccine strains. These variations identified here will also be helpful in discriminating different EV76 derivatives.

  12. Reciprocal regulation of Yersinia pestis biofilm formation and virulence by RovM and RovA.

    PubMed

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-03-01

    RovA is known to enhance Yersinia pestis virulence by directly upregulating the psa loci. This work presents a complex gene regulatory paradigm involving the reciprocal regulatory action of RovM and RovA on the expression of biofilm and virulence genes as well as on their own genes. RovM and RovA enhance and inhibit Y. pestis biofilm production, respectively, whereas RovM represses virulence in mice. RovM directly stimulates the transcription of hmsT, hmsCDE and rovM, while indirectly enhancing hmsHFRS transcription. It also indirectly represses hmsP transcription. By contrast, RovA directly represses hmsT transcription and indirectly inhibits waaAE-coaD transcription, while RovM inhibits psaABC and psaEF transcription by directly repressing rovA transcription. rovM expression is significantly upregulated at 26°C (the temperature of the flea gut) relative to 37°C (the warm-blooded host temperature). We speculate that upregulation of rovM together with downregulation of rovA in the flea gut would promote Y. pestis biofilm formation while inhibiting virulence gene expression, leading to a more transmissible infection of this pathogen in fleas. Once the bacterium shifts to a lifestyle in the warm-blooded hosts, inhibited RovM production accompanied by recovered RovA synthesis would encourage virulence factor production and inhibit biofilm gene expression. PMID:26984293

  13. Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

    PubMed

    Bugrysheva, Julia V; Lascols, Christine; Sue, David; Weigel, Linda M

    2016-06-01

    Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in <4 h for B. anthracis and <6 h for Y. pestis and B. pseudomallei One exception was B. pseudomallei in the presence of ceftazidime, which required >10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods. PMID:26984973

  14. Evaluation of the Murine Immune Response to Xenopsylla cheopis Flea Saliva and Its Effect on Transmission of Yersinia pestis

    PubMed Central

    Bosio, Christopher F.; Viall, Austin K.; Jarrett, Clayton O.; Gardner, Donald; Rood, Michael P.; Hinnebusch, B. Joseph

    2014-01-01

    Background/Aims Arthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites. Methods/Principal Findings The immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36–45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period. Conclusions Unlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea

  15. Znu is the predominant zinc importer in Yersinia pestis during in vitro growth but is not essential for virulence.

    PubMed

    Desrosiers, Daniel C; Bearden, Scott W; Mier, Ildefonso; Abney, Jennifer; Paulley, James T; Fetherston, Jacqueline D; Salazar, Juan C; Radolf, Justin D; Perry, Robert D

    2010-12-01

    Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter

  16. Proteolytic processing of the Yersinia pestis YapG autotransporter by the omptin protease Pla and the contribution of YapG to murine plague pathogenesis

    PubMed Central

    Lane, M. Chelsea; Lenz, Jonathan D.

    2013-01-01

    Autotransporter protein secretion represents one of the simplest forms of secretion across Gram-negative bacterial membranes. Once secreted, autotransporter proteins either remain tethered to the bacterial surface or are released following proteolytic cleavage. Autotransporters possess a diverse array of virulence-associated functions such as motility, cytotoxicity, adherence and autoaggregation. To better understand the role of autotransporters in disease, our research focused on the autotransporters of Yersinia pestis, the aetiological agent of plague. Y. pestis strain CO92 has nine functional conventional autotransporters, referred to as Yaps for Yersinia autotransporter proteins. Three Yaps have been directly implicated in virulence using established mouse models of plague infection (YapE, YapJ and YapK). Whilst previous studies from our laboratory have shown that most of the CO92 Yaps are cell associated, YapE and YapG are processed and released by the omptin protease Pla. In this study, we identified the Pla cleavage sites in YapG that result in many released forms of YapG in Y. pestis, but not in the evolutionarily related gastrointestinal pathogen, Yersinia pseudotuberculosis, which lacks Pla. Furthermore, we showed that YapG does not contribute to Y. pestis virulence in established mouse models of bubonic and pneumonic infection. As Y. pestis has a complex life cycle involving a wide range of mammalian hosts and a flea vector for transmission, it remains to be elucidated whether YapG has a measurable role in any other stage of plague disease. PMID:23657527

  17. Validation of Inverse Seasonal Peak Mortality in Medieval Plagues, Including the Black Death, in Comparison to Modern Yersinia pestis-Variant Diseases

    PubMed Central

    Welford, Mark R.; Bossak, Brian H.

    2009-01-01

    Background Recent studies have noted myriad qualitative and quantitative inconsistencies between the medieval Black Death (and subsequent “plagues”) and modern empirical Y. pestis plague data, most of which is derived from the Indian and Chinese plague outbreaks of A.D. 1900±15 years. Previous works have noted apparent differences in seasonal mortality peaks during Black Death outbreaks versus peaks of bubonic and pneumonic plagues attributed to Y. pestis infection, but have not provided spatiotemporal statistical support. Our objective here was to validate individual observations of this seasonal discrepancy in peak mortality between historical epidemics and modern empirical data. Methodology/Principal Findings We compiled and aggregated multiple daily, weekly and monthly datasets of both Y. pestis plague epidemics and suspected Black Death epidemics to compare seasonal differences in mortality peaks at a monthly resolution. Statistical and time series analyses of the epidemic data indicate that a seasonal inversion in peak mortality does exist between known Y. pestis plague and suspected Black Death epidemics. We provide possible explanations for this seasonal inversion. Conclusions/Significance These results add further evidence of inconsistency between historical plagues, including the Black Death, and our current understanding of Y. pestis-variant disease. We expect that the line of inquiry into the disputed cause of the greatest recorded epidemic will continue to intensify. Given the rapid pace of environmental change in the modern world, it is crucial that we understand past lethal outbreaks as fully as possible in order to prepare for future deadly pandemics. PMID:20027294

  18. Proteolytic processing of the Yersinia pestis YapG autotransporter by the omptin protease Pla and the contribution of YapG to murine plague pathogenesis.

    PubMed

    Lane, M Chelsea; Lenz, Jonathan D; Miller, Virginia L

    2013-08-01

    Autotransporter protein secretion represents one of the simplest forms of secretion across Gram-negative bacterial membranes. Once secreted, autotransporter proteins either remain tethered to the bacterial surface or are released following proteolytic cleavage. Autotransporters possess a diverse array of virulence-associated functions such as motility, cytotoxicity, adherence and autoaggregation. To better understand the role of autotransporters in disease, our research focused on the autotransporters of Yersinia pestis, the aetiological agent of plague. Y. pestis strain CO92 has nine functional conventional autotransporters, referred to as Yaps for Yersinia autotransporter proteins. Three Yaps have been directly implicated in virulence using established mouse models of plague infection (YapE, YapJ and YapK). Whilst previous studies from our laboratory have shown that most of the CO92 Yaps are cell associated, YapE and YapG are processed and released by the omptin protease Pla. In this study, we identified the Pla cleavage sites in YapG that result in many released forms of YapG in Y. pestis, but not in the evolutionarily related gastrointestinal pathogen, Yersinia pseudotuberculosis, which lacks Pla. Furthermore, we showed that YapG does not contribute to Y. pestis virulence in established mouse models of bubonic and pneumonic infection. As Y. pestis has a complex life cycle involving a wide range of mammalian hosts and a flea vector for transmission, it remains to be elucidated whether YapG has a measurable role in any other stage of plague disease.

  19. Environmental drivers of Yersinia pestis - a holistic perspective on Medieval Europe

    NASA Astrophysics Data System (ADS)

    Buentgen, U.

    2009-09-01

    Recent studies have indicated some evidence for a link between climate variability and plague (Yersinia pestis) dynamics in Central Asia and during most of the 20th century. An intensification of plague outbreaks via population peaks in its host-species, the great gerbil (Rhombomys opimus) and its fleas (Xenopsylla spp) has been found to occur during periods of warmer spring and wetter summer climate. This is important, as human epidemics of plague ultimately originate in its wildlife reservoirs. Given the fact that Medieval Europe was strongly devastated by the Black Death - the second pandemic after the Justinian plague ~540AD, and that the worldwide highest quality and quantity of climate proxy data exist for Europe, we here present, for the first time, a holistic approach to enhance understanding of the mid-14th century Black Death. This is of primary importance not only for medical/epidemiological research, but also for other scientific communities, because the Black Death disease had a sustainable impact on the socio-economic development, culture, art, and religion of Medieval Europe. Palaeoclimatic records of annually resolved European temperature and drought variability are compiled, a high-resolution time-series of anthropogenic deforestation is utilized, documentary archives of socio-economic relevance are considered, and the animal-born plague bacterium is placed in the ecological web. Considering the European/North Atlantic sector and the last millennium, periods of high solar radiation and reduced volcanic activity shift the North Atlantic Oscillation into a generally positive mode, yielding towards warmer temperatures and an intensification of the hydrological cycle. We now argue that increased internal circulation resulted in an overall wetter and warmer climate ~1350AD, which most likely was able to promote the prevalence of existing and widespread Yersinia pestis bacillus. Resulting outbreaks of bubonic plague could have been also supported by the

  20. Transmission shifts underlie variability in population responses to Yersinia pestis infection.

    PubMed

    Buhnerkempe, Michael G; Eisen, Rebecca J; Goodell, Brandon; Gage, Kenneth L; Antolin, Michael F; Webb, Colleen T

    2011-01-01

    Host populations for the plague bacterium, Yersinia pestis, are highly variable in their response to plague ranging from near deterministic extinction (i.e., epizootic dynamics) to a low probability of extinction despite persistent infection (i.e., enzootic dynamics). Much of the work to understand this variability has focused on specific host characteristics, such as population size and resistance, and their role in determining plague dynamics. Here, however, we advance the idea that the relative importance of alternative transmission routes may vary causing shifts from epizootic to enzootic dynamics. We present a model that incorporates host and flea ecology with multiple transmission hypotheses to study how transmission shifts determine population responses to plague. Our results suggest enzootic persistence relies on infection of an off-host flea reservoir and epizootics rely on transiently maintained flea infection loads through repeated infectious feeds by fleas. In either case, early-phase transmission by fleas (i.e., transmission immediately following an infected blood meal) has been observed in laboratory studies, and we show that it is capable of driving plague dynamics at the population level. Sensitivity analysis of model parameters revealed that host characteristics (e.g., population size and resistance) vary in importance depending on transmission dynamics, suggesting that host ecology may scale differently through different transmission routes enabling prediction of population responses in a more robust way than using either host characteristics or transmission shifts alone.

  1. Adhesive properties of the purified plasminogen activator Pla of Yersinia pestis.

    PubMed

    Lobo, Leandro Araujo

    2006-09-01

    The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays. PMID:16923070

  2. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

    PubMed

    Skottman, T; Piiparinen, H; Hyytiäinen, H; Myllys, V; Skurnik, M; Nikkari, S

    2007-03-01

    This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention. PMID:17294160

  3. Transmission Shifts Underlie Variability in Population Responses to Yersinia pestis Infection

    PubMed Central

    Buhnerkempe, Michael G.; Eisen, Rebecca J.; Goodell, Brandon; Gage, Kenneth L.; Antolin, Michael F.; Webb, Colleen T.

    2011-01-01

    Host populations for the plague bacterium, Yersinia pestis, are highly variable in their response to plague ranging from near deterministic extinction (i.e., epizootic dynamics) to a low probability of extinction despite persistent infection (i.e., enzootic dynamics). Much of the work to understand this variability has focused on specific host characteristics, such as population size and resistance, and their role in determining plague dynamics. Here, however, we advance the idea that the relative importance of alternative transmission routes may vary causing shifts from epizootic to enzootic dynamics. We present a model that incorporates host and flea ecology with multiple transmission hypotheses to study how transmission shifts determine population responses to plague. Our results suggest enzootic persistence relies on infection of an off-host flea reservoir and epizootics rely on transiently maintained flea infection loads through repeated infectious feeds by fleas. In either case, early-phase transmission by fleas (i.e., transmission immediately following an infected blood meal) has been observed in laboratory studies, and we show that it is capable of driving plague dynamics at the population level. Sensitivity analysis of model parameters revealed that host characteristics (e.g., population size and resistance) vary in importance depending on transmission dynamics, suggesting that host ecology may scale differently through different transmission routes enabling prediction of population responses in a more robust way than using either host characteristics or transmission shifts alone. PMID:21799873

  4. Derivatives of Salicylic Acid as Inhibitors of YopH in Yersinia pestis

    PubMed Central

    Huang, Zunnan; He, Yantao; Zhang, Xian; Gunawan, Andrea; Wu, Li; Zhang, Zhong-Yin; Wong, Chung F.

    2010-01-01

    Yersinia pestis causes diseases ranging from gastrointestinal syndromes to bubonic plague and could be misused as a biological weapon. As its protein tyrosine phosphatase YopH has already been demonstrated as a potential drug target, we have developed two series of forty salicylic acid derivatives and found sixteen to have micromolar inhibitory activity. We designed these ligands to have two chemical moieties connected by a flexible hydrocarbon linker to target two pockets in the active site of the protein to achieve binding affinity and selectivity. One moiety possessed the salicylic acid core intending to target the phosphotyrosine-binding pocket. The other moiety contained different chemical fragments meant to target a nearby secondary pocket. The two series of compounds differed by having hydrocarbon linkers with different lengths. Before experimental co-crystal structures are available, we have performed molecular docking to predict how these compounds might bind to the protein and to generate structural models for performing binding affinity calculation to aid future optimization of these series of compounds. PMID:20560978

  5. Reconstruction of an ancestral Yersinia pestis genome and comparison with an ancient sequence

    PubMed Central

    2015-01-01

    Background We propose the computational reconstruction of a whole bacterial ancestral genome at the nucleotide scale, and its validation by a sequence of ancient DNA. This rare possibility is offered by an ancient sequence of the late middle ages plague agent. It has been hypothesized to be ancestral to extant Yersinia pestis strains based on the pattern of nucleotide substitutions. But the dynamics of indels, duplications, insertion sequences and rearrangements has impacted all genomes much more than the substitution process, which makes the ancestral reconstruction task challenging. Results We use a set of gene families from 13 Yersinia species, construct reconciled phylogenies for all of them, and determine gene orders in ancestral species. Gene trees integrate information from the sequence, the species tree and gene order. We reconstruct ancestral sequences for ancestral genic and intergenic regions, providing nearly a complete genome sequence for the ancestor, containing a chromosome and three plasmids. Conclusion The comparison of the ancestral and ancient sequences provides a unique opportunity to assess the quality of ancestral genome reconstruction methods. But the quality of the sequencing and assembly of the ancient sequence can also be questioned by this comparison. PMID:26450112

  6. Isothermal solid-phase amplification system for detection of Yersinia pestis.

    PubMed

    Mayboroda, Olena; Gonzalez Benito, Angel; Sabaté del Rio, Jonathan; Svobodova, Marketa; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K; Katakis, Ioanis

    2016-01-01

    DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.

  7. Isothermal solid-phase amplification system for detection of Yersinia pestis.

    PubMed

    Mayboroda, Olena; Gonzalez Benito, Angel; Sabaté del Rio, Jonathan; Svobodova, Marketa; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K; Katakis, Ioanis

    2016-01-01

    DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets. PMID:26563112

  8. Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity.

    PubMed

    Bengoechea, J A; Brandenburg, K; Seydel, U; Díaz, R; Moriyón, I

    1998-06-01

    The hydrophobic probe N-phenyl-1-naphthylamine accumulated less in non-pathogenic Yersinia spp. and non-pathogenic and pathogenic Yersinia enterocolitica than in Yersinia pseudotuberculosis or Yersinia pestis. This was largely due to differences in the activity of efflux systems, but also to differences in outer membrane permeability because uptake of the probe in KCN/arsenate-poisoned cells was slower in the former group than in Y. pseudotuberculosis and Y. pestis. The probe accumulation rate was higher in Y. pseudotuberculosis and Y. pestis grown at 37 degrees C than at 26 degrees C and was always highest in Y. pestis. These yersiniae had LPSs with shorter polysaccharides than Y. enterocolitica, particularly when grown at 37 degrees C. Gel<-->liquid-crystalline phase transitions (Tc 28-31 degrees C) were observed in LPS aggregates of Y. enterocolitica grown at 26 and 37 degrees C, with no differences between non-pathogenic and pathogenic strains. Y. pseudotuberculosis and Y. pestis LPSs showed no phase transitions and, although the fluidity of LPSs of Y. pseudotuberculosis and Y. enterocolitica grown at 26 degrees C were close below the Tc of the latter, they were always in a more fluid state than Y. enterocolitica LPS. Comparison with previous studies of Salmonella choleraesuis subsp. choleraesuis serotype minnesota rough LPS showed that the increased fluidity and absence of transition of Y. pseudotuberculosis and Y. pestis LPSs cannot be explained by their shorter polysaccharides and suggested differences at the lipid A/core level. It is proposed that differences in LPS-LPS interactions and efflux activity explain the above observations and reflect the adaptation of Yersinia spp. to different habitats.

  9. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    PubMed

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  10. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD

    PubMed Central

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3′,5′-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  11. Genome Sequence of the Deep-Rooted Yersinia pestis Strain Angola Reveals New Insights into the Evolution and Pangenome of the Plague Bacterium▿ †

    PubMed Central

    Eppinger, Mark; Worsham, Patricia L.; Nikolich, Mikeljon P.; Riley, David R.; Sebastian, Yinong; Mou, Sherry; Achtman, Mark; Lindler, Luther E.; Ravel, Jacques

    2010-01-01

    To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies. PMID:20061468

  12. Analysis of the three Yersinia pestis CRISPR loci provides new tools for phylogenetic studies and possibly for the investigation of ancient DNA.

    PubMed

    Vergnaud, Gilles; Li, Yanjun; Gorgé, Olivier; Cui, Yujun; Song, Yajun; Zhou, Dongsheng; Grissa, Ibtissem; Dentovskaya, Svetlana V; Platonov, Mikhail E; Rakin, Alexander; Balakhonov, Sergey V; Neubauer, Heinrich; Pourcel, Christine; Anisimov, Andrey P; Yang, Ruifu

    2007-01-01

    The precise nature of the pathogen having caused early plague pandemics is uncertain. Although Yersinia pestis is a likely candidate for all three plague pandemics, the very rare direct evidence that can be deduced from ancient DNA (aDNA) analysis is controversial. Moreover, which of the three biovars, Antiqua, Medievalis or Orientalis, was associated with these pandemics is still debated. There is a need for phylogenetic analysis performed on Y. pestis strains isolated from countries from which plague probably arose and is still endemic. In addition there exist technical difficulties inherent to aDNA investigations and a lack of appropriate genetic targets. The recently described CRISPRs (clustered regularly interspaced short palindromic repeats) may represent such a target. CRISPR loci consist of a succession of highly conserved regions separated by specific "spacers" usually of viral origin. To be of use, data describing the mechanisms of evolution and diversity of CRISPRs in Y. pestis, its closest neighbors, and other species which might contaminate ancient DNA, are necessary. The investigation of closely related Y. pestis isolates has revealed recent mutation events in which elements constituting CRISPRs were acquired or lost, providing essential insight on their evolution. Rules deduced represent the basis for subsequent interpretation. In the present study, the CRISPR loci from representative Y. pestis and Yersinia pseudotuberculosis strains were investigated by PCR amplification and sequence analysis. The investigation of this wider panel of strains, including other subspecies or ecotypes within Y. pestis and also Y. pseudotuberculosis strains provides a database of the existing CRISPR spacers and helps predict the expected CRISPR structure of the Y. pestis ancestor. This knowledge will open the way to the development of a spoligotyping assay, in which spacers can be amplified even from highly degraded DNA samples. The data obtained show that CRISPR

  13. Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis.

    PubMed

    Anderson, G W; Leary, S E; Williamson, E D; Titball, R W; Welkos, S L; Worsham, P L; Friedlander, A M

    1996-11-01

    The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

  14. Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ.

    PubMed

    Lilo, Sarit; Zheng, Ying; Bliska, James B

    2008-09-01

    Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.

  15. Caspase-1 Activation in Macrophages Infected with Yersinia pestis KIM Requires the Type III Secretion System Effector YopJ▿

    PubMed Central

    Lilo, Sarit; Zheng, Ying; Bliska, James B.

    2008-01-01

    Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1β (IL-1β) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1β was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1β following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1β were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages. PMID:18559430

  16. Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis

    PubMed Central

    Nanson, Jeffrey D.; Forwood, Jade K.

    2015-01-01

    Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections. PMID:26539719

  17. Systematic analysis of cyclic di-GMP signaling enzymes and their role in biofilm formation and virulence in Yersinia pestis

    PubMed Central

    Bobrov, Alexander G.; Kirillina, Olga; Ryjenkov, Dmitri A.; Waters, Christopher M.; Price, Paul A.; Fetherston, Jacqueline D.; Mack, Dietrich; Goldman, William E.; Gomelsky, Mark; Perry, Robert D.

    2011-01-01

    Cyclic di-GMP (c-di-GMP) is a signaling molecule that governs the transition between planktonic and biofilm states. Previously we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-β-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-β-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signaling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signaling network likely resulting from the different disease cycles of these human pathogens. PMID:21219468

  18. The effects of low-shear mechanical stress on Yersinia pestis virulence.

    PubMed

    Lawal, Abidat; Jejelowo, Olufisayo A; Rosenzweig, Jason A

    2010-11-01

    Manned space exploration has created a need to evaluate the effects of spacelike stress on pathogenic and opportunistic microbes astronauts could carry with them to the International Space Station and beyond. Yersinia pestis (YP) causes bubonic, septicemic, and pneumonic plague and is capable of killing infected patients within 3-7 days. In this study, low-shear modeled microgravity (LSMMG), a spacelike stress, was used to physically stress YP; and its effects on proliferation, cold growth, and type III secretion system (T3SS) function were evaluated. YP was grown to saturation in either LSMMG or normal gravity (NG) conditions prior to being used for RAW 246.7 cell infections, HeLa cell infections, and Yop secretion assays. A mutant strain of YP (ΔyopB) that lacks the ability to inject Yersinia outer membrane proteins (Yops) into the host cell was used as a negative control in cell infection experiments. Our experimental results indicate that YP cultivated under LSMMG resulted in reduced YopM production and secretion compared to its NG-grown counterpart. Similarly, NG-grown YP induced more cell rounding in HeLa cells than did the LSMMG-grown YP, which suggests that LSMMG somehow impairs T3SS optimum function. Also, LSMMG-grown YP used to infect cultured RAW 246.7 cells showed a similar pattern of dysfunction in that it proliferated less than did its NG-grown counterpart during an 8-hour infection period. This study suggests that LSMMG can attenuate bacterial virulence contrary to previously published data that have demonstrated LSMMG-induced hypervirulence of other Gram-negative enterics.

  19. Detection of Yersinia pestis fraction 1 antigen with a fiber optic biosensor.

    PubMed Central

    Cao, L K; Anderson, G P; Ligler, F S; Ezzell, J

    1995-01-01

    A fiber optic biosensor was used to detect the fraction 1 (F1) antigen from Yersinia pestis, the etiologic agent of plague. The instrument employs an argon ion laser (514 nm) to launch light into a long-clad fiber and measures the fluorescence produced by an immunofluorescent complex formed in the evanescent wave region. This sensing area is a short section (12.5 cm) at the end of the optical fiber from which the cladding has been removed and in which the silica core has been tapered. Capture antibodies, which bind to F1 antigen, were immobilized on the core surface to form the basis of the sandwich fluoroimmunoassay. The ability to detect bound F1 antigen was provided by adding tetramethylrhodamine-labeled anti-plaque antibody to form fluorescent complexes. The evanescent wave has a limited penetration depth (< 1 lambda), which restricts detection of the fluorescent complexes bound to the fiber's surface. The direct correlation between the F1 antigen concentration and the signal provided an effective method for sample quantitation. This method achieved a high level of accuracy for determining F1 antigen concentrations from 50 to 400 ng/ml in phosphate-buffered saline, serum, plasma, and whole blood, with a 5-ng/ml limit of detection. Subsequent blind studies, which included serum samples from patients, yielded results in good agreement with measurements by enzyme-linked immunosorbent assay. A major advantage of the fiber optic biosensor is that results can be generated within minutes while isolating the user from hazardous samples. These factors favor development of this biosensor into a facile and rapid diagnostic device. PMID:7714189

  20. Highly Effective Soluble and Bacteriophage T4 Nanoparticle Plague Vaccines Against Yersinia pestis.

    PubMed

    Tao, Pan; Mahalingam, Marthandan; Rao, Venigalla B

    2016-01-01

    Plague caused by Yersinia pestis is an ancient disease, responsible for millions of deaths in human history. Unfortunately, there is no FDA-approved vaccine available. Recombinant subunit vaccines based on two major antigens, Caf 1 (F1) and LcrV (V), have been under investigation and showed promise. However, there are two main problems associated with these vaccines. First, the Yersinia capsular protein F1 has high propensity to aggregate, particularly when expressed in heterologous systems such as Escherichia coli, thus affecting vaccine quality and efficacy. Second, the subunit vaccines do not induce adequate cell-mediated immune responses that also appear to be essential for optimal protection against plague. We have developed two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that may overcome these problems. First, by engineering F1 protein, we generated a monomeric and soluble F1V mutant (F1mutV) which has similar immunogenicity as wild-type F1V. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to retain a key CD4(+) T cell epitope. Second, we generated a nanoparticle plague vaccine that can induce balanced antibody- and cell-mediated immune responses. This was done by arraying the F1mutV on phage T4 via the small outer capsid (Soc) protein which binds to T4 capsid at nanomolar affinity. Preparation of these vaccines is described in detail and we hope that these would be considered as candidates for licensing a next-generation plague vaccine. PMID:27076150

  1. Highly Effective Soluble and Bacteriophage T4 Nanoparticle Plague Vaccines Against Yersinia pestis.

    PubMed

    Tao, Pan; Mahalingam, Marthandan; Rao, Venigalla B

    2016-01-01

    Plague caused by Yersinia pestis is an ancient disease, responsible for millions of deaths in human history. Unfortunately, there is no FDA-approved vaccine available. Recombinant subunit vaccines based on two major antigens, Caf 1 (F1) and LcrV (V), have been under investigation and showed promise. However, there are two main problems associated with these vaccines. First, the Yersinia capsular protein F1 has high propensity to aggregate, particularly when expressed in heterologous systems such as Escherichia coli, thus affecting vaccine quality and efficacy. Second, the subunit vaccines do not induce adequate cell-mediated immune responses that also appear to be essential for optimal protection against plague. We have developed two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that may overcome these problems. First, by engineering F1 protein, we generated a monomeric and soluble F1V mutant (F1mutV) which has similar immunogenicity as wild-type F1V. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to retain a key CD4(+) T cell epitope. Second, we generated a nanoparticle plague vaccine that can induce balanced antibody- and cell-mediated immune responses. This was done by arraying the F1mutV on phage T4 via the small outer capsid (Soc) protein which binds to T4 capsid at nanomolar affinity. Preparation of these vaccines is described in detail and we hope that these would be considered as candidates for licensing a next-generation plague vaccine.

  2. The Effects of Low-Shear Mechanical Stress on Yersinia pestis Virulence

    NASA Astrophysics Data System (ADS)

    Lawal, Abidat; Jejelowo, Olufisayo A.; Rosenzweig, Jason A.

    2010-11-01

    Manned space exploration has created a need to evaluate the effects of spacelike stress on pathogenic and opportunistic microbes astronauts could carry with them to the International Space Station and beyond. Yersinia pestis (YP) causes bubonic, septicemic, and pneumonic plague and is capable of killing infected patients within 3-7 days. In this study, low-shear modeled microgravity (LSMMG), a spacelike stress, was used to physically stress YP; and its effects on proliferation, cold growth, and type III secretion system (T3SS) function were evaluated. YP was grown to saturation in either LSMMG or normal gravity (NG) conditions prior to being used for RAW 246.7 cell infections, HeLa cell infections, and Yop secretion assays. A mutant strain of YP (ΔyopB) that lacks the ability to inject Yersinia outer membrane proteins (Yops) into the host cell was used as a negative control in cell infection experiments. Our experimental results indicate that YP cultivated under LSMMG resulted in reduced YopM production and secretion compared to its NG-grown counterpart. Similarly, NG-grown YP induced more cell rounding in HeLa cells than did the LSMMG-grown YP, which suggests that LSMMG somehow impairs T3SS optimum function. Also, LSMMG-grown YP used to infect cultured RAW 246.7 cells showed a similar pattern of dysfunction in that it proliferated less than did its NG-grown counterpart during an 8-hour infection period. This study suggests that LSMMG can attenuate bacterial virulence contrary to previously published data that have demonstrated LSMMG-induced hypervirulence of other Gram-negative enterics.

  3. Protective immunity against respiratory tract challenge with Yersinia pestis in mice immunized with an adenovirus-based vaccine vector expressing V antigen.

    PubMed

    Chiuchiolo, Maria J; Boyer, Julie L; Krause, Anja; Senina, Svetlana; Hackett, Neil R; Crystal, Ronald G

    2006-11-01

    The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine.

  4. Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

    PubMed Central

    2010-01-01

    Background Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. Results When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. Conclusion These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates. PMID:21073689

  5. Thirty-Two Complete Genome Assemblies of Nine Yersinia Species, Including Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica

    SciTech Connect

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; Jaissle, James; Frey, Kenneth G.; Ladner, Jason T.; Broomall, Stacey M.; Bishop-Lilly, Kimberly A.; Bruce, David C.; Coyne, Susan R.; Gibbons, Henry S.; Lo, Chien-Chi; Munk, A. Christine; Rosenzweig, C. Nicole; Koroleva, Galina I.; Palacios, Gustavo F.; Redden, Cassie L.; Xu, Yan; Minogue, Timothy D.; Chain, Patrick S.

