Science.gov

Sample records for pestis em roedores

  1. Intraspecific Diversity of Yersinia pestis

    PubMed Central

    Anisimov, Andrey P.; Lindler, Luther E.; Pier, Gerald B.

    2004-01-01

    Increased interest in the pathogenic potential of Yersinia pestis has emerged because of the potential threats from bioterrorism. Pathogenic potential is based on genetic factors present in a population of microbes, yet most studies evaluating the role of specific genes in virulence have used a limited number of strains. For Y. pestis this issue is complicated by the fact that most strains available for study in the Americas are clonally derived and thus genetically restricted, emanating from a strain of Y. pestis introduced into the United States in 1902 via marine shipping and subsequent spread of this strain throughout North and South America. In countries from the former Soviet Union (FSU), Mongolia, and China there are large areas of enzootic foci of Y. pestis infection containing genetically diverse strains that have been intensely studied by scientists in these countries. However, the results of these investigations are not generally known outside of these countries. Here we describe the variety of methods used in the FSU to classify Y. pestis strains based on genetic and phenotypic variation and show that there is a high level of diversity in these strains not reflected by ones obtained from sylvatic areas and patients in the Americas. PMID:15084509

  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 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

  5. Omics strategies for revealing Yersinia pestis virulence

    PubMed Central

    Yang, Ruifu; Du, Zongmin; Han, Yanping; Zhou, Lei; Song, Yajun; Zhou, Dongsheng; Cui, Yujun

    2012-01-01

    Omics has remarkably changed the way we investigate and understand life. Omics differs from traditional hypothesis-driven research because it is a discovery-driven approach. Mass datasets produced from omics-based studies require experts from different fields to reveal the salient features behind these data. In this review, we summarize omics-driven studies to reveal the virulence features of Yersinia pestis through genomics, trascriptomics, proteomics, interactomics, etc. These studies serve as foundations for further hypothesis-driven research and help us gain insight into Y. pestis pathogenesis. PMID:23248778

  6. Developing live vaccines against Yersinia pestis

    PubMed Central

    Sun, Wei; Roland, Kenneth L.; Curtiss, Roy

    2014-01-01

    Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom. However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague. This vaccine has not gained general acceptance because of safety concerns. In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment. In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plague. PMID:21918302

  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. [Yersinia pestis as a dangerous biological weapon].

    PubMed

    Grygorczuk, Sambor; Hermanowska-Szpakowicz, Teresa

    2002-01-01

    Plague is an infectious disease caused by the Yersinia pestis microorganism, which is transmitted to the human host from a natural reservoir (different rodent species) by a flea bite. Plague is still encountered in humans in the areas of its enzootic prevalence in local rodent populations. Infection by flea bite results in a bubonic or septicemic plague, possibly complicated by secondary pneumonia. The person with pneumonic symptoms may be a source of a droplet-borne inhalatory infection for other people who consequently develop primary pneumonic plague. Despite a clinical form, plague is a severe infection characterized by a short incubation period, rapid onset and quick progress with mortality exceeding 50% if not treated properly. The pneumonic plague is associated with a particularly rapid progress and the mortality rate of almost 100% if not treated properly. As Yersinia pestis can be easily obtained and cultured and is highly pathogenic for humans, it poses a serious threat of being used for bioterrorism purposes. Artificially created aerosol containing plague bacilli can cause numerous and almost simultaneous cases of primary pulmonic plague in an exposed population. Persons exposed would most likely develop severe pneumonia with rapidly progressing respiratory and circulatory failure. The use of the Yersinia pestis strains resistant to antibiotics typically applied cannot be excluded. PMID:12474416

  9. Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

    PubMed

    Tsui, Pei-Yi; Tsai, Hui-Ping; Chiao, Der-Jiang; Liu, Cheng-Che; Shyu, Rong-Hwa

    2015-09-01

    Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge. PMID:25994256

  10. 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.

  11. 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.

  12. [Genetic analysis of biochemical differences of Yersinia pestis strains].

    PubMed

    Eroshenko, G A; Odinokov, G N; Kukleva, L M; Kutyrev, V V

    2012-01-01

    Literature data and results of our experimental studies on genetic base of biochemical differentiation of Yersinia pestis strains of various subspecies and biovars are summarized in the review. Data on variability of genes coding biochemical features (sugar and alcohol fermentation, nitrate reduction), the differential development of which are the base of existing phenotypic schemes of Y. pestis strains classification, are presented. Variability of these genes was shown to have possible use for the development of genetic classification of Y. pestis strains of various subspecies and biovars. PMID:22830282

  13. 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

  14. Distinct Clones of Yersinia pestis Caused the Black Death

    PubMed Central

    Haensch, Stephanie; Bianucci, Raffaella; Signoli, Michel; Rajerison, Minoarisoa; Schultz, Michael; Kacki, Sacha; Vermunt, Marco; Weston, Darlene A.; Hurst, Derek; Achtman, Mark; Carniel, Elisabeth; Bramanti, Barbara

    2010-01-01

    From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease. PMID:20949072

  15. Pasteurella pestis detection in fleas by fluorescent antibody staining*

    PubMed Central

    Hudson, Bruce W.; Kartman, Leo; Prince, Frank M.

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study. This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation. The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  16. 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. PMID:26698952

  17. Survival protein A is essential for virulence in Yersinia pestis.

    PubMed

    Southern, Stephanie J; Scott, Andrew E; Jenner, Dominic C; Ireland, Philip M; Norville, Isobel H; Sarkar-Tyson, Mitali

    2016-03-01

    Plague is a highly pathogenic disease caused by the bacterium Yersinia pestis. There is currently no vaccine available for prophylaxis and antibiotic resistant strains have been isolated, thus there is a need for the development of new countermeasures to treat this disease. Survival protein A (SurA) is a chaperone that has been linked to virulence in several species of bacteria, including the close relative Yersinia pseudotuberculosis. In this study, we aimed to evaluate the role of SurA in virulence of the highly pathogenic Y. pestis by creating an unmarked surA deletion mutant. The Y. pestis ΔsurA mutant was found to be more susceptible to membrane perturbing agents and was completely avirulent in a mouse infection model when delivered up to 2.1 × 10(5) CFU by the subcutaneous route. This provides strong evidence that SurA would make a promising antimicrobial target. PMID:26724738

  18. Recent emergence of new variants of Yersinia pestis in Madagascar.

    PubMed Central

    Guiyoule, A; Rasoamanana, B; Buchrieser, C; Michel, P; Chanteau, S; Carniel, E

    1997-01-01

    Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain. PMID:9350742

  19. 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

  20. 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

  1. 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

  2. A transport medium for specimens containing Pasteurella pestis

    PubMed Central

    Cavanaugh, D. C.; Vivona, S.; Do-Van-Quy; Gibson, F. L.; Deuber, G. L.; Rust, J. H.

    1967-01-01

    A medium, originally designed by Stuart and co-workers and later modified by Cary & Blair, for the maintenance and transport, without multiplication, of pathogenic bacteria contained in bacteriological specimens was tested in the laboratory and in the field in Viet-Nam to determine its effectiveness in preserving specimens known to contain Pasteurella pestis. The results indicate that this medium should be useful in diagnostic plague studies in areas where transport facilities are inadequate. Properly collected clinical specimens, sent to a central laboratory by any means and under any climatic conditions likely to be encountered in the hot tropics, should yield viable Pasteurella pestis for at least 30 days. PMID:5301387

  3. Immune responses of two Mastomys sibling species to Yersinia pestis.

    PubMed Central

    Arntzen, L; Wadee, A A; Isaäcson, M

    1991-01-01

    This study assessed the in vitro cell-mediated immune responses of Mastomys natalensis, with a diploid chromosome number of 2n = 32, and Mastomys coucha, with a diploid chromosome number of 2n = 36, to Yersinia pestis. Splenic mononuclear (MN) cells of uninfected M. natalensis proliferated in response to crude fraction 1 of Y. pestis and two subfractions derived from fraction 1 in vitro. Proliferation was dose dependent and followed the time kinetics of other well-known mitogens. Further characterization of the two fractions revealed similar protein profiles in sodium dodecyl sulfide-polyacrylamide gel electrophoresis and indicated a heat-stable protein of 25 kDa responsible for the mitogenic activity. No such response was observed with MN cells from M. coucha. The unresponsiveness of M. coucha-derived MN cells appears to be related to an inability to respond to Y. pestis organisms. The results may help explain the relative resistance and susceptibility of M. natalensis and M. coucha to Y. pestis infection. Images PMID:2037358

  4. In vitro antimicrobial susceptibilities of strains of Yersinia pestis.

    PubMed Central

    Smith, M D; Vinh, D X; Nguyen, T T; Wain, J; Thung, D; White, N J

    1995-01-01

    The in vitro activities of 14 antimicrobial agents were determined for 78 strains of Yersinia pestis. The most active antibiotics were ceftriaxone and ciprofloxacin, followed by ofloxacin and ampicillin. The agents traditionally used for the treatment of plague (streptomycin, tetracycline, and chloramphenicol) were considerably less active. Azithromycin showed poor activity against all strains. PMID:8540736

  5. Rational Considerations about Development of Live Attenuated Y. pestis Vaccines

    PubMed Central

    Sun, Wei; Curtiss, Roy

    2014-01-01

    The risk of plague as a bioweapon has prompted increasing research efforts to develop plague vaccines due to its extreme virulence and the ease of its transmission. Subunit vaccines that contain F1 and LcrV antigens of Y. pestis have been tested for safety and immunogenicity, but doubts have been raised about whether subunit vaccines that engender antibody responses will protect against pneumonic plague, which requires both humeral and cellular immune responses for protection. The live, attenuated vaccine EV76, a pgm locus deficient Y. pestis strain, has been used for a long time in the Former Soviet Union and some Asian countries, but is not commercially available in the US and Europe due to safety concerns. However, the live attenuated Y. pestis vaccines are still considered to be the most effective way to prevent plague. In this review, we present our opinions about rationally creating live, safe and immunogenic Y. pestis vaccines with potential use for human based on established researches. PMID:24372254

  6. 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.

  7. 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

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

    PubMed

    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

    2015-10-01

    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

  9. 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.

  10. A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium.

    PubMed

    Rashid, Mohammed H; Revazishvili, Tamara; Dean, Timothy; Butani, Amy; Verratti, Kathleen; Bishop-Lilly, Kimberly A; Sozhamannan, Shanmuga; Sulakvelidze, Alexander; Rajanna, Chythanya

    2012-07-01

    Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a "phage cocktail" (tentatively designated "YPP-100") capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 10(9), 10(8) and 10(7) PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 10(6) PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis. PMID:23275868

  11. 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

  12. Pasteurella pestis detection in Fleas by fluorescent antibody staining.

    PubMed

    Hudson, B W; Kartman, L; Prince, F M

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study.This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation.The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  13. 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

  14. 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...

  15. Nineteen Whole-Genome Assemblies of Yersinia pestis subsp. microtus, Including Representatives of Biovars caucasica, talassica, hissarica, altaica, xilingolensis, and ulegeica.

    PubMed

    Kislichkina, Angelina A; Bogun, Aleksandr G; Kadnikova, Lidiya A; Maiskaya, Nadezhda V; Platonov, Mikhail E; Anisimov, Nikolai V; Galkina, Elena V; Dentovskaya, Svetlana V; Anisimov, Andrey P

    2015-01-01

    The etiologic agent of plague, Yersinia pestis, includes two subspecies, of which Y. pestis subsp. microtus contains the strains that cause only occasional diseases in humans that are not accompanied by human-to-human transmission. Here, we report the draft genome sequences of 19 Y. pestis strains (across 6 biovars of Y. pestis subsp. microtus). PMID:26634751

  16. BEHAVIOR OF YERSINIA PESTIS STRAINS KIM5 AND CDC A1122 IN RAW GROUND BEEF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although Yersinia pestis, the causative agent of bubonic and pneumonic plague, has not been implicated in a foodborne outbreak, there is ongoing concern about the potential use of this pathogen as a foodborne biological weapon. There are no reports of the behavior of Y. pestis in food, and hence li...

  17. The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis.

    PubMed

    Hänsch, Stephanie; Cilli, Elisabetta; Catalano, Giulio; Gruppioni, Giorgio; Bianucci, Raffaella; Stenseth, Nils C; Bramanti, Barbara; Pallen, Mark J

    2015-01-01

    Here we present evidence to show that the pla gene, previously thought to be specific to Yersinia pestis, occurs in some strains of Citrobacter koseri and Escherichia coli. This means that detection of this gene on its own can no longer be taken as evidence of detection of Y. pestis. PMID:26438258

  18. 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...

  19. 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...

  20. 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.

  1. 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...

  2. 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...

  3. 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.

  4. Dynamics of CRISPR Loci in Microevolutionary Process of Yersinia pestis Strains

    PubMed Central

    Barros, Maria Paloma S.; França, Camila T.; Lins, Rosanny Holanda F. B.; Santos, Milena Danda V.; Silva, Ednaldo J.; Oliveira, Maria Betânia M.; Silveira-Filho, Vladimir M.; Rezende, Antônio M.; Balbino, Valdir Q.; Leal-Balbino, Tereza Cristina

    2014-01-01

    The potential use of CRISPR loci genotyping to elucidate population dynamics and microevolution of 146 Yersinia pestis strains from different biovars and locations was investigated in this work. The majority of strains from the Orientalis biovar presented specific spacer arrays, allowing for the establishment of a CRISPR signature for their respective isolates. Twenty-one new spacers were found in the Y. pestis strains from plague foci in Brazil. Ninety-three (64%) strains were grouped in the G1 genotype, whereas the others were distributed in 35 genotypes. This study allowed observing a microevolutionary process in a group of Y. pestis isolated from Brazil. We also identified specific genotypes of Y. pestis that were important for the establishment of the bacteria in plague foci in Brazil. The data have provided supporting evidence for the diversity and dynamics of CRISPR loci present in the genome of Y. pestis strains from plague foci in Brazil. PMID:25265542

  5. 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

  6. Immunomodulatory Effects of Yersinia pestis Lipopolysaccharides on Human Macrophages ▿

    PubMed Central

    Matsuura, Motohiro; Takahashi, Hideyuki; Watanabe, Haruo; Saito, Shinji; Kawahara, Kazuyoshi

    2010-01-01

    In the current study, we investigated the activity of lipopolysaccharide (LPS) purified from Yersinia pestis grown at either 27°C or 37°C (termed LPS-27 and LPS-37, respectively). LPS-27 containing hexa-acylated lipid A, similar to the LPS present in usual gram-negative bacteria, stimulated an inflammatory response in human U937 cells through Toll-like receptor 4 (TLR4). LPS-37, which did not contain hexa-acylated lipid A, exhibited strong antagonistic activity to the TLR4-mediated inflammatory response. The phagocytic activity in the cells was not affected by LPS-37. To estimate the activity of LPS in its bacterial binding form, formalin-killed bacteria (FKB) were prepared from Y. pestis cells grown at 27°C or 37°C (termed FKB-27 and FKB-37, respectively). FKB-27 strongly stimulated the inflammatory response. This activity was suppressed in the presence of an anti-TLR4 antibody but not an anti-TLR2 antibody. In addition, this activity was almost completely suppressed by LPS-37, indicating that the activity of FKB-27 is predominantly derived from the LPS-27 bacterial binding form. In contrast, FKB-37 showed no antagonistic activity. The results arising from the current study indicate that Y. pestis causes infection in humans without stimulating the TLR4-based defense system via bacterial binding of LPS-37, even when bacterial free LPS-37 is not released to suppress the defense system. This is in contrast to the findings for bacteria that possess agonistic LPS types, which are easily recognized by the defense system via the bacterial binding forms. PMID:19889939

  7. 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.

  8. 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

  9. 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

  10. 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.

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

    PubMed

    Paoli, George C; Sommers, Christopher H; Scullen, O Joseph; Wijey, Chandi

    2014-12-01

    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 with this deadly pathogen. Here we report the inactivation of avirulent (pYV-minus) strains of Y. pestis by ultraviolet light (UV-C, 254 nm). Two strains of Y. pestis containing an intact pgm virulence locus (pgm(+)) and strains from which the pgm locus was spontaneously deleted (Δpgm) were tested using cells grown in both logarithmic and stationary phase. The D10 values for inactivation (the UV-C dose required to inactivate one log of bacterial cells) of Y. pestis on the surface of agar plates ranged from 0.69 to 1.09 mJ/cm(2). A significant difference was observed between the inactivation of cells of Y. pestis strain Yokohama grown in logarithmic and stationary phases, but no significant difference between growth phase sensitivity to UV-C was observed in Y. pestis strain Kuma. No difference in D10 values was observed between pgm(+) and Δpgm strains of Yokohama grown to either logarithmic or stationary phase. A measurable difference was observed between the D10 of Kuma pgm(+) and Kuma Δpgm grown in logarithmic phase, but this difference was diminished in the Kuma strains grown to stationary phase. Though strain variations exist, the results showing that UV-C can inactivate Y. pestis cells on agar surfaces suggest that UV-C would be effect in inactivating Y. pestis on food surfaces, particularly foods with a smooth surface. PMID:25084659

  12. 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

  13. 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.