    2015-04-30

    The genus Yersinia includes three human pathogens, of which Yersinia pestis is responsible for >2,000 illnesses each year. To aid in the development of detection assays as well as aid further phylogenetic elucidation, we sequenced and assembled the complete genomes of 32 strains (across 9 Yersinia species).

  6. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    PubMed Central

    Nanson, Jeffrey D.; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  7. Yersinia pestis requires the 2-component regulatory system OmpR-EnvZ to resist innate immunity during the early and late stages of plague.

    PubMed

    Reboul, Angéline; Lemaître, Nadine; Titecat, Marie; Merchez, Maud; Deloison, Gaspard; Ricard, Isabelle; Pradel, Elizabeth; Marceau, Michaël; Sebbane, Florent

    2014-11-01

    Plague is transmitted by fleas or contaminated aerosols. To successfully produce disease, the causal agent (Yersinia pestis) must rapidly sense and respond to rapid variations in its environment. Here, we investigated the role of 2-component regulatory systems (2CSs) in plague because the latter are known to be key players in bacterial adaptation to environmental change. Along with the previously studied PhoP-PhoQ system, OmpR-EnvZ was the only one of Y. pestis' 23 other 2CSs required for production of bubonic, septicemic, and pneumonic plague. In vitro, OmpR-EnvZ was needed to counter serum complement and leukocytes but was not required for the secretion of antiphagocyte exotoxins. In vivo, Y. pestis lacking OmpR-EnvZ did not induce an early immune response in the skin and was fully virulent in neutropenic mice. We conclude that, throughout the course of Y. pestis infection, OmpR-EnvZ is required to counter toxic effectors secreted by polymorphonuclear leukocytes in the tissues.

  8. Prevalence of Yersinia pestis in rodents and fleas associated with black-tailed prairie dogs (Cynomys ludovicianus) at Thunder Basin National Grassland, Wyoming

    USGS Publications Warehouse

    Thiagarajan, B.; Bal, Y.; Gage, K.L.; Cully, J.F.

    2008-01-01

    Rodents (and their fleas) that are associated with prairie dogs are considered important for the maintenance and transmission of the bacterium (Yersinia pestis) that causes plague. Our goal was to identify rodent and flea species that were potentially involved in a plague epizootic in black-tailed prairie dogs at Thunder Basin National Grassland. We collected blood samples and ectoparasites from rodents trapped at off- and on-colony grids at Thunder Basin National Grassland between 2002 and 2004. Blood samples were tested for antibodies to Y. pestis F-1 antigen by a passive hemagglutination assay, and fleas were tested by a multiplex polymerase chain reaction, for the presence of the plague bacterium. Only one of 1,421 fleas, an Oropsylla hirsuta collected in 2002 from a deer mouse, Peromyscus maniculatus, tested positive for Y. pestis. Blood samples collected in summer 2004 from two northern grasshopper mice, Onychomys leucogaster, tested positive for Y. pestis antibodies. All three positive samples were collected from on-colony grids shortly after a plague epizootic occurred. This study confirms that plague is difficult to detect in rodents and fleas associated with prairie dog colonies, unless samples are collected immediately after a prairie dog die-off. ?? Wildlife Disease Association 2008.

  9. Thirty-Two Complete Genome Assemblies of Nine Yersinia Species, Including Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica

    PubMed Central

    Daligault, Hajnalka E.; Davenport, Karen W.; Jaissle, James; Frey, Kenneth G.; Ladner, Jason T.; Broomall, Stacey M.; Bishop-Lilly, Kimberly A.; Bruce, David C.; Coyne, Susan R.; Gibbons, Henry S.; Lo, Chien-Chi; Munk, A. Christine; Rosenzweig, C. Nicole; Koroleva, Galina I.; Palacios, Gustavo F.; Redden, Cassie L.; Xu, Yan; Minogue, Timothy D.; Chain, Patrick S.

    2015-01-01

    The genus Yersinia includes three human pathogens, of which Yersinia pestis is responsible for >2,000 illnesses each year. To aid in the development of detection assays and aid further phylogenetic elucidation, we sequenced and assembled the complete genomes of 32 strains (across 9 Yersinia species). PMID:25931590

  10. Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi

    PubMed Central

    Zhao, Xiangna; Cui, Yujun; Yan, Yanfeng; Du, Zongmin; Tan, Yafang; Yang, Huiying; Bi, Yujing; Zhang, Pingping; Zhou, Lei; Zhou, Dongsheng; Han, Yanping; Song, Yajun; Wang, Xiaoyi

    2013-01-01

    Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions. PMID:24006436

  11. Complete Genome Sequence of Yersinia pestis Strains Antiqua andNepal516: Evidence of Gene Reduction in an Emerging Pathogen

    SciTech Connect

    Chain, Patrick S.G.; Hu, Ping; Malfatti, Stephanie A.; Radnedge,Lyndsay; Larimer, Frank; Vergez, Lisa M.; Worsham, Patricia; Chu, May C.; Andersen, Gary L.

    2006-01-16

    Yersinia pestis, the causative agent of bubonic andpneumonicplague, has undergone detailed study at the molecular level. Tofurther investigate the genomic diversity among this group and to helpcharacterize lineages of the plague organism that have no sequencedmembers, we present here the genomes of two isolates of the "classical"Antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua andNepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open readingframes respectively. Though both strains belong to one of the threeclassical biovars, they represent separate lineages defined by recentphylogenetic studies. We compare all five currently sequenced Y. pestisgenomes and the corresponding features in Y. pseudotuberculosis. Thereare strain-specific rearrangements, insertions, deletions, singlenucleotide polymorphisms and a unique distribution of insertionsequences. We found 453 single nucleotide polymorphisms in protein codingregions, which were used to assess evolutionary relationships of these Y.pestis strains. Gene reduction analysis revealed that the gene deletionprocesses are under selective pressure and many of the inactivations areprobably related to the organism s interaction with its host environment.The results presented here clearly demonstrate the differences betweenthe two Antiqua lineages and support the notion that grouping Y. pestisstrains based strictly on the classical definition of biovars (predicatedupon two biochemical assays) does not accurately reflect the phylogeneticrelationships within this species. Comparison of four virulent Y. pestisstrains with the human-avirulent strain 91001 provides further insightinto the genetic basis of virulence to humans.

  12. Resistance of Mice of the 129 Background to Yersinia pestis Maps to Multiple Loci on Chromosome 1.

    PubMed

    Tencati, Michael; Tapping, Richard I

    2016-10-01

    Yersinia pestis is a Gram-negative bacterium that is the causative agent of bubonic and pneumonic plague. It is commonly acquired by mammals such as rodents and humans via the bite of an infected flea. We previously reported that multiple substrains of the 129 mouse background are resistant to pigmentation locus-negative (pgm(-)) Yersinia pestis and that this phenotype maps to a 30-centimorgan (cM) region located on chromosome 1. In this study, we have further delineated this plague resistance locus to a region of less than 20 cM through the creation and phenotyping of recombinant offspring arising from novel crossovers in this region. Furthermore, our experiments have revealed that there are at least two alleles in this initial locus, both of which are required for resistance on a susceptible C57BL/6 background. These two alleles work in trans since resistance is restored in offspring possessing one allele contributed by each parent. Our studies also indicated that the Slc11a1 gene (formerly known as Nramp1) located within the chromosome1 locus is not responsible for conferring resistance to 129 mice. PMID:27481241

  13. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  14. The importance of the magnesium transporter MgtB for virulence of Yersinia pseudotuberculosis and Yersinia pestis.

    PubMed

    Ford, Donna C; Joshua, George W P; Wren, Brendan W; Oyston, Petra C F

    2014-12-01

    Mg(2+) has been shown to be an important signal controlling gene regulation via the PhoPQ two-component regulatory system for a range of Gram-negative bacteria, including Yersinia pestis and Yersinia pseudotuberculosis. The magnesium ion transporter MgtB is part of the complex PhoPQ regulon, being upregulated in response to low Mg(2+). Despite the presence of other Mg(2+) transport systems in Yersinia, inactivation of mgtB had a significant effect on the ability of the bacteria to scavenge this crucial ion. Whereas inactivation of PhoPQ is reported to adversely affect intracellular survival, we show that Y. pestis and Y. pseudotuberculosis ΔmgtB mutants survived equally as well as the respective parent strain within macrophages, although they were more sensitive to killing in the Galleria model of infection. Surprisingly, despite MgtB being only one member of the Mg(2+) stimulon and PhoPQ controlling the expression levels of a range of genes including mgtB, the Yersinia ΔmgtB mutants were more highly attenuated than the equivalent Yersinia ΔphoP mutants in mouse models of infection. MgtB may be a suitable target for development of novel antimicrobials, and investigation of its role may help elucidate the contribution of this component of the PhoPQ regulon to pathogenesis.

  15. Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

    SciTech Connect

    Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.; Ulrich, Robert G.; Burke, Terrence R. Jr Waugh, David S.

    2011-07-01

    The first X-ray crystal structure of the Y. pestis protein tyrosine phosphatase YopH in complex with an isothiazolidinone-based lead-fragment compound is reported. Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors.

  16. Structure of the Yersinia pestis tip protein LcrV refined to 1.65 Å resolution

    PubMed Central

    Chaudhury, Sukanya; Battaile, Kevin P.; Lovell, Scott; Plano, Gregory V.; De Guzman, Roberto N.

    2013-01-01

    The human pathogen Yersinia pestis requires the assembly of the type III secretion system (T3SS) for virulence. The structural component of the T3SS contains an external needle and a tip complex, which is formed by LcrV in Y. pestis. The structure of an LcrV triple mutant (K40A/D41A/K42A) in a C273S background has previously been reported to 2.2 Å resolution. Here, the crystal structure of LcrV without the triple mutation in a C273S background is reported at a higher resolution of 1.65 Å. Overall the two structures are similar, but there are also notable differences, particularly near the site of the triple mutation. The refined structure revealed a slight shift in the backbone positions of residues Gly28–Asn43 and displayed electron density in the loop region consisting of residues Ile46–Val63, which was disordered in the original structure. In addition, the helical turn region spanning residues Tyr77–Gln95 adopts a different orientation. PMID:23695558

  17. Direct transcriptional control of the plasminogen activator gene of Yersinia pestis by the cyclic AMP receptor protein.

    PubMed

    Kim, Tae-Jong; Chauhan, Sadhana; Motin, Vladimir L; Goh, Ee-Been; Igo, Michele M; Young, Glenn M

    2007-12-01

    Horizontal gene transfer events followed by proper regulatory integration of a gene drive rapid evolution of bacterial pathogens. A key event in the evolution of the highly virulent plague bacterium Yersinia pestis was the acquisition of plasmid pPCP1, which carries the plasminogen activator gene, pla. This promoted the bubonic form of the disease by increasing bacterial dissemination from flea bite sites and incidentally enhanced replication in respiratory airways during pneumonic infection. We determined that expression of pla is controlled by the global regulator cyclic AMP (cAMP) receptor protein (Crp). This transcription factor is well conserved among distantly related bacteria, where it acts as a soluble receptor for the ubiquitous signaling molecule cAMP and controls a global network of metabolic and stress-protective genes. Crp has a similar physiological role in Y. pestis since loss of its function resulted in an inability to metabolize a variety of nonglucose substrates. Activation of pla expression requires a transcription activation element of the pla promoter that serves as a Crp binding site. Crp interaction with this site was demonstrated to occur only in the presence of cAMP. Alteration of the Crp binding site nucleotide sequence prevented in vitro formation of Crp-DNA complexes and inhibited in vivo expression of pla. The placement of pla under direct regulatory control of Crp highlights how highly adapted pathogens integrate laterally acquired genes to coordinate virulence factor expression with global gene networks to maintain homeostasis through the infectious life cycle.

  18. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

    PubMed

    Rocke, Tonie E; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

  19. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    USGS Publications Warehouse

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  20. Yersinia pestis biovar Microtus strain 201, an avirulent strain to humans, provides protection against bubonic plague in rhesus macaques.

    PubMed

    Zhang, Qingwen; Wang, Qiong; Tian, Guang; Qi, Zhizhen; Zhang, Xuecan; Wu, Xiaohong; Qiu, Yefeng; Bi, Yujing; Yang, Xiaoyan; Xin, Youquan; He, Jian; Zhou, Jiyuan; Zeng, Lin; Yang, Ruifu; Wang, Xiaoyi

    2014-01-01

    Yersinia pestis biovar Microtus is considered to be a virulent to larger mammals, including guinea pigs, rabbits and humans. It may be used as live attenuated plague vaccine candidates in terms of its low virulence. However, the Microtus strain's protection against plague has yet to be demonstrated in larger mammals. In this study, we evaluated the protective efficacy of the Microtus strain 201 as a live attenuated plague vaccine candidate. Our results show that this strain is highly attenuated by subcutaneous route, elicits an F1-specific antibody titer similar to the EV and provides a protective efficacy similar to the EV against bubonic plague in Chinese-origin rhesus macaques. The Microtus strain 201 could induce elevated secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4, IL-5, and IL-6), as well as chemokines MCP-1 and IL-8. However, the protected animals developed skin ulcer at challenge site with different severity in most of the immunized and some of the EV-immunized monkeys. Generally, the Microtus strain 201 represented a good plague vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses as well as its good protection against high dose of subcutaneous virulent Y. pestis challenge.

  1. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

    PubMed

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  2. Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis

    PubMed Central

    Peterson, Johnny W; Moen, Scott T; Healy, Daniel; Pawlik, Jennifer E; Taormina, Joanna; Hardcastle, Jason; Thomas, John M; Lawrence, William S; Ponce, Cindy; Chatuev, Bagram M; Gnade, Bryan T; Foltz, Sheri M; Agar, Stacy L; Sha, Jian; Klimpel, Gary R; Kirtley, Michelle L; Eaves-Pyles, Tonyia; Chopra, Ashok K

    2010-01-01

    Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities. PMID:21127743

  3. A LysR-Type Transcriptional Regulator, RovM, Senses Nutritional Cues Suggesting that It Is Involved in Metabolic Adaptation of Yersinia pestis to the Flea Gut.

    PubMed

    Vadyvaloo, Viveka; Hinz, Angela K

    2015-01-01

    Yersinia pestis has evolved as a clonal variant of Yersinia pseudotuberculosis to cause flea-borne biofilm-mediated transmission of the bubonic plague. The LysR-type transcriptional regulator, RovM, is highly induced only during Y. pestis infection of the flea host. RovM homologs in other pathogens regulate biofilm formation, nutrient sensing, and virulence; including in Y. pseudotuberculosis, where RovM represses the major virulence factor, RovA. Here the role that RovM plays during flea infection was investigated using a Y. pestis KIM6+ strain deleted of rovM, ΔrovM. The ΔrovM mutant strain was not affected in characteristic biofilm gut blockage, growth, or survival during single infection of fleas. Nonetheless, during a co-infection of fleas, the ΔrovM mutant exhibited a significant competitive fitness defect relative to the wild type strain. This competitive fitness defect was restored as a fitness advantage relative to the wild type in a ΔrovM mutant complemented in trans to over-express rovM. Consistent with this, Y. pestis strains, producing elevated transcriptional levels of rovM, displayed higher growth rates, and differential ability to form biofilm in response to specific nutrients in comparison to the wild type. In addition, we demonstrated that rovA was not repressed by RovM in fleas, but that elevated transcriptional levels of rovM in vitro correlated with repression of rovA under specific nutritional conditions. Collectively, these findings suggest that RovM likely senses specific nutrient cues in the flea gut environment, and accordingly directs metabolic adaptation to enhance flea gut colonization by Y. pestis.

  4. A LysR-Type Transcriptional Regulator, RovM, Senses Nutritional Cues Suggesting that It Is Involved in Metabolic Adaptation of Yersinia pestis to the Flea Gut

    PubMed Central

    Vadyvaloo, Viveka; Hinz, Angela K.

    2015-01-01

    Yersinia pestis has evolved as a clonal variant of Yersinia pseudotuberculosis to cause flea-borne biofilm–mediated transmission of the bubonic plague. The LysR-type transcriptional regulator, RovM, is highly induced only during Y. pestis infection of the flea host. RovM homologs in other pathogens regulate biofilm formation, nutrient sensing, and virulence; including in Y. pseudotuberculosis, where RovM represses the major virulence factor, RovA. Here the role that RovM plays during flea infection was investigated using a Y. pestis KIM6+ strain deleted of rovM, ΔrovM. The ΔrovM mutant strain was not affected in characteristic biofilm gut blockage, growth, or survival during single infection of fleas. Nonetheless, during a co-infection of fleas, the ΔrovM mutant exhibited a significant competitive fitness defect relative to the wild type strain. This competitive fitness defect was restored as a fitness advantage relative to the wild type in a ΔrovM mutant complemented in trans to over-express rovM. Consistent with this, Y. pestis strains, producing elevated transcriptional levels of rovM, displayed higher growth rates, and differential ability to form biofilm in response to specific nutrients in comparison to the wild type. In addition, we demonstrated that rovA was not repressed by RovM in fleas, but that elevated transcriptional levels of rovM in vitro correlated with repression of rovA under specific nutritional conditions. Collectively, these findings suggest that RovM likely senses specific nutrient cues in the flea gut environment, and accordingly directs metabolic adaptation to enhance flea gut colonization by Y. pestis. PMID:26348850

  5. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

    SciTech Connect

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.

    2014-04-01

    The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

  6. Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice.

    PubMed

    Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M; Branger, Christine G; Tinge, Steven A; Curtiss, Roy

    2010-06-01

    A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

  7. Enhanced Recovery of Airborne T3 Coliphage and Pasteurella pestis Bacteriophage by Means of a Presampling Humidification Technique

    PubMed Central

    Hatch, M. T.; Warren, J. C.

    1969-01-01

    This paper reports a series of experiments in which two methods of collecting airborne bacteriophage particles were compared. A standard aerosol sampler, the AGI-30, was evaluated for its competence in measuring the content of bacteriophage aerosols. It was used alone or with a prewetting or humidification device (humidifier bulb) to recover T3 coliphage and Pasteurella pestis bacteriophage particles from aerosols maintained at 21 C and varied relative humidity. Collection of bacteriophage particles via the humidifier bulb altered both the initial recovery level and the apparent biological decay. Sampling airborne bacteriophage particles by the AGI-30 alone yielded data that apparently underestimated the maximal number of potentially viable particles within the aerosol, sometimes by as much as 3 logs. PMID:4891719

  8. Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

    PubMed Central

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2012-01-01

    The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1–121), a transmembrane linker (122–142) and a large periplasmic domain (143–419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain. PMID:22349221

  9. Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2012-09-17

    The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.

  10. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    SciTech Connect

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Straub, Tim M.; Victry, Kristin D.; Hutchison, Janine R.; Valentine, Nancy B.; Bruckner-Lea, Cindy J.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.

  11. [Macro- and microevolution as related to the problem of origin and global expansion of the plague pathogen Yersinia pestis].

    PubMed

    Suntsov, V V; Suntsova, N I

    2008-01-01

    The ratio of macro- and microevolutionary processes is considered with reference to the ecological scenario of the origin of the plague pathogen and its subsequent natural and anthropogenic global expansion. The macroevolutionary transformation of the ancestral pseudotuberculosis microbe clone into the initial plague microbe Yersinia pestis tarbagani occurred in Central Asia at the end of the Late Pleistocene by a "vertical" Darwinian way in an inadaptive heterothermal continual intermediate environment--the Mongolian marmot Marmota sibirica-flea Oropsylla silantiewi system--via a sequence of unstable and currently extinct intermediate forms. Its natural geographic expansion on the "oil spot" principle in the postglacial time led to the microevolutionary formation of 20-30 hostal subspecies circulating in populations of the background species of burrowing rodents and pikas in arid areas of Eurasia. The intercontinental spread of the "marmot" and "rat" pathogen subspecies in the past few centuries has been exclusively anthropogenic, with the involvement of synanthropic (ship) rats.

  12. Genome-level transcription data of Yersinia pestis analyzed with a New metabolic constraint-based approach

    PubMed Central

    2012-01-01

    Background Constraint-based computational approaches, such as flux balance analysis (FBA), have proven successful in modeling genome-level metabolic behavior for conditions where a set of simple cellular objectives can be clearly articulated. Recently, the necessity to expand the current range of constraint-based methods to incorporate high-throughput experimental data has been acknowledged by the proposal of several methods. However, these methods have rarely been used to address cellular metabolic responses to some relevant perturbations such as antimicrobial or temperature-induced stress. Here, we present a new method for combining gene-expression data with FBA (GX-FBA) that allows modeling of genome-level metabolic response to a broad range of environmental perturbations within a constraint-based framework. The method uses mRNA expression data to guide hierarchical regulation of cellular metabolism subject to the interconnectivity of the metabolic network. Results We applied GX-FBA to a genome-scale model of metabolism in the gram negative bacterium Yersinia pestis and analyzed its metabolic response to (i) variations in temperature known to induce virulence, and (ii) antibiotic stress. Without imposition of any a priori behavioral constraints, our results show strong agreement with reported phenotypes. Our analyses also lead to novel insights into how Y. pestis uses metabolic adjustments to counter different forms of stress. Conclusions Comparisons of GX-FBA predicted metabolic states with fluxomic measurements and different reported post-stress phenotypes suggest that mass conservation constraints and network connectivity can be an effective representative of metabolic flux regulation in constraint-based models. We believe that our approach will be of aid in the in silico evaluation of cellular goals under different conditions and can be used for a variety of analyses such as identification of potential drug targets and their action. PMID:23216785

  13. Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

    2001-05-01

    Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

  14. Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    PubMed Central

    Zolotova, Anna; Tan, Eugene; Selden, Richard F.

    2013-01-01

    Background The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. Methodology/Principal Findings We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. Conclusions/Significance The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The

  15. A live attenuated strain of Yersinia pestis ΔyscB provides protection against bubonic and pneumonic plagues in mouse model.

    PubMed

    Zhang, Xuecan; Qi, Zhizhen; Du, Zongmin; Bi, Yujing; Zhang, Qingwen; Tan, Yafang; Yang, Huiying; Xin, Youquan; Yang, Ruifu; Wang, Xiaoyi

    2013-05-24

    To develop a safe and effective live plague vaccine, the ΔyscB mutant was constructed based on Yersinia pestis biovar Microtus strain 201 that is avirulent to humans, but virulent to mice. The virulence, immunogenicity and protective efficacy of the ΔyscB mutant were evaluated in this study. The results showed that the ΔyscB mutant was severely attenuated, elicited a higher F1-specific antibody titer and provided protective efficacy against bubonic and pneumonic plague in mouse model. The ΔyscB mutant could induce the secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4 and IL-10). Taken together, the ΔyscB mutant represented a potential vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses and to provide good protection against both subcutaneous and intranasal Y. pestis challenge.

  16. The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease.

    PubMed

    Flashner, Yehuda; Fisher, Morly; Tidhar, Avital; Mechaly, Adva; Gur, David; Halperin, Gideon; Zahavy, Eran; Mamroud, Emanuelle; Cohen, Sara

    2010-07-01

    Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

  17. Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens

    PubMed Central

    Feodorova, V.A.; Pan'kina, L.N.; Savostina, E.P.; Kuznetsov, O.S.; Konnov, N.P.; Sayapina, L.V.; Dentovskaya, S.V.; Shaikhutdinova, R.Z.; Ageev, S.A.; Lindner, B.; A.N.Kondakova; Bystrova, O.V.; Kocharova, N.A.; Senchenkova, S.N.; Holst, O.; Pier, G.B.; Knirel, Y.A.; Anisimov, A.P.; Motin, V.L.

    2010-01-01

    Deletion mutants in the lpxM gene in two Y. pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV ΔlpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation. PMID:19428838

  18. Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens.

    PubMed

    Feodorova, V A; Pan'kina, L N; Savostina, E P; Kuznetsov, O S; Konnov, N P; Sayapina, L V; Dentovskaya, S V; Shaikhutdinova, R Z; Ageev, S A; Lindner, B; Kondakova, A N; Bystrova, O V; Kocharova, N A; Senchenkova, S N; Holst, O; Pier, G B; Knirel, Y A; Anisimov, A P; Motin, V L

    2009-04-01

    Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV DeltalpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation. PMID:19428838

  19. Manipulation of Interleukin-1β and Interleukin-18 Production by Yersinia pestis Effectors YopJ and YopM and Redundant Impact on Virulence.

    PubMed

    Ratner, Dmitry; Orning, M Pontus A; Starheim, Kristian K; Marty-Roix, Robyn; Proulx, Megan K; Goguen, Jon D; Lien, Egil

    2016-05-01

    Innate immunity plays a central role in resolving infections by pathogens. Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. These cytokines are produced by a two-step mechanism involving NF-κB-mediated pro-cytokine production and inflammasome-driven maturation into bioactive inflammatory mediators. Because of the anti-microbial effects induced by IL-1β/IL-18, it may be desirable for pathogens to manipulate their production. Y. pestis type III secretion system effectors YopJ and YopM can interfere with different parts of this process. Both effectors have been reported to influence inflammasome caspase-1 activity; YopJ promotes caspase-8-dependent cell death and caspase-1 cleavage, whereas YopM inhibits caspase-1 activity via an incompletely understood mechanism. However, neither effector appears essential for full virulence in vivo Here we report that the sum of influences by YopJ and YopM on IL-1β/IL-18 release is suppressive. In the absence of YopM, YopJ minimally affects caspase-1 cleavage but suppresses IL-1β, IL-18, and other cytokines and chemokines. Importantly, we find that Y. pestis containing combined deletions of YopJ and YopM induces elevated levels of IL-1β/IL-18 in vitro and in vivo and is significantly attenuated in a mouse model of bubonic plague. The reduced virulence of the YopJ-YopM mutant is dependent on the presence of IL-1β, IL-18, and caspase-1. Thus, we conclude that Y. pestis YopJ and YopM can both exert a tight control of host IL-1β/IL-18 production to benefit the bacteria, resulting in a redundant impact on virulence. PMID:26884330

  20. Genomic Insights into a New Citrobacter koseri Strain Revealed Gene Exchanges with the Virulence-Associated Yersinia pestis pPCP1 Plasmid.

    PubMed

    Armougom, Fabrice; Bitam, Idir; Croce, Olivier; Merhej, Vicky; Barassi, Lina; Nguyen, Ti-Thien; La Scola, Bernard; Raoult, Didier

    2016-01-01

    The history of infectious diseases raised the plague as one of the most devastating for human beings. Far too often considered an ancient disease, the frequent resurgence of the plague has led to consider it as a reemerging disease in Madagascar, Algeria, Libya, and Congo. The genetic factors associated with the pathogenicity of Yersinia pestis, the causative agent of the plague, involve the acquisition of the pPCP1 plasmid that promotes host invasion through the expression of the virulence factor Pla. The surveillance of plague foci after the 2003 outbreak in Algeria resulted in a positive detection of the specific pla gene of Y. pestis in rodents. However, the phenotypic characterization of the isolate identified a Citrobacter koseri. The comparative genomics of our sequenced C. koseri URMITE genome revealed a mosaic gene structure resulting from the lifestyle of our isolate and provided evidence for gene exchanges with different enteric bacteria. The most striking was the acquisition of a continuous 2 kb genomic fragment containing the virulence factor Pla of the Y. pestis pPCP1 plasmid; however, the subcutaneous injection of the CKU strain in mice did not produce any pathogenic effect. Our findings demonstrate that fast molecular detection of plague using solely the pla gene is unsuitable and should rather require Y. pestis gene marker combinations. We also suggest that the evolutionary force that might govern the expression of pathogenicity can occur through the acquisition of virulence genes but could also require the loss or the inactivation of resident genes such as antivirulence genes. PMID:27014253

  1. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

    DOE PAGESBeta

    Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.; Gao, Tingjuan; Arroyo, Erin S.; Hinz, Angela K.; Bourguet, Feliza A.; Segelke, Brent; Hoeprich, Paul D.; Huser, Thomas; et al

    2016-03-25

    Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

  2. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

    PubMed Central

    Blanchette, Craig D.; Gao, Tingjuan; Arroyo, Erin S.; Hinz, Angela K.; Bourguet, Feliza A.; Segelke, Brent; Hoeprich, Paul D.; Huser, Thomas; Laurence, Ted A.; Motin, Vladimir L.; Chromy, Brett A.