  14. 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

  15. Yersinia pestis targets neutrophils via complement receptor 3.

    PubMed

    Merritt, Peter M; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S; Marketon, Melanie M

    2015-05-01

    Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet, the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins because of reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria towards neutrophils during plague infection. PMID:25359083

  16. Yersinia pestis targets neutrophils via complement receptor 3

    PubMed Central

    Merritt, Peter M.; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S.; Marketon, Melanie M.

    2015-01-01

    Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins due to reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria toward neutrophils during plague infection. PMID:25359083

  17. Iron uptake and iron-repressible polypeptides in Yersinia pestis.

    PubMed Central

    Lucier, T S; Fetherston, J D; Brubaker, R R; Perry, R D

    1996-01-01

    Pigmented (Pgm+) cells of Yersinia pestis are virulent, are sensitive to pesticin, adsorb exogenous hemin at 26 degrees C (Hms+), produce iron-repressible outer membrane proteins, and grow at 37 degrees C in iron-deficient media. These traits are lost upon spontaneous deletion of a chromosomal 102-kb pgm locus (Pgm-). Here we demonstrate that an Hms+ but pesticin-resistant (Pst(r)) mutant acquired a 5-bp deletion in the pesticin receptor gene (psn) encoding IrpB to IrpD. Growth and assimilation of iron by Pgm- and Hms+ Pst(r) mutants were markedly inhibited by ferrous chelators at 37 degrees C; inhibition by ferric and ferrous chelators was less effective at 26 degrees C. Iron-deficient growth at 26 degrees C induced iron-regulated outer membrane proteins of 34, 28.5, and 22.5 kDa and periplasmic polypeptides of 33.5 and 30 kDa. These findings provide a basis for understanding the psn-driven system of iron uptake, indicate the existence of at least one additional 26 degrees C-dependent iron assimilation system, and define over 30 iron-repressible proteins in Y. pestis. PMID:8757829

  18. 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

  19. 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

  20. 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. PMID:26496604

  1. 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

  2. A review of methods for subtyping Yersinia pestis: From phenotypes to whole genome sequencing.

    PubMed

    Vogler, Amy J; Keim, Paul; Wagner, David M

    2016-01-01

    Numerous subtyping methods have been applied to Yersinia pestis with varying success. Here, we review the various subtyping methods that have been applied to Y. pestis and their capacity for answering questions regarding the population genetics, phylogeography, and molecular epidemiology of this important human pathogen. Methods are evaluated in terms of expense, difficulty, transferability among laboratories, discriminatory power, usefulness for different study questions, and current applicability in light of the advent of whole genome sequencing. PMID:26518910

  3. 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

  4. Draft Genome Sequences of Yersinia pestis Isolates from Natural Foci of Endemic Plague in China ▿

    PubMed Central

    Eppinger, Mark; Guo, Zhaobiao; Sebastian, Yinong; Song, Yajun; Lindler, Luther E.; Yang, Ruifu; Ravel, Jacques

    2009-01-01

    To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague. PMID:19820101

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

    PubMed

    Eisen, Rebecca J; Dennis, David T; Gage, Kenneth L

    2015-11-01

    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

  6. 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

  7. 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.

  8. 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. PMID:25998808

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Genotyping and Phylogenetic Analysis of Yersinia pestis by MLVA: Insights into the Worldwide Expansion of Central Asia Plague Foci

    PubMed Central

    Hauck, Yolande; Platonov, Mikhail E.; Dai, Erhei; Song, Yajun; Guo, Zhaobiao; Pourcel, Christine; Dentovskaya, Svetlana V.; Anisimov, Andrey P.; Yang, Ruifu; Vergnaud, Gilles

    2009-01-01

    Background The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar. Methodology/Principal Findings Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci. Conclusions/Significance The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an

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

    PubMed Central

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

    2015-01-01

    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

  15. Evaluation of the Infectiousness to Mice of Soil Contaminated with Yersinia pestis-Infected Blood

    PubMed Central

    Boegler, Karen A.; Graham, Christine B.; Montenieri, John A.; MacMillan, Katherine; Holmes, Jennifer L.; Petersen, Jeannine M.; Gage, Kenneth L.; Eisen, Rebecca J.

    2015-01-01

    Plague, an often-fatal zoonotic disease caused by Yersinia pestis, is characterized by epizootic and quiescent periods. How Y. pestis is maintained during inter-epizootic periods is poorly understood, but soil has been implicated as a potential reservoir. Although previous studies have suggested that Y. pestis is able to survive in soil for weeks or months, it is unclear whether or not it is infectious to susceptible hosts. Here we investigate the potential for Y. pestis to infect mice through close contact with contaminated soil under laboratory conditions. In an attempt to approximate the natural conditions under which animals would be exposed to Y. pestis-contaminated soil, mouse cages filled with soil from a plague-endemic region were held at temperature and humidity ranges observed in ground squirrel burrows. These laboratory “burrows” were contaminated with highly bacteremic blood (> 108 cfu/mL) to simulate the introduction of infectious material from a dying animal during an epizootic. Outbred Swiss-Webster mice with scarified skin patches were held on contaminated soil for 10 days and monitored for signs of illness. Following exposure to contaminated soil, one animal of 104 became infected with Y. pestis. None of the remaining animals seroconverted following a 21-day holding period. Under our experimental conditions, which maximized the likelihood of contact between susceptible mice and contaminated soil, transmission efficiency from soil to mice was 0.96% (95% CI 0.17, 5.25%). This suggests that although transmission of Y. pestis from contaminated soils is possible, it is not likely a major transmission route under natural conditions. PMID:22925020

  16. Role of a new intimin/invasin-like protein in Yersinia pestis virulence.

    PubMed

    Seo, Keun Seok; Kim, Jong Wan; Park, Joo Youn; Viall, Austin K; Minnich, Scott S; Rohde, Harold N; Schnider, Darren R; Lim, Seung Yong; Hong, Joon Bae; Hinnebusch, B Joseph; O'Loughlin, Jason L; Deobald, Claudia F; Bohach, Gregory A; Hovde, Carolyn J; Minnich, Scott A

    2012-10-01

    A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis. PMID:22851752

  17. 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...

  18. 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...

  19. 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

  20. Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing.

    PubMed

    Amoako, Kingsley K; Shields, Michael J; Goji, Noriko; Paquet, Chantal; Thomas, Matthew C; Janzen, Timothy W; Bin Kingombe, Cesar I; Kell, Arnold J; Hahn, Kristen R

    2012-01-01

    Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10 CFU/g). The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence. PMID:23091729

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

    PubMed

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

    2015-10-01

    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

  6. Yersinia pestis pFra Shows Biovar-Specific Differences and Recent Common Ancestry with a Salmonella enterica Serovar Typhi Plasmid

    PubMed Central

    Prentice, Michael B.; James, Keith D.; Parkhill, Julian; Baker, Stephen G.; Stevens, Kim; Simmonds, Mark N.; Mungall, Karen L.; Churcher, Carol; Oyston, Petra C. F.; Titball, Richard W.; Wren, Brendan W.; Wain, John; Pickard, Derek; Hien, Tran Tinh; Farrar, Jeremy J.; Dougan, Gordon

    2001-01-01

    Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi. PMID:11274119

  7. Pulmonary infection by Yersinia pestis rapidly establishes a permissive environment for microbial proliferation

    PubMed Central

    Price, Paul A.; Jin, Jianping; Goldman, William E.

    2012-01-01

    Disease progression of primary pneumonic plague is biphasic, consisting of a preinflammatory and a proinflammatory phase. During the long preinflammatory phase, bacteria replicate to high levels, seemingly uninhibited by normal pulmonary defenses. In a coinfection model of pneumonic plague, it appears that Yersinia pestis quickly creates a localized, dominant anti-inflammatory state that allows for the survival and rapid growth of both itself and normally avirulent organisms. Yersinia pseudotuberculosis, the relatively recent progenitor of Y. pestis, shows no similar trans-complementation effect, which is unprecedented among other respiratory pathogens. We demonstrate that the effectors secreted by the Ysc type III secretion system are necessary but not sufficient to mediate this apparent immunosuppression. Even an unbiased negative selection screen using a vast pool of Y. pestis mutants revealed no selection against any known virulence genes, demonstrating the transformation of the lung from a highly restrictive to a generally permissive environment during the preinflammatory phase of pneumonic plague. PMID:22308352

  8. 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

  9. 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

  10. 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

  11. Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

    PubMed

    Bartra, Sara Schesser; Ding, Yi; Fujimoto, L Miya; Ring, Joshua G; Jain, Vishal; Ram, Sanjay; Marassi, Francesca M; Plano, Gregory V

    2015-11-01

    Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs. PMID:26377177

  12. Phosphoglucomutase of Yersinia pestis Is Required for Autoaggregation and Polymyxin B Resistance▿

    PubMed Central

    Felek, Suleyman; Muszyński, Artur; Carlson, Russell W.; Tsang, Tiffany M.; Hinnebusch, B. Joseph; Krukonis, Eric S.

    2010-01-01

    Yersinia pestis, the causative agent of plague, autoaggregates within a few minutes of cessation of shaking when grown at 28°C. To identify the autoaggregation factor of Y. pestis, we performed mariner-based transposon mutagenesis. Autoaggregation-defective mutants from three different pools were identified, each with a transposon insertion at a different position within the gene encoding phosphoglucomutase (pgmA; y1258). Targeted deletion of pgmA in Y. pestis KIM5 also resulted in loss of autoaggregation. Given the previously defined role for phosphoglucomutase in antimicrobial peptide resistance in other organisms, we tested the KIM5 ΔpgmA mutant for antimicrobial peptide sensitivity. The ΔpgmA mutant displayed >1,000-fold increased sensitivity to polymyxin B compared to the parental Y. pestis strain, KIM5. This sensitivity is not due to changes in lipopolysaccharide (LPS) since the LPSs from both Y. pestis KIM5 and the ΔpgmA mutant are identical based on a comparison of their structures by mass spectrometry (MS), tandem MS, and nuclear magnetic resonance analyses. Furthermore, the ability of polymyxin B to neutralize LPS toxicity was identical for LPS purified from both KIM5 and the ΔpgmA mutant. Our results indicate that increased polymyxin B sensitivity of the ΔpgmA mutant is due to changes in surface structures other than LPS. Experiments with mice via the intravenous and intranasal routes did not demonstrate any virulence defect for the ΔpgmA mutant, nor was flea colonization or blockage affected. Our findings suggest that the activity of PgmA results in modification and/or elaboration of a surface component of Y. pestis responsible for autoaggregation and polymyxin B resistance. PMID:20028810

  13. The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

    PubMed Central

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

    2009-01-01

    The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague. PMID:19701461

  14. 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

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

    PubMed

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

    2016-01-01

    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

  16. Insight into Microevolution of Yersinia pestis by Clustered Regularly Interspaced Short Palindromic Repeats

    PubMed Central

    Gorgé, Olivier; Platonov, Mikhail E.; Yan, Yanfeng; Guo, Zhaobiao; Pourcel, Christine; Dentovskaya, Svetlana V.; Balakhonov, Sergey V.; Wang, Xiaoyi; Song, Yajun; Anisimov, Andrey P.; Vergnaud, Gilles; Yang, Ruifu

    2008-01-01

    Background Yersinia pestis, the pathogen of plague, has greatly influenced human history on a global scale. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), an element participating in immunity against phages' invasion, is composed of short repeated sequences separated by unique spacers and provides the basis of the spoligotyping technology. In the present research, three CRISPR loci were analyzed in 125 strains of Y. pestis from 26 natural plague foci of China, the former Soviet Union and Mongolia were analyzed, for validating CRISPR-based genotyping method and better understanding adaptive microevolution of Y. pestis. Methodology/Principal Findings Using PCR amplification, sequencing and online data processing, a high degree of genetic diversity was revealed in all three CRISPR elements. The distribution of spacers and their arrays in Y. pestis strains is strongly region and focus-specific, allowing the construction of a hypothetic evolutionary model of Y. pestis. This model suggests transmission route of microtus strains that encircled Takla Makan Desert and ZhunGer Basin. Starting from Tadjikistan, one branch passed through the Kunlun Mountains, and moved to the Qinghai-Tibet Plateau. Another branch went north via the Pamirs Plateau, the Tianshan Mountains, the Altai Mountains and the Inner Mongolian Plateau. Other Y. pestis lineages might be originated from certain areas along those routes. Conclusions/significance CRISPR can provide important information for genotyping and evolutionary research of bacteria, which will help to trace the source of outbreaks. The resulting data will make possible the development of very low cost and high-resolution assays for the systematic typing of any new isolate. PMID:18612419

  17. Defective Innate Cell Response and Lymph Node Infiltration Specify Yersinia pestis Infection

    PubMed Central

    Guinet, Françoise; Avé, Patrick; Jones, Louis; Huerre, Michel; Carniel, Elisabeth

    2008-01-01

    Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen. PMID:18301765

  18. Development of Yersinia pestis F1 antigen-loaded microspheres vaccine against plague

    PubMed Central

    Huang, Shih-shiung; Li, I-Hsun; Hong, Po-da; Yeh, Ming-kung

    2014-01-01

    Yersinia pestis F1 antigen-loaded poly(DL-lactide-co-glycolide)/polyethylene glycol (PEG) (PLGA/PEG) microspheres were produced using a water-in-oil-in-water emulsion/solvent extraction technique and assayed for their percent yield, entrapment efficiency, surface morphology, particle size, zeta potential, in vitro release properties, and in vivo animal protect efficacy. The Y. pestis F1 antigen-loaded microspheres (mean particle size 3.8 μm) exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (38.1%), and presented a controlled in vitro release profile with a low initial burst (18.5%), then continued to release Y. pestis F1 antigen over 70 days. The distribution (%) of Y. pestis F1 on the microspheres surface, outer layer, and core was 3.1%, 28.9%, and 60.7%, respectively. A steady release rate was noticed to be 0.55 μg Y. pestis F1 antigen/mg microspheres/day of Y. pestis F1 antigen release maintained for 42 days. The cumulative release amount at the 1st, 28th, and 42nd days was 8.2, 26.7, and 31.0 μg Y. pestis F1 antigen/mg microspheres, respectively. The 100 times median lethal dose 50% (LD50) of Y. pestis Yokohama-R strain by intraperitoneal injection challenge in mice test, in which mice received one dose of 40 μg F1 antigen content of PLGA/PEG microspheres, F1 antigen in Al(OH)3, and in comparison with F1 antigen in Al(OH)3 vaccine in two doses, was evaluated after given by subcutaneous immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with one dose of F1 antigen-loaded PLGA/PEG microspheres, and two doses of F1 antigen in Al(OH)3 vaccine (100%). In vivo vaccination studies also demonstrated that F1 vaccines microspheres had a protective ability; its steady-state IgG immune protection in mice plasma dramatic increased from 2 weeks (18,764±3,124) to 7 weeks (126,468±19,176) after vaccination. These findings strongly suggest that F1-antigen loaded

  19. Development of Yersinia pestis F1 antigen-loaded microspheres vaccine against plague.

    PubMed

    Huang, Shih-shiung; Li, I-Hsun; Hong, Po-da; Yeh, Ming-kung

    2014-01-01

    Yersinia pestis F1 antigen-loaded poly(DL-lactide-co-glycolide)/polyethylene glycol (PEG) (PLGA/PEG) microspheres were produced using a water-in-oil-in-water emulsion/solvent extraction technique and assayed for their percent yield, entrapment efficiency, surface morphology, particle size, zeta potential, in vitro release properties, and in vivo animal protect efficacy. The Y. pestis F1 antigen-loaded microspheres (mean particle size 3.8 μm) exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (38.1%), and presented a controlled in vitro release profile with a low initial burst (18.5%), then continued to release Y. pestis F1 antigen over 70 days. The distribution (%) of Y. pestis F1 on the microspheres surface, outer layer, and core was 3.1%, 28.9%, and 60.7%, respectively. A steady release rate was noticed to be 0.55 μg Y. pestis F1 antigen/mg microspheres/day of Y. pestis F1 antigen release maintained for 42 days. The cumulative release amount at the 1st, 28th, and 42nd days was 8.2, 26.7, and 31.0 μg Y. pestis F1 antigen/mg microspheres, respectively. The 100 times median lethal dose 50% (LD50) of Y. pestis Yokohama-R strain by intraperitoneal injection challenge in mice test, in which mice received one dose of 40 μg F1 antigen content of PLGA/PEG microspheres, F1 antigen in Al(OH)3, and in comparison with F1 antigen in Al(OH)3 vaccine in two doses, was evaluated after given by subcutaneous immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with one dose of F1 antigen-loaded PLGA/PEG microspheres, and two doses of F1 antigen in Al(OH)3 vaccine (100%). In vivo vaccination studies also demonstrated that F1 vaccines microspheres had a protective ability; its steady-state IgG immune protection in mice plasma dramatic increased from 2 weeks (18,764 ± 3,124) to 7 weeks (126,468 ± 19,176) after vaccination. These findings strongly suggest that F1-antigen

  20. 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.

  1. 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

  2. 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

  3. 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

  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 LD; Lin, Andy; Hutchison, Janine R.; Melville, Angela M.; Wagner, David M.; Keim, Paul S.; Foster, Jeff; Kreuzer, Helen W.