    2016-01-01

    Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses. PMID:27015536

  3. A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague.

    PubMed

    Sha, Jian; Rosenzweig, Jason A; Kirtley, Michelle L; van Lier, Christina J; Fitts, Eric C; Kozlova, Elena V; Erova, Tatiana E; Tiner, Bethany L; Chopra, Ashok K

    2013-02-01

    The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD(50). Intranasal infection of mice with 15 LD(50) of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24-72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy

  4. Genomic Insights into a New Citrobacter koseri Strain Revealed Gene Exchanges with the Virulence-Associated Yersinia pestis pPCP1 Plasmid

    PubMed Central

    Armougom, Fabrice; Bitam, Idir; Croce, Olivier; Merhej, Vicky; Barassi, Lina; Nguyen, Ti-Thien; La Scola, Bernard; Raoult, Didier

    2016-01-01

    The history of infectious diseases raised the plague as one of the most devastating for human beings. Far too often considered an ancient disease, the frequent resurgence of the plague has led to consider it as a reemerging disease in Madagascar, Algeria, Libya, and Congo. The genetic factors associated with the pathogenicity of Yersinia pestis, the causative agent of the plague, involve the acquisition of the pPCP1 plasmid that promotes host invasion through the expression of the virulence factor Pla. The surveillance of plague foci after the 2003 outbreak in Algeria resulted in a positive detection of the specific pla gene of Y. pestis in rodents. However, the phenotypic characterization of the isolate identified a Citrobacter koseri. The comparative genomics of our sequenced C. koseri URMITE genome revealed a mosaic gene structure resulting from the lifestyle of our isolate and provided evidence for gene exchanges with different enteric bacteria. The most striking was the acquisition of a continuous 2 kb genomic fragment containing the virulence factor Pla of the Y. pestis pPCP1 plasmid; however, the subcutaneous injection of the CKU strain in mice did not produce any pathogenic effect. Our findings demonstrate that fast molecular detection of plague using solely the pla gene is unsuitable and should rather require Y. pestis gene marker combinations. We also suggest that the evolutionary force that might govern the expression of pathogenicity can occur through the acquisition of virulence genes but could also require the loss or the inactivation of resident genes such as antivirulence genes. PMID:27014253

  5. The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    PubMed Central

    Osei-Owusu, Patrick; Jessen Condry, Danielle L.; Toosky, Melody; Roughead, William; Bradley, David S.

    2015-01-01

    The type III secretion system is employed by many pathogens, including the genera Yersinia, Shigella, Pseudomonas, and Salmonella, to deliver effector proteins into eukaryotic cells. The injectisome needle is formed by the polymerization of a single protein, e.g., YscF (Yersinia pestis), PscF (Pseudomonas aeruginosa), PrgI (Salmonella enterica SPI-1), SsaG (Salmonella enterica SPI-2), or MxiH (Shigella flexneri). In this study, we demonstrated that the N termini of some needle proteins, particularly the N terminus of YscF from Yersinia pestis, influences host immune responses. The N termini of several needle proteins were truncated and tested for the ability to induce inflammatory responses in a human monocytic cell line (THP-1 cells). Truncated needle proteins induced proinflammatory cytokines to different magnitudes than the corresponding wild-type proteins, except SsaG. Notably, N-terminally truncated YscF induced significantly higher activation of NF-κB and/or AP-1 and higher induction of proinflammatory cytokines, suggesting that a function of the N terminus of YscF is interference with host sensing of YscF, consistent with Y. pestis pathogenesis. To directly test the ability of the N terminus of YscF to suppress cytokine induction, a YscF-SsaG chimera with 15 N-terminal amino acids from YscF added to SsaG was constructed. The chimeric YscF-SsaG induced lower levels of cytokines than wild-type SsaG. However, the addition of 15 random amino acids to SsaG had no effect on NF-κB/AP-1 activation. These results suggest that the N terminus of YscF can function to decrease cytokine induction, perhaps contributing to a favorable immune environment leading to survival of Y. pestis within the eukaryotic host. PMID:25644012

  6. Manipulation of Interleukin-1β and Interleukin-18 Production by Yersinia pestis Effectors YopJ and YopM and Redundant Impact on Virulence.

    PubMed

    Ratner, Dmitry; Orning, M Pontus A; Starheim, Kristian K; Marty-Roix, Robyn; Proulx, Megan K; Goguen, Jon D; Lien, Egil

    2016-05-01

    Innate immunity plays a central role in resolving infections by pathogens. Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. These cytokines are produced by a two-step mechanism involving NF-κB-mediated pro-cytokine production and inflammasome-driven maturation into bioactive inflammatory mediators. Because of the anti-microbial effects induced by IL-1β/IL-18, it may be desirable for pathogens to manipulate their production. Y. pestis type III secretion system effectors YopJ and YopM can interfere with different parts of this process. Both effectors have been reported to influence inflammasome caspase-1 activity; YopJ promotes caspase-8-dependent cell death and caspase-1 cleavage, whereas YopM inhibits caspase-1 activity via an incompletely understood mechanism. However, neither effector appears essential for full virulence in vivo Here we report that the sum of influences by YopJ and YopM on IL-1β/IL-18 release is suppressive. In the absence of YopM, YopJ minimally affects caspase-1 cleavage but suppresses IL-1β, IL-18, and other cytokines and chemokines. Importantly, we find that Y. pestis containing combined deletions of YopJ and YopM induces elevated levels of IL-1β/IL-18 in vitro and in vivo and is significantly attenuated in a mouse model of bubonic plague. The reduced virulence of the YopJ-YopM mutant is dependent on the presence of IL-1β, IL-18, and caspase-1. Thus, we conclude that Y. pestis YopJ and YopM can both exert a tight control of host IL-1β/IL-18 production to benefit the bacteria, resulting in a redundant impact on virulence.

  7. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles.

    PubMed

    Coleman, Matthew A; Cappuccio, Jenny A; Blanchette, Craig D; Gao, Tingjuan; Arroyo, Erin S; Hinz, Angela K; Bourguet, Feliza A; Segelke, Brent; Hoeprich, Paul D; Huser, Thomas; Laurence, Ted A; Motin, Vladimir L; Chromy, Brett A

    2016-01-01

    Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses. PMID:27015536

  8. Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

    PubMed Central

    Quintard, Kévin; Dewitte, Amélie; Reboul, Angéline; Madec, Edwige; Bontemps-Gallo, Sébastien; Dondeyne, Jacqueline; Marceau, Michaël; Simonet, Michel

    2015-01-01

    The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary. PMID:26150539

  9. Vaccination with F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against plague upon oral challenge with Yersinia pestis

    USGS Publications Warehouse

    Rocke, T.E.; Smith, S.; Marinari, P.; Kreeger, J.; Enama, J.T.; Powell, B.S.

    2008-01-01

    Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody postvaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862, respectively) were found in all vaccinates up to 2 yr postvaccination, whereas seven control animals remained antibody negative throughout the same time period. ?? Wildlife Disease Association 2008.

  10. Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague.

    PubMed

    Sebbane, Florent; Jarrett, Clayton O; Gardner, Donald; Long, Daniel; Hinnebusch, B Joseph

    2006-04-01

    Yersinia pestis is transmitted by fleas and causes bubonic plague, characterized by severe local lymphadenitis that progresses rapidly to systemic infection and life-threatening septicemia. Here, we show that although flea-borne transmission usually leads to bubonic plague in mice, it can also lead to primary septicemic plague. However, intradermal injection of Y. pestis, commonly used to mimic transmission by fleabite, leads only to bubonic plague. A Y. pestis strain lacking the plasmid-encoded cell-surface plasminogen activator, which is avirulent by intradermal or s.c. injection, was able to cause fatal primary septicemic plague at low incidence, but not bubonic plague, when transmitted by fleas. The results clarify a long-standing uncertainty about the etiology of primary septicemic plague and support an evolutionary scenario in which plague first emerged as a flea-borne septicemic disease of limited transmissibility. Subsequent acquisition of the plasminogen activator gene by horizontal transfer enabled the bubonic form of disease and increased the potential for epidemic spread.

  11. STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : VI. ON THE VIRULENCE OF THE OVERGROWTH IN THE LYSED CULTURES OF BACILLUS PESTIS CAVIAE (M. T. II).

    PubMed

    Bronfenbrenner, J; Muckenfuss, R S; Korb, C

    1926-10-31

    Resistants isolated from the overgrowth of cultures of B. pestis caviae (M. T. II) lysed by various strains of specific bacteriophage proved to be avirulent when administered to mice by feeding, or by intraperitoneal injection. These cultures remained resistant to the action of bacteriophage so long as they were carried on agar. When transferred to broth, however, one group of resistants, namely, those isolated by means of "weak" phages, became susceptible to lysis after five to seven daily passages. The other group of resistants, isolated from the cultures lysed by one of the "strong" phages, failed to become susceptible to lysis even after nearly 200 passages in broth. Simultaneously with the recovery of susceptibility, the cultures of the first group regained a degree of virulence comparable to that of the parent culture of B. pestis caviae. The cultures of the second group of resistants have failed thus far to recover virulence (10 months after isolation). The latter cultures, apart from lack of both virulence and susceptibility to lysis, are identical with the parent culture of B. pestis caviae, as indicated by biochemical and antigenic properties. Our findings offer evidence in favor of the view that resistant strains result from selection among variants already existing in the parent culture and do not arise through the inheritance of specific immunity properties produced by the action of phage.

  12. Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae)

    PubMed Central

    Hinnebusch, B. Joseph; Boegler, Karen A.; Graham, Christine B.; MacMillan, Katherine; Montenieri, John A.; Bearden, Scott W.; Gage, Kenneth L.; Eisen, Rebecca J.

    2014-01-01

    Plague, caused by Yersinia pestis, is characterized by quiescent periods punctuated by rapidly spreading epizootics. The classical ‘blocked flea’ paradigm, by which a blockage forms in the flea’s proventriculus on average 1–2 weeks post-infection (p.i.), forces starving fleas to take multiple blood meals, thus increasing opportunities for transmission. Recently, the importance of early-phase transmission (EPT), which occurs prior to blockage formation, has been emphasized during epizootics. Whilst the physiological and molecular mechanisms of blocked flea transmission are well characterized, the pathogen–vector interactions have not been elucidated for EPT. Within the blocked flea model, Yersinia murine toxin (Ymt) has been shown to be important for facilitating colonization of the midgut within the flea. One proposed mechanism of EPT is the regurgitation of infectious material from the flea midgut during feeding. Such a mechanism would require bacteria to colonize and survive for at least brief periods in the midgut, a process that is mediated by Ymt. Two key bridging vectors of Y. pestis to humans, Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae), were used in our study to test this hypothesis. Fleas were infected with a mutant strain of Y. pestis containing a non-functional ymt that was shown previously to be incapable of colonizing the midgut and were then allowed to feed on SKH-1 mice 3 days p.i. Our results show that Ymt was not required for EPT by either flea species. PMID:25187626

  13. Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms.

    PubMed

    Kopylov, Pavel Kh; Platonov, Mikhail E; Ablamunits, Vitaly G; Kombarova, Tat'yana I; Ivanov, Sergey A; Kadnikova, Lidiya A; Somov, Aleksey N; Dentovskaya, Svetlana V; Uversky, Vladimir N; Anisimov, Andrey P

    2016-01-01

    Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4-7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection. PMID:27606595

  14. Protective immunity against a lethal respiratory Yersinia pestis challenge induced by V antigen or the F1 capsular antigen incorporated into adenovirus capsid.

    PubMed

    Boyer, Julie L; Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Chiuchiolo, Maria J; Senina, Svetlana; Perlin, David; Crystal, Ronald G

    2010-07-01

    The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective

  15. Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms

    PubMed Central

    Kopylov, Pavel Kh.; Platonov, Mikhail E.; Ablamunits, Vitaly G.; Kombarova, Tat’yana I.; Ivanov, Sergey A.; Kadnikova, Lidiya A.; Somov, Aleksey N.; Dentovskaya, Svetlana V.; Uversky, Vladimir N.

    2016-01-01

    Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4–7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection. PMID:27606595

  16. lcrR, a low-Ca2(+)-response locus with dual Ca2(+)-dependent functions in Yersinia pestis.

    PubMed Central

    Barve, S S; Straley, S C

    1990-01-01

    The low-Ca2+ response (Lcr) of Yersinia includes a regulatory cascade and a set of virulence-related proteins, one of which is the V antigen. The regulatory genes modulate both bacterial growth and expression of the virulence-related proteins in response to temperature and the presence of Ca2+ and nucleotides. In this study we defined a new Lcr locus, lcrR, in Yersinia pestis KIM. An lcrR mutant, obtained by insertion mutagenesis, failed to grow at 37 degrees C whether Ca2+ was present or not. However, it grew normally in the presence of ATP, showing that the Ca2(+)- and nucleotide-responsive mechanisms are separate in Y. pestis. The lcrR mutant was avirulent in mice, probably due to its compromised growth at 37 degrees C. beta-Galactosidase measurements and Northern (RNA blot) analysis revealed that lcrR transcription was regulated primarily by temperature. The DNA sequence of the lcrR locus contained a single open reading frame of 441 bases that could encode a protein with a molecular weight of 16,470 and a pI of 10.73. Expression of an lcrR-containing clone in Escherichia coli yielded a 16,000-molecular-weight protein. At 37 degrees C, the lcrR mutant strongly expressed V antigen and initiated lcrGVH transcription whether Ca2+ was present or not, indicating that this mutant had lost the transcriptional downregulation of lcrGVH shown by the parent in the presence of Ca2+. In the absence of Ca2+, the mutant failed to express LcrG, even though lcrGVH mRNA initiated upstream of lcrG at the normal sites. These data suggest that the lcrR locus is necessary for the regulation of LcrG expression in the absence of Ca2+. Therefore, this locus has a dual regulatory role in the low-Ca2+ response. Images PMID:1695896

  17. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

    PubMed Central

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.

    2014-01-01

    Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenyl­alanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully understand the function of the acyl-group binding pocket in substrate specificity. PMID:24699651

  18. Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis as next generation plague vaccines.

    PubMed

    Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L; van Lier, Christina J; Sha, Jian; Yeager, Linsey A; Chopra, Ashok K; Rao, Venigalla B

    2013-01-01

    Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH₂-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

  19. Development and Evaluation of Two Simple, Rapid Immunochromatographic Tests for the Detection of Yersinia pestis Antibodies in Humans and Reservoirs

    PubMed Central

    Rajerison, Minoarisoa; Dartevelle, Sylvie; Ralafiarisoa, Lalao A.; Bitam, Idir; Tuyet, Dinh Thi Ngoc; Andrianaivoarimanana, Voahangy; Nato, Faridabano; Rahalison, Lila

    2009-01-01

    Background Tools for plague diagnosis and surveillance are not always available and affordable in most of the countries affected by the disease. Yersinia pestis isolation for confirmation is time-consuming and difficult to perform under field conditions. Serologic tests like ELISA require specific equipments not always available in developing countries. In addition to the existing rapid test for antigen detection, a rapid serodiagnostic assay may be useful for plague control. Methods/Principal Findings We developed two rapid immunochromatography-based tests for the detection of antibodies directed against F1 antigen of Y. pestis. The first test, SIgT, which detects total Ig (IgT) anti-F1 in several species (S) (human and reservoirs), was developed in order to have for the field use an alternative method to ELISA. The performance of the SIgT test was evaluated with samples from humans and animals for which ELISA was used to determine the presumptive diagnosis of plague. SIgT test detected anti-F1 Ig antibodies in humans with a sensitivity of 84.6% (95% CI: 0.76–0.94) and a specificity of 98% (95% CI: 0.96–1). In evaluation of samples from rodents and other small mammals, the SlgT test had a sensitivity of 87.8% (95% CI: 0.80–0.94) and a specificity of 90.3% (95% CI: 0.86–0.93). Improved performance was obtained with samples from dogs, a sentinel animal, with a sensitivity of 93% (95% CI: 0.82–1) and a specificity of 98% (95% CI: 0.95–1.01). The second test, HIgM, which detects human (H) IgM anti-F1, was developed in order to have another method for plague diagnosis. Its sensitivity was 83% (95% CI: 0.75–0.90) and its specificity about 100%. Conclusion/Significance The SIgT test is of importance for surveillance because it can detect Ig antibodies in a range of reservoir species. The HIgM test could facilitate the diagnosis of plague during outbreaks, particularly when only a single serum sample is available. PMID:19399164

  20. [COMPARATIVE ANALYSIS OF THE MLVA25- AND MLVA7-TYPING ACCORDING TO THEIR ABILITY TO ASCERTAIN FOCAL AFFILIATION OF YERSINIA PESTIS STRAINS BY THE EXAMPLE OF ISOLATES FROM THE CENTRAL-CAUCASIAN HIGHLAND NATURAL PLAGUE FOCUS].

    PubMed

    Evseeva, V V; Platonov, M E; Govorunov, I G; Efremenko, D V; Kuznetsova, I V; Dentovskaya, S V; Kulichenko, A N; Anisimov, A P

    2016-01-01

    Comparative analysis of the MLVA25- and MLVA7-typing ability to evaluate focal belonging of Y. pestis strains by the example of bv. medievalis isolates from the Central-Caucasian highland natural plague focus was carried out. The MLVA25-types of-82 isolates from this area were determined and included into the database containing information on 949 Y. pestis strains from other natural foci of Russia and other countries. Categorical-UPGMA dendrograms were created on the bases of the data concerning all 25 VNTR loci or only seven of them, which were recommended by the experts of the Russian Research Anti-Plague Institute "Microbe" for differentiation of the Y. pestis strains according to their affiliation to specific foci. The obtained data indicated greater possibility of diagnostic mistakes in the case of the MLVA7-typing and supported expediency of division of the Central-Caucasian highland natural plague focus into two sub-foci. PMID:27183721

  1. Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis.

    PubMed

    Ding, Yi; Fujimoto, L Miya; Yao, Yong; Plano, Gregory V; Marassi, Francesca M

    2015-02-01

    The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.

  2. Evaluation of Yersinia pestis Transmission Pathways for Sylvatic Plague in Prairie Dog Populations in the Western U.S.

    PubMed

    Richgels, Katherine L D; Russell, Robin E; Bron, Gebbiena M; Rocke, Tonie E

    2016-06-01

    Sylvatic plague, caused by the bacterium Yersinia pestis, is periodically responsible for large die-offs in rodent populations that can spillover and cause human mortalities. In the western US, prairie dog populations experience nearly 100% mortality during plague outbreaks, suggesting that multiple transmission pathways combine to amplify plague dynamics. Several alternate pathways in addition to flea vectors have been proposed, such as transmission via direct contact with bodily fluids or inhalation of infectious droplets, consumption of carcasses, and environmental sources of plague bacteria, such as contaminated soil. However, evidence supporting the ability of these proposed alternate pathways to trigger large-scale epizootics remains elusive. Here we present a short review of potential plague transmission pathways and use an ordinary differential equation model to assess the contribution of each pathway to resulting plague dynamics in black-tailed prairie dogs (Cynomys ludovicianus) and their fleas (Oropsylla hirsuta). Using our model, we found little evidence to suggest that soil contamination was capable of producing plague epizootics in prairie dogs. However, in the absence of flea transmission, direct transmission, i.e., contact with bodily fluids or inhalation of infectious droplets, could produce enzootic dynamics, and transmission via contact with or consumption of carcasses could produce epizootics. This suggests that these pathways warrant further investigation. PMID:27234457

  3. Evaluation of Yersinia pestis transmission pathways for sylvatic plague in prairie dog populations in the western U.S.

    USGS Publications Warehouse

    Richgels, Katherine L. D.; Russell, Robin E.; Bron, Gebbiena; Rocke, Tonie E.

    2016-01-01

    Sylvatic plague, caused by the bacterium Yersinia pestis, is periodically responsible for large die-offs in rodent populations that can spillover and cause human mortalities. In the western US, prairie dog populations experience nearly 100% mortality during plague outbreaks, suggesting that multiple transmission pathways combine to amplify plague dynamics. Several alternate pathways in addition to flea vectors have been proposed, such as transmission via direct contact with bodily fluids or inhalation of infectious droplets, consumption of carcasses, and environmental sources of plague bacteria, such as contaminated soil. However, evidence supporting the ability of these proposed alternate pathways to trigger large-scale epizootics remains elusive. Here we present a short review of potential plague transmission pathways and use an ordinary differential equation model to assess the contribution of each pathway to resulting plague dynamics in black-tailed prairie dogs (Cynomys ludovicianus) and their fleas (Oropsylla hirsuta). Using our model, we found little evidence to suggest that soil contamination was capable of producing plague epizootics in prairie dogs. However, in the absence of flea transmission, direct transmission, i.e., contact with bodily fluids or inhalation of infectious droplets, could produce enzootic dynamics, and transmission via contact with or consumption of carcasses could produce epizootics. This suggests that these pathways warrant further investigation.

  4. Evaluation of Yersinia pestis Transmission Pathways for Sylvatic Plague in Prairie Dog Populations in the Western U.S.

    PubMed

    Richgels, Katherine L D; Russell, Robin E; Bron, Gebbiena M; Rocke, Tonie E

    2016-06-01

    Sylvatic plague, caused by the bacterium Yersinia pestis, is periodically responsible for large die-offs in rodent populations that can spillover and cause human mortalities. In the western US, prairie dog populations experience nearly 100% mortality during plague outbreaks, suggesting that multiple transmission pathways combine to amplify plague dynamics. Several alternate pathways in addition to flea vectors have been proposed, such as transmission via direct contact with bodily fluids or inhalation of infectious droplets, consumption of carcasses, and environmental sources of plague bacteria, such as contaminated soil. However, evidence supporting the ability of these proposed alternate pathways to trigger large-scale epizootics remains elusive. Here we present a short review of potential plague transmission pathways and use an ordinary differential equation model to assess the contribution of each pathway to resulting plague dynamics in black-tailed prairie dogs (Cynomys ludovicianus) and their fleas (Oropsylla hirsuta). Using our model, we found little evidence to suggest that soil contamination was capable of producing plague epizootics in prairie dogs. However, in the absence of flea transmission, direct transmission, i.e., contact with bodily fluids or inhalation of infectious droplets, could produce enzootic dynamics, and transmission via contact with or consumption of carcasses could produce epizootics. This suggests that these pathways warrant further investigation.

  5. The role of transition metal transporters for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis.

    PubMed

    Perry, Robert D; Bobrov, Alexander G; Fetherston, Jacqueline D

    2015-06-01

    Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.

  6. A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

    PubMed

    Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

    2012-07-01

    Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague.

  7. Recombinant F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against virulent Yersinia pestis infection

    USGS Publications Warehouse

    Rocke, T.E.; Mencher, J.; Smith, S.R.; Friedlander, A.M.; Andrews, G.P.; Baeten, L.A.

    2004-01-01

    Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague, caused by the bacterium Yersinia pestis, and this disease has severely hampered efforts to restore ferrets to their historic range. A study was conducted to assess the efficacy of vaccination of black-footed ferrets against plague using a recombinant protein vaccine, designated F1-V, developed by personnel at the U.S. Army Medical Research Institute of Infectious Diseases. Seven postreproductive black-footed ferrets were immunized with the vaccine, followed by two booster immunizations on days 23 and 154; three control black-footed ferrets received a placebo. After the second immunization, antibody titers to both F1 and V antigen were found to be significantly higher in vaccinates than controls. On challenge with 7,800 colony-forming units of virulent plague by s.c. injection, the three control animals died within 3 days, but six of seven vaccinates survived with no ill effects. The seventh vaccinate died on day 8. These results indicate that black-footed ferrets can be immunized against plague induced by the s.c. route, similar to fleabite injection.

  8. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis

    PubMed Central

    Seiner, DR; Colburn, HA; Baird, C; Bartholomew, RA; Straub, T; Victry, K; Hutchison, JR; Valentine, N; Bruckner-Lea, CJ

    2013-01-01

    Aims To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray® system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results DNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray® Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs. Conclusions Our results indicate that the FilmArray® Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp. Significance and Impact of the Study The FilmArray® platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training. PMID:23279070

  9. The Role of Transition Metal Transporters for Iron, Zinc, Manganese, and Copper in the Pathogenesis of Yersinia pestis

    PubMed Central

    Perry, Robert D.; Bobrov, Alexander G.; Fetherston, Jacqueline D.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent. PMID:25891079

  10. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

  11. Growth Curve Models for the Analysis of Phenotype Arrays for a Systems Biology Overview of Yersinia pestis

    SciTech Connect

    Fodor, I K; Holtz-Morris, A E; McCutchen-Maloney, S L

    2005-09-08

    The Phenotype MicroArray technology of Biolog, Inc. (Hayward, CA) measures the respiration of cells as a function of time in thousands of microwells simultaneously, and thus provides a high-throughput means of studying cellular phenotypes. The microwells contain compounds involved in a number of biochemical pathways, as well as chemicals that test the sensitivity of cells against antibiotics and stress. While the PM experimental workflow is completely automated, statistical methods to analyze and interpret the data are lagging behind. To take full advantage of the technology, it is essential to develop efficient analytical methods to quantify the information in the complex datasets resulting from PM experiments. We propose the use of statistical growth-curve models to rigorously quantify observed differences in PM experiments, in the context of the growth and metabolism of Yersinia pestis cells grown under different physiological conditions. The information from PM experiments complement genomic and proteomic results and can be used to identify gene function and in drug development. Successful coupling of phenomics results with genomics and proteomics will lead to an unprecedented ability to characterize bacterial function at a systems biology level.

  12. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops. PMID:26894530

  13. A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague.

    PubMed

    Spear, Abigail M; Rana, Rohini R; Jenner, Dominic C; Flick-Smith, Helen C; Oyston, Petra C F; Simpson, Peter; Matthews, Stephen J; Byrne, Bernadette; Atkins, Helen S

    2012-06-01

    The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

  14. [On the origin of Yersinia pestis, a causative agent of the plague: A concept of population-genetic macroevolution in transitive environment].

    PubMed

    Suntsov, V V

    2015-01-01

    An ecological scenario is proposed for the origin of causative agent of the plague (the bacterium Yersenia pestis) from the clone of pseudotuberculous microbe of the first serotype Y. pseudotuberculosis O:1b. Disclosed are the conditions of gradual intrusion of psychrophile saprozoonosis ancestor into the blood of the primary host, Mongolian tarbagan marmot Marmota sibirica. As an inductor of speciation acted the Sartan cooling that occurred in the end of late Pleistocene under conditions of arid ultra-continental climate in Central Asia. Soil freezing down to the level of hibernating chambers in marmot burrows initiated the transition of marmot flea, Oropsylla silantiewi, larvae to optional hemophagy on the mucous coat inside the mouth cavity of sleeping marmots. In its turn, this promoted the conditions of mass traumatic intrusion of Y pseudotuberculosis into marmots bloodstream from faecal particles getting in their mouth cavity in course of building up a plug in a burrow for hibernating. In marmot populations, the selection of bacteria underwent under conditions of heterothermy with repeated changes of hibernating marmots body temperature within the range of 5-37 degrees C (torpor-euthermy). During the warm season, when pseudotuberculous microbes are totally eliminated from the bloodstream of healthy marmots with body temperature about 37 degrees C, bacteria could survive in fleas' digestive tract in the form of biofilm developing in proventriculus as a so called blockage. Final isolation between ancestral and daughter species was helped by the development of intrapopulation antagonism related with the beginning of full-scale synthesis of bacteriocin pesticin. Population-genetic processes in the "marmot-flea" system have led to a macroevolutionary event, that is, to passage of bacteria in a new ecological niche and adaptive zone that are principally different from those of the ancestor. All the present intraspecies forms of Y. pestis that appeared due to

  15. [On the origin of Yersinia pestis, a causative agent of the plague: A concept of population-genetic macroevolution in transitive environment].

    PubMed

    Suntsov, V V

    2015-01-01

    An ecological scenario is proposed for the origin of causative agent of the plague (the bacterium Yersenia pestis) from the clone of pseudotuberculous microbe of the first serotype Y. pseudotuberculosis O:1b. Disclosed are the conditions of gradual intrusion of psychrophile saprozoonosis ancestor into the blood of the primary host, Mongolian tarbagan marmot Marmota sibirica. As an inductor of speciation acted the Sartan cooling that occurred in the end of late Pleistocene under conditions of arid ultra-continental climate in Central Asia. Soil freezing down to the level of hibernating chambers in marmot burrows initiated the transition of marmot flea, Oropsylla silantiewi, larvae to optional hemophagy on the mucous coat inside the mouth cavity of sleeping marmots. In its turn, this promoted the conditions of mass traumatic intrusion of Y pseudotuberculosis into marmots bloodstream from faecal particles getting in their mouth cavity in course of building up a plug in a burrow for hibernating. In marmot populations, the selection of bacteria underwent under conditions of heterothermy with repeated changes of hibernating marmots body temperature within the range of 5-37 degrees C (torpor-euthermy). During the warm season, when pseudotuberculous microbes are totally eliminated from the bloodstream of healthy marmots with body temperature about 37 degrees C, bacteria could survive in fleas' digestive tract in the form of biofilm developing in proventriculus as a so called blockage. Final isolation between ancestral and daughter species was helped by the development of intrapopulation antagonism related with the beginning of full-scale synthesis of bacteriocin pesticin. Population-genetic processes in the "marmot-flea" system have led to a macroevolutionary event, that is, to passage of bacteria in a new ecological niche and adaptive zone that are principally different from those of the ancestor. All the present intraspecies forms of Y. pestis that appeared due to

  16. Functional and Structural Analysis of HicA3-HicB3, a Novel Toxin-Antitoxin System of Yersinia pestis

    PubMed Central

    Bibi-Triki, Sabrina; Li de la Sierra-Gallay, Inès; Lazar, Noureddine; Leroy, Arnaud; Van Tilbeurgh, Herman

    2014-01-01

    The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif. PMID:25112480

  17. Applications of docking and molecular dynamic studies on the search for new drugs against the biological warfare agents Bacillus anthracis and Yersinia pestis.

    PubMed

    França, Tanos Celmar Costa; Guimarães, Ana Paula; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; Ramalho, Teodorico Castro

    2013-12-01

    The fear of biological warfare agents (BWA) use by terrorists is the major concern of the security agencies and health authorities worldwide today. The non-existence of vaccines or drugs against most BWA and the possibility of genetic modified strains has turned the search for new drugs to a state of urgency. Fast in silico techniques are, therefore, perfect tools for this task once they can quickly provide structures of several new lead compounds for further experimental work. Here we try to present a mini-review on docking and molecular dynamics simulations studies applied to the drug design against the BWA Bacillus anthracis and Yersinia pestis.

  18. [Pilot-scale purification of rF1-V fusion protein of Yersinia pestis and characterization of its immunogenicity].

    PubMed

    Fang, Ting; Ren, Jun; Zhang, Jinlong; Yin, Kexin; Yang, Xiuxu; Yu, Rui; Zhang, Xiaopeng; Yu, Changming

    2016-01-01

    Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine. PMID:27363202

  19. Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase.

    PubMed

    Neckles, Carla; Pschibul, Annica; Lai, Cheng-Tsung; Hirschbeck, Maria; Kuper, Jochen; Davoodi, Shabnam; Zou, Junjie; Liu, Nina; Pan, Pan; Shah, Sonam; Daryaee, Fereidoon; Bommineni, Gopal R; Lai, Cristina; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2016-05-31

    The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.

  20. Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase.

    PubMed

    Neckles, Carla; Pschibul, Annica; Lai, Cheng-Tsung; Hirschbeck, Maria; Kuper, Jochen; Davoodi, Shabnam; Zou, Junjie; Liu, Nina; Pan, Pan; Shah, Sonam; Daryaee, Fereidoon; Bommineni, Gopal R; Lai, Cristina; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2016-05-31

    The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156. PMID:27136302

  1. Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis

    PubMed Central

    Chapman, David A. G.; Zavialov, Anton V.; Chernovskaya, Tatiana V.; Karlyshev, Andrey V.; Zav’yalova, Galina A.; Vasiliev, Anatoly M.; Dudich, Igor V.; Abramov, Vyacheslav M.; Zav’yalov, Vladimir P.; MacIntyre, Sheila

    1999-01-01

    The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 β-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 β-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones. PMID:10198004

  2. Yersinia pestis YopJ suppresses tumor necrosis factor alpha induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague.

    PubMed

    Lemaître, Nadine; Sebbane, Florent; Long, Daniel; Hinnebusch, B Joseph

    2006-09-01

    The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system that transfers six Yop effector proteins into host cells. One of these proteins, YopJ, has been shown to disrupt host cell signaling pathways involved in proinflammatory cytokine production and to induce macrophage apoptosis in vitro. YopJ-dependent apoptosis in mesenteric lymph nodes has also been demonstrated in a mouse model of Yersinia pseudotuberculosis infection. These results suggest that YopJ attenuates the host innate and adaptive immune response during infection, but the role of YopJ during bubonic plague has not been completely established. We evaluated the role of Yersinia pestis YopJ in a rat model of bubonic plague following intradermal infection with a fully virulent Y. pestis strain and an isogenic yopJ mutant. Deletion of yopJ resulted in a twofold decrease in the number of apoptotic immune cells in the bubo and a threefold increase in serum tumor necrosis factor alpha levels but did not result in decreased virulence, systemic spread, or colonization levels in the spleen and blood. Our results indicate that YopJ is not essential for bubonic plague pathogenesis, even after peripheral inoculation of low doses of Y. pestis. Instead, the effects of YopJ appear to overlap and augment the immunomodulatory effects of other Y. pestis virulence factors.

  3. Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge.

    PubMed

    Branger, Christine G; Sun, Wei; Torres-Escobar, Ascención; Perry, Robert; Roland, Kenneth L; Fetherston, Jacqueline; Curtiss, Roy

    2010-12-16

    We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131-326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.

  4. Multiple roles of Myd88 in the immune response to the plague F1-V vaccine and in protection against an aerosol challenge of Yersinia pestis CO92 in mice.

    PubMed

    Dankmeyer, Jennifer L; Fast, Randy L; Cote, Christopher K; Worsham, Patricia L; Fritz, David; Fisher, Diana; Kern, Steven J; Merkel, Tod; Kirschning, Carsten J; Amemiya, Kei

    2014-01-01

    The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host's innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.

  5. Human single-chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.

    PubMed

    Järvinen, Hanna M; Laakkonen, Liisa; Haiko, Johanna; Johansson, Tiira; Juuti, Katri; Suomalainen, Marjo; Buchrieser, Carmen; Kalkkinen, Nisse; Korhonen, Timo K

    2013-08-01

    Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis.

  6. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

    PubMed Central

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-01-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

  7. Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia pestis Outer Protein H (YopH) Phosphatase

    SciTech Connect

    Bahta, Medhanit; Lountos, George T.; Dyas, Beverly; Kim, Sung-Eun; Ulrich, Robert G.; Waugh, David S.; Burke, Jr., Terrence R.

    2012-08-10

    Our current study reports the first K{sub M} optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (K{sub M} = 80 {micro}M) was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC{sub 50} = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.

  8. Using Comparative Genomics for Inquiry-Based Learning to Dissect Virulence of Escherichia coli O157:H7 and Yersinia pestis

    PubMed Central

    Baumler, David J.; Banta, Lois M.; Hung, Kai F.; Schwarz, Jodi A.; Cabot, Eric L.; Glasner, Jeremy D.; Perna, Nicole T.

    2012-01-01

    Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples related to bacterial pathogenesis. Students first examine alignments of genomes of Escherichia coli O157:H7 strains isolated from three food-poisoning outbreaks using the multiple-genome alignment tool Mauve. Students investigate conservation of virulence factors using the Mauve viewer and by browsing annotations available at the A Systematic Annotation Package for Community Analysis of Genomes database. In the second module, students use an alignment of five Yersinia pestis genomes to analyze single-nucleotide polymorphisms of three genes to classify strains into biovar groups. Students are then given sequences of bacterial DNA amplified from the teeth of corpses from the first and second pandemics of the bubonic plague and asked to classify these new samples. Learning-assessment results reveal student improvement in self-efficacy and content knowledge, as well as students' ability to use BLAST to identify genomic islands and conduct analyses of virulence factors from E. coli O157:H7 or Y. pestis. Each of these educational modules offers educators new ready-to-implement resources for integrating comparative genomic topics into their curricula. PMID:22383620

  9. Structure and Activity of Yersinia pestis 6-hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as a Novel Target for the Development of Antiplague Therapeutics

    SciTech Connect

    Blaszczyk,J.; Li, Y.; Cherry, S.; Alexandratos, J.; Wu, Y.; Shaw, G.; Tropea, J.; Waugh, D.; Yan, H.; Ji, X.

    2007-01-01

    6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate-biosynthetic pathway and is essential for microorganisms but absent from mammals. HPPK catalyzes Mg2+-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Previously, three-dimensional structures of Escherichia coli HPPK (EcHPPK) have been determined at almost every stage of its catalytic cycle and the reaction mechanism has been established. Here, the crystal structure of Yersinia pestis HPPK (YpHPPK) in complex with HP and an ATP analog is presented together with thermodynamic and kinetic characterizations. The two HPPK molecules differ significantly in a helix-loop area ([alpha]2-Lp3). YpHPPK has lower affinities than EcHPPK for both nucleotides and HP, but its rate constants for the mechanistic steps of both chemical transformation and product release are comparable with those of EcHPPK. Y. pestis, which causes plague, is a category A select agent according to the Centers for Disease Control and Prevention (CDC). Therefore, these structural and biochemical data are valuable for the design of novel medical countermeasures against plague.

  10. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    PubMed

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

  11. A Recombinant Trivalent Fusion Protein F1-LcrV-HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis.

    PubMed

    Verma, Shailendra K; Batra, Lalit; Tuteja, Urmil

    2016-01-01

    Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model. PMID:27458447

  12. Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge.

    PubMed

    Van Blarcom, T J; Sofer-Podesta, C; Ang, J; Boyer, J L; Crystal, R G; Georgiou, G

    2010-07-01

    Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K(D))=3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K(D) values (H8, K(D)=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdalphaV, giving rise to AdalphaV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10(9), 10(10) or 10(11) particle units (pu). After intranasal challenge with 363 LD(50) (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10(10) pu of AdalphaV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdalphaV expressing the lower-affinity 2C12.4 (P<0.04; AdalphaV versus AdalphaV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis

  13. A Recombinant Trivalent Fusion Protein F1–LcrV–HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis

    PubMed Central

    Verma, Shailendra K.; Batra, Lalit; Tuteja, Urmil

    2016-01-01

    Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1–LcrV and F1–LcrV–HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1–LcrV and F1–LcrV–HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it’s isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1–LcrV–HSP70(II) sera in comparison to anti-F1–LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1–LcrV–HSP(II) immunized mice in comparison to F1–LcrV. Both F1–LcrV and F1–LcrV–HSP70(II) provided 100% protection. Our research findings suggest that F1–LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model. PMID:27458447

  14. High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

    PubMed

    Ponnusamy, Duraisamy; Fitts, Eric C; Sha, Jian; Erova, Tatiana E; Kozlova, Elena V; Kirtley, Michelle L; Tiner, Bethany L; Andersson, Jourdan A; Chopra, Ashok K

    2015-05-01

    The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20

  15. High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

    PubMed Central

    Ponnusamy, Duraisamy; Fitts, Eric C.; Erova, Tatiana E.; Kozlova, Elena V.; Kirtley, Michelle L.; Tiner, Bethany L.; Andersson, Jourdan A.

    2015-01-01

    The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20

  16. Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics.

    PubMed

    Jabbour, Rabih E; Wade, Mary Margaret; Deshpande, Samir V; Stanford, Michael F; Wick, Charles H; Zulich, Alan W; Snyder, A Peter

    2010-07-01

    Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and nonpathogenic strains of Yersinia pestis and Escherichia coli using ultracentrifugation and a sarkosyl extraction method followed by protein digestion and analysis using liquid chromatography tandem mass spectrometry (MS). Whole cell protein extracts contain many different types of proteins resident in an organism at a given phase in its growth cycle. OMPs, however, are often associated with virulence in Gram-negative pathogens and could prove to be model biomarkers for strain differentiation among bacteria. The mass spectra of bacterial peptides were searched, using the SEQUEST algorithm, against a constructed proteome database of microorganisms in order to determine the identity and number of unique peptides for each bacterial sample. Data analysis was performed with the in-house BACid software. It calculated the probabilities that a peptide sequence assignment to a product ion mass spectrum was correct and used accepted spectrum-to-sequence matches to generate a sequence-to-bacterium (STB) binary matrix of assignments. Validated peptide sequences, either present or absent in various strains (STB matrices), were visualized as assignment bitmaps and analyzed by the BACid module that used phylogenetic relationships among bacterial species as part of a decision tree process. The bacterial classification and identification algorithm used assignments of organisms to taxonomic groups (phylogenetic classification) based on an organized scheme that begins at the phylum level and follows through the class, order, family, genus, and species to the strain level. For both Gram-negative organisms, the number of unique distinguishing proteins arrived at by the whole

  17. The Hemophore HasA from Yersinia pestis (HasAyp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change

    PubMed Central

    Kumar, Ritesh; Lovell, Scott; Matsumura, Hirotoshi; Battaile, Kevin P.; Moënne-Loccoz, Pierre; Rivera, Mario

    2015-01-01

    Hemophores from Serratia marcescens (HasAsm) and Pseudomonas aeruginosa (HasAp) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because Yersinia pestis HasA (HasAyp) presents a Gln at position 32, we determined the structures of apo-and holo-HasAyp. Surprisingly, the Q32 loop in apo-HasAyp is already in the closed conformation but no residue from the Q32 loop binds hemin in holo-HasAyp. In agreement with the minimal reorganization between the apo-and holo-structures, the hemin on-rate is too fast to detect by conventional stopped-flow measurements. PMID:23578210

  18. A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

    PubMed

    Bozue, Joel; Cote, Christopher K; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J; Kijek, Todd K; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G; Bell, Todd; Worsham, Patricia

    2014-01-01

    Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

  19. Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

    PubMed

    van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

    2014-06-01

    Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

  20. Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

    PubMed

    Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

    2015-04-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  1. Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

    PubMed Central

    Tiner, Bethany L.; Kirtley, Michelle L.; Erova, Tatiana E.; Popov, Vsevolod L.; Baze, Wallace B.; van Lier, Christina J.; Ponnusamy, Duraisamy; Andersson, Jourdan A.; Motin, Vladimir L.; Chauhan, Sadhana

    2015-01-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  2. Annual seroprevalence of Yersinia pestis in coyotes as predictors of interannual variation in reports of human plague cases in Arizona, United States.

    PubMed

    Brown, Heidi E; Levy, Craig E; Enscore, Russell E; Schriefer, Martin E; DeLiberto, Thomas J; Gage, Kenneth L; Eisen, Rebecca J

    2011-11-01

    Although several health departments collect coyote blood samples for plague surveillance, the association between reported human cases and coyote seroprevalence rates remains anecdotal. Using data from an endemic region of the United States, we sought to quantify this association. From 1974 to 1998, about 2,276 coyote blood samples from four Arizona counties were tested for serological evidence of exposure to Yersinia pestis, the causative agent of plague. Using a titer threshold presumed to be indicative of recent infection (serum titers of ≥1:256), we found a statistically significant relationship between years with >17% sero-positive coyotes and years with two or more human cases reported. Moreover, when the annual coyote seroprevalence rates were dichotomized at 17%, 84% of the years were correctly classified using four biologically relevant meteorological variables in a linear regression. This is the first time a statistically significant temporal association between human plague cases and coyote seroprevalence rates has been shown. However, issues with data resolution and surveillance effort that potentially limit the public health utility of using coyote seroprevalence rates are discussed.

  3. Structural analysis of Pla protein from the biological warfare agent Yersinia pestis: docking and molecular dynamics of interactions with the mammalian plasminogen system.

    PubMed

    Ruback, Eduardo; Lobo, Leandro Araujo; França, Tanos Celmar Costa; Pascutti, Pedro Geraldo

    2013-01-01

    Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.

  4. The Yersinia pestis Siderophore, Yersiniabactin, and the ZnuABC system both contribute to Zinc acquisition and the development of lethal septicemic plague in mice

    PubMed Central

    Bobrov, Alexander G.; Kirillina, Olga; Fetherston, Jacqueline D.; Miller, M. Clarke; Burlison, Joseph A.; Perry, Robert D.

    2014-01-01

    Summary Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA∷lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, that are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicemic plague mouse model. PMID:24979062

  5. CpG oligodeoxynucleotides augment the murine immune response to the Yersinia pestis F1-V vaccine in bubonic and pneumonic models of plague.

    PubMed

    Amemiya, Kei; Meyers, Jennifer L; Rogers, Taralyn E; Fast, Randy L; Bassett, Anthony D; Worsham, Patricia L; Powell, Bradford S; Norris, Sarah L; Krieg, Arthur M; Adamovicz, Jeffrey J

    2009-04-01

    The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P<0.0001).

  6. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

    PubMed

    McGillick, Brian E; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency. PMID:26818694

  7. Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen capture-based dipsticks.

    PubMed

    Sha, Jian; Endsley, Janice J; Kirtley, Michelle L; Foltz, Sheri M; Huante, Matthew B; Erova, Tatiana E; Kozlova, Elena V; Popov, Vsevolod L; Yeager, Linsey A; Zudina, Irina V; Motin, Vladimir L; Peterson, Johnny W; DeBord, Kristin L; Chopra, Ashok K

    2011-05-01

    We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

  8. EMS Student Handbook.

    ERIC Educational Resources Information Center

    Ogle, Patrick

    This student guide is one of a series of self-contained materials for students enrolled in an emergency medical services (EMS) training program. Discussed in the individual sections of the guide are the following topics: the purpose and history of EMS professionals; EMS training, certification and examinations (national and state certification and…

  9. EM International. Volume 1

    SciTech Connect

    Not Available

    1993-07-01

    It is the intent of EM International to describe the Office of Environmental Restoration and Waste Management`s (EM`s) various roles and responsibilities within the international community. Cooperative agreements and programs, descriptions of projects and technologies, and synopses of visits to international sites are all highlighted in this semiannual journal. Focus on EM programs in this issue is on international collaboration in vitrification projects. Technology highlights covers: in situ sealing for contaminated sites; and remote sensors for toxic pollutants. Section on profiles of countries includes: Arctic contamination by the former Soviet Union, and EM activities with Germany--cooperative arrangements.

  10. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    PubMed

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.

  11. Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-05-03

    The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

  12. Host response during Yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways

    PubMed Central

    Alem, Farhang; Yao, Kuan; Lane, Douglas; Calvert, Valerie; Petricoin, Emanuel F.; Kramer, Liana; Hale, Martha L.; Bavari, Sina; Panchal, Rekha G.; Hakami, Ramin M.

    2015-01-01

    Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and

  13. Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Strips for Rapid Detection of Bacillus anthracis Spore, Brucella spp., and Yersinia pestis

    PubMed Central

    Zhao, Yong; Hua, Fei; Li, Chunfeng; Yang, Ruifu; Zhou, Lei

    2014-01-01

    Bacillus anthracis, Brucella spp., and Yersinia pestis are zoonotic pathogens and biowarfare- or bioterrorism-associated agents that must be detected rapidly on-site from various samples (e.g., viscera and powders). An up-converting phosphor technology-based lateral flow (UPT–LF) strip was developed as a point-of-care testing (POCT) to satisfy the requirements of first-level emergency response. We developed UPT–LF POCT to quantitatively detect the three pathogens within 15 min. Sample and operation-error tolerances of the assay were comprehensively evaluated. The sensitivity of UPT–LF assay to bacterial detection reached 104 cfu·mL−1 (100 cfu/test), with a linear quantitative range of 4 to 6 orders of magnitude. Results revealed that the UPT–LF assay exhibited a high specificity with the absence of false-positive results even at 109 cfu·mL−1 of non-specific bacterial contamination. The assay could tolerate samples with a wide pH range (2 to 12), high ion strengths (≥4 mol·L−1 of NaCl), high viscosities (≤25 mg·mL−1 of PEG20000 or ≥20% of glycerol), and high concentrations of bio-macromolecule (≤200 mg·mL−1 of bovine serum albumin or ≥80 mg·mL−1 of casein). The influence of various types of powders and viscera (fresh and decomposed) on the performance of UPT–LF assay was determined. The operational error of liquid measurement exhibited few effects on sensitivity and specificity. The developed UPT–LF POCT assay is applicable under field conditions with excellent tolerance to sample complexity and operational error. PMID:25144726

  14. Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

    PubMed Central

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-01-01

    Summary The plague-causing bacterium Yersinia pestis utilizes a Type III Secretion System (T3SS) to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as "chaperones" to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa T3SS, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat (TPR) family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal TPR motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the N-terminal 49 residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex. PMID:18281060

  15. Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

    SciTech Connect

    Camarero, J A

    2006-12-07

    Protein arrays are ideal tools for the rapid analysis of whole proteomes as well as for the development of reliable and cheap biosensors. The objective of this proposal is to develop a new ligand assisted ligation method based in the naturally occurring protein trans-splicing process. This method has been used for the generation of spatially addressable arrays of multiple protein components by standard micro-lithographic techniques. Key to our approach is the use of the protein trans-splicing process. This naturally occurring process allows the development of a truly generic and highly efficient method for the covalent attachment of proteins through its C-terminus to any solid support. This technology has been used for the creation of protein chips containing several virulence factors from the human pathogen Y. pestis.

  16. EMS in Mauritius.

    PubMed

    Ramalanjaona, Georges; Brogan, Gerald X

    2009-02-01

    Mauritius lies in the southwest Indian Ocean about 1250 miles from the African coast and 500 miles from Madagascar. Mauritius (estimated population 1,230,602) became independent from the United Kingdom in 1968 and has one of the highest GDP per capita in Africa. Within Mauritius there is a well established EMS system with a single 999 national dispatch system. Ambulances are either publicly or privately owned. Public ambulances are run by the Government (SAMU). Megacare is a private subscriber only ambulance service. The Government has recently invested in new technology such as telemedicine to further enhance the role of EMS on the island. This article describes the current state of EMS in Mauritius and depicts its development in the context of Government effort to decentralise and modernise the healthcare system.

  17. The effect of low shear force on the virulence potential of Yersinia pestis: new aspects that space-like growth conditions and the final frontier can teach us about a formidable pathogen.

    PubMed

    Rosenzweig, Jason A; Chopra, Ashok K

    2012-01-01

    Manned space exploration has created a need to evaluate the effects of space-like stress (SLS) on pathogenic and opportunistic microbes. Interestingly, several Gram-negative enteric pathogens, e.g., Salmonella enterica serovar Typhimurium, have revealed a transient hyper-virulent phenotype following simulated microgravity (SMG) or actual space flight exposures. We have explored the virulence potential of Yersinia pestis KIM/D27 (YP) following exposure to mechanical low shear forces associated with SMG. Our experimental results demonstrated that SMG-grown YP was decreased in its induced HeLa cell cytotoxicity, suggesting that SMG somehow compromises T3SS functions. This was confirmed by an actual reduced amount of effector protein production and secretion through the T3SS injectisome. Also, SMG-grown YP proliferated less than their NG-grown counterparts did during an 8-h macrophage infection. Presently, we are evaluating the influence of SMG on various KIM/D27 mutant strains to further understanding of our initial phenomenology described above. Taken together, characterizing YP grown under the low shear forces of SMG can provide new insights into its pathogenesis and potentially uncover new targets that could be exploited for the development of novel antimicrobials as well as potential live-attenuated vaccines.

  18. Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

    PubMed Central

    Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

    2001-01-01

    The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

  19. The EM Earthquake Precursor

    NASA Astrophysics Data System (ADS)

    Jones, K. B., II; Saxton, P. T.

    2013-12-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After the 1989 Loma Prieta Earthquake, American earthquake investigators predetermined magnetometer use and a minimum earthquake magnitude necessary for EM detection. This action was set in motion, due to the extensive damage incurred and public outrage concerning earthquake forecasting; however, the magnetometers employed, grounded or buried, are completely subject to static and electric fields and have yet to correlate to an identifiable precursor. Secondly, there is neither a networked array for finding any epicentral locations, nor have there been any attempts to find even one. This methodology needs dismissal, because it is overly complicated, subject to continuous change, and provides no response time. As for the minimum magnitude threshold, which was set at M5, this is simply higher than what modern technological advances have gained. Detection can now be achieved at approximately M1, which greatly improves forecasting chances. A propagating precursor has now been detected in both the field and laboratory. Field antenna testing conducted outside the NE Texas town of Timpson in February, 2013, detected three strong EM sources along with numerous weaker signals. The antenna had mobility, and observations were noted for recurrence, duration, and frequency response. Next, two

  20. Identified EM Earthquake Precursors

    NASA Astrophysics Data System (ADS)

    Jones, Kenneth, II; Saxton, Patrick

    2014-05-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After a number of custom rock experiments, two hypotheses were formed which could answer the EM wave model. The first hypothesis concerned a sufficient and continuous electron movement either by surface or penetrative flow, and the second regarded a novel approach to radio transmission. Electron flow along fracture surfaces was determined to be inadequate in creating strong EM fields, because rock has a very high electrical resistance making it a high quality insulator. Penetrative flow could not be corroborated as well, because it was discovered that rock was absorbing and confining electrons to a very thin skin depth. Radio wave transmission and detection worked with every single test administered. This hypothesis was reviewed for propagating, long-wave generation with sufficient amplitude, and the capability of penetrating solid rock. Additionally, fracture spaces, either air or ion-filled, can facilitate this concept from great depths and allow for surficial detection. A few propagating precursor signals have been detected in the field occurring with associated phases using custom-built loop antennae. Field testing was conducted in Southern California from 2006-2011, and outside the NE Texas town of Timpson in February, 2013. The antennae have mobility and observations were noted for

  1. EPA LABORATORIES IMPLEMENT EMS PROGRAM

    EPA Science Inventory

    This paper highlights the breadth and magnitude of carrying out an effective Environmental Management System (EMS) program at the U.S. EPA's research and development laboratories. Federal research laboratories have unique operating challenges compared to more centralized industr...

  2. A Comparative Biochemical and Structural Analysis of the Intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H(37)R(v) and the Secreted chorismate mutase (y2828) from Yersinia pestis

    SciTech Connect

    S Kim; S Reddy; B Nelson; H Robinson; P Reddy; J Ladner

    2011-12-31

    The Rv0948c gene from Mycobacterium tuberculosis H{sub 37}R{sub v} encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 5.5 {+-} 0.2 s{sup -1} and a K{sub m} of 1500 {+-} 100 {micro}m at 37 C and pH 7.5. The 2.0 {angstrom} X-ray structure shows that 90-MtCM is an all {alpha}-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k{sub cat}. Hence, 90-MtCM belongs to a subfamily of {alpha}-helical AroQ CMs termed AroQ{delta}. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 70 {+-} 5 s{sup -1} and Km of 500 {+-} 50 {micro}m at 37 C and pH 7.5. The 2.1 {angstrom} X-ray structure shows that *YpCM is an all {alpha}-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ{gamma} class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.

  3. A comparative biochemical and structural analysis of the intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H37Rv and the secreted chorismate mutase (y2828) from Yersinia pestis

    SciTech Connect

    Kim, S.K.; Robinson, H.; Reddy, S. K.; Nelson, B. C.; Reddy, P. T.; Ladner, J. E.

    2008-10-01

    The Rv0948c gene from Mycobacterium tuberculosis H{sub 37}R{sub v} encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 5.5 {+-} 0.2 s{sup -1} and a K{sub m} of 1500 {+-} 100 {mu}m at 37 C and pH 7.5. The 2.0 {angstrom} X-ray structure shows that 90-MtCM is an all {alpha}-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k{sub cat}. Hence, 90-MtCM belongs to a subfamily of {alpha}-helical AroQ CMs termed AroQ{sub {delta}}. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k{sub cat} of 70 {+-} 5 s{sup -1} and K{sub m} of 500 {+-} 50 {mu}m at 37 C and pH 7.5. The 2.1 {angstrom} X-ray structure shows that *YpCM is an all {alpha}-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ{sub {gamma}} class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.

  4. The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

    PubMed

    Lawal, Abidat; Kirtley, Michelle L; van Lier, Christina J; Erova, Tatiana E; Kozlova, Elena V; Sha, Jian; Chopra, Ashok K; Rosenzweig, Jason A

    2013-09-01

    Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

  5. The Effects of Modeled Microgravity on Growth Kinetics, Antibiotic Susceptibility, Cold Growth, and the Virulence Potential of a Yersinia pestis ymoA-Deficient Mutant and Its Isogenic Parental Strain

    PubMed Central

    Lawal, Abidat; Kirtley, Michelle L.; van Lier, Christina J.; Erova, Tatiana E.; Kozlova, Elena V.; Sha, Jian; Chopra, Ashok K.

    2013-01-01

    Abstract Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent. Key Words: Type three secretion system (T3SS

  6. Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

    PubMed Central

    Tiner, Bethany L.; Ponnusamy, Duraisamy; Baze, Wallace B.; Fitts, Eric C.; Popov, Vsevolod L.; van Lier, Christina J.; Erova, Tatiana E.

    2015-01-01

    Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 106 CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD50) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but with

  7. Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

    PubMed

    Tiner, Bethany L; Sha, Jian; Ponnusamy, Duraisamy; Baze, Wallace B; Fitts, Eric C; Popov, Vsevolod L; van Lier, Christina J; Erova, Tatiana E; Chopra, Ashok K

    2015-12-01

    Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but

  8. Busca de estruturas em grandes escalas em altos redshifts

    NASA Astrophysics Data System (ADS)

    Boris, N. V.; Sodrã©, L., Jr.; Cypriano, E.

    2003-08-01

    A busca por estruturas em grandes escalas (aglomerados de galáxias, por exemplo) é um ativo tópico de pesquisas hoje em dia, pois a detecção de um único aglomerado em altos redshifts pode por vínculos fortes sobre os modelos cosmológicos. Neste projeto estamos fazendo uma busca de estruturas distantes em campos contendo pares de quasares próximos entre si em z Â3 0.9. Os pares de quasares foram extraídos do catálogo de Véron-Cetty & Véron (2001) e estão sendo observados com os telescópios: 2,2m da University of Hawaii (UH), 2,5m do Observatório de Las Campanas e com o GEMINI. Apresentamos aqui a análise preliminar de um par de quasares observado nos filtros i'(7800 Å) e z'(9500 Å) com o GEMINI. A cor (i'-z') mostrou-se útil para detectar objetos "early-type" em redshifts menores que 1.1. No estudo do par 131046+0006/J131055+0008, com redshift ~ 0.9, o uso deste método possibilitou a detecção de sete objetos candidatos a galáxias "early-type". Num mapa da distribuição projetada dos objetos para 22 < i' < 25 observou-se que estas galáxias estão localizadas próximas a um dos quasares e há indícios de que estejam aglomeradas dentro de um área de ~ 6 arcmin2. Se esse for o caso, estes objetos seriam membros de uma estrutura em grande escala. Um outro argumento em favor dessa hipótese é que eles obedecem uma relação do tipo Kormendy (raio equivalente X brilho superficial dentro desse raio), como a apresentada pelas galáxias elípticas em z = 0.

  9. The National EMS Research strategic plan.

    PubMed

    Sayre, Michael R; White, Lynn J; Brown, Lawrence H; McHenry, Susan D

    2005-01-01

    One of the eight major recommendations put forth by the National EMS Research Agenda Implementation Project in 2002 was the development of an emergency medical services (EMS) research strategic plan. Using a modified Delphi technique along with a consensus conference approach, a strategic plan for EMS research was created. The plan includes recommendations for concentrating efforts by EMS researchers, policy makers, and funding resources with the ultimate goal of improving clinical outcomes. Clinical issues targeted for additional research efforts include evaluation and treatment of patients with asthma, acute cardiac ischemia, circulatory shock, major injury, pain, acute stroke, and traumatic brain injury. The plan calls for developing, evaluating, and validating improved measurement tools and techniques. Additional research to improve the education of EMS personnel as well as system design and operation is also suggested. Implementation of the EMS research strategic plan will improve both the delivery of services and the care of individuals who access the emergency medical system.