    2015-11-24

    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. 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.

  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. 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.

  8. 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

  9. EFFECT OF TEMPERATURE ON THE RADIATION RESISTANCE OF YERSINIA PESTIS SUSPENDED IN RAW GROUND PORK

    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. Ionizing rad...

  10. 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

  11. 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.

  12. Genome Assemblies for 11 Yersinia pestis Strains Isolated in the Caucasus Region.

    PubMed

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

    2015-01-01

    Yersinia pestis, the causative agent of plague, is endemic to the Caucasus region but few reference strain genome sequences from that region are available. Here, we present the improved draft or finished assembled genomes from 11 strains isolated in the nation of Georgia and surrounding countries. PMID:26383663

  13. 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

  14. 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

  15. 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

  16. Identification and Characterization of the Hemophore-Dependent Heme Acquisition System of Yersinia pestis

    PubMed Central

    Rossi, Maria-Silvia; Fetherston, Jacqueline D.; Létoffé, Sylvie; Carniel, Elisabeth; Perry, Robert D.; Ghigo, Jean-Marc

    2001-01-01

    Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens. This locus consisted of the hasRyp receptor gene, the hasAyp hemophore gene, and genes encoding components of the HasAyp dedicated ABC transporter factor (hasDEyp), as well as a tonB homologue (hasByp). By using a reconstituted secretion system in Escherichia coli, we showed that HasAyp is a secreted heme-binding protein and that expression of HasAyp is iron regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37°C. Hemoglobin utilization via the Hasyp system was studied with both E. coli and Y. pestis, for which has and has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu− Has− double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity. PMID:11598042

  17. 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.

  18. 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

  19. 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

  20. 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.

  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. 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

  3. 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

  4. 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

  5. Integrating High-Content Imaging and Chemical Genetics to Probe Host Cellular Pathways Critical for Yersinia Pestis Infection

    PubMed Central

    Kota, Krishna P.; Eaton, Brett; Lane, Douglas; Ulrich, Melanie; Ulrich, Ricky; Peyser, Brian D.; Robinson, Camenzind G.; Jaissle, James G.; Pegoraro, Gianluca; Bavari, Sina; Panchal, Rekha G.

    2013-01-01

    The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics. PMID:23383093

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. [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. PMID:25876503

  14. Transcriptional regulation mechanism of ter operon by OxyR in Yersinia pestis.

    PubMed

    Ni, Bin; Zhang, Yiquan; Huang, Xinxiang; Yang, Ruifu; Zhou, Dongsheng

    2014-07-01

    The aim of this work was to study the transcriptional regulation mechanism of ter operon by OxyR in Yersinia pestis. Total RNAs were extracted from the wild-type (WT) strain and the oxyR null mutant (ΔoxyR) strain. Primer extension assay was employed to detect the promoter activity (the amount of primer extension product) of terZ in WT and ΔoxyR. terZ promoter-proximal region was cloned into the pRW50 plasmid containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔoxyR, respectively, to measure the promoter activity (the β-galactosidase activity) of terZ in WT and ΔoxyR by using the β-galactosidase enzyme assay system. The entire promoter-proximal region of the terZ gene was amplified by PCR from Y. pestis strain 201, and the over-expressed His-OxyR was also purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay was applied to analyze the DNA-binding activity of His-OxyR to terZ promoter region in vitro. Primer extension assay detected only one transcriptional start site located at 50 bp upstream of terZ, whose transcript was directly activated by OxyR in Y. pestis. The EMSA result shows that His-OxyR has the ability to bind to the upstream DNA region of terZ. The transcription of ter operon was found to be directly activated by OxyR in Y. pestis. PMID:24577613

  15. 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

  16. [Genetic bases of methionine dependence in Yersinia pestis strains of major and non-major subspecies].

    PubMed

    Odinokov, G N; Eroshenko, G A; Krasnov, Ia M; Kukleva, L M; Shavina, N Iu; Pavlova, A I; Kutyrev, V V

    2011-03-01

    Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis. PMID:21542303

  17. 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

  18. Retracing the evolutionary path that led to flea-borne transmission of Yersinia pestis.

    PubMed

    Sun, Yi-Cheng; Jarrett, Clayton O; Bosio, Christopher F; Hinnebusch, B Joseph

    2014-05-14

    Yersinia pestis is an arthropod-borne bacterial pathogen that evolved recently from Yersinia pseudotuberculosis, an enteric pathogen transmitted via the fecal-oral route. This radical ecological transition can be attributed to a few discrete genetic changes from a still-extant recent ancestor, thus providing a tractable case study in pathogen evolution and emergence. Here, we determined the genetic and mechanistic basis of the evolutionary adaptation of Y. pestis to flea-borne transmission. Remarkably, only four minor changes in the bacterial progenitor, representing one gene gain and three gene losses, enabled transmission by flea vectors. All three loss-of-function mutations enhanced cyclic-di-GMP-mediated bacterial biofilm formation in the flea foregut, which greatly increased transmissibility. Our results suggest a step-wise evolutionary model in which Y. pestis emerged as a flea-borne clone, with each genetic change incrementally reinforcing the transmission cycle. The model conforms well to the ecological theory of adaptive radiation. PMID:24832452

  19. Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis

    PubMed Central

    Cui, Yujun; Yu, Chang; Yan, Yanfeng; Li, Dongfang; Li, Yanjun; Jombart, Thibaut; Weinert, Lucy A.; Wang, Zuyun; Guo, Zhaobiao; Xu, Lizhi; Zhang, Yujiang; Zheng, Hancheng; Qin, Nan; Xiao, Xiao; Wu, Mingshou; Wang, Xiaoyi; Zhou, Dongsheng; Qi, Zhizhen; Du, Zongmin; Wu, Honglong; Yang, Xianwei; Cao, Hongzhi; Wang, Hu; Wang, Jing; Yao, Shusen; Rakin, Alexander; Li, Yingrui; Falush, Daniel; Balloux, Francois; Achtman, Mark; Song, Yajun; Wang, Jun; Yang, Ruifu

    2013-01-01

    The genetic diversity of Yersinia pestis, the etiologic agent of plague, is extremely limited because of its recent origin coupled with a slow clock rate. Here we identified 2,326 SNPs from 133 genomes of Y. pestis strains that were isolated in China and elsewhere. These SNPs define the genealogy of Y. pestis since its most recent common ancestor. All but 28 of these SNPs represented mutations that happened only once within the genealogy, and they were distributed essentially at random among individual genes. Only seven genes contained a significant excess of nonsynonymous SNP, suggesting that the fixation of SNPs mainly arises via neutral processes, such as genetic drift, rather than Darwinian selection. However, the rate of fixation varies dramatically over the genealogy: the number of SNPs accumulated by different lineages was highly variable and the genealogy contains multiple polytomies, one of which resulted in four branches near the time of the Black Death. We suggest that demographic changes can affect the speed of evolution in epidemic pathogens even in the absence of natural selection, and hypothesize that neutral SNPs are fixed rapidly during intermittent epidemics and outbreaks. PMID:23271803

  20. Comparative Genomics of 2009 Seasonal Plague (Yersinia pestis) in New Mexico

    PubMed Central

    Gibbons, Henry S.; Onischuk, Lisa; Leonard, Pascale; Broomall, Stacey; Sickler, Todd; Betters, Janet L.; McGregor, Paul; Donarum, Greg; Liem, Alvin; Fochler, Ed; McNew, Lauren; Rosenzweig, C. Nicole; Skowronski, Evan

    2012-01-01

    Plague disease caused by the Gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19th century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen. PMID:22359605

  1. 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

  2. Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome.

    PubMed

    Klevytska, A M; Price, L B; Schupp, J M; Worsham, P L; Wong, J; Keim, P

    2001-09-01

    Yersinia pestis, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the Y. pestis genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the Y. pestis genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (R = 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world. PMID:11526147

  3. 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.

  4. 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.

  5. 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

  6. 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...

  7. Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins.

    PubMed Central

    Straley, S C; Bowmer, W S

    1986-01-01

    Yersinia pestis, the causative agent of plague, has a virulence determinant called the low-Ca2+ response (Lcr+ phenotype) that confers on the bacterium Ca2+ dependence for growth at 37 degrees C and expression of V antigen. This virulence determinant is common to the three species of Yersinia and is mediated by Lcr plasmids (called pCD in Y. pestis). In this study, we generated insertions of Mu dI1(Ap lac) in pCD1 of Y. pestis KIM, screened for cells showing transcriptional regulation by Ca2+, and obtained inserts that define at least four pCD1 genes. Their patterns of transcription under different growth conditions closely paralleled the pattern of expression of the V antigen. We tested for expression of Lcr-specific yersinial outer membrane proteins (Yops) by the pCD1::Mu dI1(Ap lac) plasmids. Four of the inserts each eliminated expression of a different Yop; one of these Yops was unique to Y. pestis. Two of the insertions affecting Yops caused avirulence, and one caused strongly decreased virulence of Y. pestis in mice. These data indicate that Yops, like the V antigen, are virulence attributes regulated in the low-Ca2+ response. Images PMID:3002984

  8. 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.

  9. The Outer Membrane Protein A (OmpA) of Y. pestis promotes intracellular survival and virulence in mice

    PubMed Central

    Bartra, Sara Schesser; Gong, Xin; Lorica, Cherish D.; Jain, Chaitanya; Nair, Manoj K. M.; Schifferli, Dieter; Qian, Lianfen; Li, Zhongwei; Plano, Gregory V.; Schesser, Kurt

    2011-01-01

    The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 105 Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Y. pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was outcompeted by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS. PMID:22023991

  10. 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

  11. Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins.

    PubMed Central

    Sodeinde, O A; Sample, A K; Brubaker, R R; Goguen, J D

    1988-01-01

    The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid. Images PMID:2843471

  12. Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens.

    PubMed

    Glynn, Audrey; Roy, Chad J; Powell, Bradford S; Adamovicz, Jeffrey J; Freytag, Lucy C; Clements, John D

    2005-08-01

    A Yersinia pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examined different prime-boost regimens with F1-V and demonstrate that (i) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (ii) parenteral immunization is not required to protect mice against aerosolized plague challenge, (iii) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the immunoglobulin G1 (IgG1)/IgG2a ratio, and (iv) inclusion of an appropriate adjuvant is critical for nonparenteral immunization. PMID:16041052

  13. Conserved Filamentous Prophage in Escherichia coli O18:K1:H7 and Yersinia pestis Biovar orientalis

    PubMed Central

    Gonzalez, Mark D.; Lichtensteiger, Carol A.; Caughlan, Ruth; Vimr, Eric R.

    2002-01-01

    Microbial virulence is known to emerge by horizontal gene transfer mechanisms. Here we describe the discovery of a novel filamentous prophage, designated CUS-1, which is integrated into the chromosomal dif homologue of the high-virulence clone Escherichia coli O18:K1:H7. An homologous chromosomal element (CUS-2) in Yersinia pestis biovar orientalis is integrated at the same relative location as CUS-1; both lysogenic E. coli and Y. pestis strains produce particles with properties expected of single-stranded DNA virions. CUSφ is epidemiologically correlated with the emergence of K1 strains with increased virulence and with the Y. pestis biovar responsible for the current (third) plague pandemic. PMID:12374839

  14. 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. PMID:26435220

  15. Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

    PubMed Central

    Hoe, N P; Goguen, J D

    1993-01-01

    The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented. Images PMID:7504666

  16. 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

  17. 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

  18. 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.

  19. Virtual screening, docking, and dynamics of potential new inhibitors of dihydrofolate reductase from Yersinia pestis.

    PubMed

    Bastos, Leonardo da Costa; de Souza, Felipe Rodrigues; Guimarães, Ana Paula; Sirouspour, Mehdi; Cuya Guizado, Teobaldo Ricardo; Forgione, Pat; Ramalho, Teodorico Castro; França, Tanos Celmar Costa

    2016-10-01

    In the present work, we propose to design drugs that target the enzyme dihydrofolate redutase (DHFR) as a means of a novel drug therapy against plague. Potential inhibitors of DHFR from Yersinia pestis (YpDHFR) were selected by virtual screening and subjected to docking, molecular dynamics (MD) simulations, and Poisson-Boltzmann surface area method, in order to evaluate their interactions in the active sites of YpDHFR and human DHFR (HssDHFR). The results suggested selectivity for three compounds that were further used to propose the structures of six new potential selective inhibitors for YpDHFR. PMID:26494420

  20. 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

  1. Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent.

    PubMed Central

    Munier-Lehmann, Hélène; Chenal-Francisque, Viviane; Ionescu, Mihaela; Chrisova, Petya; Foulon, Jeannine; Carniel, Elisabeth; Bârzu, Octavian

    2003-01-01

    Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals. PMID:12879903

  2. 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

  3. 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

  4. 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

  5. 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...

  6. 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

  7. 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...

  8. 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

    ... of a Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector AGENCY: Animal and Plant Health Inspection..., Live Raccoon Poxvirus Vector. The environmental assessment, which is based on a risk analysis prepared...-2013-0020, Regulatory Analysis and Development, PPD, APHIS, Station 3A-03.8, 4700 River Road Unit...

  9. The hmsT 3' untranslated region mediates c-di-GMP metabolism and biofilm formation in Yersinia pestis.

    PubMed

    Zhu, Hui; Mao, Xu-Jian; Guo, Xiao-Peng; Sun, Yi-Cheng

    2016-03-01

    Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment. PMID:26711808

  10. [Genetic basis of the variability of nitrate reduction trait in Yersinia pestis strains].

    PubMed

    Eroshenko, G A; Odinokov, G N; Kukleva, L M; Shavina, N Iu; Guseva, N P; Kutyrev, V V

    2014-05-01

    The genetic basis of the varying ability to reduce nitrate in strains belonging to different biovars and subspecies of plague-causing microbe has been investigated and the inability to reduce nitrate observed in different intraspecies groups of Yersinia pestis has been shown to stem from mutations in different genes involved in the expression of this trait. The absence of denitrifying activity in strains of altaica and hissarica subspecies was not due to a mutation at position 613 of the periplasmic reductase napA observed in the strains of the biovar medievalis of the main subspecies, but rather was due to a mutation in the sequence encoding the nitrate-binding domain of the ABC transporter protein SsuA; a thymine insertion (+T) was detected at position 302 from the start of the ssuA gene. Five strains of biovar antiqua isolated at different times in Mongolia, China, and Africa were shown to lack the ability to reduce nitrate. A PCR test targeting two chromosomal regions containing deletions of 19 and 24 bp in size has been developed for the identification of strains of the biovar medievalis. This test can be combined with the test for the marker mutation in the napA gene for a more reliable detection of Y. pestis strains belonging to this biovar. PMID:25715468

  11. 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

  12. 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.

  13. 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.

  14. Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature.

    PubMed

    Kawahara, Kazuyoshi; Tsukano, Hiroko; Watanabe, Haruo; Lindner, Buko; Matsuura, Motohiro

    2002-08-01

    Yersinia pestis strain Yreka was grown at 27 or 37 degrees C, and the lipid A structures (lipid A-27 degrees C and lipid A-37 degrees C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27 degrees C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37 degrees C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27 degrees C than in lipid A-37 degrees C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-alpha). In both cell lines the LPS and the lipid A from bacteria grown at 27 degrees C were stronger inducers of TNF-alpha than those from bacteria grown at 37 degrees C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37 degrees C. PMID:12117916

  15. Diversity in a Variable-Number Tandem Repeat from Yersinia pestis

    PubMed Central

    Adair, D. M.; Worsham, P. L.; Hill, K. K.; Klevytska, A. M.; Jackson, P. J.; Friedlander, A. M.; Keim, P.

    2000-01-01

    We have identified a tetranucleotide repeat sequence, (CAAA)N, in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague. PMID:10747136

  16. Plasmid-like properties of the four virulence-associated factors of Yersinia pestis.

    PubMed

    Tsukano, H; Wake, A; Sakakibara, Y

    1986-01-01

    The virulence-associated factors of Yersinia pestis, which determine the abilities to produce pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) and a cell-surface component for adsorption of exogenous pigments (Pgm+), were independently eliminated by cultivation of the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at a subinhibitory concentration. A virulent Y. pestis strain, Yreka, harbored at least five extrachromosomal DNA molecules of different sizes. In these molecules, a novel 13-megadalton DNA which was cured concomitantly with the elimination of the Fra factor was found, in addition to the known species of 7 and 44 megadaltons which were lost with the conversions to Pst- and Vwa-, respectively. Although the conversion to Pgm- could not be correlated with the lack of any proper extra-chromosomal DNA, the factor was transmitted to Pgm- cells with the aid of self-conjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice. PMID:3796319

  17. Natural History of Yersinia pestis Pneumonia in Aerosol-Challenged BALB/c Mice

    PubMed Central

    Chuvala, Lara; Riggins, Renaldo; Hurteau, Gregory; Cirz, Ryan; Cass, Robert; Louie, Arnold; Drusano, G. L.