  10. DOE/EM Criticality Safety Needs Assessment

    SciTech Connect

    Westfall, Robert Michael; Hopper, Calvin Mitchell

    2011-02-01

    The issue of nuclear criticality safety (NCS) in Department of Energy Environmental Management (DOE/EM) fissionable material operations presents challenges because of the large quantities of material present in the facilities and equipment that are committed to storage and/or material conditioning and dispositioning processes. Given the uncertainty associated with the material and conditions for many DOE/EM fissionable material operations, ensuring safety while maintaining operational efficiency requires the application of the most-effective criticality safety practices. In turn, more-efficient implementation of these practices can be achieved if the best NCS technologies are utilized. In 2002, DOE/EM-1 commissioned a survey of criticality safety technical needs at the major EM sites. These needs were documented in the report Analysis of Nuclear Criticality Safety Technology Supporting the Environmental Management Program, issued May 2002. Subsequent to this study, EM safety management personnel made a commitment to applying the best and latest criticality safety technology, as described by the DOE Nuclear Criticality Safety Program (NCSP). Over the past 7 years, this commitment has enabled the transfer of several new technologies to EM operations. In 2008, it was decided to broaden the basis of the EM NCS needs assessment to include not only current needs for technologies but also NCS operational areas with potential for improvements in controls, analysis, and regulations. A series of NCS workshops has been conducted over the past years, and needs have been identified and addressed by EM staff and contractor personnel. These workshops were organized and conducted by the EM Criticality Safety Program Manager with administrative and technical support by staff at Oak Ridge National Laboratory (ORNL). This report records the progress made in identifying the needs, determining the approaches for addressing these needs, and assimilating new NCS technologies into EM

  11. EM international activities. February 1997 highlights

    SciTech Connect

    1997-02-01

    EM International Highlights is a brief summary of on-going international projects within the Department of Energy`s Office of Environmental Management (EM). This document contains sections on: Global Issues, activities in Western Europe, activities in central and Eastern Europe, activities in Russia, activities in Asia and the Pacific Rim, activities in South America, activities in North America, and International Organizations.

  12. School Budget Hold'em Facilitator's Guide

    ERIC Educational Resources Information Center

    Education Resource Strategies, 2012

    2012-01-01

    "School Budget Hold'em" is a game designed to help school districts rethink their budgeting process. It evolved out of Education Resource Strategies' (ERS) experience working with large urban districts around the country. "School Budget Hold'em" offers a completely new approach--one that can turn the budgeting process into a long-term visioning…

  13. EMS: part three. Preventive medicine for EMS maladies

    SciTech Connect

    Hyfantis, J.F.

    1983-05-01

    Shakeout problems in the fast-growing field of energy-management systems (EMS) are gorwing pains and neither unexpected nor insoluble. A National Bureau of Standards (NBS) survey found user complaints ranging from problems with food spoilage to poorly trained service people. But blame can be placed on users, manufacturers, and distributors. Involving operators and managers in load-control strategies can alleviate some problems and save some money. Allowing for realistic testing time in the installation phase can also save in the long run, as will initiating a maintenance schedule and maintaining operating logs. Software maintenance can present a serious problem. Another NBS study result indicates there is perceptible improvement in system performance when good training accompanies installation, particularly when the training is provided at the user facility. User education is probably the best key to avoiding most of the potential pitfalls. 2 figures, 2 tables.

  14. EM International, July 1994, Volume 2

    SciTech Connect

    Not Available

    1994-10-01

    The Office of Environmental Management (EM) at the Department of Energy (DOE) is seeking out and leveraging foreign technology, data, and resources in keeping with EM`s mandate to protect public health and the environment through the safe and cost-effective remediation of the Department`s nuclear weapons sites. EM works closely with foreign governments, industry, and universities to obtain innovative environmental technologies, scientific and engineering expertise, and operations experience that will support EM`s objectives. Where appropriate, these international resources are used to manage the more urgent risks at our sites, secure a safe workplace, help build consensus on critical issues, and strengthen our technology development program. Through international agreements EM engages in cooperative exchange of information, technology, and individuals. Currently, we are managing agreements with a dozen countries in Europe, Latin America, and Asia. These agreements focus on environmental restoration, waste management, transportation of radioactive wastes, and decontamination and decommissioning. This publication contains the following articles: in situ remediation integrated program; in-situ characterization and inspection of tanks; multimedia environmental pollutant assessment system (MEPAS); LLNL wet oxidation -- AEA technology. Besides these articles, this publication covers: EU activities with Russia; technology transfer activities; and international organization activities.

  15. Project X RFQ EM Design

    SciTech Connect

    Romanov, Gennady; Hoff, Matthew; Li, Derun; Staples, John; Virostek, Steve; /LBNL

    2012-05-09

    Project X is a proposed multi-MW proton facility at Fermi National Accelerator Laboratory (FNAL). The Project X front-end would consist of an H- ion source, a low-energy beam transport (LEBT), a CW 162.5 MHz radio-frequency quadrupole (RFQ) accelerator, and a medium-energy beam transport (MEBT). Lawrence Berkeley National Laboratory (LBNL) and FNAL collaboration is currently developing the designs for various components in the Project X front end. This paper reports the detailed EM design of the CW 162.5 MHz RFQ that provides bunching of the 1-10 mA H- beam with acceleration from 30 keV to 2.1 MeV.

  16. Unified Data Resource for CryoEM

    PubMed Central

    Lawson, Catherine L.

    2010-01-01

    3D cryo-electron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Data Bank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB), and the National Center for Macromolecular Imaging (NCMI), is a “one-stop shop” resource for global deposition and retrieval of cryoEM map, model and associated metadata. The resource unifies public access to the two major EM Structural Data archives: EM Data Bank (EMDB) and Protein Data Bank (PDB), and facilitates use of EM structural data of macromolecules and macromolecular complexes by the wider scientific community. PMID:20888470

  17. EMS offshore. A new horizon for paramedics.

    PubMed

    Mallard, A S

    1991-10-01

    The difficulty in getting medical aid to offshore drilling platforms can be a source of life-threatening delays. Recently, some companies have charted new waters by actually stationing EMS crews on their rigs.

  18. EMS offshore. A new horizon for paramedics.

    PubMed

    Mallard, A S

    1991-10-01

    The difficulty in getting medical aid to offshore drilling platforms can be a source of life-threatening delays. Recently, some companies have charted new waters by actually stationing EMS crews on their rigs. PMID:10116023

  19. EMS adaptation for climate change

    NASA Astrophysics Data System (ADS)

    Pan, C.; Chang, Y.; Wen, J.; Tsai, M.

    2010-12-01

    The purpose of this study was to find an appropriate scenario of pre-hospital transportation of an emergency medical service (EMS) system for burdensome casualties resulting from extreme climate events. A case of natural catastrophic events in Taiwan, 88 wind-caused disasters, was reviewed and analyzed. A sequential-conveyance method was designed to shorten the casualty transportation time and to promote the efficiency of ambulance services. A proposed mobile emergency medical center was first constructed in a safe area, but nearby the disaster area. The Center consists of professional medical personnel who process the triage of incoming patients and take care of casualties with minor injuries. Ambulances in the Center were ready to sequentially convey the casualties with severer conditions to an assigned hospital that is distant from the disaster area for further treatment. The study suggests that if we could construct a spacious and well-equipped mobile emergency medical center, only a small portion of casualties would need to be transferred to distant hospitals. This would reduce the over-crowding problem in hospital ERs. First-line ambulances only reciprocated between the mobile emergency medical center and the disaster area, saving time and shortening the working distances. Second-line ambulances were highly regulated between the mobile emergency medical center and requested hospitals. The ambulance service of the sequential-conveyance method was found to be more efficient than the conventional method and was concluded to be more profitable and reasonable on paper in adapting to climate change. Therefore, additional practical work should be launched to collect more precise quantitative data.

  20. 10 CFR Appendixes E-M to Part 52 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false E Appendixes E-M to Part 52 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Appendixes E-M to Part 52...

  1. 10 CFR Appendixes E-M to Part 52 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false E Appendixes E-M to Part 52 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Appendixes E-M to Part 52...

  2. 10 CFR Appendixes E-M to Part 52 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false E Appendixes E-M to Part 52 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Appendixes E-M to Part 52...

  3. 10 CFR Appendixes E-M to Part 52 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false E Appendixes E-M to Part 52 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Appendixes E-M to Part 52...

  4. 10 CFR Appendixes E-M to Part 52 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false E Appendixes E-M to Part 52 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Appendixes E-M to Part 52...

  5. Processing of Cryo-EM Movie Data.

    PubMed

    Ripstein, Z A; Rubinstein, J L

    2016-01-01

    Direct detector device (DDD) cameras dramatically enhance the capabilities of electron cryomicroscopy (cryo-EM) due to their improved detective quantum efficiency (DQE) relative to other detectors. DDDs use semiconductor technology that allows micrographs to be recorded as movies rather than integrated individual exposures. Movies from DDDs improve cryo-EM in another, more surprising, way. DDD movies revealed beam-induced specimen movement as a major source of image degradation and provide a way to partially correct the problem by aligning frames or regions of frames to account for this specimen movement. In this chapter, we use a self-consistent mathematical notation to explain, compare, and contrast several of the most popular existing algorithms for computationally correcting specimen movement in DDD movies. We conclude by discussing future developments in algorithms for processing DDD movies that would extend the capabilities of cryo-EM even further.

  6. Processing of Cryo-EM Movie Data.

    PubMed

    Ripstein, Z A; Rubinstein, J L

    2016-01-01

    Direct detector device (DDD) cameras dramatically enhance the capabilities of electron cryomicroscopy (cryo-EM) due to their improved detective quantum efficiency (DQE) relative to other detectors. DDDs use semiconductor technology that allows micrographs to be recorded as movies rather than integrated individual exposures. Movies from DDDs improve cryo-EM in another, more surprising, way. DDD movies revealed beam-induced specimen movement as a major source of image degradation and provide a way to partially correct the problem by aligning frames or regions of frames to account for this specimen movement. In this chapter, we use a self-consistent mathematical notation to explain, compare, and contrast several of the most popular existing algorithms for computationally correcting specimen movement in DDD movies. We conclude by discussing future developments in algorithms for processing DDD movies that would extend the capabilities of cryo-EM even further. PMID:27572725

  7. Risk Communication Within the EM Program

    SciTech Connect

    Edelson, M.

    2003-02-26

    The U.S. Department of Energy Environmental Management program (EM) conducts the most extensive environmental remediation effort in the world. The annual EM budgets have exceeded $6,000,000,000 for approximately ten years and EM has assumed responsibility for the cleanup of the largest DOE reservations (i.e., at Hanford, Washington, Aiken, South Carolina, and Idaho Falls, Idaho) as well as the facilities at Rocky Flats, Colorado and in Ohio. Each of these sites has areas of extensive radioactive and chemical contamination, numerous surplus facilities that require decontamination and removal, while some have special nuclear material that requires secure storage. The EM program has been criticized for being ineffective (1) and has been repeatedly reorganized to address perceived shortcomings. The most recent reorganization was announced in 2001 to become effective at the beginning of the 2003 Federal Fiscal Year (i.e., October 2002). It was preceded by a ''top to bottom'' review (TTBR) of the program (2) that identified several deficiencies that were to be corrected as a result of the reorganization. One prominent outcome of the TTBR was the identification of ''risk reduction'' as an organizing principle to prioritize the activities of the new EM program. The new program also sought to accelerate progress by identifying a set of critical activities at each site that could be accelerated and result in more rapid site closure, with attendant risk, cost, and schedule benefits. This paper investigates how the new emphasis on risk reduction in the EM program has been communicated to EM stakeholders and regulators. It focuses on the Rocky Flats Environmental Technology Site (RFETS) as a case study and finds that there is little evidence for a new emphasis on risk reduction in EM communications with RFETS stakeholders. Discussions between DOE and RFETS stakeholders often refer to ''risk,'' but the word serves as a placeholder for other concepts. Thus ''risk'' communication

  8. Modelling and design for PM/EM magnetic bearings

    NASA Technical Reports Server (NTRS)

    Pang, D.; Kirk, J. A.; Anand, D. K.; Johnson, R. G.; Zmood, R. B.

    1992-01-01

    A mathematical model of a permanent magnet/electromagnet (PM/EM) radially active bearing is presented. The bearing is represented by both a reluctance model and a stiffness model. The reluctance model analyzes the magnetic circuit of the PM/EM bearings. By combining the two models, the performance of the bearing can be predicted given geometric dimensions, permanent magnet strength, and the parameters of the EM coils. The overall bearing design including the PM and EM design is subject to the performance requirement and physical constraints. A study of these requirements and constraints is discussed. The PM design is based on the required magnetic flux for proper geometric dimensions and magnet strength. The EM design is based on the stability and force slew rate consideration, and dictates the number of turns for the EM coils and the voltage and current of the power amplifier. An overall PM/EM bearing design methodology is proposed and a case study is also demonstrated.

  9. The E-MS Algorithm: Model Selection with Incomplete Data

    PubMed Central

    Jiang, Jiming; Nguyen, Thuan; Rao, J. Sunil

    2014-01-01

    We propose a procedure associated with the idea of the E-M algorithm for model selection in the presence of missing data. The idea extends the concept of parameters to include both the model and the parameters under the model, and thus allows the model to be part of the E-M iterations. We develop the procedure, known as the E-MS algorithm, under the assumption that the class of candidate models is finite. Some special cases of the procedure are considered, including E-MS with the generalized information criteria (GIC), and E-MS with the adaptive fence (AF; Jiang et al. 2008). We prove numerical convergence of the E-MS algorithm as well as consistency in model selection of the limiting model of the E-MS convergence, for E-MS with GIC and E-MS with AF. We study the impact on model selection of different missing data mechanisms. Furthermore, we carry out extensive simulation studies on the finite-sample performance of the E-MS with comparisons to other procedures. The methodology is also illustrated on a real data analysis involving QTL mapping for an agricultural study on barley grains. PMID:26783375

  10. Crosshole EM in steel-cased boreholes

    SciTech Connect

    Wilt, M.; Lee, K.H.; Becker, A.; Spies, B.; Wang, B.

    1996-07-01

    The application of crosshole EM methods through steel well-casing was investigated in theoretical, laboratory and field studies. A numerical code was developed that calculates the attenuation and phase delay of an EM dipole signal propagated through a steel well casing lodged in a homogeneous medium. The code was validated with a scale model and used for sensitivity studies of casing and formation properties. Finally, field measurements were made in an oil field undergoing waterflooding. Our most important findings are that (1) crosshole surveys are feasible using a well pair with one metallic and one non-metallic casing. (2) The casing effect seems be localized within the pipe section that includes the sensor. (3) The effects of the casing can be corrected using simple means and (4) crosshole field data that are sensitive to both formation and casing were acquired in a working environment.

  11. Generation and identification of Arabidopsis EMS mutants.

    PubMed

    Qu, Li-Jia; Qin, Genji

    2014-01-01

    EMS mutant analysis is a routine experiment to identify new players in a specific biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A mutant with a phenotype of interest (mpi) is obtained by screening plants of the M2 generation under a specific condition. Once the phenotype of the mpi is confirmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants (Arabidopsis Columbia-0 (Col-0) ecotype background) are first crossed with Arabidopsis Landsberg erecta (Ler) ecotype, and the presence or absence of the phenotype in the F1 hybrids indicates whether the mpi is recessive or dominant. F2 plants with phenotypes similar to the mpi, if the mpi is recessive, or those without the phenotype, if the mpi is dominant, are used as the mapping population. As few as 24 such plants are selected for rough mapping. After finding one marker (MA) linked to the mpi locus or mutant phenotype, more markers near MA are tested to identify recombinants. The recombinants indicate the interval in which the mpi is located. Additional recombinants and molecular markers are then required to narrow down the interval. This is an iterative process of narrowing down the mapping interval until no further recombinants or molecular markers are available. The genes in the mapping interval are then sequenced to look for the mutation. In the last step, the wild-type or mutated gene is cloned to generate binary constructs. Complementation or recapitulation provides the most convincing evidence in determining the mutation that causes the phenotype of the mpi. Here, we describe the procedures for generating mutants with EMS and analyzing EMS mutations by map-based cloning.

  12. Leukocyte Recognition Using EM-Algorithm

    NASA Astrophysics Data System (ADS)

    Colunga, Mario Chirinos; Siordia, Oscar Sánchez; Maybank, Stephen J.

    This document describes a method for classifying images of blood cells. Three different classes of cells are used: Band Neutrophils, Eosinophils and Lymphocytes. The image pattern is projected down to a lower dimensional sub space using PCA; the probability density function for each class is modeled with a Gaussian mixture using the EM-Algorithm. A new cell image is classified using the maximum a posteriori decision rule.

  13. Evaluation of the Ems Estuary ecosystem model

    NASA Astrophysics Data System (ADS)

    Baretta, J. W.; Ruardij, P.

    1987-11-01

    An ecosystem model is used to calculate and summarize carbon budgets within the Ems Estuary, The Netherlands. The similarity between model calculations and field data is established using a validation procedure. Model results show that the seaward boundary concentration for suspended matter is important in determining whether an estuary is an importer or exporter of carbon. Lowered boundary concentrations of suspended matter enhance pelagic primary production, but reduce sedimentation and hence the carbon flux from pelagic to benthic systems.

  14. TandEM: Titan and Enceladus mission

    USGS Publications Warehouse

    Coustenis, A.; Atreya, S.K.; Balint, T.; Brown, R.H.; Dougherty, M.K.; Ferri, F.; Fulchignoni, M.; Gautier, D.; Gowen, R.A.; Griffith, C.A.; Gurvits, L.I.; Jaumann, R.; Langevin, Y.; Leese, M.R.; Lunine, J.I.; McKay, C.P.; Moussas, X.; Muller-Wodarg, I.; Neubauer, F.; Owen, T.C.; Raulin, F.; Sittler, E.C.; Sohl, F.; Sotin, C.; Tobie, G.; Tokano, T.; Turtle, E.P.; Wahlund, J.-E.; Waite, J.H.; Baines, K.H.; Blamont, J.; Coates, A.J.; Dandouras, I.; Krimigis, T.; Lellouch, E.; Lorenz, R.D.; Morse, A.; Porco, C.C.; Hirtzig, M.; Saur, J.; Spilker, T.; Zarnecki, J.C.; Choi, E.; Achilleos, N.; Amils, R.; Annan, P.; Atkinson, D.H.; Benilan, Y.; Bertucci, C.; Bezard, B.; Bjoraker, G.L.; Blanc, M.; Boireau, L.; Bouman, J.; Cabane, M.; Capria, M.T.; Chassefiere, E.; Coll, P.; Combes, M.; Cooper, J.F.; Coradini, A.; Crary, F.; Cravens, T.; Daglis, I.A.; de Angelis, E.; De Bergh, C.; de Pater, I.; Dunford, C.; Durry, G.; Dutuit, O.; Fairbrother, D.; Flasar, F.M.; Fortes, A.D.; Frampton, R.; Fujimoto, M.; Galand, M.; Grasset, O.; Grott, M.; Haltigin, T.; Herique, A.; Hersant, F.; Hussmann, H.; Ip, W.; Johnson, R.; Kallio, E.; Kempf, S.; Knapmeyer, M.; Kofman, W.; Koop, R.; Kostiuk, T.; Krupp, N.; Kuppers, M.; Lammer, H.; Lara, L.-M.; Lavvas, P.; Le, Mouelic S.; Lebonnois, S.; Ledvina, S.; Li, J.; Livengood, T.A.; Lopes, R.M.; Lopez-Moreno, J. -J.; Luz, D.; Mahaffy, P.R.; Mall, U.; Martinez-Frias, J.; Marty, B.; McCord, T.; Salvan, C.M.; Milillo, A.; Mitchell, D.G.; Modolo, R.; Mousis, O.; Nakamura, M.; Neish, C.D.; Nixon, C.A.; Mvondo, D.N.; Orton, G.; Paetzold, M.; Pitman, J.; Pogrebenko, S.; Pollard, W.; Prieto-Ballesteros, O.; Rannou, P.; Reh, K.; Richter, L.; Robb, F.T.; Rodrigo, R.; Rodriguez, S.; Romani, P.; Bermejo, M.R.; Sarris, E.T.; Schenk, P.; Schmitt, B.; Schmitz, N.; Schulze-Makuch, D.; Schwingenschuh, K.; Selig, A.; Sicardy, B.; Soderblom, L.; Spilker, L.J.; Stam, D.; Steele, A.; Stephan, K.; Strobel, D.F.; Szego, K.; Szopa,

    2009-01-01

    TandEM was proposed as an L-class (large) mission in response to ESA's Cosmic Vision 2015-2025 Call, and accepted for further studies, with the goal of exploring Titan and Enceladus. The mission concept is to perform in situ investigations of two worlds tied together by location and properties, whose remarkable natures have been partly revealed by the ongoing Cassini-Huygens mission. These bodies still hold mysteries requiring a complete exploration using a variety of vehicles and instruments. TandEM is an ambitious mission because its targets are two of the most exciting and challenging bodies in the Solar System. It is designed to build on but exceed the scientific and technological accomplishments of the Cassini-Huygens mission, exploring Titan and Enceladus in ways that are not currently possible (full close-up and in situ coverage over long periods of time). In the current mission architecture, TandEM proposes to deliver two medium-sized spacecraft to the Saturnian system. One spacecraft would be an orbiter with a large host of instruments which would perform several Enceladus flybys and deliver penetrators to its surface before going into a dedicated orbit around Titan alone, while the other spacecraft would carry the Titan in situ investigation components, i.e. a hot-air balloon (Montgolfi??re) and possibly several landing probes to be delivered through the atmosphere. ?? Springer Science + Business Media B.V. 2008.

  15. Test beam performance of CDF plug upgrade EM calorimeter

    SciTech Connect

    Fukui, Y.; CDF Upgrade Group

    1998-01-01

    CDF Plug Upgrade(tile-fiber) EM Calorimeter performed resolution of 15%/{radical}E{circle_plus}0.7% with non-linearity less than 1% in a energy range of 5-180 GeV at Fermilab Test Beam. Transverse uniformity of inside-tower-response of the EM Calorimeter was 2.2% with 56 GeV positron, which was reduced to 1.0% with response map correction. We observed 300 photo electron/GeV in the EM Calorimeter. Ratios of EM Calorimeter response to positron beam to that to {sup 137}Cs Source was stable within 1% in the period of 8 months.

  16. 2. HI PAR (ACQUISITION RADAR) TOWER AND ENLISTED MEN (EM) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. HI PAR (ACQUISITION RADAR) TOWER AND ENLISTED MEN (EM) BARRACKS WITH RADAR ATTACHED. - Nike Hercules Missile Battery Summit Site, Battery Control Administration & Barracks Building, Anchorage, Anchorage, AK

  17. Communication - An Effective Tool for Implementing ISO 14001/EMS

    SciTech Connect

    Rachel Damewood; Bowen Huntsman

    2004-04-01

    The Idaho National Engineering and Environmental Laboratory (INEEL) received ISO 14001/EMS certification in June 2002. Communication played an effective role in implementing ISO 14001/EMS at the INEEL. This paper describes communication strategies used during the implementation and certification processes. The INEEL achieved Integrated Safety Management System (ISMS) and Voluntary Protection Program (VPP) Star status in 2001. ISMS implemented a formal process to plan and execute work. VPP facilitated worker involvement by establishing geographic units at various facilities with employee points of contact and management champions. The INEEL Environmental Management System (EMS) was developed to integrate the environmental functional area into its ISMS and VPP. Since the core functions of ISMS, VPP, and EMS are interchangeable, they were easy to integrate. Communication is essential to successfully implement an EMS. (According to ISO 14001 requirements, communication interacts with 12 other elements of the requirements.) We developed communication strategies that integrated ISMS, VPP, and EMS. For example, the ISMS, VPP, and EMS Web sites communicated messages to the work force, such as “VPP emphasizes the people side of doing business, ISMS emphasizes the system side of doing business, and EMS emphasizes the systems to protect the environment; but they all define work, identify and analyze hazards, and mitigate the hazards.” As a result of this integration, the work force supported and implemented the EMS. In addition, the INEEL established a cross-functional communication team to assist with implementing the EMS. The team included members from the Training and Communication organizations, VPP office, Pollution Prevention, Employee and Media Relations, a union representative, facility environmental support, and EMS staff. This crossfunctional team used various communication strategies to promote our EMS to all organization levels and successfully implemented EMS

  18. EM threat analysis for wireless systems.

    SciTech Connect

    Burkholder, R. J. (Ohio State University Electroscience Laboratory); Mariano, Robert J.; Schniter, P. (Ohio State University Electroscience Laboratory); Gupta, I. J. (Ohio State University Electroscience Laboratory)

    2006-06-01

    Modern digital radio systems are complex and must be carefully designed, especially when expected to operate in harsh propagation environments. The ability to accurately predict the effects of propagation on wireless radio performance could lead to more efficient radio designs as well as the ability to perform vulnerability analyses before and after system deployment. In this report, the authors--experts in electromagnetic (EM) modeling and wireless communication theory--describe the construction of a simulation environment that is capable of quantifying the effects of wireless propagation on the performance of digital communication.

  19. 7 CFR 1945.35 - Special EM loan training.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Designation Staff to the State to assist the State Director in conducting a training meeting(s) with State... 7 Agriculture 13 2010-01-01 2009-01-01 true Special EM loan training. 1945.35 Section 1945.35...) PROGRAM REGULATIONS (CONTINUED) EMERGENCY Disaster Assistance-General § 1945.35 Special EM loan...

  20. 7 CFR 1945.20 - Making EM loans available.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 13 2012-01-01 2012-01-01 false Making EM loans available. 1945.20 Section 1945.20...) PROGRAM REGULATIONS (CONTINUED) EMERGENCY Disaster Assistance-General § 1945.20 Making EM loans available... consideration by the Secretary in making determinations under § 1945.6(c)(3) of this subpart. The State...

  1. 7 CFR 1945.20 - Making EM loans available.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 13 2011-01-01 2009-01-01 true Making EM loans available. 1945.20 Section 1945.20...) PROGRAM REGULATIONS (CONTINUED) EMERGENCY Disaster Assistance-General § 1945.20 Making EM loans available... consideration by the Secretary in making determinations under § 1945.6(c)(3) of this subpart. The State...

  2. 7 CFR 1945.20 - Making EM loans available.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 13 2010-01-01 2009-01-01 true Making EM loans available. 1945.20 Section 1945.20...) PROGRAM REGULATIONS (CONTINUED) EMERGENCY Disaster Assistance-General § 1945.20 Making EM loans available... consideration by the Secretary in making determinations under § 1945.6(c)(3) of this subpart. The State...

  3. CryoEM at IUCrJ: a new era

    PubMed Central

    Subramaniam, Sriram; Kühlbrandt, Werner; Henderson, Richard

    2016-01-01

    In this overview, we briefly outline recent advances in electron cryomicroscopy (cryoEM) and explain why the journal IUCrJ, published by the International Union of Crystallography, could provide a natural home for publications covering many present and future developments in the cryoEM field. PMID:26870375

  4. 7 CFR 759.6 - EM to be made available.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... designations. When production losses meet the requirements in § 759.5 and the county has been designated as a disaster area for that reason, or when the discretionary exception to production losses for EM under § 759... eligible producers can receive EM loans. (2) Physical loss notification. When only qualifying...

  5. Cryptosporidium tyzzeri and Cryptosporidium pestis: which name is valid?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The validity of the name Cryptosporidium tyzzeri has been questioned because this name was previously used for a Cryptosporidium species in chickens in the original description by E. E. Tyzzer in 1929 which was later given the name by N.D. Levine in 1961. To further complicate matters, this specie...

  6. Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment

    PubMed Central

    Joseph, Agnel Praveen; Malhotra, Sony; Burnley, Tom; Wood, Chris; Clare, Daniel K.; Winn, Martyn; Topf, Maya

    2016-01-01

    As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5–4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite. PMID:26988127

  7. Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.

    PubMed

    Joseph, Agnel Praveen; Malhotra, Sony; Burnley, Tom; Wood, Chris; Clare, Daniel K; Winn, Martyn; Topf, Maya

    2016-05-01

    As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders' overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.

  8. Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.

    PubMed

    Joseph, Agnel Praveen; Malhotra, Sony; Burnley, Tom; Wood, Chris; Clare, Daniel K; Winn, Martyn; Topf, Maya

    2016-05-01

    As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders' overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite. PMID:26988127

  9. Electromagnetic optimization of EMS-MAGLEV systems

    SciTech Connect

    Andriollo, M.; Martinelli, G.; Morini, A.; Tortella, A.

    1998-07-01

    In EMS-MAGLEV high-speed transport systems, devices for propulsion, levitation and contactless on-board electric power transfer are combined in a single electromagnetic structure. The strong coupling among the windings affects the performance of each device and requires the utilization of numerical codes. The paper describes an overall optimization procedure, based on a suitable mathematical model of the system, which takes into account several items of the system performance. The parameters of the model are calculated by an automated sequence of FEM analyses of the configuration. Both the linear generator output characteristics and the propulsion force ripple are improved applying the procedure to a reference configuration. The results are compared with the results obtained by a sequence of partial optimizations operating separately on two different subsets of the geometric parameters.

  10. Online EM with weight-based forgetting.

    PubMed

    Celaya, Enric; Agostini, Alejandro

    2015-05-01

    In the online version of the EM algorithm introduced by Sato and Ishii ( 2000 ), a time-dependent discount factor is introduced for forgetting the effect of the old estimated values obtained with an earlier, inaccurate estimator. In their approach, forgetting is uniformly applied to the estimators of each mixture component depending exclusively on time, irrespective of the weight attributed to each unit for the observed sample. This causes an excessive forgetting in the less frequently sampled regions. To address this problem, we propose a modification of the algorithm that involves a weight-dependent forgetting, different for each mixture component, in which old observations are forgotten according to the actual weight of the new samples used to replace older values. A comparison of the time-dependent versus the weight-dependent approach shows that the latter improves the accuracy of the approximation and exhibits much greater stability. PMID:25710091

  11. Online EM with weight-based forgetting.

    PubMed

    Celaya, Enric; Agostini, Alejandro

    2015-05-01

    In the online version of the EM algorithm introduced by Sato and Ishii ( 2000 ), a time-dependent discount factor is introduced for forgetting the effect of the old estimated values obtained with an earlier, inaccurate estimator. In their approach, forgetting is uniformly applied to the estimators of each mixture component depending exclusively on time, irrespective of the weight attributed to each unit for the observed sample. This causes an excessive forgetting in the less frequently sampled regions. To address this problem, we propose a modification of the algorithm that involves a weight-dependent forgetting, different for each mixture component, in which old observations are forgotten according to the actual weight of the new samples used to replace older values. A comparison of the time-dependent versus the weight-dependent approach shows that the latter improves the accuracy of the approximation and exhibits much greater stability.

  12. Processing of Structurally Heterogeneous Cryo-EM Data in RELION.

    PubMed

    Scheres, S H W

    2016-01-01

    This chapter describes algorithmic advances in the RELION software, and how these are used in high-resolution cryo-electron microscopy (cryo-EM) structure determination. Since the presence of projections of different three-dimensional structures in the dataset probably represents the biggest challenge in cryo-EM data processing, special emphasis is placed on how to deal with structurally heterogeneous datasets. As such, this chapter aims to be of practical help to those who wish to use RELION in their cryo-EM structure determination efforts. PMID:27572726

  13. Processing of Structurally Heterogeneous Cryo-EM Data in RELION.

    PubMed

    Scheres, S H W

    2016-01-01

    This chapter describes algorithmic advances in the RELION software, and how these are used in high-resolution cryo-electron microscopy (cryo-EM) structure determination. Since the presence of projections of different three-dimensional structures in the dataset probably represents the biggest challenge in cryo-EM data processing, special emphasis is placed on how to deal with structurally heterogeneous datasets. As such, this chapter aims to be of practical help to those who wish to use RELION in their cryo-EM structure determination efforts.

  14. Application of the Chameleon Model to EM Field Momentum

    NASA Astrophysics Data System (ADS)

    Robertson, Glen A.

    2008-01-01

    The Chameleon scalar field model proposed by Khoury and Weltman presents an alternative mechanism for circumventing the constraints from local tests of gravity by mediating a fifth force for cosmological expansion, which could result in experimental signatures detectable through modest improvements of current laboratory set-ups in the vicinity of oscillating matter. In this paper, the oscillation of a dielectric by a crossed EM field is investigated in light of the Chameleon model. An EM excited Chameleon field-force equation is developed and compared to several EM experiments using the Barium Titanate based dielectric material.

  15. Environmental Education and Development Division (EM-522). Annual report, Fiscal year 1993

    SciTech Connect

    Not Available

    1993-12-31

    The Environmental Education and Development Division (EM-522) is one of three divisions within the Office of Technology Integration and Environmental Education and Development (EM-52) in Environmental Restoration and Waste Management`s (EM`s) Office of Technology Development (EM-50). The primary design criterion for EM-522 education activities is directly related to meeting EM`s goal of environmental compliance on an accelerated basis and cleanup of the 1989 inventory of inactive sites and facilities by the year 2019. Therefore, EM-522`s efforts are directed specifically toward stimulating knowledge and capabilities to achieve the goals of EM while contributing to DOE`s overall goal of increasing scientific, mathematical, and technical literacy and competency. This report discusses fiscal year 1993 activities.

  16. 7 CFR 759.6 - EM to be made available.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Agriculture under the Plant Protection Act or the animal quarantine laws, as defined in section 2509 of the Food, Agriculture, Conservation, and Trade Act of 1990, automatically authorizes EM for production...

  17. Antibody-based affinity cryo-EM grid.

    PubMed

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

  18. E.M. and Hadronic Shower Simulation with FLUKA

    SciTech Connect

    Battistoni, G.; Fasso, A.; Ferrari, A.; Ranft, J.; Rubbia, A.; Sala, P.R.; /INFN, Milan /SLAC /CERN /Siegen U. /Zurich, ETH

    2005-10-03

    A description of the main features of e.m. and hadronic shower simulation models used in the FLUKA code is summarized and some recent applications are discussed. The general status of the FLUKA project is also reported.

  19. Near-atomic-resolution cryo-EM for molecular virology.

    PubMed

    Hryc, Corey F; Chen, Dong-Hua; Chiu, Wah

    2011-08-01

    Electron cryo-microscopy (cryo-EM) is a technique in structural biology that is widely used to solve the three-dimensional structures of macromolecular assemblies, close to their biological and solution conditions. Recent improvements in cryo-EM and single-particle reconstruction methodologies have led to the determination of several virus structures at near-atomic resolution (3.3 - 4.6 Å). These cryo-EM structures not only resolve the Cα backbones and side-chain densities of viral capsid proteins, but also suggest functional roles that the protein domains and some key amino acid residues play. This paper reviews the recent advances in near-atomic-resolution cryo-EM for probing the mechanisms of virus assembly and morphogenesis.

  20. NASA EM Followup of LIGO-Virgo Candidate Events

    NASA Technical Reports Server (NTRS)

    Blackburn, Lindy L.

    2011-01-01

    We present a strategy for a follow-up of LIGO-Virgo candidate events using offline survey data from several NASA high-energy photon instruments aboard RXTE, Swift, and Fermi. Time and sky-location information provided by the GW trigger allows for a targeted search for prompt and afterglow EM signals. In doing so, we expect to be sensitive to signals which are too weak to be publicly reported as astrophysical EM events.

  1. Reducing stress factors in EMS: report of a national survey.

    PubMed

    Brownstone, J E; Shatoff, D K; Duckro, P N

    1983-01-01

    The existence of stress, coping with stress, and the effects of stress in Emergency Medical Services (EMS) are highly current and hotly debated subjects. This article describes in part the results of a national survey of 25 EMS systems in 24 large metropolitan areas. The portion of the survey reported here focused on sources of stress and programs available to promote more effective coping with stres. Results are discussed generally and in terms of the services' organizational affiliations.

  2. EM Telemetry Tool for Deep Well Drilling Applications

    SciTech Connect

    Jeffrey M. Gabelmann

    2005-11-15

    This final report discusses the successful development and testing of a deep operational electromagnetic (EM) telemetry system, produced under a cooperative agreement with the United States Department of Energy's National Energy Technology Laboratory. This new electromagnetic telemetry system provides a wireless communication link between sensors deployed deep within oil and gas wells and data acquisition equipment located on the earth's surface. EM based wireless telemetry is a highly appropriate technology for oil and gas exploration in that it avoids the need for thousands of feet of wired connections. In order to achieve the project performance objectives, significant improvements over existing EM telemetry systems were made. These improvements included the development of new technologies that have improved the reliability of the communications link while extending operational depth. A key element of the new design is the incorporation of a data-fusion methodology which enhances the communication receiver's ability to extract very weak signals from large amounts of ambient environmental noise. This innovative data-fusion receiver based system adapts advanced technologies, not normally associated with low-frequency communications, and makes them work within the harsh drilling environments associated with the energy exploration market. Every element of a traditional EM telemetry system design, from power efficiency to reliability, has been addressed. The data fusion based EM telemetry system developed during this project is anticipated to provide an EM tool capability that will impact both onshore and offshore oil and gas exploration operations, for conventional and underbalanced drilling applications.

  3. A HF EM installation allowing simultaneous whole body and deep local EM hyperthermia.

    PubMed

    Mazokhin, V N; Kolmakov, D N; Lucheyov, N A; Gelvich, E A; Troshin, I I

    1999-01-01

    The structure and main features of a HF EM installation based upon a new approach for creating electromagnetic fields destined for whole body (WBH) and deep local (DLH) hyperthermia are discussed. The HF EM field, at a frequency of 13.56 MHz, is created by a coplanar capacity type applicator positioned under a distilled water filled bolus that the patient is lying on. The EM energy being released directly in the deep tissues ensures effective whole body heating to required therapeutic temperatures of up to 43.5 degrees C, whereas the skin temperature can be maintained as low as 39-40.5 degrees C. For DLH, the installation is equipped with additional applicators and a generator operating at a frequency of 40.68 MHz. High efficiency of the WBH applicator makes it possible to carry out the WBH procedure without any air-conditioning cabin. Due to this, a free access to the patient's body during the WBH treatment is provided and a simultaneous WBH/DLH or WBH/LH procedure by means of additional applicators is possible. Controllable power output in the range of 100-800 W at a frequency of 13.56 MHz and 50-350 W at a frequency of 40.68 MHz allows accurate temperature control during WBH, DLH and WBH/DLH procedures. SAR patterns created by the WBH and DLH applicators in a liquid muscle phantom and measured by means of a non-perturbing E-dipole are investigated. The scattered EM field strength measured in the vicinity of the operating installation during the WBH, DLH and WBH/DLH procedures does not exceed security standards. Examples of temperature versus time graphs in the course of WBH, DLH and WBH/DLH procedures in clinics are presented. The installation is successfully used in leading oncological institutions of Russia and Belarus, though combined WBH/DLH procedures are evidently more complicated and demand thorough planning and temperature measurements to avoid overheating. PMID:10458570

  4. A History and Informal Assessment of the <em>Slacker Astronomyem> Podcast

    NASA Astrophysics Data System (ADS)

    Price, Aaron; Gay, Pamela; Searle, Travis; Brissenden, Gina

    Slacker Astronomyem> is a weekly podcast that covers a recent astronomical news event or discovery. The show has a unique style consisting of irreverent, over-the-top humor combined with a healthy dose of hard science. According to our demographic analysis, the combination of this style and the unique podcasting distribution mechanism allows the show to reach audiences younger and busier than those reached via traditional channels. We report on the successes and challenges of the first year of the show, and provide an informal assessment of its role as a source for astronomical news and concepts for its approximately 15,500 weekly listeners.

  5. Databases and Archiving for CryoEM.

    PubMed

    Patwardhan, A; Lawson, C L

    2016-01-01

    CryoEM in structural biology is currently served by three public archives-EMDB for 3DEM reconstructions, PDB for models built from 3DEM reconstructions, and EMPIAR for the raw 2D image data used to obtain the 3DEM reconstructions. These archives play a vital role for both the structural community and the wider biological community in making the data accessible so that results may be reused, reassessed, and integrated with other structural and bioinformatics resources. The important role of the archives is underpinned by the fact that many journals mandate the deposition of data to PDB and EMDB on publication. The field is currently undergoing transformative changes where on the one hand high-resolution structures are becoming a routine occurrence while on the other hand electron tomography is enabling the study of macromolecules in the cellular context. Concomitantly the archives are evolving to best serve their stakeholder communities. In this chapter, we describe the current state of the archives, resources available for depositing, accessing, searching, visualizing and validating data, on-going community-wide initiatives and opportunities, and challenges for the future. PMID:27572735

  6. DOE EM industry programs robotics development

    SciTech Connect

    Staubly, R.; Kothari, V.

    1998-12-31

    The Office of Science and Technology (OST) manages an aggressive program for RD and D, as well as testing and evaluation for the Department of Energy`s (DOE`s) Environmental Management (EM) organization. The goal is to develop new and improved environmental restoration and waste management technologies to clean up the inventory of the DOE weapons complex faster, safer, and cheaper than is possible with currently available technologies. Robotic systems reduce worker exposure to the absolute minimum, while providing proven, cost-effective, and, for some applications, the only acceptable technique for addressing challenging problems. Development of robotic systems for remote operations occurs in three main categories: tank waste characterization and retrieval; decontamination and dismantlement; and characterization, mapping, and inspection systems. In addition, the Federal Energy Technology Center (FETC) has some other projects which fall under the heading of supporting R and D. The central objective of all FETC robotic projects is to make robotic systems more attractive by reducing costs and health risks associated with the deployment of robotic technologies in the cleanup of the nuclear weapons complex. This will be accomplished through development of robots that are cheaper, faster, safer, and more reliable, as well as more straightforward to modify/adapt and more intuitive to operate with autonomous capabilities and intelligent controls that prevent accidents and optimize task execution.

  7. Elected medical staff leaders: who needs 'em?

    PubMed

    Thompson, R E

    1994-03-01

    Authority, influence, and power are not synonyms. In working with elected medical staff leaders, a physician executive who chooses to exert authority may soon find him- or herself relatively powerless. But one who chooses to downplay authority, to influence through persuasion, and to coach leaders to lead effectively soon generates support for his or her ideas. The need to coax, cajole, explain, persuade, and "seek input" frustrates many leaders in all kinds of organizations. It would be much easier just to order people about. It's so tempting to think: "Who needs 'em? I'm the 'chief physician.' I know what needs to be done. Let's weigh anchor, take her out, and do what it takes to sail those rough, uncharted seas." If you really enjoy sailing a large ship in rough seas without a crew, go right ahead. Or if you think it makes sense to run an organization with only an executive staff and no knowledgeable middle managers, by all means let clinician leaders know that, now that you're aboard, they're just window-dressing. If you can make this approach work, well and good. Your life will be much less complicated, each day will have far fewer frustrations, and progress toward established goals will be much faster. However, given the reality of traditionally thinking physicians, it would be best to keep an up-dated resume in the locked lower left-hand drawer of your desk.

  8. Persistent topology for cryo-EM data analysis.

    PubMed

    Xia, Kelin; Wei, Guo-Wei

    2015-08-01

    In this work, we introduce persistent homology for the analysis of cryo-electron microscopy (cryo-EM) density maps. We identify the topological fingerprint or topological signature of noise, which is widespread in cryo-EM data. For low signal-to-noise ratio (SNR) volumetric data, intrinsic topological features of biomolecular structures are indistinguishable from noise. To remove noise, we employ geometric flows that are found to preserve the intrinsic topological fingerprints of cryo-EM structures and diminish the topological signature of noise. In particular, persistent homology enables us to visualize the gradual separation of the topological fingerprints of cryo-EM structures from those of noise during the denoising process, which gives rise to a practical procedure for prescribing a noise threshold to extract cryo-EM structure information from noise contaminated data after certain iterations of the geometric flow equation. To further demonstrate the utility of persistent homology for cryo-EM data analysis, we consider a microtubule intermediate structure Electron Microscopy Data (EMD 1129). Three helix models, an alpha-tubulin monomer model, an alpha-tubulin and beta-tubulin model, and an alpha-tubulin and beta-tubulin dimer model, are constructed to fit the cryo-EM data. The least square fitting leads to similarly high correlation coefficients, which indicates that structure determination via optimization is an ill-posed inverse problem. However, these models have dramatically different topological fingerprints. Especially, linkages or connectivities that discriminate one model from another, play little role in the traditional density fitting or optimization but are very sensitive and crucial to topological fingerprints. The intrinsic topological features of the microtubule data are identified after topological denoising. By a comparison of the topological fingerprints of the original data and those of three models, we found that the third model is

  9. Emergency medical service (EMS): A unique flight environment

    NASA Technical Reports Server (NTRS)

    Shively, R. Jay

    1993-01-01

    The EMS flight environment is unique in today's aviation. The pilots must respond quickly to emergency events and often fly to landing zones where they have never been before . The time from initially receiving a call to being airborne can be as little as two to three minutes. Often the EMS pilot is the only aviation professional on site, they have no operations people or other pilots to aid them in making decisons. Further, since they are often flying to accident scenes, not airports, there is often complete weather and condition information. Therefore, the initial decision that the pilot must make, accepting or declining a flight, can become very difficult. The accident rate of EMS helicopters has been relatively high over the past years. NASA-Ames research center has taken several steps in an attempt to aid EMS pilots in their decision making and situational awareness. A preflight risk assessment system (SAFE) was developed to aid pilots in their decision making, and was tested at an EMS service. The resutls of the study were promising and a second version incorporating the lessons learned is under development. A second line of research was the development of a low cost electronic chart display (ECD). This is a digital map display to help pilots maintain geographical orientation. Another thrust was undertaken in conjunction with the Aviation Safety Reporting System (ASRS). This involved publicizing the ASRS to EMS pilots and personnel, and calling each of the reporters back to gather additional information. This paper will discuss these efforts and how they may positively impact the safety of EMS operations.

  10. Persistent topology for cryo-EM data analysis.

    PubMed

    Xia, Kelin; Wei, Guo-Wei

    2015-08-01

    In this work, we introduce persistent homology for the analysis of cryo-electron microscopy (cryo-EM) density maps. We identify the topological fingerprint or topological signature of noise, which is widespread in cryo-EM data. For low signal-to-noise ratio (SNR) volumetric data, intrinsic topological features of biomolecular structures are indistinguishable from noise. To remove noise, we employ geometric flows that are found to preserve the intrinsic topological fingerprints of cryo-EM structures and diminish the topological signature of noise. In particular, persistent homology enables us to visualize the gradual separation of the topological fingerprints of cryo-EM structures from those of noise during the denoising process, which gives rise to a practical procedure for prescribing a noise threshold to extract cryo-EM structure information from noise contaminated data after certain iterations of the geometric flow equation. To further demonstrate the utility of persistent homology for cryo-EM data analysis, we consider a microtubule intermediate structure Electron Microscopy Data (EMD 1129). Three helix models, an alpha-tubulin monomer model, an alpha-tubulin and beta-tubulin model, and an alpha-tubulin and beta-tubulin dimer model, are constructed to fit the cryo-EM data. The least square fitting leads to similarly high correlation coefficients, which indicates that structure determination via optimization is an ill-posed inverse problem. However, these models have dramatically different topological fingerprints. Especially, linkages or connectivities that discriminate one model from another, play little role in the traditional density fitting or optimization but are very sensitive and crucial to topological fingerprints. The intrinsic topological features of the microtubule data are identified after topological denoising. By a comparison of the topological fingerprints of the original data and those of three models, we found that the third model is

  11. Degradation of Benzodiazepines after 120 Days of EMS Deployment

    PubMed Central

    McMullan, Jason T.; Jones, Elizabeth; Barnhart, Bruce; Denninghoff, Kurt; Spaite, Daniel; Zaleski, Erin; Silbergleit, Robert

    2014-01-01

    Introduction EMS treatment of status epilepticus improves outcomes, but the benzodiazepine best suited for EMS use is unclear, given potential high environmental temperature exposures. Objective To describe the degradation of diazepam, lorazepam, and midazolam as a function of temperature exposure and time over 120 days of storage on active EMS units. Methods Study boxes containing vials of diazepam, lorazepam, and midazolam were distributed to 4 active EMS units in each of 2 EMS systems in the southwestern United States during May–August 2011. The boxes logged temperature every minute and were stored in EMS units per local agency policy. Two vials of each drug were removed from each box at 30-day intervals and underwent high-performance liquid chromatography to determine drug concentration. Concentration was analyzed as mean (and 95%CI) percent of initial labeled concentration as a function of time and mean kinetic temperature (MKT). Results 192 samples were collected (2 samples of each drug from each of 4 units per city at 4 time-points). After 120 days, the mean relative concentration (95%CI) of diazepam was 97.0% (95.7–98.2%) and of midazolam was 99.0% (97.7–100.2%). Lorazepam experienced modest degradation by 60 days (95.6% [91.6–99.5%]) and substantial degradation at 90 days (90.3% [85.2-95.4%]) and 120 days (86.5% [80.7–92.3%]). Mean MKT was 31.6°C (95%CI 27.1–36.1). Increasing MKT was associated with greater degradation of lorazepam, but not midazolam or diazepam. Conclusions Midazolam and diazepam experienced minimal degradation throughout 120 days of EMS deployment in high-heat environments. Lorazepam experienced significant degradation over 120 days and appeared especially sensitive to higher MKT exposure. PMID:24548058

  12. Colloidal Oatmeal <em>(Avena Sativa)em> Improves Skin Barrier Through Multi-Therapy Activity.

    PubMed

    Ilnytska, Olha; Kaur, Simarna; Chon, Suhyoun; Reynertson, Kurt A; Nebus, Judith; Garay, Michelle; Mahmood, Khalid; Southall, Michael D

    2016-06-01

    Oats (Avena sativa) are a centuries-old topical treatment for a variety of skin barrier conditions, including dry skin, skin rashes, and eczema; however, few studies have investigated the actual mechanism of action for the skin barrier strengthening activity of colloidal oatmeal. Four extracts of colloidal oatmeal were prepared with various solvents and tested in vitro for skin barrier related gene expression and activity. Extracts of colloidal oatmeal were found to induce the expression of genes related to epidermal differentiation, tight junctions and lipid regulation in skin, and provide pH-buffering capacity. Colloidal oatmeal boosted the expression of multiple target genes related to skin barrier, and resulted in recovery of barrier damage in an in vitro model of atopic dermatitis. In addition, an investigator-blinded study was performed with 50 healthy female subjects who exhibited bilateral moderate to severe dry skin on their lower legs. Subjects were treated with a colloidal oatmeal skin protectant lotion. Clinically, the colloidal oatmeal lotion showed significant clinical improvements in skin dryness, moisturization, and barrier. Taken together, these results demonstrate that colloidal oatmeal can provide clinically effective benefits for dry and compromised skin by strengthening skin barrier.

    <em>J Drugs Dermatolem>. 2016;15(6):684-690. PMID:27272074

  13. Colloidal Oatmeal <em>(Avena Sativa)em> Improves Skin Barrier Through Multi-Therapy Activity.

    PubMed

    Ilnytska, Olha; Kaur, Simarna; Chon, Suhyoun; Reynertson, Kurt A; Nebus, Judith; Garay, Michelle; Mahmood, Khalid; Southall, Michael D

    2016-06-01

    Oats (Avena sativa) are a centuries-old topical treatment for a variety of skin barrier conditions, including dry skin, skin rashes, and eczema; however, few studies have investigated the actual mechanism of action for the skin barrier strengthening activity of colloidal oatmeal. Four extracts of colloidal oatmeal were prepared with various solvents and tested in vitro for skin barrier related gene expression and activity. Extracts of colloidal oatmeal were found to induce the expression of genes related to epidermal differentiation, tight junctions and lipid regulation in skin, and provide pH-buffering capacity. Colloidal oatmeal boosted the expression of multiple target genes related to skin barrier, and resulted in recovery of barrier damage in an in vitro model of atopic dermatitis. In addition, an investigator-blinded study was performed with 50 healthy female subjects who exhibited bilateral moderate to severe dry skin on their lower legs. Subjects were treated with a colloidal oatmeal skin protectant lotion. Clinically, the colloidal oatmeal lotion showed significant clinical improvements in skin dryness, moisturization, and barrier. Taken together, these results demonstrate that colloidal oatmeal can provide clinically effective benefits for dry and compromised skin by strengthening skin barrier.

    <em>J Drugs Dermatolem>. 2016;15(6):684-690.

  14. FitEM2EM--tools for low resolution study of macromolecular assembly and dynamics.

    PubMed

    Frankenstein, Ziv; Sperling, Joseph; Sperling, Ruth; Eisenstein, Miriam

    2008-01-01

    Studies of the structure and dynamics of macromolecular assemblies often involve comparison of low resolution models obtained using different techniques such as electron microscopy or atomic force microscopy. We present new computational tools for comparing (matching) and docking of low resolution structures, based on shape complementarity. The matched or docked objects are represented by three dimensional grids where the value of each grid point depends on its position with regard to the interior, surface or exterior of the object. The grids are correlated using fast Fourier transformations producing either matches of related objects or docking models depending on the details of the grid representations. The procedures incorporate thickening and smoothing of the surfaces of the objects which effectively compensates for differences in the resolution of the matched/docked objects, circumventing the need for resolution modification. The presented matching tool FitEM2EMin successfully fitted electron microscopy structures obtained at different resolutions, different conformers of the same structure and partial structures, ranking correct matches at the top in every case. The differences between the grid representations of the matched objects can be used to study conformation differences or to characterize the size and shape of substructures. The presented low-to-low docking tool FitEM2EMout ranked the expected models at the top. PMID:18974836

  15. FitEM2EM--tools for low resolution study of macromolecular assembly and dynamics.

    PubMed

    Frankenstein, Ziv; Sperling, Joseph; Sperling, Ruth; Eisenstein, Miriam

    2008-01-01

    Studies of the structure and dynamics of macromolecular assemblies often involve comparison of low resolution models obtained using different techniques such as electron microscopy or atomic force microscopy. We present new computational tools for comparing (matching) and docking of low resolution structures, based on shape complementarity. The matched or docked objects are represented by three dimensional grids where the value of each grid point depends on its position with regard to the interior, surface or exterior of the object. The grids are correlated using fast Fourier transformations producing either matches of related objects or docking models depending on the details of the grid representations. The procedures incorporate thickening and smoothing of the surfaces of the objects which effectively compensates for differences in the resolution of the matched/docked objects, circumventing the need for resolution modification. The presented matching tool FitEM2EMin successfully fitted electron microscopy structures obtained at different resolutions, different conformers of the same structure and partial structures, ranking correct matches at the top in every case. The differences between the grid representations of the matched objects can be used to study conformation differences or to characterize the size and shape of substructures. The presented low-to-low docking tool FitEM2EMout ranked the expected models at the top.

  16. Learning when to Hold'em and When to Fold'em: ERS's Budget Hold'em Game Facilitates the Budget Development Process in Memphis

    ERIC Educational Resources Information Center

    Education Resource Strategies, 2010

    2010-01-01

    If your school district is facing a budget issue, it might surprise you to learn that the solution might very well lie in a game of cards. That certainly was the case earlier this year for the city schools of Memphis, Tennessee. The game is called Budget Hold'em, and it was developed by Education Resource Strategies (ERS) of Watertown,…

  17. Refinement of Atomic Structures Against cryo-EM Maps.

    PubMed

    Murshudov, G N

    2016-01-01

    This review describes some of the methods for atomic structure refinement (fitting) against medium/high-resolution single-particle cryo-EM reconstructed maps. Some of the tools developed for macromolecular X-ray crystal structure analysis, especially those encapsulating prior chemical and structural information can be transferred directly for fitting into cryo-EM maps. However, despite the similarities, there are significant differences between data produced by these two techniques; therefore, different likelihood functions linking the data and model must be used in cryo-EM and crystallographic refinement. Although tools described in this review are mostly designed for medium/high-resolution maps, if maps have sufficiently good quality, then these tools can also be used at moderately low resolution, as shown in one example. In addition, the use of several popular crystallographic methods is strongly discouraged in cryo-EM refinement, such as 2Fo-Fc maps, solvent flattening, and feature-enhanced maps (FEMs) for visualization and model (re)building. Two problems in the cryo-EM field are overclaiming resolution and severe map oversharpening. Both of these should be avoided; if data of higher resolution than the signal are used, then overfitting of model parameters into the noise is unavoidable, and if maps are oversharpened, then at least parts of the maps might become very noisy and ultimately uninterpretable. Both of these may result in suboptimal and even misleading atomic models.

  18. International Space Station (ISS) Emergency Mask (EM) Development

    NASA Technical Reports Server (NTRS)

    Toon, Katherine P.; Hahn, Jeffrey; Fowler, Michael; Young, Kevin

    2011-01-01

    The Emergency Mask (EM) is considered a secondary response emergency Personal Protective Equipment (PPE) designed to provide respiratory protection to the International Space Station (ISS) crewmembers in response to a post-fire event or ammonia leak. The EM is planned to be delivered to ISS in 2012 to replace the current air purifying respirator (APR) onboard ISS called the Ammonia Respirator (AR). The EM is a one ]size ]fits ]all model designed to fit any size crewmember, unlike the APR on ISS, and uses either two Fire Cartridges (FCs) or two Commercial Off-the-Shelf (COTS) 3M(Trademark). Ammonia Cartridges (ACs) to provide the crew with a minimum of 8 hours of respiratory protection with appropriate cartridge swap ]out. The EM is designed for a single exposure event, for either post ]fire or ammonia, and is a passive device that cannot help crewmembers who cannot breathe on their own. The EM fs primary and only seal is around the wearer fs neck to prevent a crewmember from inhaling contaminants. During the development of the ISS Emergency Mask, several design challenges were faced that focused around manufacturing a leak free mask. The description of those challenges are broadly discussed but focuses on one key design challenge area: bonding EPDM gasket material to Gore(Registered Trademark) fabric hood.