    2013-01-01

    After a relatively short untreated interval, pneumonic plague has a mortality approaching 100%. We employed a murine model of aerosol challenge with Yersinia pestis to investigate the early course of pneumonic plague in the lung, blood, and spleen. We fit a mathematical model to all data simultaneously. The model fit to the data was acceptable. The number of organisms in the lung at baseline was estimated to be 135 (median) or 1,184 (mean) CFU/g. The doubling time was estimated as 1.5 to 1.7 h. Between 1 and 12 h postexposure, counts declined, but they then increased by 24 h, a finding hypothesized to be due to innate immunity. The model predicted that innate immunity declined with a half-time of 3 to 3.8 h. The threshold for bacteremia was 6.4 × 104 to 1.52 × 106 CFU/g. By 42 to 48 h, stationary phase was obtained. Lung bacterial burdens exceeded 10 log CFU/g. Obviating early defenses allows for rapid amplification of Y. pestis in bacteremia, making the rapid course with high mortality understandable. PMID:23403418

  18. 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. PMID:25711606

  19. 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

  20. Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

    PubMed Central

    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. PMID:21436885

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Palindromic unit-independent transposition of IS1397 in Yersinia pestis.

    PubMed

    Wilde, Caroline; Bachellier, Sophie; Hofnung, Maurice; Carniel, Elisabeth; Clément, Jean-Marie

    2002-09-01

    Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes of Escherichia coli and several other enterobacteria. In the latter, IS1397, an E. coli insertion sequence specific to PUs, transposes into PUs with sequences close to the E. coli consensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that for Yersinia pestis, a species deprived of PUs, IS1397 can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397 transposase. PMID:12169598

  9. [Studies of Yersinia pestis in wild animals captured in Ankara, Konya and Nevsehir].

    PubMed

    Ozsan, K; Fazli, A; Aktan, M; Beyoğlu, K

    1976-01-01

    No Yersinia pestis could be isolated, by culturing and by inoculations to 1212 guinea-pigs and 150 mice; from 623 citellus, 41 Mus musculus, 55 Microtus, 442 Meriones, 70 Rattus rattus, 56 turtle, 89 hare, 1 hamster, 1 hedgehog, 1 sea snake, altogether 790 dead, 589 alive, i.e. 1379. wild animals captured in Ankara, Konya (Karapinar), Urfa (Akçakale) and in Nevşehir. In 141 sera taken from citellus captured alive, and in 174 sera taken from guinea-pigs inoculated with spleen, liver and kidney suspensions of wild animals, 1/20 - 1/80 agglutination titers (one of the sera from a guinea-pig inoculated with hare organ suspension) were obtained. These findings, probably were due to Yersinia pseudotuberculosis, because this organism was isolated from citellus captured in Ankara and Konya. PMID:933892

  10. 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.

  11. Purification and biochemical characterisation of GlmU from Yersinia pestis.

    PubMed

    Patin, Delphine; Bayliss, Marc; Mengin-Lecreulx, Dominique; Oyston, Petra; Blanot, Didier

    2015-04-01

    Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents. PMID:25417006

  12. Host stress and immune responses during aerosol challenge of Brown Norway rats with Yersinia pestis

    PubMed Central

    Gater, Susan T.; Peters, Kristen N.; Kocsis, Andrew G.; Dhariwala, Miqdad O.; Anderson, Deborah M.; Anderson, Paul E.

    2012-01-01

    Inhalation exposure models are becoming the preferred method for the comparative study of respiratory infectious diseases due to their resemblance to the natural route of infection. To enable precise delivery of pathogen to the lower respiratory tract in a manner that imposes minimal biosafety risk, nose-only exposure systems have been developed. Early inhalation exposure technology for infectious disease research grew out of technology used in asthma research where predominantly the Collison nebulizer is used to generate an aerosol by beating a liquid sample against glass. Although infectious aerosol droplets of 1–5 μm in size can be generated, the Collison often causes loss of viability. In this work, we evaluate a gentler method for aerosolization of living cells and describe the use of the Sparging Liquid Aerosol Generator (SLAG) in a rat pneumonic plague model. The SLAG creates aerosols by continuous dripping of liquid sample on a porous metal disc. We show the generation of 0.5–1 μm Yersinia pestis aerosol particles using the SLAG with spray factors typically ranging from 10−7 to 10−8 with no detectable loss of bacterial viability. Delivery of these infectious particles via nose-only exposure led to the rapid development of lethal pneumonic plague. Further, we evaluated the effect of restraint-stress imposed by the nose-only exposure chamber on early inflammatory responses and bacterial deposition. Elevated serum corticosterone which peaked at 2 h post-procedure indicated the animals experienced stress as a result of restraint in the nose-only chamber. However, we observed no correlation between elevated corticosterone and the amount of bacterial deposition or inflammation in the lungs. Together these data demonstrate the utility of the SLAG and the nose-only chamber for aerosol challenge of rodents by Y. pestis. PMID:23226684

  13. Modification of the Structure and Activity of Lipid A in Yersinia pestis Lipopolysaccharide by Growth Temperature

    PubMed Central

    Kawahara, Kazuyoshi; Tsukano, Hiroko; Watanabe, Haruo; Lindner, Buko; Matsuura, Motohiro

    2002-01-01

    Yersinia pestis strain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-α). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-α than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37°C. PMID:12117916

  14. 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

  15. MAP of F1 and V antigens from Yersinia pestis astride innate and adaptive immune response.

    PubMed

    Rai, Reeta; Das, Baijnath; Choudhary, Nageshwar; Talukdar, Ayantika; Rao, Donthamsetty Nageswara

    2015-10-01

    Yersinia pestis, a causative agent of plague, has a plethora of armors to fight against major components of innate immunity and survive within host cells. Dendritic cells and macrophages are important antigen presenting cells for effective immune response. This report is focused on the changes in DC activation and TLR2 and TLR4 expression on macrophages induced by MAP of F1 and V antigens of Y. pestis. F1 and V MAPs bear potential synthetic T and B cell epitopes from F1 and V protein respectively. We evaluated these parameters in DC's isolated from spleen and lamina propria and macrophages isolated from peritoneal lavage of mice after intranasal immunization. F1 MAP and V MAP significantly increased the expression of CD80 and CD86 on CD11c(+) dendritic cells isolated from spleen and lamina propria as well as intracellular IL-12 levels. Similarly, in macrophages derived from peritoneal cavity, the above formulation enhanced TLR2 and TLR4 expression. Again after in vitro stimulation with F1 and V MAP these macrophages produced significantly high IL12 and TNFα. The study clearly indicates involvement of DC and macrophages for efficient antigen presentation to immune cells. From this study we conclude that F1MAP and VMAP ameliorate innate immune mechanism. These two synthetic constructs exert their effect via TLR2 and TLR4, leading to the production of proinflammatory cytokines by macrophages and are able to increase DC activation, that could be helpful in generation of adaptive immunity as well as is important strong immune response. PMID:26188288

  16. 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

  17. 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

  18. 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.

  19. Manganese transporters Yfe and MntH are Fur-regulated and important for the virulence of Yersinia pestis

    PubMed Central

    Craig, Susannah K.; Abney, Jennifer; Bobrov, Alexander G.; Kirillina, Olga; Mier, Ildefonso; Truszczynska, Helena; Fetherston, Jacqueline D.

    2012-01-01

    Yersinia pestis has a flea-mammal-flea transmission cycle, and is a zoonotic pathogen that causes the systemic diseases bubonic and septicaemic plague in rodents and humans, as well as pneumonic plague in humans and non-human primates. Bubonic and pneumonic plague are quite different diseases that result from different routes of infection. Manganese (Mn) acquisition is critical for the growth and pathogenesis of a number of bacteria. The Yfe/Sit and/or MntH systems are the two prominent Mn transporters in Gram-negative bacteria. Previously we showed that the Y. pestis Yfe system transports Fe and Mn. Here we demonstrate that a mutation in yfe or mntH did not significantly affect in vitro aerobic growth under Mn-deficient conditions. A yfe mntH double mutant did exhibit a moderate growth defect which was alleviated by supplementation with Mn. No short-term energy-dependent uptake of 54Mn was observed in this double mutant. Like the yfeA promoter, the mntH promoter was repressed by both Mn and Fe via Fur. Sequences upstream of the Fur binding sequence in the yfeA promoter converted an iron-repressible promoter to one that is also repressed by Mn and Fe. To our knowledge, this is the first report identifying cis promoter elements needed to alter cation specificities involved in transcriptional repression. Finally, the Y. pestis yfe mntH double mutant had an ~133-fold loss of virulence in a mouse model of bubonic plague but no virulence loss in the pneumonic plague model. This suggests that Mn availability, bacterial Mn requirements or Mn transporters used by Y. pestis are different in the lungs (pneumonic plague) compared with systemic disease. PMID:22222497

  20. Cethromycin-Mediated Protection against the Plague Pathogen Yersinia pestis in a Rat Model of Infection and Comparison with Levofloxacin ▿

    PubMed Central

    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.

    2011-01-01

    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. PMID:21859946

  1. 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

  2. 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

  3. Homology Analysis of Pathogenic Yersinia Species Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis Based on Multilocus Sequence Typing

    PubMed Central

    Duan, Ran; Liang, Junrong; Shi, Guoxiang; Cui, Zhigang; Hai, Rong; Wang, Peng; Xiao, Yuchun; Li, Kewei; Qiu, Haiyan; Gu, Wenpeng; Du, Xiaoli

    2014-01-01

    We developed a multilocus sequence typing (MLST) scheme and used it to study the population structure and evolutionary relationships of three pathogenic Yersinia species. MLST of these three Yersinia species showed a complex of two clusters, one composed of Yersinia pseudotuberculosis and Yersinia pestis and the other composed of Yersinia enterocolitica. Within the first cluster, the predominant Y. pestis sequence type 90 (ST90) was linked to Y. pseudotuberculosis ST43 by one locus difference, and 81.25% of the ST43 strains were from serotype O:1b, supporting the hypothesis that Y. pestis descended from the O:1b serotype of Y. pseudotuberculosis. We also found that the worldwide-prevalent serotypes O:1a, O:1b, and O:3 were predominated by specific STs. The second cluster consisted of pathogenic and nonpathogenic Y. enterocolitica strains, two of which may not have identical STs. The pathogenic Y. enterocolitica strains formed a relatively conserved group; most strains clustered within ST186 and ST187. Serotypes O:3, O:8, and O:9 were separated into three distinct blocks. Nonpathogenic Y. enterocolitica STs were more heterogeneous, reflecting genetic diversity through evolution. By providing a better and effective MLST procedure for use with the Yersinia community, valuable information and insights into the genetic evolutionary differences of these pathogens were obtained. PMID:24131695

  4. A refrigeration temperature of 4 degrees C does not prevent static growth of Yersinia pestis in heart infusion broth.

    PubMed

    Torosian, Stephen D; Regan, Patrick M; Doran, Tara; Taylor, Michael A; Margolin, Aaron

    2009-09-01

    Multiple barriers such as inspections, testing, and proper storage conditions are used to minimize the risk of contaminated food. Knowledge of which barriers, such as refrigeration, are effective in preventing pathogen growth and persistence, can help direct the focus of efforts during food sampling. In this study, the doubling times were evaluated for 10 strains of Yersinia pestis of different genetic background cultured in heart infusion broth (HIB) kept at 4 degrees C +/- 1 degrees C under static conditions. Nine out of the 10 strains were able to grow at 4 degrees C +/- 1 degrees C. Apparent doubling times for 7 of the strains ranged from 41 to 50 h. Strain Harbin and strain D1 had apparent doubling times of 65 and 35 h, respectively, and strain O19 Ca-6 did not grow at all. Analysis of variance showed that the averaged growth data (colony forming units per mL) between strains that grew were not significantly different. The data presented here demonstrate that refrigeration alone is not an effective barrier to prevent static growth of Y. pestis in HIB. These findings provide the preliminary impetus to investigate Y. pestis growth in a variety of food matrices that may provide a similar environment as HIB. PMID:19898555

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Temperature-controlled plasmid regulon associated with low calcium response in Yersinia pestis.

    PubMed Central

    Yother, J; Chamness, T W; Goguen, J D

    1986-01-01

    Both the low calcium response and virulence in Yersinia pestis strain KIM5 are mediated by genes located on the 75.4-kilobase plasmid pCD1. The results presented here demonstrate the existence of two new genetic loci of pCD1 whose expression is regulated in response to temperature. Levels of transcription in the trtA and trtB loci were elevated 12- to 16-fold at 37 degrees C compared with levels of transcription at 30 degrees C. In addition, the absolute levels of transcription were the highest that have been reported for genes on pCD1. Mutations in trtB also abolished production of the V antigen. Thermal induction at these loci was dramatically reduced in strains harboring a Tn5 insertion in the lcrF locus of pCD1. lcrF lies 33 and 13 kilobases from the trtA and trtB loci, respectively. Thus, lcrF is a positive regulatory gene responsible for temperature-induced transcription of genes required for the low calcium response. PMID:3944056

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed Central

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

    1996-01-01

    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. PMID:8890210

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

    PubMed

    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

  20. 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

  1. 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.

  2. Kinetic epitope mapping of monoclonal antibodies raised against the Yersinia pestis virulence factor LcrV.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2014-10-01

    Five monoclonal antibodies, mAb7.3, mAb29.3, mAb46.3, mAb12.3 and mAb36.3, raised to the LcrV virulence factor from Yersinia pestis were characterised for their Fab affinity against the purified protein and their Fc affinity to Protein A/G as a proxy for the FcγR receptor. Kinetic measurements were performed label-free in a localised particle plasmon array reader. The Fc-ProteinA/G complex first-order half-life was determined for each antibody and fell in the range of 0.8-3.8h. The Fab first-order half-lives had ranged from 3.4 to 9.2h although two antibodies, mAb12.3 and mAb36.3, showed low affinity interactions. Competitive binding studies of mixtures of the Fab-active antibodies were performed to measure the relative binding efficiency of one antibody in the presence of the other. A geometric relative positioning of the epitopes of mAb7.3, mAb29.3 and mAb46.3 was determined based on the footprint locus of the antibody and the percentage of competitive binding. The two known protective antibodies mAb7.3 and mAb29.3 showed greater interference, indicating epitopes close to one another compared to the non-protective mAb46.3 antibody. The Fab-Fc complex half-life screen and epitope mapping are potentially useful tools in the screening of therapeutic antibodies or vaccine candidates. PMID:25461137

  3. 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

  4. 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

  5. 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., Jr.

    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.

  6. Evaluation of a Yersinia pestis Mutant Impaired in a Thermoregulated Type VI-Like Secretion System in Flea, Macrophage and Murine Models

    PubMed Central

    Robinson, Jennilee B.; Telepnev, Maxim V.; Zudina, Irina V.; Bouyer, Donald; Montenieri, John A.; Bearden, Scott W.; Gage, Kenneth L.; Agar, Stacy L.; Foltz, Sheri M.; Chauhan, Sadhana; Chopra, Ashok K.; Motin, Vladimir L.

    2009-01-01

    Type VI secretion systems (T6SS) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26° C in comparison to 37° C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 hours postinfection for bacterial cells pre-grown at 26° C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37°C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain. PMID:19716410

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

    PubMed

    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

    2015-01-01

    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

  8. 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

  9. Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea, macrophage and murine models.

    PubMed

    Robinson, Jennilee B; Telepnev, Maxim V; Zudina, Irina V; Bouyer, Donald; Montenieri, John A; Bearden, Scott W; Gage, Kenneth L; Agar, Stacy L; Foltz, Sheri M; Chauhan, Sadhana; Chopra, Ashok K; Motin, Vladimir L

    2009-11-01

    Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 degrees C in comparison to 37 degrees C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 degrees C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 degrees C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain. PMID:19716410

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

    PubMed

    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

  11. 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).

  12. Molecular identification by “suicide PCR” of Yersinia pestis as the agent of Medieval Black Death

    PubMed Central

    Raoult, Didier; Aboudharam, Gérard; Crubézy, Eric; Larrouy, Georges; Ludes, Bertrand; Drancourt, Michel

    2000-01-01

    Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new “suicide PCR” protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer—its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague. PMID:11058154

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

    PubMed Central

    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

  14. 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

  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. 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.

  17. 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

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

    PubMed Central

    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. PMID:24225642

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Biochemical, Structural and Molecular Dynamics Analyses of the Potential Virulence Factor RipA from Yersinia pestis

    PubMed Central

    Torres, Rodrigo; Swift, Robert V.; Chim, Nicholas; Wheatley, Nicole; Lan, Benson; Atwood, Brian R.; Pujol, Céline; Sankaran, Banu; Bliska, James B.; Amaro, Rommie E.; Goulding, Celia W.

    2011-01-01

    Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in

  5. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    PubMed Central

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

    2015-01-01

    Solid-state NMR studies of sedimented soluble proteins has been recently developed as an attractive approach for overcoming the size limitations of solution NMR spectroscopy and bypassing the need for sample crystallization or precipitation (Bertini et al. 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales. PMID:25578899

  6. 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.

  7. 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

  8. 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.

  9. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation.

    PubMed

    Ding, Yi; Fujimoto, L Miya; Yao, Yong; Marassi, Francesca M

    2015-04-01

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales. PMID:25578899

  10. 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.

  11. 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

  12. 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

  13. Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J.; Patel, Trishal; Bases, Jessica; Jose, Geraldine San; Jackson, Emily R.; Dowd, Cynthia S.; Couch, Robin D.

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

  14. Evaluation of Imipenem for Prophylaxis and Therapy of Yersinia pestis Delivered by Aerosol in a Mouse Model of Pneumonic Plague

    PubMed Central

    Louie, Arnold; Adamovicz, Jeffrey J.; Amemiya, Kei; Fast, Randy L.; Miller, Lynda; Opal, Steven M.; Palardy, John; Parejo, Nicolas A.; Sörgel, Fritz; Kinzig-Schippers, Martina; Drusano, George L.

    2014-01-01

    It has been previously shown that mice subjected to an aerosol exposure to Yersinia pestis and treated with β-lactam antibiotics after a delay of 42 h died at an accelerated rate compared to controls. It was hypothesized that endotoxin release in antibiotic-treated mice accounted for the accelerated death rate in the mice exposed to aerosol Y. pestis. Imipenem, a β-lactam antibiotic, binds to penicillin binding protein 2 with the highest affinity and produces rounded cells. The binding of imipenem causes cells to lyse quickly and thereby to release less free endotoxin. Two imipenem regimens producing fractions of time that the concentration of free, unbound drug was above the MIC (fT>MIC) of approximately 25% (6/24 h) and 40% (9.5/24 h) were evaluated. In the postexposure prophylaxis study, the 40% and 25% regimens produced 90% and 40% survivorship, respectively. In the 42-h treatment study, both regimens demonstrated a 40 to 50% survivorship at therapy cessation and some deaths thereafter, resulting in a 30% survivorship. As this was an improvement over the results with other β-lactams, a comparison of both endotoxin and cytokine levels in mice treated with imipenem and ceftazidime (a β-lactam previously demonstrated to accelerate death in mice during treatment) was performed and supported the original hypotheses; however, the levels observed in animals treated with ciprofloxacin (included as an unrelated antibiotic that is also bactericidal but should cause little lysis due to a different mode of action) were elevated and significantly (7-fold) higher than those with ceftazidime. PMID:24687492

  15. Structural and functional characterization of TesB from Yersinia pestis reveals a unique octameric arrangement of hotdog domains

    PubMed Central

    Swarbrick, C. M. D.; Perugini, M. A.; Cowieson, N.; Forwood, J. K.

    2015-01-01

    Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on the Escherichia coli structure. Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of the Y. pestis TesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family. PMID:25849407

  16. An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

    PubMed Central

    Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

    1998-01-01

    The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

  17. Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

    PubMed

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J; Patel, Trishal; Bases, Jessica; San Jose, Geraldine; Jackson, Emily R; Dowd, Cynthia S; Couch, Robin D

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

  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. 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

  20. A New Clade of African Body and Head Lice Infected by Bartonella quintana and Yersinia pestis-Democratic Republic of the Congo.

    PubMed

    Drali, Rezak; Shako, Jean-Christophe; Davoust, Bernard; Diatta, Georges; Raoult, Didier

    2015-11-01

    The human body louse is known as a vector for the transmission of three serious diseases-specifically, epidemic typhus, trench fever, and relapsing fever caused by Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis, respectively-that have killed millions of people. It is also suspected in the transmission of a fourth pathogen, Yersinia pestis, which is the etiologic agent of plague. To date, human lice belonging to the genus Pediculus have been classified into three mitochondrial clades: A, B, and C. Here, we describe a fourth mitochondrial clade, Clade D, comprising head and body lice. Clade D may be a vector of B. quintana and Y. pestis, which is prevalent in a highly plague-endemic area near the Rethy Health District, Orientale Province, Democratic Republic of the Congo. PMID:26392158

  1. 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

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

    PubMed

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

    2015-09-01

    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

  3. A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis.

    PubMed Central

    Fields, K A; Plano, G V; Straley, S C

    1994-01-01

    The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulence-related stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lcrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. The sequence of a 2-kb region revealed three open reading frames, designated yscQ, yscR, and yscS, predicted to encode acidic proteins of 34.4, 24.4, and 8.5 kDa. All three proteins were homologous to proteins involved in flagellar function or virulence. An antipeptide antibody specific for YscR was used to localize YscR to the inner membrane of Y. pestis. Analysis of yscR-phoA fusions supported a model for yscR which predicts four transmembrane regions and a large, central hydrophilic domain. In-frame deletion mutations of yscQ and yscR were constructed and moved into Y. pestis. Both mutants failed to show the restriction of growth that normally accompanies maximal LCRS induction. Unlike the parent Y. pestis, the yscR mutant did not respond to the absence of Ca2+ by increasing the net transcription or translation of the LCRS-encoded V antigen, YopM, or LcrG. The yscR mutant also was defective for secretion of V antigen, YopM, and LcrG. These findings implicate a dual role for YscR in regulation of LCRS operons and secretion of LCRS proteins and add to the developing picture of how secretion of virulence proteins may be coupled to transcriptional regulation in yersiniae. Images PMID:8300512

  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. 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

  6. 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

  7. 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

  8. 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

  9. Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP receptor protein-cyclic AMP complex by PhoP in Yersinia pestis.

    PubMed

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

    2013-03-01

    Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter -10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP. PMID:23264579

  10. 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

  11. 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

  12. 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.

  13. Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant

    PubMed Central

    Galindo, Cristi L.; Moen, Scott T.; Kozlova, Elena V.; Sha, Jian; Garner, Harold R.; Agar, Stacy L.; Chopra, Ashok K.

    2009-01-01

    We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Δlpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Δlpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague. PMID:20145715

  14. 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

  15. 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

  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. [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

  18. Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants.

    PubMed Central

    Goguen, J D; Yother, J; Straley, S C

    1984-01-01

    Yersinia pestis strain KIM requires plasmid pCD1 for expression of the low calcium response, plague virulence antigen V, and virulence. We constructed Mu d1(Ap lac) insertion mutants of this plasmid which were unable to express the low calcium response. The insertions mapped to a 17-kilobase region of the plasmid. By determining the orientation of the insertions and examining beta-galactosidase production from the Mu d1 lac genes, we determined that this region contains three units of transcription, one of which is transcribed in a direction opposite the direction of transcription of the other two. Transcription of at least two of these units was induced significantly at 37 degrees C compared with 26 degrees C. Ca2+ (2.5 mM) and ATP (18 mM) had no significant effect on the level of expression of the Mu d1 lac genes of these mutants. All insertions in the region strongly reduced production of the V antigen. Insertions from each unit of transcription also reduced virulence in mice. PMID:6094509

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Katz, Anna; Niemimaa, Jukka; Forbes, Kristian M; Huitu, Otso; Stuart, Peter; Henttonen, Heikki; Nikkari, Simo

    2015-08-01

    In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods. PMID:26043838

  6. Global gene expression profiling of Yersinia pestis replicating inside macrophages reveals the roles of a putative stress-induced operon in regulating type III secretion and intracellular cell division.

    PubMed

    Fukuto, Hana S; Svetlanov, Anton; Palmer, Lance E; Karzai, A Wali; Bliska, James B

    2010-09-01

    Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Deltay2313-y2316 or DeltaorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment. PMID:20566693

  7. Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

    PubMed Central

    Motin, Vladimir L.; Georgescu, Anca M.; Elliott, Jeffrey M.; Hu, Ping; Worsham, Patricia L.; Ott, Linda L.; Slezak, Tomas R.; Sokhansanj, Bahrad A.; Regala, Warren M.; Brubaker, Robert R.; Garcia, Emilio

    2002-01-01

    A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed. PMID:11807062

  8. [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

  9. 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. PMID:24341424

  10. [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

  11. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. PMID:26259880

  12. 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

  13. 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. PMID:23763588

  14. 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

  15. 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.

  16. Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium.

    PubMed

    Sodeinde, O A; Goguen, J D

    1989-05-01

    We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene. PMID:2651310

  17. 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.

  18. Repertoire of HLA-DR1-Restricted CD4 T-Cell Responses to Capsular Caf1 Antigen of Yersinia pestis in Human Leukocyte Antigen Transgenic Mice▿

    PubMed Central

    Musson, Julie A.; Ingram, Rebecca; Durand, Guillaume; Ascough, Stephanie; Waters, Emma L.; Hartley, M. Gillian; Robson, Timothy; Maillere, Bernard; Williamson, E. Diane; Sriskandan, Shiranee; Altmann, Daniel; Robinson, John H.

    2010-01-01

    Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in “humanized” HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans. PMID:20660611

  19. Development of real-time PCR assays for specific detection of hmsH, hmsF, hmsR, and irp2 located within the 102-kb pgm locus of Yersinia pestis.

    PubMed

    Gaddy, Charla E; Cuevas, Pedro F; Hartman, Laurie J; Howe, Gerald B; Worsham, Patricia L; Minogue, Timothy D

    2014-01-01

    Virulent isolates of three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a 102-kb chromosomal region which encodes elements critical for virulence. A 35-kb high pathogenicity island is contained in this region, is a known virulence determinant, contains irp1 and irp2 iron-regulating genes. An additional segment, the 68-kb high pathogenicity island, contains genetic elements responsible for conferring the Y. pestis pigmentation phenotype on Congo red agar at 28 °C. Collectively, these contiguous segments are referred to as the pigmentation (pgm) locus, the absence of which results in strain attenuation and exemption from CDC Select Agent status. In this study, we developed a set of four real-time PCR assays to detect the presence or absence of multiple virulence genes located within this region. Specifically, we designed TaqMan(®) PCR assays to individually detect three hemin storage genes (hmsH, hmsF, and hmsR) which are genetic elements that confer the pigmentation phenotype, as well as the iron-regulating status of 25 Y. pestis isolates (representing 23 different strains), thus establishing a molecular based assay capable of determining the pgm status of candidate Y. pestis isolates. Included in the validation process, was a comparison of these real-time PCR assays and newly developed conventional PCR assays targeting much larger areas of the 102-kb region (including one assay spanning hmsR and hmsF, one spanning hmsH and hsmF, one targeting hmsF, and one targeting irp2). There was high concordance between the conventional and real-time PCR assays for all Y. pestis strains tested. The results from the comparative analysis document the specificity and sensitivity of the real-time PCR assays and further solidify the ostensible benefits of real-time PCR over conventional PCR. PMID:25261118

  20. 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

  1. 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

  2. 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

  3. Validation of cooking times and temperatures for thermal inactivation of Yersinia pestis strains KIM5 and CDC-A1122 in irradiated ground beef.

    PubMed

    Porto-Fett, Anna C S; Juneja, Vijay K; Tamplin, Mark L; Luchansky, John B

    2009-03-01

    Irradiated ground beef samples (ca. 3-g portions with ca. 25% fat) inoculated with Yersina pestis strain KIM5 (ca. 6.7 log CFU/g) were heated in a circulating water bath stabilized at 48.9, 50, 52.5, 55, 57.5, or 60 degrees C (120, 122, 126.5, 131, 135.5, and 140 degrees F, respectively). Average D-values were 192.17, 34.38, 17.11, 3.87, 1.32, and 0.56 min, respectively, with a corresponding z-value of 4.67 degrees C (8.41 degrees F). In related experiments, irradiated ground beef patties (ca. 95 g per patty with ca. 25% fat) were inoculated with Y. pestis strains KIMS or CDC-A1122 (ca. 6.0 log CFU/g) and cooked on an open-flame gas grill or on a clam-shell type electric grill to internal target temperatures of 48.9, 60, and 71.1 degrees C (120, 140, and 160 degrees F, respectively). For patties cooked on the gas grill, strain KIM5 populations decreased from ca. 6.24 to 4.32, 3.51, and < or = 0.7 log CFU/g at 48.9, 60, and 71.1 degrees C, respectively, and strain CDC-A1122 populations decreased to 3.46 log CFU/g at 48.9 degrees C and to < or = 0.7 log CFU/g at both 60 and 71.1 degrees C. For patties cooked on the clam-shell grill, strain KIM5 populations decreased from ca. 5.96 to 2.53 log CFU/g at 48.9 degrees C and to < or = 0.7 log CFU/g at 60 or 71.1 degrees C, and strain CDC-A1122 populations decreased from ca. 5.98 to < or = 0.7 log CFU/g at all three cooking temperatures. These data confirm that cooking ground beef on an open-flame gas grill or on a clam-shell type electric grill to the temperatures and times recommended by the U.S. Department of Agriculture and the U.S. Food and Drug Administration Food Code, appreciably lessens the likelihood, severity, and/or magnitude of consumer illness if the ground beef were purposefully contaminated even with relatively high levels of Y. pestis. PMID:19343945

  4. Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

    PubMed Central

    Drancourt, Michel; Aboudharam, Gérard; Signoli, Michel; Dutour, Olivier; Raoult, Didier

    1998-01-01

    Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks. PMID:9770538

  5. The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha.

    PubMed Central

    Leung, K Y; Straley, S C

    1989-01-01

    In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM. Images PMID:2670888

  6. 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 Central

    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. PMID:25101850

  7. Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

    PubMed Central

    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.

    2014-01-01

    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. PMID:24686064

  8. The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

    PubMed Central

    Price, S B; Cowan, C; Perry, R D; Straley, S C

    1991-01-01

    The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein. Images PMID:1901573

  9. Crystallization and preliminary X-ray diffraction analysis of PsaA, the adhesive pilin subunit that forms the pH 6 antigen on the surface of Yersinia pestis.

    PubMed

    Bao, Rui; Esser, Lothar; Sadhukhan, Annapurna; Nair, Manoj K M; Schifferli, Dieter M; Xia, Di

    2012-10-01

    Yersinia pestis has been responsible for a number of high-mortality epidemics throughout human history. Like all other bacterial infections, the pathogenesis of Y. pestis begins with the attachment of bacteria to the surface of host cells. At least five surface proteins from Y. pestis have been shown to interact with host cells. Psa, the pH 6 antigen, is one of them and is deployed on the surface of bacteria as thin flexible fibrils that are the result of the polymerization of a single PsaA pilin subunit. Here, the crystallization of recombinant donor-strand complemented PsaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected to 1.9 Å resolution from a native crystal and to 1.5 Å resolution from a bromide-derivatized crystal. These crystals displayed the symmetry of the orthorhombic space group P222(1), with unit-cell parameters a = 26.3, b = 54.6, c = 102.1 Å. Initial phases were derived from single isomorphous replacement with anomalous scattering experiments, resulting in an electron-density map that showed a single molecule in the crystallographic asymmetric unit. Sequence assignment was aided by residues binding to bromide ions of the heavy-atom derivative. PMID:23027758

  10. Caspase-3 Mediates the Pathogenic Effect of Yersinia pestis YopM in Liver of C57BL/6 Mice and Contributes to YopM's Function in Spleen

    PubMed Central

    Ye, Zhan; Gorman, Amanda A.; Uittenbogaard, Annette M.; Myers-Morales, Tanya; Kaplan, Alan M.; Cohen, Donald A.; Straley, Susan C.

    2014-01-01

    The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G+, F4/80+, and iNOS+ cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs. PMID:25372388

  11. Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

    PubMed Central

    2010-01-01

    Background In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination

  12. 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

  13. 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

  14. Aurintricarboxylic acid blocks in vitro and in vivo activity of YopH, an essential virulent factor of Yersinia pestis, the agent of plague.

    PubMed

    Liang, Fubo; Huang, Zhonghui; Lee, Seung-Yub; Liang, Jiao; Ivanov, Maya I; Alonso, Andres; Bliska, James B; Lawrence, David S; Mustelin, Tomas; Zhang, Zhong-Yin

    2003-10-24

    Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to Bubonic Plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. YopH is an essential virulence factor whose protein-tyrosine phosphatase (PTP) activity is required for Yersinia pathogenicity. Consequently, there is considerable interest in developing potent and selective YopH inhibitors as novel anti-plague agents. We have screened a library of 720 structurally diverse commercially available carboxylic acids and identified 26 YopH inhibitors with IC50 values below 100 mum. The most potent and specific YopH inhibitor is aurintricarboxylic acid (ATA), which exhibits a Ki value of 5 nm for YopH and displays 6-120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in human Jurkat cells transfected with YopH. We show that YopH severely decreases the T-cell receptor-induced cellular tyrosine phosphorylation, ERK1/2 activity, and interleukin-2 transcriptional activity. We demonstrate that ATA can effectively block the inhibitory activity of YopH and restore normal T-cell function. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. In addition, it is expected that potent and selective YopH inhibitors, such as ATA, should be useful reagents to delineate YopH's cellular targets in plague and other pathogenic conditions caused by Yersinia infection. PMID:12888560

  15. 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

  16. Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

    PubMed

    Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

    2015-02-01

    The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples. PMID:25551670

  17. 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.

  18. A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

    PubMed Central

    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.

    2014-01-01

    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. PMID:25332120

  19. 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

  20. Pilot Study on the Use of DNA Priming Immunization to Enhance Y. pestis LcrV-Specific B Cell Responses Elicited by a Recombinant LcrV Protein Vaccine

    PubMed Central

    Li, Wei; Wang, Shixia; Lu, Shan

    2013-01-01

    Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis). Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems. PMID:26344467

  1. 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.

  2. Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice.

    PubMed

    Uppada, Srijayaprakash Babu; Bhat, Ajaz Ahmed; Sah, Anil; Donthamshetty, Rao Nageswara

    2011-11-21

    Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p<0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p<0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio>1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for

  3. 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.