  19. Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

    PubMed

    Merk, Alan; Bartesaghi, Alberto; Banerjee, Soojay; Falconieri, Veronica; Rao, Prashant; Davis, Mindy I; Pragani, Rajan; Boxer, Matthew B; Earl, Lesley A; Milne, Jacqueline L S; Subramaniam, Sriram

    2016-06-16

    Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

  20. Refinement of Atomic Structures Against cryo-EM Maps.

    PubMed

    Murshudov, G N

    2016-01-01

    This review describes some of the methods for atomic structure refinement (fitting) against medium/high-resolution single-particle cryo-EM reconstructed maps. Some of the tools developed for macromolecular X-ray crystal structure analysis, especially those encapsulating prior chemical and structural information can be transferred directly for fitting into cryo-EM maps. However, despite the similarities, there are significant differences between data produced by these two techniques; therefore, different likelihood functions linking the data and model must be used in cryo-EM and crystallographic refinement. Although tools described in this review are mostly designed for medium/high-resolution maps, if maps have sufficiently good quality, then these tools can also be used at moderately low resolution, as shown in one example. In addition, the use of several popular crystallographic methods is strongly discouraged in cryo-EM refinement, such as 2Fo-Fc maps, solvent flattening, and feature-enhanced maps (FEMs) for visualization and model (re)building. Two problems in the cryo-EM field are overclaiming resolution and severe map oversharpening. Both of these should be avoided; if data of higher resolution than the signal are used, then overfitting of model parameters into the noise is unavoidable, and if maps are oversharpened, then at least parts of the maps might become very noisy and ultimately uninterpretable. Both of these may result in suboptimal and even misleading atomic models. PMID:27572731

  1. Analyses of Subnanometer Resolution Cryo-EM Density Maps

    PubMed Central

    Baker, Matthew L.; Baker, Mariah R.; Hryc, Corey F.; DiMaio, Frank

    2011-01-01

    Today, electron cryomicroscopy (cryo-EM) can routinely achieve subnanometer resolutions of complex macromolecular assemblies. From a density map, one can extract key structural and functional information using a variety of computational analysis tools. At subnanometer resolution, these tools make it possible to isolate individual subunits, identify secondary structures, and accurately fit atomic models. With several cryo-EM studies achieving resolutions beyond 5 Å, computational modeling and feature recognition tools have been employed to construct backbone and atomic models of the protein components directly from a density map. In this chapter, we describe several common classes of computational tools that can be used to analyze and model subnanometer resolution reconstructions from cryo-EM. A general protocol for analyzing subnanometer resolution density maps is presented along with a full description of steps used in analyzing the 4.3 Å resolution structure of Mm-cpn. PMID:20888467

  2. EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.).

    PubMed

    Srivastava, P; Kumar, G

    2011-01-01

    Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.

  3. Virus particle dynamics derived from CryoEM studies.

    PubMed

    Doerschuk, Peter C; Gong, Yunye; Xu, Nan; Domitrovic, Tatiana; Johnson, John E

    2016-06-01

    The direct electron detector has revolutionized electron cryo-microscopy (CryoEM). Icosahedral virus structures are routinely produced at 4Å resolution or better and the approach has largely displaced virus crystallography, as it requires less material, less purity and often produces a structure more rapidly. Largely ignored in this new era of CryoEM is the dynamic information in the data sets that was not available in X-ray structures. Here we review an approach that captures the dynamic character of viruses displayed in the CryoEM ensemble of particles at the moment of freezing. We illustrate the approach with a simple model, briefly describe the details and provide a practical application to virus particle maturation. PMID:27085980

  4. Recent technical advancements enabled atomic resolution CryoEM

    NASA Astrophysics Data System (ADS)

    Xueming, Li

    2016-01-01

    With recent breakthroughs in camera and image processing technologies single-particle electron cryo-microscopy (CryoEM) has suddenly gained the attention of structural biologists as a powerful tool able to solve the atomic structures of biological complexes and assemblies. Compared with x-ray crystallography, CryoEM can be applied to partially flexible structures in solution and without the necessity of crystallization, which is especially important for large complexes and assemblies. This review briefly explains several key bottlenecks for atomic resolution CryoEM, and describes the corresponding solutions for these bottlenecks based on the recent technical advancements. The review also aims to provide an overview about the technical differences between its applications in biology and those in material science. Project supported by Tsinghua-Peking Joint Center for Life Sciences, China.

  5. The effective molarity (EM)--a computational approach.

    PubMed

    Karaman, Rafik

    2010-08-01

    The effective molarity (EM) for 12 intramolecular S(N)2 processes involving the formation of substituted aziridines and substituted epoxides were computed using ab initio and DFT calculation methods. Strong correlation was found between the calculated effective molarity and the experimentally determined values. This result could open a door for obtaining EM values for intramolecular processes that are difficult to be experimentally provided. Furthermore, the calculation results reveal that the driving forces for ring-closing reactions in the two different systems are proximity orientation of the nucleophile to the electrophile and the ground strain energies of the products and the reactants.

  6. Geospatial Analysis of Pediatric EMS Run Density and Endotracheal Intubation

    PubMed Central

    Hansen, Matthew; Loker, William; Warden, Craig

    2016-01-01

    Introduction The association between geographic factors, including transport distance, and pediatric emergency medical services (EMS) run clustering on out-of-hospital pediatric endotracheal intubation is unclear. The objective of this study was to determine if endotracheal intubation procedures are more likely to occur at greater distances from the hospital and near clusters of pediatric calls. Methods This was a retrospective observational study including all EMS runs for patients less than 18 years of age from 2008 to 2014 in a geographically large and diverse Oregon county that includes densely populated urban areas near Portland and remote rural areas. We geocoded scene addresses using the automated address locator created in the cloud-based mapping platform ArcGIS, supplemented with manual address geocoding for remaining cases. We then use the Getis-Ord Gi spatial statistic feature in ArcGIS to map statistically significant spatial clusters (hot spots) of pediatric EMS runs throughout the county. We then superimposed all intubation procedures performed during the study period on maps of pediatric EMS-run hot spots, pediatric population density, fire stations, and hospitals. We also performed multivariable logistic regression to determine if distance traveled to the hospital was associated with intubation after controlling for several confounding variables. Results We identified a total of 7,797 pediatric EMS runs during the study period and 38 endotracheal intubations. In univariate analysis we found that patients who were intubated were similar to those who were not in gender and whether or not they were transported to a children’s hospital. Intubated patients tended to be transported shorter distances and were older than non-intubated patients. Increased distance from the hospital was associated with reduced odds of intubation after controlling for age, sex, scene location, and trauma system entry status in a multivariate logistic regression. The

  7. Speaker verification using combined acoustic and EM sensor signal processing

    SciTech Connect

    Ng, L C; Gable, T J; Holzrichter, J F

    2000-11-10

    Low Power EM radar-like sensors have made it possible to measure properties of the human speech production system in real-time, without acoustic interference. This greatly enhances the quality and quantity of information for many speech related applications. See Holzrichter, Burnett, Ng, and Lea, J. Acoustic. SOC. Am . 103 ( 1) 622 (1998). By combining the Glottal-EM-Sensor (GEMS) with the Acoustic-signals, we've demonstrated an almost 10 fold reduction in error rates from a speaker verification system experiment under a moderate noisy environment (-10dB).

  8. Low Bandwidth Vocoding using EM Sensor and Acoustic Signal Processing

    SciTech Connect

    Ng, L C; Holzrichter, J F; Larson, P E

    2001-10-25

    Low-power EM radar-like sensors have made it possible to measure properties of the human speech production system in real-time, without acoustic interference [1]. By combining these data with the corresponding acoustic signal, we've demonstrated an almost 10-fold bandwidth reduction in speech compression, compared to a standard 2.4 kbps LPC10 protocol used in the STU-III (Secure Terminal Unit, third generation) telephone. This paper describes a potential EM sensor/acoustic based vocoder implementation.

  9. Waste fuel, EMS may save plant $1M yearly

    SciTech Connect

    Barber, J.

    1982-05-24

    A mixture of paper trash and coal ash fueling an Erie, Pa. General Electric plant and a Network 90 microprocessor-based energy-management system (EMS) to optimize boiler efficiency will cost about $3 million and have a three-to-four-year payback. Over half the savings will come from the avoided costs of burning plant-generated trash. The EMS system will monitor fuel requirements in the boiler and compensate for changes in steam demand. It will also monitor plant electrical needs and control the steam diverted for cogeneration. (DCK)

  10. A emissão em 8mm e as bandas de Merrill-Sanford em estrelas carbonadas

    NASA Astrophysics Data System (ADS)

    de Mello, A. B.; Lorenz-Martins, S.

    2003-08-01

    Estrelas carbonadas possuem bandas moleculares em absorção no visível e, no infravermelho (IR) as principais características espectrais se devem a emissão de grãos. Recentemente foi detectada a presença de bandas de SiC2 (Merrill-Sanford, MS) em emissão sendo atribuída à presença de um disco rico em poeira. Neste trabalho analisamos uma amostra de 14 estrelas carbonadas, observadas no telescópio de 1.52 m do ESO em 4 regiões espectrais diferentes, a fim de detectar as bandas de MS em emissão. Nossa amostra é composta de estrelas que apresentam além da emissão em 11.3 mm, outra em 8 mm. Esta última emissão, não usual nestes objetos, tem sido atribuída ou a moléculas de C2H2, ou a um composto sólido ainda indefinido. A detecção de emissões de MS e aquelas no IR, simultaneamente, revelaria um cenário mais complexo que o habitualmente esperado para os ventos destes objetos. No entanto como primeiro resultado, verificamos que as bandas de Merrill-Sanford encontram-se em absorção, não revelando nenhuma conexão com a emissão a 8 mm. Assim, temos duas hipóteses: (a) a emissão a 8 mm se deve à molécula C2H2 ou (b) essa emissão é resultado da emissão térmica de grãos. Testamos a segunda hipótese modelando a amostra com grãos não-homogêneos de SiC e quartzo, o qual emite em aproximadamente 8mm. Este grão seria produzido em uma fase evolutiva anterior a das carbonadas (estrelas S) e por terem uma estrutura cristalina são destruídos apenas na presença de campos de radiação ultravioleta muito intensos. Os modelos para os envoltórios utilizam o método de Monte Carlo para descrever o problema do transporte da radiação. As conclusões deste trabalho são: (1) as bandas de Merrill-Sanford se encontram em absorção, sugerindo um cenário usual para os ventos das estrelas da amostra; (2) neste cenário, a emissão em 8 mm seria resultado de grãos de quartzo com mantos de SiC, indicando que o quartzo poderia sobreviver a fase

  11. A modified EM algorithm for estimation in generalized mixed models.

    PubMed

    Steele, B M

    1996-12-01

    Application of the EM algorithm for estimation in the generalized mixed model has been largely unsuccessful because the E-step cannot be determined in most instances. The E-step computes the conditional expectation of the complete data log-likelihood and when the random effect distribution is normal, this expectation remains an intractable integral. The problem can be approached by numerical or analytic approximations; however, the computational burden imposed by numerical integration methods and the absence of an accurate analytic approximation have limited the use of the EM algorithm. In this paper, Laplace's method is adapted for analytic approximation within the E-step. The proposed algorithm is computationally straightforward and retains much of the conceptual simplicity of the conventional EM algorithm, although the usual convergence properties are not guaranteed. The proposed algorithm accommodates multiple random factors and random effect distributions besides the normal, e.g., the log-gamma distribution. Parameter estimates obtained for several data sets and through simulation show that this modified EM algorithm compares favorably with other generalized mixed model methods.

  12. Functionalized anatomical models for EM-neuron Interaction modeling

    NASA Astrophysics Data System (ADS)

    Neufeld, Esra; Cassará, Antonino Mario; Montanaro, Hazael; Kuster, Niels; Kainz, Wolfgang

    2016-06-01

    The understanding of interactions between electromagnetic (EM) fields and nerves are crucial in contexts ranging from therapeutic neurostimulation to low frequency EM exposure safety. To properly consider the impact of in vivo induced field inhomogeneity on non-linear neuronal dynamics, coupled EM-neuronal dynamics modeling is required. For that purpose, novel functionalized computable human phantoms have been developed. Their implementation and the systematic verification of the integrated anisotropic quasi-static EM solver and neuronal dynamics modeling functionality, based on the method of manufactured solutions and numerical reference data, is described. Electric and magnetic stimulation of the ulnar and sciatic nerve were modeled to help understanding a range of controversial issues related to the magnitude and optimal determination of strength-duration (SD) time constants. The results indicate the importance of considering the stimulation-specific inhomogeneous field distributions (especially at tissue interfaces), realistic models of non-linear neuronal dynamics, very short pulses, and suitable SD extrapolation models. These results and the functionalized computable phantom will influence and support the development of safe and effective neuroprosthetic devices and novel electroceuticals. Furthermore they will assist the evaluation of existing low frequency exposure standards for the entire population under all exposure conditions.

  13. Texas Hold 'em Online Poker: A Further Examination

    ERIC Educational Resources Information Center

    Hopley, Anthony A. B.; Dempsey, Kevin; Nicki, Richard

    2012-01-01

    Playing Texas Hold 'em Online Poker (THOP) is on the rise. However, there is relatively little research examining factors that contribute to problem gambling in poker players. The aim of this study was to extend the research findings of Hopley and Nicki (2010). The negative mood states of depression, anxiety and stress were found to be linked to…

  14. Signs and Guides: Wayfinding Alternatives for the EMS Library.

    ERIC Educational Resources Information Center

    Johnson, Johanna H.

    Concerned with increasing the accessibility of the collection of the Engineering/Math Sciences (EMS) Library at the University of California at Los Angeles through the use of self guidance systems, this practical study focused on the problem context, general library guides, and library signage in reviewing the literature, and conducted a survey of…

  15. A shape constrained MAP-EM algorithm for colorectal segmentation

    NASA Astrophysics Data System (ADS)

    Wang, Huafeng; Li, Lihong; Song, Bowen; Han, Fangfang; Liang, Zhengrong

    2013-02-01

    The task of effectively segmenting colon areas in CT images is an important area of interest in medical imaging field. The ability to distinguish the colon wall in an image from the background is a critical step in several approaches for achieving larger goals in automated computer-aided diagnosis (CAD). The related task of polyp detection, the ability to determine which objects or classes of polyps are present in a scene, also relies on colon wall segmentation. When modeling each tissue type as a conditionally independent Gaussian distribution, the tissue mixture fractions in each voxel via the modeled unobservable random processes of the underlying tissue types can be estimated by maximum a posteriori expectation-maximization (MAP-EM) algorithm in an iterative manner. This paper presents, based on the assumption that the partial volume effect (PVE) could be fully described by a tissue mixture model, a theoretical solution to the MAP-EM segmentation algorithm. However, the MAP-EM algorithm may miss some small regions which also belong to the colon wall. Combining with the shape constrained model, we present an improved algorithm which is able to merge similar regions and reserve fine structures. Experiment results show that the new approach can refine the jagged-like boundaries and achieve better results than merely exploited our previously presented MAP-EM algorithm.

  16. Airborne EM for geothermal and hydrogeological mapping

    NASA Astrophysics Data System (ADS)

    Menghini, A.; Manzella, A.; Viezzoli, A.; Montanari, D.; Maggi, S.

    2012-12-01

    Within the "VIGOR" project, aimed at assessing the geothermal potential of four regions in southern Italy, Airborne EM data have been acquired, modeled and interpreted. The system deployed was SkyTEM, a time-domain helicopter electromagnetic system designed for hydrogeophysical, environmental and mineral investigations. The AEM data provide, after data acquisition, analysis, processing, and modeling, a distribution volume of electrical resistivity, spanning an investigation depth from ground surface of few hundred meters, depending on resistivity condition. Resistivity is an important physical parameter for geothermal investigation, since it proved to be very effective in mapping anomalies due to hydrothermal fluid circulation, which usually has high salt content and produces clayey alteration minerals. Since the project required, among other issues, to define geothermal resources at shallow level, it was decided to perform a test with an airborne electromagnetic geophysical survey, to verify the advantages offered by the system in covering large areas in a short time. The geophysical survey was carried out in Sicily, Italy, in late 2011, over two test sites named "Termini" and "Western Sicily". The two areas were chosen on different basis. "Termini" area is covered by extensive geological surveys, and was going to be investigated also by means of electrical tomography in its northern part. Since geological condition of Sicily, even at shallow depth, is very complex, this area provided a good place for defining the resistivity values of the main geological units outcropping in the region. "Termini" survey has been also an occasion to define relations between resistivity distribution, lithological units and thermal conductivity. The "Western Sicily" area cover the main thermal manifestations of western Sicily, and the research target was to establish whether they are characterized by common hydrogeological or tectonic features that could be mapped by resistivity

  17. <em>An Amphibious Magnetotelluric Investigation of the Cascadian Seismogenic and ETS zones.em>

    NASA Astrophysics Data System (ADS)

    Parris, B. A.; Livelybrooks, D.; Bedrosian, P.; Egbert, G. D.; Key, K.; Schultz, A.; Cook, A.; Kant, M.; Wogan, N.; Zeryck, A.

    2015-12-01

    The amphibious Magnetotelluric Observations of Cascadia using a Huge Array (MOCHA) experiment seeks to address unresolved questions about the seismogenic locked zone and down-dip transition zone where episodic tremor and slip (ETS) originates. The presence of free fluids is thought to be one of the primary controls on ETS behavior within the Cascadia margin. Since the bulk electrical conductivity in the crust and mantle can be greatly increased by fluids, magnetotelluric(MT) observations can offer unique insights on the fluid distribution and its relation to observed ETS behavior. Here we present preliminary results from the 146 MT stations collected for the MOCHA project. MOCHA is unique in that it is the first amphibious array of MT stations occupied to provide for 3-D interpretation of conductivity structure of a subduction zone. The MOCHA data set comprises 75 onshore stations and 71 offshore stations, accumulated over a two-year period, and located on an approximate 25km grid, spanning from the trench to the Eastern Willamette Valley, and from central Oregon into middle Washington. We present the results of a series of east-west (cross-strike) oriented, two-dimensional inversions created using the MARE2DEM software that provide an initial picture of the conductivity structure of the locked and ETS zones and its along strike variations. Our models can be used to identify correlations between ETS occurrence rates and inferred fluid concentrations. Our modeling explores the impact of various parameterizations on 2-D inversion results, including inclusion of a smoothness penalty reduction along the inferred slab interface. This series of 2-D inversions can then be used collectively to help make and guide an a priori 3-D inversion. In addition we will present a preliminary 3-D inversion of the onshore stations created using the ModEM software. We are currently working on modifying ModEM to support inversion of offshore data. The more computationally intensive 3-D

  18. The B and Be States of the Star EM Cepheus

    NASA Astrophysics Data System (ADS)

    Kjurkchieva, Diana; Marchev, Dragomir; Sigut, T. A. A.; Dimitrov, Dinko

    2016-09-01

    We present 11 yr of high-resolution, spectroscopic observations for the star EM Cep. EM Cep switches between B and Be star states, as revealed by the level of Hα emission, but spends most of its time in the B star state. EM Cep has been considered to be an eclipsing, near-contact binary of nearly equal-mass B stars in order to reproduce regular photometric variations; however, this model is problematic due to the lack of any observed Doppler shift in the spectrum. Our observations confirm that there are no apparent Doppler shifts in the wide spectral lines Hα and He i λ6678 in either the B or Be star states. The profiles of He i λ6678 typically exhibited a filled-in absorption core, but we detected weak emission in this line during the highest Be state. Given the lack of observed Doppler shifts, we model EM Cep as an isolated Be star with a variable circumstellar disk. We can reproduce the observed Hα emission profiles over the 11 yr period reasonably well with disk masses on the order of 3{--}10× {10}-11 {M}* in the Be state with the circumstellar disk seen at an inclination of 78° to the line of sight. From a disk ejection episode in 2014, we estimate a mass-loss rate of ≈ 3× {10}-9 {M}⊙ {{yr}}-1. The derived disk density parameters are typical of those found for the classical Be stars. We therefore suggest that the EM Cep is a classical Be star and that its photometric variations are the result of β Cep or nonradial pulsations.

  19. Signals Attenuation and Application of EM-MWD in China

    NASA Astrophysics Data System (ADS)

    Wang, R.; Liu, H.; Yang, Q.; Li, J.; Wu, D.

    2012-12-01

    In the course of drilling utilizes directional well and horizontal well, it is indispensable to master timely and exact geological guide information, and offering services for such complicated drilling is electromagnetic Measurement While Drilling (EM-MWD) which can measure guide data of drilling tool in the hole and formation data while drilling. This knowledge allows the directional driller to make appropriate mechanical corrections in drill string orientation that will allow the advancing drill bit to hit an intended subsurface target area. Based on electromagnetic field theory, the paper has studied the propagation particularity and attenuation regularity that the signals of electromagnetic wave for EM-MWD transmit in stratum. The paper also gives a brief introduction of the containing, the work principle and the main technology parameter of EM-MWD. To check up the performance of EM-MWD, the field test of 5 wells were carried out in Shengli Oilfield and Liaohe Oilfield. Numerical simulation results indicate the signal attenuation will be added with the decrease of the stratum resistibility. In the frequency range from 1 to 10 Hz the stratum absorptivity is tiny and does not add noticeably with the increase of the electromagnetic wave frequency and the decrease of the stratum resistibility. In the frequency range from 1 to 10000 Hz the stratum absorptivity does not increase noticeably with the decrease of the dielectric constant of the stratum. Field test demonstrate that EM-MWD receives successfully signal emitted from depth underground 1600 meters in Shengli oilfield and 2400 meters in Liaohe oilfield. Our results indicate that numerical simulation methods are comparable to field test.

  20. The B and Be States of the Star EM Cepheus

    NASA Astrophysics Data System (ADS)

    Kjurkchieva, Diana; Marchev, Dragomir; Sigut, T. A. A.; Dimitrov, Dinko

    2016-09-01

    We present 11 yr of high-resolution, spectroscopic observations for the star EM Cep. EM Cep switches between B and Be star states, as revealed by the level of Hα emission, but spends most of its time in the B star state. EM Cep has been considered to be an eclipsing, near-contact binary of nearly equal-mass B stars in order to reproduce regular photometric variations; however, this model is problematic due to the lack of any observed Doppler shift in the spectrum. Our observations confirm that there are no apparent Doppler shifts in the wide spectral lines Hα and He i λ6678 in either the B or Be star states. The profiles of He i λ6678 typically exhibited a filled-in absorption core, but we detected weak emission in this line during the highest Be state. Given the lack of observed Doppler shifts, we model EM Cep as an isolated Be star with a variable circumstellar disk. We can reproduce the observed Hα emission profiles over the 11 yr period reasonably well with disk masses on the order of 3{--}10× {10}-11 {M}* in the Be state with the circumstellar disk seen at an inclination of 78° to the line of sight. From a disk ejection episode in 2014, we estimate a mass-loss rate of ≈ 3× {10}-9 {M}ȯ {{yr}}-1. The derived disk density parameters are typical of those found for the classical Be stars. We therefore suggest that the EM Cep is a classical Be star and that its photometric variations are the result of β Cep or nonradial pulsations.

  1. Composting of rice straw with effective microorganisms (EM) and its influence on compost quality

    PubMed Central

    2013-01-01

    This study aims to assess the effect of EM application on the composting process of rice straw with goat manure and green waste and to evaluate the quality of both compost treatments. There are two treatment piles in this study, in which one pile was applied with EM and another pile without EM. Each treatment was replicated three times with 90 days of composting duration. The parameters for the temperature, pH, TOC and C/N ratio, show that decomposition of organic matter occurs during the 90-day period. The t-test conducted shows that there is a significant difference between compost with EM and compost without EM. The application of EM in compost increases the macro and micronutrient content. The following parameters support this conclusion: compost applied with EM has more N, P and K content (P < 0.05) compared to compost without EM. Although the Fe in compost with EM is much higher (P < 0.05) than in the compost without EM, for Zn and Cu, there is no significant difference between treatments. This study suggests that the application of EM is suitable to increase the mineralization in the composting process. The final resultant compost indicated that it was in the range of the matured level and can be used without any restriction. PMID:23390930

  2. Application of Electromagnetic (EM) Separation Technology to Metal Refining Processes: A Review

    NASA Astrophysics Data System (ADS)

    Zhang, Lifeng; Wang, Shengqian; Dong, Anping; Gao, Jianwei; Damoah, Lucas Nana Wiredu

    2014-12-01

    Application of electromagnetic (EM) force to metal processing has been considered as an emerging technology for the production of clean metals and other advanced materials. In the current paper, the principle of EM separation was introduced and several schemes of imposing EM field, such as DC electric field with a crossed steady magnetic field, AC electric field, AC magnetic field, and traveling magnetic field were reviewed. The force around a single particle or multi-particles and their trajectories in the conductive liquid under EM field were discussed. Applications of EM technique to the purification of different liquid metals such as aluminum, zinc, magnesium, silicon, copper, and steel were summarized. Effects of EM processing parameters, such as the frequency of imposed field, imposed magnetic flux density, processing time, particle size, and the EM unit size on the EM purification efficiency were discussed. Experimental and theoretical investigations have showed that the separation efficiency of inclusions from the molten aluminum using EM purification could as high as over 90 pct. Meanwhile, the EM purification was also applied to separate intermetallic compounds from metal melt, such as α-AlFeMnSi-phase from the molten aluminum. And then the potential industrial application of EM technique was proposed.

  3. Effects of caffeine or EDTA post-treatment on EMS mutagenesis in soybean.

    PubMed

    Zhu, B; Gu, A; Deng, X; Geng, Y; Lu, Z

    1995-04-01

    Seeds of soybean cultivar LD4 were mutagenically treated with EMS (0.3, 0.5, 0.6, 0.9, 1.5 and 1.8%) for 3 h only or plus caffeine (50 mM) or EDTA (1 mM) post-treatment for 5 h. The experimental results indicated that: (1) of the different concentrations of EMS treatment, the M2 mutation frequency induced with 0.6% EMS was the highest (9.7%). When the EMS concentration was over 0.9%, the mutation frequency decreased rapidly. (2) Of the EMS treatments plus caffeine or EDTA post-treatment, the mutagenic effect of 0.6% EMS was the best for inducing morphological variations. Caffeine post-treatment decreased notably the mutation frequency of EMS treatment; when concentrations of EMS were very high (1.5% and 1.8%), mutation frequencies of EDTA post-treatment were still 5.0% and 4.88%, but no mutants were found in EMS treatment or plus caffeine post-treatment. (3) In the M2 mutation spectrum, 11 kinds of mutant types were observed in EMS treatment or plus caffeine or EDTA post-treatment. Relative frequencies of some mutant types (growth period, plant height, grain size, leaf shape and sterility, etc.) were similar among the three treatments, but EDTA post-treatment could change the relative frequencies of yield characteristics (number of pods and grains, grain weight/plant) induced by EMS treatment only.

  4. Development of the Emergency Medical Services Role Identity Scale (EMS-RIS).

    PubMed

    Donnelly, Elizabeth A; Siebert, Darcy; Siebert, Carl

    2015-01-01

    This article describes the development and validation of the theoretically grounded Emergency Medical Services Role Identity Scale (EMS-RIS), which measures four domains of EMS role identity. The EMS-RIS was developed using a mixed methods approach. Key informants informed item development and the scale was validated using a representative probability sample of EMS personnel. Factor analyses revealed a conceptually consistent, four-factor solution with sound psychometric properties as well as evidence of convergent and discriminant validities. Social workers work with EMS professionals in crisis settings and as their counselors when they are distressed. The EMS-RIS provides useful information for the assessment of and intervention with distressed EMS professionals, as well as how role identity may influence occupational stress. PMID:25760489

  5. Similarity-regulation of OS-EM for accelerated SPECT reconstruction

    NASA Astrophysics Data System (ADS)

    Vaissier, P. E. B.; Beekman, F. J.; Goorden, M. C.

    2016-06-01

    Ordered subsets expectation maximization (OS-EM) is widely used to accelerate image reconstruction in single photon emission computed tomography (SPECT). Speedup of OS-EM over maximum likelihood expectation maximization (ML-EM) is close to the number of subsets used. Although a high number of subsets can shorten reconstruction times significantly, it can also cause severe image artifacts such as improper erasure of reconstructed activity if projections contain few counts. We recently showed that such artifacts can be prevented by using a count-regulated OS-EM (CR-OS-EM) algorithm which automatically adapts the number of subsets for each voxel based on the estimated number of counts that the voxel contributed to the projections. While CR-OS-EM reached high speed-up over ML-EM in high-activity regions of images, speed in low-activity regions could still be very slow. In this work we propose similarity-regulated OS-EM (SR-OS-EM) as a much faster alternative to CR-OS-EM. SR-OS-EM also automatically and locally adapts the number of subsets, but it uses a different criterion for subset regulation: the number of subsets that is used for updating an individual voxel depends on how similar the reconstruction algorithm would update the estimated activity in that voxel with different subsets. Reconstructions of an image quality phantom and in vivo scans show that SR-OS-EM retains all of the favorable properties of CR-OS-EM, while reconstruction speed can be up to an order of magnitude higher in low-activity regions. Moreover our results suggest that SR-OS-EM can be operated with identical reconstruction parameters (including the number of iterations) for a wide range of count levels, which can be an additional advantage from a user perspective since users would only have to post-filter an image to present it at an appropriate noise level.