  4. Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models.

    PubMed

    Weller, Simon A; Cox, Victoria; Essex-Lopresti, Angela; Hartley, Margaret G; Parsons, Tanya M; Rachwal, Phillip A; Stapleton, Helen L; Lukaszewski, Roman A

    2012-11-01

    Two multiplex PCR screening capabilities (TaqMan Array Cards and FilmArray) were evaluated for their ability to detect Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples obtained from respective murine infection models. Blood samples were obtained from infected mice at 24 h intervals after exposure. Multiplex PCR results were compared with standard blood culture and singleplex real-time PCR. Across all three models, 71 mice were tested in total, within which a subset of 43 samples was shown to contain an infecting agent by at least one of the detection technologies. Within this subset of positive samples, for each model studied, the detection rates of each technology were compared. The B. anthracis model blood culture (14 of 15 agent-containing samples tested) and FilmArray PCR (12 of 15) were shown to have equivalent detection rates, which were significantly higher (at the 95 % confidence level) than singleplex (five of 14) or Array Card (two of 14) PCRs. The F. tularensis model blood culture (12 of 12) was shown to have a significantly higher (at 95 % confidence level) detection rate than all PCR technologies, with FilmArray (seven of 11) and singleplex (seven of 12) PCRs shown to have significantly higher (at 95 % confidence level) detection rates than the Array Card PCR (two of 11). Within the Y. pestis model, there was no significant difference in detection rates between blood culture (10 of 16), singleplex PCR (14 of 16), Array Card PCR (10 of 16) and FilmArray PCR (10 of 13). PMID:22899777

  5. Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

    PubMed Central

    Zav'yalov, V P; Chernovskaya, T V; Chapman, D A; Karlyshev, A V; MacIntyre, S; Zavialov, A V; Vasiliev, A M; Denesyuk, A I; Zav'yalova, G A; Dudich, I V; Korpela, T; Abramov, V M

    1997-01-01

    The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed. PMID:9182720

  6. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    PubMed

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  7. 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

  8. emGain: Determination of EM gain of CCD

    NASA Astrophysics Data System (ADS)

    Daigle, Olivier; Carignan, Claude; Blais-Ouellette, Sebastien

    2012-01-01

    The determination of the EM gain of the CCD is best done by fitting the histogram of many low-light frames. Typically, the dark+CIC noise of a 30ms frame itself is a sufficient amount of signal to determine accurately the EM gain with about 200 512x512 frames. The IDL code emGain takes as an input a cube of frames and fit the histogram of all the pixels with the EM stage output probability function. The function returns the EM gain of the frames as well as the read-out noise and the mean signal level of the frames.

  9. 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

  10. Multiple antigen peptide consisting of B- and T-cell epitopes of F1 antigen of Y. pestis showed enhanced humoral and mucosal immune response in different strains of mice.

    PubMed

    Ali, Riyasat; Kumar, Sudhir; Naqvi, Raza Ali; Sheikh, Ishfaq Ahmed; Rao, D N

    2013-01-01

    Yersinia pestis is a causative agent of plague. F1 and V antigen based vaccines have shown remarkable protection in experimental animals. In order to develop epitope based immunogen, three B and one T-cell epitopes of F1 antigen with palmitate residue at amino terminal were assembled on a lysine backbone as multiple antigen peptide (MAP or F1-MAP). MAP was characterized by SDS-PAGE, immunoblot and immunoreactivity with anti F1 sera. MAP was entrapped in PLGA (polylactide-co-glycolide) microparticles and humoral, mucosal immune responses were studied after intranasal immunization with/without CpG ODN 1826 (CpG)/murabutide in different strains of mice. Serum and mucosal washes were measured for MAP specific IgG, IgA, sIgA and IgG subclasses in three strains of mice. F1-MAP showed high serum antibody and mucosal IgG and IgA peak antibody titers. MAP with CpG showed significantly high (p<0.001) peak antibody titer ranging from 102,400 to 204,800 for IgG and 6400 to 12,800 for IgA. High mucosal sIgA and its secretary component detection confirmed generation of mucosal response in intestinal and lung washes. MAP antisera also showed significant immunoreactivity with individual peptides. Moreover, antibody specific activity (IgG, IgA and sIgA) positively correlates with peak antibody titers. Predominantly IgG2a/IgG2b subclass was observed with CpG formulation but in other formulation a mixed IgG1 and IgG2a response was observed. The present study highlights the importance of multiple antigen peptide approach of F1-antigen with CpG as an alternative approach for subunit vaccine. PMID:23174507

  11. Bioterrorism awareness for EMS.

    PubMed

    Patrick, Richard W

    2004-04-01

    It is important to understand that the issues surrounding bioterrorism and all weapons of mass destruction are complex. In an effort to enhance response to such events, EMS should handle all incidents from the perspective of an all-hazards approach. Prevention, preparation, response and recovery are essential to the safe mitigation of all incidents. Organizations must be prepared. Plan now for a safer tomorrow. Your personnel and communities depend on you. PMID:15131906

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca2+ response.

    PubMed Central

    Nilles, M L; Williams, A W; Skrzypek, E; Straley, S C

    1997-01-01

    Yersinia pestis contains a virulence plasmid, pCD1, that encodes many virulence-associated traits, such as the Yops (Yersinia outer proteins) and the bifunctional LcrV, which has both regulatory and antihost functions. In addition to LcrV and the Yops, pCD1 encodes a type III secretion system that is responsible for Yop and LcrV secretion. The Yop-LcrV secretion mechanism is believed to regulate transcription of lcrV and yop operons indirectly by controlling the intracellular concentration of a secreted repressor. The activity of the secretion mechanism and consequently the expression of LcrV and Yops are negatively regulated in response to environmental conditions such as Ca2+ concentration by LcrE and, additionally, by LcrG, both of which have been proposed to block the secretion mechanism. This block is removed by the absence of Ca2+ or by contact with eukaryotic cells, and some Yops are then translocated into the cells. Regulation of LcrV and Yop expression also is positively affected by LcrV. Previously, LcrG was shown to be secreted from bacterial cells when the growth medium lacks added Ca2+, although most of the LcrG remains cell associated. In the present study, we showed that the cell-associated LcrG is cytoplasmically localized. We demonstrated that LcrG interacts with LcrV to form a heterodimeric complex by using chemical cross-linking and copurification of LcrG and LcrV. Additionally, we found that small amounts of LcrV and YopE can be detected in periplasmic fractions isolated by cold osmotic shock and spheroplast formation, indicating that their secretion pathway is accessible to the periplasm or to these procedures for obtaining periplasmic fractions. We propose that the cytoplasmically localized LcrG blocks the Yop secretion apparatus from the cytoplasmic side and that LcrV is required to remove the LcrG secretion block to yield full induction of Yop and LcrV secretion and expression. PMID:9023216

  19. 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...

  20. Is EMS communicating with the FCC?

    PubMed

    Johnson, M S; VanCott, C; Glass, C; Anderson, P B

    1989-07-01

    Radio communication problems in EMS run the spectrum from annoying to deadly. Dedicated radio frequencies for EMS, much like those exclusive to police and fire departments, are long overdue. PMID:10293680

  1. Alveolar Echinococcosis: Characterization of Diagnostic Antigen Em18 and Serological Evaluation of Recombinant Em18

    PubMed Central

    Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Yamasaki, Hiroshi; Gottstein, Bruno; Lightowers, Marshall W.; Schantz, Peter M.; Ito, Akira

    2002-01-01

    The Echinococcus multilocularis protein Em18 is one of the most promising antigens for use in serodiagnosis of alveolar echinococcosis in human patients. Here we identify an antigenic relationship between Em18 and a 65-kDa immunodominant E. multilocularis surface protein previously identified as either EM10 or EmII/3. The NH2-terminal sequence of native Em18 was determined, revealing it to be a fragment of EM10. Experiments were undertaken to investigate the effect of proteinase inhibitors on the degradation of EM10 in crude extracts of E. multilocularis protoscoleces. Em18 was found to be the product of degradation of EM10 by cysteine proteinase. A recombinant Em18 (RecEm18, derived from 349K to 508K of EM10) was successfully expressed by using Escherichia coli expression system and then evaluated for use in serodiagnosis of alveolar echinococcosis. RecEm18 was recognized by 27 (87.1%) and 28 (90.3%) of 31 serum samples from clinically and/or pathologically confirmed alveolar echinococcosis patients by enzyme-linked immunosorbent assay and immunoblotting, respectively. Of 33 serum samples from cystic echinococcosis patients, 1 was recorded as having a weak positive reaction to RecEm18; however, none of the serum samples which were tested from neurocysticercosis patients (n = 10) or healthy people (n = 15) showed positive reactions. RecEm18 has the potential for use in the differential serodiagnosis of alveolar echinococcosis. PMID:12149326

  2. The European Mobile System (EMS)

    NASA Technical Reports Server (NTRS)

    Jongejans, A.; Rogard, R.; Mistretta, I.; Ananasso, F.

    1993-01-01

    The European Space Agency is presently procuring an L band payload in order to promote a regional European L band system coping with the specific needs of the European market. The payload, and the two communications systems to be supported, are described below. The potential market for EMS in Europe is discussed.

  3. 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

  4. 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…

  5. View of Spacelab engineering Model (EM)

    NASA Technical Reports Server (NTRS)

    1981-01-01

    View of Spacelab engineering Model (EM) as it is being brought in the O and C bldg at Kenndey Space Center (27464); view of the EM as it is being offloaded from the C-54 aircraft. Kennedy Space Center alternative photo number is 108-KSC-80-OC-666 (27465); model taken out to launch pad (27466).

  6. 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.

  7. 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.

  8. Many factors complicate EM susceptibility tests

    NASA Astrophysics Data System (ADS)

    Richards, R. J.

    1982-09-01

    Procedures and apparatus currently employed for assaying the EM susceptibility of communication, navigation, and EW equipment are described. Susceptibility is examined in either conducted susceptibility tests, where signals are introduced into the input port of the device under test, or in radiated modes, where the entire device is exposed to an EM field to test for component and system failure. Noting that military standards require up to 10 times the EM resistance as commercial standards, the use of shielded enclosures in both commercial and military testing facilities is explored. RF-tight enclosures are filled with a homogeneous EM field produced by, optimally, broadband generators which emit signals which are amplified to desired levels. Sweep functions permit testing under broadband conditions. Attention is given to radiator selection and antenna choice to produce satisfactory test conditions at all frequencies.

  9. 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

  10. EMS in Taiwan: past, present, and future.

    PubMed

    Chiang, Wen-Chu; Ko, Patrick Chow-In; Wang, Hui-Chih; Yang, Chi-Wei; Shih, Fuh-Yuan; Hsiung, Kuang-Hua; Ma, Matthew Huei-Ming

    2009-01-01

    Taiwan is a small island country located in East Asia. From around 1995 modern concepts of the EMS were imported and supported by legislation. Considerable progress has since been made towards the construction of an effective pre-hospital care system. This article introduces the current status of the EMS in Taiwan, including the systems, response configurations, funding, personnel, medical directorship, and outcome research. The features and problems of in-hospital emergency care are also discussed. Key areas for further development in the country vary depending on regional differences in available resource and population density. An analysis of the strength, weakness, opportunity, and threats of the evolving EMS in Taiwan could be an example for other countries where the EMS is undergoing a similar process of development and optimisation. PMID:19059690

  11. 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.

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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

  18. 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

  19. Do earthquakes generate EM signals?

    NASA Astrophysics Data System (ADS)

    Walter, Christina; Onacha, Stephen; Malin, Peter; Shalev, Eylon; Lucas, Alan

    2010-05-01

    study areas, large swarms of earthquakes were located very close to the electromagnetic coils. This abstract focuses on the data from the Wairakei area. Preliminary data analysis has been carried out by band pass filtering and removing of the harmonics of the 50 Hz power line frequency. The initial results clearly show that electromagnetic signals accompany the seismic P and S waves (coseismic signal). Further data analysis involves the extraction of the seismoelectric signal generated at the onset of the earthquake and at interfaces from the coseismic signal and other ‘noise' sources. This processing step exhibits a major challenge in seismoelectric data processing. Unlike in other studies we measured the EM field and the seismic field at one location. Therefore the seismoelectric wave travelling at the speed of light cannot be determined as easily in the arrival times as when an array of coils is used. This makes the determination of the origin time much more difficult. Hence other processing techniques need to be explored.

  20. 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

  1. Percutaneous absorption and disposition of Tinopal EMS.

    PubMed

    Black, J G; Moule, R C; Philp, J

    1977-08-01

    A cotton-substantive, anionic, fluorescent whitening agent manufactured by several suppliers under various trade names e.g. Tinopal EMS, has been synthesized in radioactive form. Intubation of detergent or aqueous solution into rats resulted in little absorption from the intestinal tract as evidenced by low radioactivity in the urine and tissues. Most of the dose was excreted rapidly in the faeces. After parenteral administration to rats, the radioactivity was rapidly excreted in the faeces with small amounts remaining in tissues and organs. There was slight evidence of retention of radioactivity in the kidneys. Very small amounts of Tinopal EMS in detergent were absorbed through rat skin, but only when concentrations greater than those normally used by the consumer, together with occlusion of the skin were employed. Small amounts were absorbed throught skin when applied in ethanol. It is concluded that the possibility of systemic toxic effects in man as a result of percutaneous absorption is remote. PMID:929616

  2. 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.

  3. 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.

  4. Helicopter EMS: Research Endpoints and Potential Benefits

    PubMed Central

    Thomas, Stephen H.; Arthur, Annette O.

    2012-01-01

    Patients, EMS systems, and healthcare regions benefit from Helicopter EMS (HEMS) utilization. This article discusses these benefits in terms of specific endpoints utilized in research projects. The endpoint of interest, be it primary, secondary, or surrogate, is important to understand in the deployment of HEMS resources or in planning further HEMS outcomes research. The most important outcomes are those which show potential benefits to the patients, such as functional survival, pain relief, and earlier ALS care. Case reports are also important “outcomes” publications. The benefits of HEMS in the rural setting is the ability to provide timely access to Level I or Level II trauma centers and in nontrauma, interfacility transport of cardiac, stroke, and even sepsis patients. Many HEMS crews have pharmacologic and procedural capabilities that bring a different level of care to a trauma scene or small referring hospital, especially in the rural setting. Regional healthcare and EMS system's benefit from HEMS by their capability to extend the advanced level of care throughout a region, provide a “backup” for areas with limited ALS coverage, minimize transport times, make available direct transport to specialized centers, and offer flexibility of transport in overloaded hospital systems. PMID:22203905

  5. 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.

  6. 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

  7. 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...

  8. Siderochrome production by Yersinia pestis and its relation to virulence.

    PubMed Central

    Wake, A; Misawa, M; Matsui, A

    1975-01-01

    P+ plague strains contained more siderochrome-producing organisms than P-. Siderochrome enhanced the mouse virulence of an F1+Vw+P1+P-Pu+ strains, inhibited P1 activity, and could be assayed by a paper disk titration method. Images PMID:1104486

  9. 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

  10. 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.

  11. Stochastic EM-based TFBS motif discovery with MITSU

    PubMed Central

    Kilpatrick, Alastair M.; Ward, Bruce; Aitken, Stuart

    2014-01-01

    Motivation: The Expectation–Maximization (EM) algorithm has been successfully applied to the problem of transcription factor binding site (TFBS) motif discovery and underlies the most widely used motif discovery algorithms. In the wider field of probabilistic modelling, the stochastic EM (sEM) algorithm has been used to overcome some of the limitations of the EM algorithm; however, the application of sEM to motif discovery has not been fully explored. Results: We present MITSU (Motif discovery by ITerative Sampling and Updating), a novel algorithm for motif discovery, which combines sEM with an improved approximation to the likelihood function, which is unconstrained with regard to the distribution of motif occurrences within the input dataset. The algorithm is evaluated quantitatively on realistic synthetic data and several collections of characterized prokaryotic TFBS motifs and shown to outperform EM and an alternative sEM-based algorithm, particularly in terms of site-level positive predictive value. Availability and implementation: Java executable available for download at http://www.sourceforge.net/p/mitsu-motif/, supported on Linux/OS X. Contact: a.m.kilpatrick@sms.ed.ac.uk PMID:24931999

  12. 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

  13. 7 CFR 1945.20 - Making EM loans available.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Secretary in making a decision on the requested natural disaster determination. (4) The Secretary will... 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...

  14. 7 CFR 1945.20 - Making EM loans available.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Secretary in making a decision on the requested natural disaster determination. (4) The Secretary will... 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...

  15. Click-EM for imaging metabolically tagged nonprotein biomolecules.

    PubMed

    Ngo, John T; Adams, Stephen R; Deerinck, Thomas J; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F; Bertozzi, Carolyn R; Ellisman, Mark H; Tsien, Roger Y

    2016-06-01

    EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

  16. The Orthogonally Partitioned EM Algorithm: Extending the EM Algorithm for Algorithmic Stability and Bias Correction Due to Imperfect Data.