  6. Em polypeptide and its messenger RNA levels are modulated by abscisic acid during embryogenesis in wheat.

    PubMed

    Williamson, J D; Quatrano, R S; Cuming, A C

    1985-10-15

    The effect of abscisic acid (ABA) on the expression of the 'early-methionine-labeled' (Em) polypeptide was examined in cultured, immature wheat (Triticum aestivum, L.) embryos and in developing embryos in planta. A complementary DNA (cDNA) library was constructed from poly(A)-rich RNA from immature embryos cultured in the presence of ABA. ABA-enhanced sequences were first identified by differential colony-blot hybridization, and then verified using RNA slot-blot analysis. Dot-blot hybridization showed that one clone, p1015, was homologous to the previously isolated Em cDNA, pWG432. Electrophoretic analysis of the hybrid-select translation product of p1015 confirmed its identity as an Em sequence. Comparison of the p1015 cDNA insert size and the Em message size, from northern blot analysis, showed that p1015 contained about 87% of the Em sequence. RNA slot-blot analysis and protein electrophoresis showed that Em message, but not Em protein, accumulated at a low, basal level in immature embryos in the absence of ABA. Neither Em message nor Em protein was seen in three-day germinated seedlings. Steady-state levels of Em message and protein increased in immature embryos in the presence of ABA, both in culture and in planta. Regulation appeared to be primarily at the level of transcription or specific message stability. Regulation may also involve specific protein stability, since synthesis of Em protein continued in immature embryos in the absence of ABA, but Em protein did not accumulate in detectable amounts. We conclude that ABA specifically modulates Em message and protein levels in immature embryos, but is probably not responsible for the embryogenic specificity of Em expression.

  7. Magen David Adom--the EMS in Israel.

    PubMed

    Ellis, Daniel Y; Sorene, Eliot

    2008-01-01

    Israel is a small country with a population of around 7 million. The sole EMS provider for Israel is Magen David Adom (MDA) (translated as 'Red Shield of David'). MDA also carries out the functions of a National Society (similar to the Red Cross) and provides all the blood and blood product services for the country. Nationwide, the organisation responds to over 1000 emergency calls a day and uses doctors, paramedics, emergency medical technicians and volunteers. Local geopolitics has meant that MDA has to be prepared for anything from everyday emergency calls to suicide bombings and regional wars. MDA also prides itself in being able to rapidly assemble and dispatch mobile aid teams to scenes of international disasters. Such a broad range of activities is unusual for a single EMS organisation. PMID:17767990

  8. Magen David Adom--the EMS in Israel.

    PubMed

    Ellis, Daniel Y; Sorene, Eliot

    2008-01-01

    Israel is a small country with a population of around 7 million. The sole EMS provider for Israel is Magen David Adom (MDA) (translated as 'Red Shield of David'). MDA also carries out the functions of a National Society (similar to the Red Cross) and provides all the blood and blood product services for the country. Nationwide, the organisation responds to over 1000 emergency calls a day and uses doctors, paramedics, emergency medical technicians and volunteers. Local geopolitics has meant that MDA has to be prepared for anything from everyday emergency calls to suicide bombings and regional wars. MDA also prides itself in being able to rapidly assemble and dispatch mobile aid teams to scenes of international disasters. Such a broad range of activities is unusual for a single EMS organisation.

  9. Advanced communication infrastructure for pre-hospital EMS care.

    PubMed

    Orthner, Helmuth; Mazza, Giovanni; Mazza, Giovanni Giorgio; Shenvi, Rohit; Battles, Marcie

    2008-11-06

    The traditional communication infrastructure of the pre-hospital Emergency Medical System (EMS) is limited to voice communication using radio or cell phone technologies. With the emergence of 3rd Generation wireless networks (3G) and enhanced mobile devices capable of data communication (e.g., mobile tablets, PDAs with cell phones, or cell phones with PDA capabilities), the voice communication can be enhanced with interactive data messaging and perhaps even with interactive video communication. However, video requires substantially more bandwidth which 4th Generation (4G) systems are promising. However, their availability is limited. We present an infrastructure that allows dynamic selection of the best data transport mode in the pre-hospital EMS environment.

  10. Superconducting Electromagnetic Suspension (EMS) system for Grumman Maglev concept

    NASA Technical Reports Server (NTRS)

    Kalsi, Swarn S.

    1994-01-01

    The Grumman developed Electromagnetic Suspension (EMS) Maglev system has the following key characteristics: a large operating airgap--40 mm; levitation at all speeds; both high speed and low speed applications; no deleterious effects on SC coils at low vehicle speeds; low magnetic field at the SC coil--less than 0.35 T; no need to use non-magnetic/non-metallic rebar in the guideway structure; low magnetic field in passenger cabin--approximately 1 G; low forces on the SC coil; employs state-of-the-art NbTi wire; no need for an active magnet quench protection system; and lower weight than a magnet system with copper coils. The EMS Maglev described in this paper does not require development of any new technologies. The system could be built with the existing SC magnet technology.

  11. Range Condition and ML-EM Checkerboard Artifacts

    PubMed Central

    You, Jiangsheng; Wang, Jing; Liang, Zhengrong

    2007-01-01

    The expectation maximization (EM) algorithm for the maximum likelihood (ML) image reconstruction criterion generates severe checkerboard artifacts in the presence of noise. A classical remedy is to impose an a priori constraint for a penalized ML or maximum a posteriori probability solution. The penalty reduces the checkerboard artifacts and also introduces uncertainty because a priori information is usually unknown in clinic. Recent theoretical investigation reveals that the noise can be divided into two components: one is called null-space noise and the other is range-space noise. The null-space noise can be numerically estimated using filtered backprojection (FBP) algorithm. By the FBP algorithm, the null-space noise annihilates in the reconstruction while the range-space noise propagates into the reconstructed image. The aim of this work is to investigate the relation between the null-space noise and the checkerboard artifacts in the ML-EM reconstruction from noisy projection data. Our study suggests that removing the null-space noise from the projection data could improve the signal-to-noise ratio of the projection data and, therefore, reduce the checkerboard artifacts in the ML-EM reconstructed images. This study reveals an in-depth understanding of the different noise propagations in analytical and iterative image reconstructions, which may be useful to single photon emission computed tomography, where the noise has been a major factor for image degradation. The reduction of the ML-EM checkerboard artifacts by removing the null-space noise avoids the uncertainty of using a priori penalty. PMID:18449363

  12. Generalized single-particle cryo-EM--a historical perspective.

    PubMed

    Frank, Joachim

    2016-02-01

    This is a brief account of the earlier history of single-particle cryo-EM of biological molecules lacking internal symmetry, which goes back to the mid-seventies. The emphasis of this review is on the mathematical concepts and computational approaches. It is written as the field experiences a turning point in the wake of the introduction of digital cameras capable of single electron counting, and near-atomic resolution can be reached even for smaller molecules. PMID:26566976

  13. A Bayesian View on Cryo-EM Structure Determination

    PubMed Central

    Scheres, Sjors H.W.

    2012-01-01

    Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures. PMID:22100448

  14. EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.).

    PubMed

    Srivastava, P; Kumar, G

    2011-01-01

    Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future. PMID:21950142

  15. The US DOE-EM International Program - 13004

    SciTech Connect

    Elmetti, Rosa R.; Han, Ana M.; Williams, Alice C.

    2013-07-01

    The U.S. Department of Energy (DOE) Office of Environmental Management (EM) conducts international collaboration activities in support of U.S. policies and objectives regarding the accelerated risk reduction and remediation of environmental legacy of the nations' nuclear weapons program and government sponsored nuclear energy research. The EM International Program supported out of the EM Office of the Associate Principal Deputy Assistant Secretary pursues collaborations with foreign government organizations, educational institutions and private industry to assist in identifying technologies and promote international collaborations that leverage resources and link international experience and expertise. In fiscal year (FY) 2012, the International Program awarded eight international collaborative projects for work scope spanning waste processing, groundwater and soil remediation, deactivation and decommissioning (D and D) and nuclear materials disposition initiatives to seven foreign organizations. Additionally, the International Program's scope and collaboration opportunities were expanded to include technical as well as non-technical areas. This paper will present an overview of the on-going tasks awarded in FY 2012 and an update of upcoming international activities and opportunities for expansion into FY 2013 and beyond. (authors)

  16. Improving EM&V for Energy Efficiency Programs (Fact Sheet)

    SciTech Connect

    Not Available

    2012-07-01

    This fact sheet describes the objectives of the U.S. Department of Energy Uniform Methods Project to bring consistency to energy savings calculations in U.S. energy efficiency programs. The U.S. Department of Energy (DOE) is developing a framework and a set of protocols for determining gross energy savings from energy efficiency measures and programs. The protocols represent a refinement of the body of knowledge supporting energy efficiency evaluation, measurement, and verification (EM&V) activities. They have been written by technical experts within the field and reviewed by industry experts. Current EM&V practice allows for multiple methods for calculating energy savings. These methods were developed to meet the needs of energy efficiency program administrators and regulators. Although they served their original objectives well, they have resulted in inconsistent and incomparable savings results - even for identical measures. The goal of the Uniform Methods Project is to strengthen the credibility of energy savings determinations by improving EM&V, increasing the consistency and transparency of how energy savings are determined.

  17. Conjoined Use of EM and NMR in RNA Structure Refinement

    PubMed Central

    Gong, Zhou; Schwieters, Charles D.; Tang, Chun

    2015-01-01

    More than 40% of the RNA structures have been determined using nuclear magnetic resonance (NMR) technique. NMR mainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characterization of large RNAs can be difficult for NMR alone. Electron microscopy (EM) provides global shape information of macromolecules at nanometer resolution, which should be complementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems — U2/U6 small-nuclear RNA, genome-packing motif (ΨCD)2 from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorporation of a map restraint, either experimental or generated from known PDB structure, greatly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nucleotide in the torsion angle simulated annealing refinement. As increasing number of macromolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures. PMID:25798848

  18. Minimum-distortion isometric shape correspondence using EM algorithm.

    PubMed

    Sahillioğlu, Yusuf; Yemez, Yücel

    2012-11-01

    We present a purely isometric method that establishes 3D correspondence between two (nearly) isometric shapes. Our method evenly samples high-curvature vertices from the given mesh representations, and then seeks an injective mapping from one vertex set to the other that minimizes the isometric distortion. We formulate the problem of shape correspondence as combinatorial optimization over the domain of all possible mappings, which then reduces in a probabilistic setting to a log-likelihood maximization problem that we solve via the Expectation-Maximization (EM) algorithm. The EM algorithm is initialized in the spectral domain by transforming the sampled vertices via classical Multidimensional Scaling (MDS). Minimization of the isometric distortion, and hence maximization of the log-likelihood function, is then achieved in the original 3D euclidean space, for each iteration of the EM algorithm, in two steps: by first using bipartite perfect matching, and then a greedy optimization algorithm. The optimal mapping obtained at convergence can be one-to-one or many-to-one upon choice. We demonstrate the performance of our method on various isometric (or nearly isometric) pairs of shapes for some of which the ground-truth correspondence is available.

  19. SubspaceEM: A Fast Maximum-a-posteriori Algorithm for Cryo-EM Single Particle Reconstruction

    PubMed Central

    Dvornek, Nicha C.; Sigworth, Fred J.; Tagare, Hemant D.

    2015-01-01

    Single particle reconstruction methods based on the maximum-likelihood principle and the expectation-maximization (E–M) algorithm are popular because of their ability to produce high resolution structures. However, these algorithms are computationally very expensive, requiring a network of computational servers. To overcome this computational bottleneck, we propose a new mathematical framework for accelerating maximum-likelihood reconstructions. The speedup is by orders of magnitude and the proposed algorithm produces similar quality reconstructions compared to the standard maximum-likelihood formulation. Our approach uses subspace approximations of the cryo-electron microscopy (cryo-EM) data and projection images, greatly reducing the number of image transformations and comparisons that are computed. Experiments using simulated and actual cryo-EM data show that speedup in overall execution time compared to traditional maximum-likelihood reconstruction reaches factors of over 300. PMID:25839831

  20. Inhomogeneous Media 3D EM Modeling with Integral Equation Method

    NASA Astrophysics Data System (ADS)

    di, Q.; Wang, R.; An, Z.; Fu, C.; Xu, C.

    2010-12-01

    In general, only the half space of earth is considered in electromagnetic exploration. However, for the long bipole source, because the length is close to the height of ionosphere and also most offsets between source and receivers are equal or larger than the height of ionosphere, the effect of ionosphere on the electromagnetic (EM) field should be considered when observation is carried at a very far (about several thousands kilometers) location away from the source. At this point the problem becomes one which should contain ionosphere, atmosphere and earth that is “earth-ionosphere” case. There are a few of literatures to report the electromagnetic field results which is including ionosphere, atmosphere and earth media at the same time. We firstly calculate the electromagnetic fields with the traditional controlled source (CSEM) configuration using integral equation (IE) method for a three layers earth-ionosphere model. The modeling results agree well with the half space analytical results because the effect of ionosphere for this small scale bipole source can be ignorable. The comparison of small scale three layers earth-ionosphere modeling and half space analytical resolution shows that the IE method can be used to modeling the EM fields for long bipole large offset configuration. In order to discuss EM fields’ characteristics for complicate earth-ionosphere media excited by long bipole source in the far-field and wave-guide zones, we first modeled the decay characters of electromagnetic fields for three layers earth-ionosphere model. Because of the effect of ionosphere, the earth-ionosphere electromagnetic fields’ decay curves with given frequency show that there should be an extra wave guide zone for long bipole artificial source, and there are many different characters between this extra zone and far field zone. They are: 1) the amplitudes of EM fields decay much slower; 2) the polarization patterns change; 3) the positions better to measure Zxy and

  1. Modeling of MHD edge containment in strip casting with ELEKTRA and CaPS-EM codes

    SciTech Connect

    Chang, F. C.

    2000-01-12

    This paper presents modeling studies of magnetohydrodynamics analysis in twin-roll casting. Argonne National Laboratory (ANL) and ISPAT Inland Inc. (Inland), formerly Inland Steel Co., have worked together to develop a three-dimensional (3-D) computer model that can predict eddy currents, fluid flows, and liquid metal containment of an electromagnetic (EM) edge containment device. The model was verified by comparing predictions with experimental results of liquid metal containment and fluid flow in EM edge dams (EMDs) that were designed at Inland for twin-roll casting. This mathematical model can significantly shorten casting research on the use of EM fields for liquid metal containment and control. The model can optimize the EMD design so it is suitable for application, and minimize expensive time-consuming full-scale testing. Numerical simulation was performed by coupling a 3-D finite-element EM code (ELEKTRA) and a 3-D finite-difference fluids code (CaPS-EM) to solve heat transfer, fluid flow, and turbulence transport in a casting process that involves EM fields. ELEKTRA can predict the eddy-current distribution and the EM forces in complex geometries. CaPS-EM can model fluid flows with free surfaces. The computed 3-D magnetic fields and induced eddy currents in ELEKTRA are used as input to temperature- and flow-field computations in CaPS-EM. Results of the numerical simulation compared well with measurements obtained from both static and dynamic tests.

  2. Emergency medical services and "psych calls": Examining the work of urban EMS providers.

    PubMed

    Prener, Christopher; Lincoln, Alisa K

    2015-11-01

    Emergency medical technicians and paramedics form the backbone of the United States' Emergency Medical Service (EMS) system. Despite the frequent involvement of EMS with people with mental health and substance abuse problems, the nature and content of this work, as well as how EMS providers think about this work, have not been fully explored. Using data obtained through observations and interviews with providers at an urban American EMS agency, this paper provides an analysis of the ways in which EMS providers interact with people with mental illness and substance abuse problems, as well as providers' experiences with the mental health care system. Results demonstrate that EMS providers share common beliefs and frustrations about "psych calls" and the types of calls that involve people with behavioral health problems. In addition, providers described their understandings of the ways in which people with mental health and substance use problems "abuse the system" and the consequences of this abuse. Finally, EMS providers discuss the system-level factors that impact their work and specific barriers and challenges to care. These results suggest that additional work is needed to expand our understanding of the role of EMS providers in the care of people with behavioral health problems and that mental health practitioners and policy makers should include consideration of the important role of EMS and prehospital care in providing community-based supports for people with behavioral health needs. (PsycINFO Database Record

  3. Volta phase plate cryo-EM of the small protein complex Prx3

    NASA Astrophysics Data System (ADS)

    Khoshouei, Maryam; Radjainia, Mazdak; Phillips, Amy J.; Gerrard, Juliet A.; Mitra, Alok K.; Plitzko, Jürgen M.; Baumeister, Wolfgang; Danev, Radostin

    2016-01-01

    Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles (<400 kDa). Here we demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. The Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates structure determination.

  4. Perda de massa em ventos empoeirados de estrelas supergigantes

    NASA Astrophysics Data System (ADS)

    Vidotto, A. A.; Jatenco-Pereira, V.

    2003-08-01

    Em praticamente todas as regiões do diagrama HR, as estrelas apresentam evidências observacionais de perda de massa. Na literatura, pode-se encontrar trabalhos que tratam tanto do diagnóstico da perda de massa como da construção de modelos que visam explicá-la. O amortecimento de ondas Alfvén tem sido utilizado como mecanismo de aceleração de ventos homogêneos. Entretanto, sabe-se que os envelopes de estrelas frias contêm grãos sólidos e moléculas. Com o intuito de estudar a interação entre as ondas Alfvén e a poeira e a sua conseqüência na aceleração do vento estelar, Falceta-Gonçalves & Jatenco-Pereira (2002) desenvolveram um modelo de perda de massa para estrelas supergigantes. Neste trabalho, apresentamos um estudo do modelo acima proposto para avaliar a dependência da taxa de perda de massa com alguns parâmetros iniciais como, por exemplo, a densidade r0, o campo magnético B0, o comprimento de amortecimento da onda L0, seu fluxo f0, entre outros. Sendo assim, aumentando f0 de 10% a partir de valores de referência, vimos que aumenta consideravelmente, enquanto que um aumento de mesmo valor em r0, B0 e L0 acarreta uma diminuição em .

  5. EM-Based Multiuser Detection in Fast Fading Multipath Environments

    NASA Astrophysics Data System (ADS)

    Borran, Mohammad Jaber; Aazhang, Behnaam

    2002-12-01

    We address the problem of multiuser detection in fast fading multipath environments for DS-CDMA systems. In fast fading scenarios, temporal variations of the channel cause significant performance degradation even with the Rake receiver. We use a previously introduced time-frequency (TF) Rake receiver based on a canonical formulation of the channel and signals to simultaneously combat fading and multipath effects. This receiver uses the Doppler spread caused by rapid time-varying channel as another means of diversity. In dealing with multiaccess interference and as an attempt to avoid the prohibitive computational complexity of the optimum maximum-likelihood (ML) detector, we use the expectation maximization (EM) algorithm to derive an approximate ML detector. The new detector turns out to have an iterative structure very similar to the well-known multistage detector with some extra parameters. At the two extreme values of these parameters, the EM detector reduces to either one-shot TF Rake or generalized multistage detector. For the intermediate values of the parameters, it combines the two estimates to obtain a better decision for the bits of the users. Because of using the EM algorithm, this detector has better convergence properties than the multistage detector; the bit estimates always converge, and if an appropriate initial vector is used, they converge to the global maximizer of the likelihood function. As a result, the new detector provides significantly improved performance while maintaining the low complexity of the multistage detector. Our simulation results confirm the expected performance improvements compared to the base case of the TF Rake as well as the multistage detector used with the TF Rake.

  6. EM-21 Retrieval Knowledge Center: Waste Retrieval Challenges

    SciTech Connect

    Fellinger, Andrew P.; Rinker, Michael W.; Berglin, Eric J.; Minichan, Richard L.; Poirier, Micheal R.; Gauglitz, Phillip A.; Martin, Bruce A.; Hatchell, Brian K.; Saldivar, Eloy; Mullen, O Dennis; Chapman, Noel F.; Wells, Beric E.; Gibbons, Peter W.

    2009-04-10

    EM-21 is the Waste Processing Division of the Office of Engineering and Technology, within the U.S. Department of Energy’s (DOE) Office of Environmental Management (EM). In August of 2008, EM-21 began an initiative to develop a Retrieval Knowledge Center (RKC) to provide the DOE, high level waste retrieval operators, and technology developers with centralized and focused location to share knowledge and expertise that will be used to address retrieval challenges across the DOE complex. The RKC is also designed to facilitate information sharing across the DOE Waste Site Complex through workshops, and a searchable database of waste retrieval technology information. The database may be used to research effective technology approaches for specific retrieval tasks and to take advantage of the lessons learned from previous operations. It is also expected to be effective for remaining current with state-of-the-art of retrieval technologies and ongoing development within the DOE Complex. To encourage collaboration of DOE sites with waste retrieval issues, the RKC team is co-led by the Savannah River National Laboratory (SRNL) and the Pacific Northwest National Laboratory (PNNL). Two RKC workshops were held in the Fall of 2008. The purpose of these workshops was to define top level waste retrieval functional areas, exchange lessons learned, and develop a path forward to support a strategic business plan focused on technology needs for retrieval. The primary participants involved in these workshops included retrieval personnel and laboratory staff that are associated with Hanford and Savannah River Sites since the majority of remaining DOE waste tanks are located at these sites. This report summarizes and documents the results of the initial RKC workshops. Technology challenges identified from these workshops and presented here are expected to be a key component to defining future RKC-directed tasks designed to facilitate tank waste retrieval solutions.

  7. Covariance Matrix Estimation for the Cryo-EM Heterogeneity Problem*

    PubMed Central

    Katsevich, E.; Katsevich, A.; Singer, A.

    2015-01-01

    In cryo-electron microscopy (cryo-EM), a microscope generates a top view of a sample of randomly oriented copies of a molecule. The problem of single particle reconstruction (SPR) from cryo-EM is to use the resulting set of noisy two-dimensional projection images taken at unknown directions to reconstruct the three-dimensional (3D) structure of the molecule. In some situations, the molecule under examination exhibits structural variability, which poses a fundamental challenge in SPR. The heterogeneity problem is the task of mapping the space of conformational states of a molecule. It has been previously suggested that the leading eigenvectors of the covariance matrix of the 3D molecules can be used to solve the heterogeneity problem. Estimating the covariance matrix is challenging, since only projections of the molecules are observed, but not the molecules themselves. In this paper, we formulate a general problem of covariance estimation from noisy projections of samples. This problem has intimate connections with matrix completion problems and high-dimensional principal component analysis. We propose an estimator and prove its consistency. When there are finitely many heterogeneity classes, the spectrum of the estimated covariance matrix reveals the number of classes. The estimator can be found as the solution to a certain linear system. In the cryo-EM case, the linear operator to be inverted, which we term the projection covariance transform, is an important object in covariance estimation for tomographic problems involving structural variation. Inverting it involves applying a filter akin to the ramp filter in tomography. We design a basis in which this linear operator is sparse and thus can be tractably inverted despite its large size. We demonstrate via numerical experiments on synthetic datasets the robustness of our algorithm to high levels of noise. PMID:25699132

  8. Epidemiology of major incidents: an EMS study from Pakistan

    PubMed Central

    2011-01-01

    Background A major incident is defined as an event that owing to the number of casualties has the potential to overwhelm the available resources. This paper attempts to describe the incidence and epidemiology of major incidents dealt with by a government-run emergency medical service (EMS) in the Punjab province of Pakistan, a developing country in South Asia. A major incident in this EMS is defined as any incident that produces three or more patients, or any incident in which extraordinary resources are needed. Methods All the calls received by an EMS Rescue 1122 were studied over a 6-month period. Calls that were defined as major incidents were identified, and further details were sought from the districts regarding these incidents. Questions specifically asked were the type of incident, time of the incident, response time for the incident, the resources needed, and the number of dead and injured casualties. Retrospective data were collected from the submitted written reports. Results Road traffic crashes (RTCs) emerged as the leading cause of a major incident in the province of Punjab and also led to the greatest number of casualties, followed by fire incidents. The total number of casualties was 3,380, out of which 73.7% were RTC victims. There was a high rate of death on the scene (10.4%). Certain other causes of major incidents also emerged, including violence, gas explosions and drowning. Conclusion Road traffic crashes are the most common cause of a major incident in developing countries such as Pakistan. Injury prevention initiatives need to focus on RTCs. PMID:21798011

  9. The effective molarity (EM) puzzle in proton transfer reactions.

    PubMed

    Karaman, Rafik

    2009-08-01

    The DFT and HF calculation results for the proton transfer reactions of three different systems reveal that the reaction mechanism (transfer of a proton to a nucleophile) is largely determined by the distance between the two reactive centers (r). Systems with relatively large r values tend to abstract a proton from a molecule of water, whereas, these with a relatively small r values prefer to be engaged intramolecularly and their interaction with water is only via hydrogen bonding. Further, the results indicate that the effective molarity (logEM) for an intramolecular process is strongly correlated with the distance between the two reacting centers (r) in accordance with Menger's "spatiotemporal hypothesis".

  10. EMS Bill of Rights: what every patient deserves.

    PubMed

    Meador, Steven R; Slovis, Corey M; Wrenn, Keith D

    2003-03-01

    Every EMT, paramedic, supervisor and EMS medical director must stress that our job is to treat every patient the way we'd like to be treated or the way we'd want one of our family members treated. Those charged with supervision must ensure that EMTs and paramedics receive the support and training necessary to allow adherence to the 10 objectives outlined in this article. Quality-assurance reviews, quality-improvement programs, provider comments and complaints from the public must all be used to improve the care we deliver. Prehospital care should be constantly improving the delivery of excellent, life-saving care.

  11. On the EM algorithm for overdispersed count data.

    PubMed

    McLachlan, G J

    1997-03-01

    In this paper, we consider the use of the EM algorithm for the fitting of distributions by maximum likelihood to overdispersed count data. In the course of this, we also provide a review of various approaches that have been proposed for the analysis of such data. As the Poisson and binomial regression models, which are often adopted in the first instance for these analyses, are particular examples of a generalized linear model (GLM), the focus of the account is on the modifications and extensions to GLMs for the handling of overdispersed count data. PMID:9185291

  12. Theme park EMS. It's no Mickey Mouse operation.

    PubMed

    Philips, J H

    1995-07-01

    In a single shift, they can rescue someone trapped in a time warp, perform CPR while menacing monsters look over their shoulders or calmly watch as a motorboat chase ends in a huge ball of fire. It's just another routine day for EMS providers in America's theme parks, where getting ready for unusual calls is the norm. JEMS went behind the scenes with theme park responders to explore the unique challenges of their service environment. What we found was a group of super-dedicated professionals who place genuine hospitality right alongside quality patient care. PMID:10143711

  13. Single-particle cryo-EM at crystallographic resolution

    PubMed Central

    Cheng, Yifan

    2015-01-01

    Until only a few years ago, single-particle electron cryo-microscopy (cryo-EM) was usually not the first choice for many structural biologists due to its limited resolution in the range of nanometer to subnanometer. Now, this method rivals X-ray crystallography in terms of resolution and can be used to determine atomic structures of macromolecules that are either refractory to crystallization or difficult to crystallize in specific functional states. In this review, I discuss the recent breakthroughs in both hardware and software that transformed cryo-microscopy, enabling understanding of complex biomolecules and their functions at atomic level. PMID:25910205

  14. State of the Art in EM Field Computation

    SciTech Connect

    Ng, C.; Akcelik, V.; Candel, A.; Chen, S.; Folwell, N.; Ge, L.; Guetz, A.; Jiang, H.; Kabel, A.; Lee, L.-Q.; Li, Z.; Prudencio, E.; Schussman, G.; Uplenchwar, R.; Xiao, L.; Ko, K.; /SLAC

    2006-09-25

    This paper presents the advances in electromagnetic (EM) field computation that have been enabled by the US DOE SciDAC Accelerator Science and Technology project which supports the development and application of a suite of electromagnetic codes based on the higher-order finite element method. Implemented on distributed memory supercomputers, this state of the art simulation capability has produced results which are of great interest to accelerator designers and with realism previously not possible with standard codes. Examples from work on the International Linear Collider (ILC) project are described.

  15. Covariance Structure Model Fit Testing under Missing Data: An Application of the Supplemented EM Algorithm

    ERIC Educational Resources Information Center

    Cai, Li; Lee, Taehun

    2009-01-01

    We apply the Supplemented EM algorithm (Meng & Rubin, 1991) to address a chronic problem with the "two-stage" fitting of covariance structure models in the presence of ignorable missing data: the lack of an asymptotically chi-square distributed goodness-of-fit statistic. We show that the Supplemented EM algorithm provides a convenient…

  16. Method for evaluating compatibility of commercial electromagnetic (EM) microsensor tracking systems with surgical and imaging tables

    NASA Astrophysics Data System (ADS)

    Nafis, Christopher; Jensen, Vern; von Jako, Ron

    2008-03-01

    Electromagnetic (EM) tracking systems have been successfully used for Surgical Navigation in ENT, cranial, and spine applications for several years. Catheter sized micro EM sensors have also been used in tightly controlled cardiac mapping and pulmonary applications. EM systems have the benefit over optical navigation systems of not requiring a line-of-sight between devices. Ferrous metals or conductive materials that are transient within the EM working volume may impact tracking performance. Effective methods for detecting and reporting EM field distortions are generally well known. Distortion compensation can be achieved for objects that have a static spatial relationship to a tracking sensor. New commercially available micro EM tracking systems offer opportunities for expanded image-guided navigation procedures. It is important to know and understand how well these systems perform with different surgical tables and ancillary equipment. By their design and intended use, micro EM sensors will be located at the distal tip of tracked devices and therefore be in closer proximity to the tables. Our goal was to define a simple and portable process that could be used to estimate the EM tracker accuracy, and to vet a large number of popular general surgery and imaging tables that are used in the United States and abroad.

  17. Global Convergence of the EM Algorithm for Unconstrained Latent Variable Models with Categorical Indicators

    ERIC Educational Resources Information Center

    Weissman, Alexander

    2013-01-01

    Convergence of the expectation-maximization (