    PubMed

    Regier, Michael D; Moodie, Erica E M

    2016-05-01

    We propose an extension of the EM algorithm that exploits the common assumption of unique parameterization, corrects for biases due to missing data and measurement error, converges for the specified model when standard implementation of the EM algorithm has a low probability of convergence, and reduces a potentially complex algorithm into a sequence of smaller, simpler, self-contained EM algorithms. We use the theory surrounding the EM algorithm to derive the theoretical results of our proposal, showing that an optimal solution over the parameter space is obtained. A simulation study is used to explore the finite sample properties of the proposed extension when there is missing data and measurement error. We observe that partitioning the EM algorithm into simpler steps may provide better bias reduction in the estimation of model parameters. The ability to breakdown a complicated problem in to a series of simpler, more accessible problems will permit a broader implementation of the EM algorithm, permit the use of software packages that now implement and/or automate the EM algorithm, and make the EM algorithm more accessible to a wider and more general audience. PMID:27227718

  17. Evaluation of Fracture Azimuth by EM Wave and Elastic Wave

    NASA Astrophysics Data System (ADS)

    Feng, X.; Wang, Q.; Liu, C.; Lu, Q.; Zeng, Z.; Liang, W.; Yu, Y.; Ren, Q.

    2013-12-01

    Fracture system plays an important role in the development of underground energy, for example enhanced geothermal system (EGS), oil shale and shale gas, etc. Therefore, it becomes more and more important to detect and evaluate the fracture system. Geophysical prospecting is an useful method to evaluate the characteristics of the subsurface fractures. Currently, micro-seismology, multi-wave seismic exploration, and electromagnetic (EM) survey are reported to be used for the purpose. We are studying a method using both elastic wave and EM wave to detect and evaluate the fracture azimuth in laboratory. First, we build a 3D horizontal transverse isotropy (HTI) model, shown in the figure 1, by dry parallel fractures system, which was constructed by plexiglass plates and papers. Then, we used the ultrasonic system to obtain reflected S-wave data. Depending on the shear wave splitting, we evaluated the fracture azimuth by the algorithm of Pearson correlation coefficient. In addition, we used the full Polarimetric ultra wide band electromagnetic (FP-UWB-EM) wave System, shown in the figure 2, to obtain full polarimetric reflected EM-wave data. Depending on the rotation of the EM wave polarimetry, we evaluated the fracture azimuth by the the ration between maximum amplitude of co-polarimetric EM wave and maximum amplitude of cross-polarimetric EM wave. Finally, we used both EM-wave data and S-wave data to evaluate the fracture azimuth by the method of cross plot and statistical mathematics. To sum up, we found that FP-UWB-EM wave can be used to evaluated the fracture azimuth and is more accurate than ultrasound wave. Also joint evaluation using both data could improve the precision.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. DOE EM industry programs robotics development

    SciTech Connect

    Staubly, R.; Kothari, V.

    1997-12-01

    The Office of Science and Technology (OST) manages an aggressive program for RD&D, as well as testing and evaluation for the U.S. Department of Energy`s (DOE) 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. OST has organized technology management activities along focus teams for each major problem area. There are currently five focus areas: decontamination and decommissioning, tanks, subsurface contaminants, mixed waste, and plutonium. In addition, OST is pursuing research and development (R&D) that cuts across these focus areas by having applications in two or more focus areas. Currently, there are three cross-cutting programs: the robotics technology development; characterization, monitoring, and sensor technologies; and efficient separations and processing.

  3. 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

  4. 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

  5. Test beam performance of CDF plug upgrade EM calorimeter

    SciTech Connect

    Fukui, Y.

    1998-11-01

    CDF Plug Upgrade(tile-fiber) EM Calorimeter performed resolution of 15{percent}/{radical} (E) {circle_plus}0.7{percent} with non-linearity less than 1{percent} in a energy range of 5{endash}180 GeV at Fermilab Test Beam. Transverse uniformity of inside-tower-response of the EM Calorimeter was 2.2{percent} with 56 GeV positron, which was reduced to 1.0{percent} 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}C{sub s} Source was stable within 1{percent} in the period of 8 months. {copyright} {ital 1998 American Institute of Physics.}

  6. 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.

  7. 7 CFR 759.6 - EM to be made available.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 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...

  8. 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...

  9. 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.

  10. 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.

  11. 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

  12. Analysis of EM dataset with several sensor configurations obtained by the loop-loop EM survey on magnetic anomalies

    NASA Astrophysics Data System (ADS)

    CHOI, J.; Yi, M. J.; Sasaki, Y.; Son, J.; Nam, M. J.

    2015-12-01

    Most of mineral mines in Korea are located in rugged mountain area embedding small-scale anomalies. Loop-loop EM survey system can be a better choice for exploring those mines because no ground contact is required and portable loops are freely positioned. Survey design is very important for detecting small amount of mineral deposits efficiently and spatial limits of survey lines should be considered. Along a same survey line, surveys with different separations between a transmitter and a receiver are applicable. EM responses are calculated in a layered-earth model embedding magnetic anomalies and analyses considering electric conductivity and magnetic permeability are made for the loop-loop EM survey data. Combining EM dataset with multi-frequency and multi-separation slightly enhanced a reconstructed image. Loop-loop EM survey using PROMOIS system was conducted on a small magnetite mine. Inversion with and without considering magnetic permeability was conducted for EM data with multi-frequency and multi-separation between a transmitter and a receiver.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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,…

  20. Topical Treatment With an Agent Disruptive to <em>P. acnesem> Biofilm Provides Positive Therapeutic Response: Results of a Randomized Clinical Trial.

    PubMed

    Bernhardt, Michael J; Myntti, Matthew F

    2016-06-01

    The traditional disease model of acne has been one of follicular plugging due to 'sticky epithelial cells' associated with increased sebum production with deep follicular anaerobic conditions favoring <em>P. acnesem>- generated inflammation. <em>P. acnesem> biofilms have been found more frequently in patients with acne than controls. Biofilms are genetically coded to create adhesion to the pilosebaceous unit followed by production of a mucopolysaccharide coating capable of binding to lipid surfaces. Traditional therapies for acne have involved mixtures of oral and topical antibiotics admixed with topical keratolytics and retinoids, which are aimed at traditional bacterial reduction as well as downregulating the inflammatory cascade. These approaches are limited by side effect and compliance/tolerability issues. As the <em>P. acnesem> biofilm may, in fact, be the instigator of this process, we studied the use of a topical agent designed to reduce the <em>P. acnesem> biofilm to see if reducing the biofilm would be therapeutically efficacious. We present data of a proprietary topical non-prescription agent with a novel pharmaco mechanism designed to attack the biofilm produced by <em>P. acnesem>. Our data shows a decrease of inflammatory lesions by 44% and non-inflammatory lesions by 32% after 12 weeks and also provided for a meaningful improvement in the quality of life of the patients in the study. These improvements were achieved with a product that was not associated with burning, chafing, irritation, or erythema, which can be seen with topical treatments. It is apparent from this study that by addressing the biofilm which protects the <em>P. acnesem> bacteria through the use of the Acne Gel, the incidence of acne symptoms can be greatly reduced, while having no negative impacts on the patients' skin (ClinicalTrials.gov registry number NCT02404285).

    <em>J Drugs Dermatol. em>2016;15(6):677-683. PMID:27272073

  1. 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

  2. Active geophysical monitoring of hydrocarbon reservoirs using EM methods

    NASA Astrophysics Data System (ADS)

    Gribenko, A.; Black, N.; Zhdanov, M. S.

    2008-12-01

    Marine controlled-source electromagnetic (MCSEM) technology has been successfully established as an effective tool for offshore hydrocarbon (HC) exploration. In this paper we consider another application of the MCSEM method for HC reservoir monitoring. We demonstrate that EM methods can be successfully used for the monitoring of producing wells in connection with the enhanced recovery of hydrocarbons. We have developed a new powerful EM modeling technique based on the integral equation method with an inhomogeneous background conductivity (IE IBC). This new method and the corresponding computer software make it possible to model the EM response over a realistic complex model of a sea-bottom HC reservoir. The numerical modeling results demonstrate that the MCSEM method has the ability to map changes in resistivity caused by the production of hydrocarbons over time. In addition, the EM data help to visualize the changes in the location of the oil-water contact within the reservoir. This result opens the possibility for practical application of the EM method in HC reservoir monitoring.

  3. Application of the EM algorithm to radiographic images.

    PubMed

    Brailean, J C; Little, D; Giger, M L; Chen, C T; Sullivan, B J

    1992-01-01

    The expectation maximization (EM) algorithm has received considerable attention in the area of positron emitted tomography (PET) as a restoration and reconstruction technique. In this paper, the restoration capabilities of the EM algorithm when applied to radiographic images is investigated. This application does not involve reconstruction. The performance of the EM algorithm is quantitatively evaluated using a "perceived" signal-to-noise ratio (SNR) as the image quality metric. This perceived SNR is based on statistical decision theory and includes both the observer's visual response function and a noise component internal to the eye-brain system. For a variety of processing parameters, the relative SNR (ratio of the processed SNR to the original SNR) is calculated and used as a metric to compare quantitatively the effects of the EM algorithm with two other image enhancement techniques: global contrast enhancement (windowing) and unsharp mask filtering. The results suggest that the EM algorithm's performance is superior when compared to unsharp mask filtering and global contrast enhancement for radiographic images which contain objects smaller than 4 mm. PMID:1435595

  4. 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. PMID:27238019

  5. 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.

  6. A Computerized Evaluation Methodology for Pre-Hospital EMS Cardiac Care

    PubMed Central

    Nagurney, Frank K.

    1980-01-01

    The computerized application of cardiac care protocols for pre-hospital EMS care is presented. The program logic is reviewed and an example of its application is provided. Uses of the results of the program in EMS management are suggested.

  7. EMS Stretcher “Misadventures” in a Large, Urban EMS System: A Descriptive Analysis of Contributing Factors and Resultant Injuries

    PubMed Central

    Goodloe, Jeffrey M.; Crowder, Christopher J.; Arthur, Annette O.; Thomas, Stephen H.

    2012-01-01

    Purpose. There is a paucity of data regarding EMS stretcher-operation-related injuries. This study describes and analyzes characteristics associated with undesirable stretcher operations, with or without resultant injury in a large, urban EMS agency. Methods. In the study agency, all stretcher-related “misadventures” are required to be documented, regardless of whether injury results. All stretcher-related reports between July 1, 2009 and June 30, 2010 were queried in retrospective analysis, avoiding Hawthorne effect in stretcher operations. Results. During the year studied, 129,110 patients were transported. 23 stretcher incidents were reported (0.16 per 1,000 transports). No patient injury occurred. Four EMS providers sustained minor injuries. Among contributing aspects, the most common involved operations surrounding the stretcher-ambulance safety latch, 14/23 (60.9%). From a personnel injury prevention perspective, there exists a significant relationship between combative patients and crew injury related to stretcher operation, Fisher's exact test 0.048. Conclusions. In this large, urban EMS system, the incidence of injury related to stretcher operations in the one-year study period is markedly low, with few personnel injuries and no patient injuries incurred. Safety for EMS personnel and patients could be advanced by educational initiatives that highlight specific events and conditions contributing to stretcher-related adverse events. PMID:22606379

  8. 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.

  9. 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

  10. 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

  11. 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).

  12. 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.

  13. Developments in the EM-CCD camera for OGRE

    NASA Astrophysics Data System (ADS)

    Tutt, James H.; McEntaffer, Randall L.; DeRoo, Casey; Schultz, Ted; Miles, Drew M.; Zhang, William; Murray, Neil J.; Holland, Andrew D.; Cash, Webster; Rogers, Thomas; O'Dell, Steve; Gaskin, Jessica; Kolodziejczak, Jeff; Evagora, Anthony M.; Holland, Karen; Colebrook, David

    2014-07-01

    The Off-plane Grating Rocket Experiment (OGRE) is a sub-orbital rocket payload designed to advance the development of several emerging technologies for use on space missions. The payload consists of a high resolution soft X-ray spectrometer based around an optic made from precision cut and ground, single crystal silicon mirrors, a module of off-plane gratings and a camera array based around Electron Multiplying CCD (EM-CCD) technology. This paper gives an overview of OGRE with emphasis on the detector array; specifically this paper will address the reasons that EM-CCDs are the detector of choice and the advantages and disadvantages that this technology offers.

  14. Item Parameter Estimation via Marginal Maximum Likelihood and an EM Algorithm: A Didactic.

    ERIC Educational Resources Information Center

    Harwell, Michael R.; And Others

    1988-01-01

    The Bock and Aitkin Marginal Maximum Likelihood/EM (MML/EM) approach to item parameter estimation is an alternative to the classical joint maximum likelihood procedure of item response theory. This paper provides the essential mathematical details of a MML/EM solution and shows its use in obtaining consistent item parameter estimates. (TJH)

  15. 7 CFR 1945.35 - Special EM loan training.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...) PROGRAM REGULATIONS (CONTINUED) EMERGENCY Disaster Assistance-General § 1945.35 Special EM loan training. (a) General. When it is evident that a large number of farmers were affected by a widespread...

  16. Reservoir characterization and steam flood monitoring with crosshole EM

    SciTech Connect

    Wilt, M.; Torres-Verdin, C.

    1995-06-01

    Crosshole electromagnetic (EM) imaging is applied to reservoir characterization and steam flood monitoring in a central California oil field. Steam was injected into three stacked eastward-dipping, unconsolidated oil sands within the upper 200 m. The steam plume is expected to develop as an ellipse aligned with the regional northwest-southeast strike. EM measurements were made from two fiberglass-cased observation wells straddling the steam injector on a northeast-southwest profile using the LLNL frequency domain crosshole EM system. Field data were collected before the initiation of a steam drive to map the distribution of the oil sands and then 6 and 12 months later to monitor the progress of the steam chest. Resistivity images derived from the EM data before steam injection clearly delineate the distribution and dipping structure on the target oil sands. Difference images, from data collected before and after steam flooding, show resistivity changes that indicate that the steam chest has developed only in the deeper oil sands although steam injection occurred in all three sand layers.

  17. 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 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS-COOPERATIVE SERVICE, RURAL UTILITIES SERVICE, AND FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE...

  18. Functionalized anatomical models for EM-neuron Interaction modeling.

    PubMed

    Neufeld, Esra; Cassará, Antonino Mario; Montanaro, Hazael; Kuster, Niels; Kainz, Wolfgang

    2016-06-21

    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. PMID:27224508

  19. 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.

  20. 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…

  1. 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

  2. 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

  3. Nonlinear Smoothing and the EM Algorithm for Positive Integral Equations of the First Kind

    SciTech Connect

    Eggermont, P. P. B.

    1999-01-15

    We study a modification of the EMS algorithm in which each step of the EMS algorithm is preceded by a nonlinear smoothing step of the form Nf-exp(S*log f) , where S is the smoothing operator of the EMS algorithm. In the context of positive integral equations (a la positron emission tomography) the resulting algorithm is related to a convex minimization problem which always admits a unique smooth solution, in contrast to the unmodified maximum likelihood setup. The new algorithm has slightly stronger monotonicity properties than the original EM algorithm. This suggests that the modified EMS algorithm is actually an EM algorithm for the modified problem. The existence of a smooth solution to the modified maximum likelihood problem and the monotonicity together imply the strong convergence of the new algorithm. We also present some simulation results for the integral equation of stereology, which suggests that the new algorithm behaves roughly like the EMS algorithm.

  4. GPS computer navigators to shorten EMS response and transport times.

    PubMed

    Ota, F S; Muramatsu, R S; Yoshida, B H; Yamamoto, L G

    2001-05-01

    GPS (global positioning satellite system to determine one's position on earth) units have become inexpensive and compact. The purpose of this study is to assess the effectiveness of a GPS enhanced computer street map navigator to improve the ability of EMS drivers in an urban setting to locate their destination and shorten response times. For part I, residential addresses in the city were randomly selected from a telephone directory. Two driver/navigator teams were assigned to drive to the address adhering to speed limits. One team used a standard street map, whereas the other team used a GPS computer navigator. The travel time and distance of the runs were compared. For part II, the computer GPS navigator was placed on an ambulance to supplement their normal methods of navigation to find the address requesting EMS. After the run was completed, EMS providers were interviewed to determine their opinion of whether the GPS navigator was helpful. For part I the results showed that in the 29 initial test runs, comparing the GPS team versus the standard map team, the mean distances traveled were 8.7 versus 9.0 kilometers (not significant) and the mean travel times were 13.5 versus 14.6 minutes (P=.02), respectively. The GPS team arrived faster in 72% runs. For part II the results showed that most EMS providers surveyed noted that the GPS computer navigator enhanced their ability to find the destination and all EMS providers acknowledged that it would enhance their ability to find a destination in an area in which they were unfamiliar. These results suggest that a portable GPS computer navigator system is helpful and can enhance the ability of prehospital care providers to locate their destination. Because these units are accurate and inexpensive, GPS computer navigators may be a valuable tool in reducing pre-hospital transport times. PMID:11326345

  5. 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.

  6. Similarity-regulation of OS-EM for accelerated SPECT reconstruction.

    PubMed

    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. PMID:27206135

  7. 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.

  8. Service Discovery Framework Supported by EM Algorithm and Bayesian Classifier

    NASA Astrophysics Data System (ADS)

    Peng, Yanbin

    Service oriented computing has become the main stream research field nowadays. Meanwhile, machine learning is a promising AI technology which can enhance the performance of traditional algorithm. Therefore, aiming at solving service discovery problem, this paper imports Bayesian classifier to web service discovery framework, which can improve service querying speed. In this framework, services in service library become training set of Bayesian classifier, service query becomes a testing sample. Service matchmaking process can be executed in related service class, which has fewer services, thus can save time. Due to don't know the class of service in training set, EM algorithm is used to estimate prior probability and likelihood functions. Experiment results show that the EM algorithm and Bayesian classifier supported method outperforms other methods in time complexity.

  9. 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

  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. Environmental Restoration and Waste Management (EM) program: An introduction

    SciTech Connect

    Not Available

    1990-12-01

    This booklet introduces the reader to the mission and functions of a major new unit within the US Department of Energy (DOE): the Office of Environmental Restoration and Waste Management (EM). The Secretary of Energy established EM in November 1989, implementing a central purpose of DOE's first annual Environmental Restoration and Waste Management Five-Year Plan, which had appeared three months earlier. The contents of this booklet, and their arrangement, reflect the annual update of the Five-Year Plan. The Five-Year Plan supports DOE's strategy for meeting its 30-year compliance and cleanup goal. This strategy involves: focusing DOE's activities on eliminating or reducing known or recognized potential risks to worker and public health and the environment, containing or isolating, removing, or detoxifying onsite and offsite contamination, and developing technology to achieve DOE's environmental goals.

  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. EMS-STARS: Emergency Medical Services "Superuser" Transport Associations: An Adult Retrospective Study.

    PubMed

    Hall, M Kennedy; Raven, Maria C; Hall, Jane; Yeh, Clement; Allen, Elaine; Rodriguez, Robert M; Tangherlini, Niels L; Sporer, Karl A; Brown, John F

    2015-01-01

    Abstract Objective. Emergency medical services (EMS) "superusers" -those who use EMS services at extremely high rates -have not been well characterized. Recent interest in the small group of individuals who account for a disproportionate share of health-care expenditures has led to research on frequent users of emergency departments and other health services, but little research has been done regarding those who use EMS services. To inform policy and intervention implementation, we undertook a descriptive analysis of EMS superusers in a large urban community. In this paper we compare EMS superusers to low, moderate, and high users to characterize factors contributing to EMS use. We also estimate the financial impact of EMS superusers. Methods. We conducted a retrospective cross-sectional study based on 1 year of data from an urban EMS system. Data for all EMS encounters with patients age ≥18 years were extracted from electronic records generated on scene by paramedics. We identified demographic and clinical variables associated with levels of EMS use. EMS users were characterized by the annual number of EMS encounters: low (1), moderate (2-4), high (5-14), and superusers (≥15). In addition, we performed a financial analysis using San Francisco Fire Department (SFFD) 2009 charge and reimbursement data. Results. A total of 31,462 adults generated 43,559 EMS ambulance encounters, which resulted in 39,107 transports (a 90% transport rate). Encounters for general medical reasons were common among moderate and high users and less frequent among superusers and low users, while alcohol use was exponentially correlated with encounter frequency. Superusers were significantly younger than moderate EMS users, and more likely to be male. The superuser group created a significantly higher financial burden/person than any other group, comprising 0.3% of the study population, but over 6% of annual EMS charges and reimbursements. Conclusions. In this retrospective study, adult

  14. 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.

  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. Volta phase plate cryo-EM of the small protein complex Prx3.

    PubMed

    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. PMID:26817416

  17. Standard errors for EM estimates in generalized linear models with random effects.

    PubMed

    Friedl, H; Kauermann, G

    2000-09-01

    A procedure is derived for computing standard errors of EM estimates in generalized linear models with random effects. Quadrature formulas are used to approximate the integrals in the EM algorithm, where two different approaches are pursued, i.e., Gauss-Hermite quadrature in the case of Gaussian random effects and nonparametric maximum likelihood estimation for an unspecified random effect distribution. An approximation of the expected Fisher information matrix is derived from an expansion of the EM estimating equations. This allows for inferential arguments based on EM estimates, as demonstrated by an example and simulations. PMID:10985213

  18. 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.

  19. 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.

  20. 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

  1. 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 .

  2. 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.

  3. 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

  4. A compulsator driven rapid-fire EM-gun

    SciTech Connect

    Pratap, S.B.; Bird, W.L.

    1984-03-01

    A compulsator-driven railgun is an attractive alternative to the homopolar generator-inductor-switch configuration, especially for repetitive duty. A conceptual design of a rapid-fire EM-gun system is presented. The generator is sized to accelerate a 0.08-kg projectile to 2 to 3 km/s at a 60 pulse-per-second repetition rate. Initial design parameters are discussed, and example current and velocity waveforms are given. The generator is discharged at the proper phase angle to provide a current zero just as the projectile exits the muzzle of the railgun.

  5. EM Modelling of RF Propagation Through Plasma Plumes

    NASA Astrophysics Data System (ADS)

    Pandolfo, L.; Bandinelli, M.; Araque Quijano, J. L.; Vecchi, G.; Pawlak, H.; Marliani, F.

    2012-05-01

    Electric propulsion is a commercially attractive solution for attitude and position control of geostationary satellites. Hall-effect ion thrusters generate a localized plasma flow in the surrounding of the satellite, whose impact on the communication system needs to be qualitatively and quantitatively assessed. An electromagnetic modelling tool has been developed and integrated into the Antenna Design Framework- ElectroMagnetic Satellite (ADF-EMS). The system is able to guide the user from the plume definition phases through plume installation and simulation. A validation activity has been carried out and the system has been applied to the plume modulation analysis of SGEO/Hispasat mission.

  6. Developing State and National Evaluation Infrastructures- Guidance for the Challenges and Opportunities of EM&V

    SciTech Connect

    Schiller, Steven R.; Goldman, Charles A.

    2011-06-24

    Evaluating the impacts and effectiveness of energy efficiency programs is likely to become increasingly important for state policymakers and program administrators given legislative mandates and regulatory goals and increasing reliance on energy efficiency as a resource. In this paper, we summarize three activities that the authors have conducted that highlight the expanded role of evaluation, measurement and verification (EM&V): a study that identified and analyzed challenges in improving and scaling up EM&V activities; a scoping study that identified issues involved in developing a national efficiency EM&V standard; and lessons learned from providing technical assistance on EM&V issues to states that are ramping up energy efficiency programs. The lessons learned are summarized in 13 EM&V issues that policy makers should address in each jurisdiction and which are listed and briefly described. The paper also discusses how improving the effectiveness and reliability of EM&V will require additional capacity building, better access to existing EM&V resources, new methods to address emerging issues and technologies, and perhaps foundational documents and approaches to improving the credibility and cross jurisdictional comparability of efficiency investments. Two of the potential foundational documents discussed are a national EM&V standard or resource guide and regional deemed savings and algorithm databases.

  7. EMS Instructor Training Program. National Standard Curriculum. Instructor Guide. Student Guide.

    ERIC Educational Resources Information Center

    National Highway Traffic Safety Administration (DOT), Washington, DC.

    This guide for teaching a course to prepare emergency medical service (EMS) trainers focuses on the skills necessary to present any of the Department of Transportation (DOT), National Highway Traffic Safety Administration (NHTSA) EMS courses. Course topics are as follows: (1) introduction; (2) instructor roles and responsibilities; (3) legal…

  8. 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.

  9. 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…

  10. The Relationship between the Bock-Aitkin Procedure and the EM Algorithm for IRT Model Estimation.

    ERIC Educational Resources Information Center

    Hsu, Yaowen; Ackerman, Terry A.; Fan, Meichu

    It has previously been shown that the Bock-Aitkin procedure (R. Bock and M. Aitkin, 1981) is an instance of the EM algorithm when trying to find the marginal maximum likelihood estimate for a discrete latent ability variable (latent trait). In this paper, it is shown that the Bock-Aitkin procedure is a numerical implementation of the EM algorithm…

  11. 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 (EM) algorithm to a global optimum of the marginal log likelihood function for unconstrained latent variable models with categorical indicators is presented. The sufficient conditions under which global convergence of the EM algorithm is attainable are provided in an information-theoretic context by…

  12. Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase

    PubMed Central

    Jia, Gengxiang; Liu, Xiaodong; Owen, Heather A.; Zhao, Dazhong

    2008-01-01

    Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction. PMID:18250314

  13. A Generalized Fast Frequency Sweep Algorithm for Coupled Circuit-EM Simulations

    SciTech Connect

    Rockway, J D; Champagne, N J; Sharpe, R M; Fasenfest, B

    2004-01-14

    Frequency domain techniques are popular for analyzing electromagnetics (EM) and coupled circuit-EM problems. These techniques, such as the method of moments (MoM) and the finite element method (FEM), are used to determine the response of the EM portion of the problem at a single frequency. Since only one frequency is solved at a time, it may take a long time to calculate the parameters for wideband devices. In this paper, a fast frequency sweep based on the Asymptotic Wave Expansion (AWE) method is developed and applied to generalized mixed circuit-EM problems. The AWE method, which was originally developed for lumped-load circuit simulations, has recently been shown to be effective at quasi-static and low frequency full-wave simulations. Here it is applied to a full-wave MoM solver, capable of solving for metals, dielectrics, and coupled circuit-EM problems.

  14. AVALIAÇÃO DA PRESENÇA DE ENDOSSIMBIONTES Cardinium em DIFERENTES ESPÉCIES DE ARTRÓPODES.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A presença de endossimbiontes do gênero Cardinium em alguns grupos de artrópodes foi recentemente relatada e relacionada com diversas alterações reprodutivas em seus hospedeiros, tais como feminilização de ácaros, partenogênese em parasitóides, incompatibilidade citoplasmática e aumento da fecundida...

  15. Aristarchus's <em>On the Sizes and Distances of the Sun and the Moonem>: Greek and Arabic Texts

    NASA Astrophysics Data System (ADS)

    Berggren, J. L.; Sidoli, N.

    2007-05-01

    In the 1920s, T. L. Heath pointed out that historians of mathematics have "given too little attention to Aristarchus". This is still true today. The Greek text of Aristarchus's On the Sizes and Distances of the Sun and the Moonem> has received little attention; the Arabic editions virtually none. Much of what this text has to tell us about ancient and medieval mathematics and the mathematical sciences has gone unnoticed. It should be taken as an important source for our understanding of the mathematical sciences of the early Hellenistic period.

  16. The EM fields in the Solid Generated by a Fault in a Porous Region

    NASA Astrophysics Data System (ADS)

    Ren, H.; Huang, Q.; Chen, X.

    2015-12-01

    Electrokinetic effect, as one of the most possible generation mechanisms of the seismo-electromagnetic phenomenons associated with natural earthquakes, has interested many researchers. Besides, it is also considered as a potential tool for the water/oil exploration. Recently, we numerically investigated the electromagnetic (EM) fields due to the electrokinetic effect in mixed layered model. The mixed model comprises not only porous layers but also solid layers. We firstly tested a two-layer mixed model. The numerical results show that, in addition to the radiation EM fields, another kind of evanescent EM fields can be generated by the seismic waves arriving at the interface with incident angles greater the critical angle. The evanescent EM fields decay faster than the radiation EM fields when getting away from the interface. For the seismic frequency band, the evanescent EM fields in the solid are still measurable at a distance of, e.g., 2km to the interface. We then tested a eight-layer mixed model. The top and bottom layers are solid and the other layers are porous. A finite fault of 20x10km is located in the porous region. The focal depth is 8km. The applied source time function is a ramp fuction with an arise time of 0.8s. Point stacking method was used to compute the wave-fields caused by the finite fault. Our nuemrical results show that, this model can generate the EM fields before the arrival of seismic waves as well as the residual EM fields. Both the two kinds of EM fields have been observed in field observations. There is a possibility that the anomalous EM activities before big earthquakes may be caused by the fluid flow in the shallow Earth as a result of the stress changes.

  17. Review of selected oceanic EM/EO scattering problems

    NASA Astrophysics Data System (ADS)

    Haller, Merrick C.

    2010-02-01

    Electromagnetic and electro-optical (EM/EO) propagation and scattering in the ocean is of interest for a wide range of science problems. For example, the biological productivity of ocean waters through photochemical processes is governed by the vertical attenuation of solar radiation. Also, EO scattering theory is the primary basis for determining biogeochemical parameters (e.g. phytoplankton, suspended sediments, and dissolved matter) from the water leaving optical radiance. In addition, EO scattering from suspended sediments and bubbles is the limiting factor for active lidar systems used to map the sea bottom. This work will review specific applications of EO/EM scattering theory with regard to the influence of bubbles and droplets on remote sensing in the nearshore ocean. The current state of understanding concerning models and applications for optical scattering from bubbles in the water column as well as microwave scattering from water droplets produced by breaking waves at the ocean surface will be discussed as well as future research directions.

  18. The Search for Gravitational Wave EM Counterparts with Swift

    NASA Astrophysics Data System (ADS)

    Kennea, Jamie; Evans, Phil; Swift GW follow-up Team

    2016-04-01

    We present the plan to search for electromagnetic counterparts of Gravitational Waves (GWs) discovered during the current and upcoming runs of the LIGO and Virgo detectors. As we enter a period where the sensitivity of the current generation of GW detectors approaches a high probability of the first detection of a real GW signal, confirmation of the reality of these triggers will be greatly improved if an EM counterpart can be found. Swift’s ability to rapidly respond to high priority target-of-opportunity observations, it’s multi-wavelength capabilities and low overhead observing make it a seemingly ideal follow-up facility. However comparing the size of the expected GW error regions with the fields of view of the Swift XRT and UVOT telescopes, we find that covering the large GW error regions would require a unreasonably large number of pointings. We present our method of meeting this challenge, by both reducing the problem using Galaxy targeting, and by operating Swift in an entirely new way in order to cover the still large number of fields needed to chase down the EM counterpart before it disappears.

  19. Chapter 19: HVAC Controls (DDC/EMS/BAS) Evaluation Protocol

    SciTech Connect

    Romberger, J.

    2014-11-01

    The HVAC Controls Evaluation Protocol is designed to address evaluation issues for direct digital controls/energy management systems/building automation systems (DDC/EMS/BAS) that are installed to control heating, ventilation, and air-conditioning (HVAC) equipment in commercial and institutional buildings. (This chapter refers to the DDC/EMS/BAS measure as HVAC controls.) This protocol may also be applicable to industrial facilities such as clean rooms and labs, which have either significant HVAC equipment or spaces requiring special environmental conditions. This protocol addresses only HVAC-related equipment and the energy savings estimation methods associated with installing such control systems as an energy efficiency measure. The affected equipment includes: Air-side equipment (air handlers, direct expansion systems, furnaces, other heating- and cooling-related devices, terminal air distribution equipment, and fans); Central plant equipment (chillers, cooling towers, boilers, and pumps). These controls may also operate or affect other end uses, such as lighting, domestic hot water, irrigation systems, and life safety systems such as fire alarms and other security systems. Considerable nonenergy benefits, such as maintenance scheduling, system component troubleshooting, equipment failure alarms, and increased equipment lifetime, may also be associated with these systems. When connected to building utility meters, these systems can also be valuable demand-limiting control tools. However, this protocol does not evaluate any of these additional capabilities and benefits.

  20. Contactless ultrasonic treatment of melts using EM induction

    NASA Astrophysics Data System (ADS)

    Bojarevics, V.; Djambazov, G.; Lebon, G. S. B.; Pericleous, K. A.

    2015-06-01

    Ultrasound Treatment (UT) is commonly used in light alloys during solidification to refine microstructure, or disperse immersed particles. A sonotrode probe introduced into the melt generates sound waves that are strong enough to produce cavitation of dissolved gases. The same method cannot be used in high temperature melts, or for highly reactive alloys, due to probe erosion and melt contamination. An alternative, contactless method of generating sound waves is proposed and investigated theoretically in this paper, using electromagnetic (EM) induction. In addition to strong vibration, the EM induction currents generate strong stirring in the melt that aids distribution of the UT effect to large volumes of material. In a typical application, the same induction coil surrounding the crucible used to melt the alloy may be adopted for UT with suitable frequency tuning. Alternatively - or in addition - a top coil may be used. For industrial use, instead of multiple sonotrodes as has been the practice in scaling up, modelling shows that one simply has to alter the coil geometry and current to suit. To reach sinusoidal pressure fluctuations suitable for cavitation it may be necessary to tune the induction coil frequency for resonance, given the crucible dimensions.

  1. Estudo em microondas do aprisionamento e precipitação de elétrons em explosões solares

    NASA Astrophysics Data System (ADS)

    Rosal, A. C.; Costa, J. E. R.

    2003-08-01

    Uma explosão solar é uma variação rápida e intensa do brilho que ocorre nas chamadas regiões ativas da atmosfera, constituídas por um plasma magnetizado com intensa indução magnética. Os modelos de explosões solares atuais, discutidos na literatura, apresentam características de aprisionamento e precipitação de elétrons em ambientes magnéticos simplificados. Neste trabalho, nos propusemos a separar a emissão dos elétrons aprisionados da emissão dos elétrons em precipitação apenas a partir da emissão em microondas, melhorando portanto o controle sobre o conjunto de parâmetros inferidos. A emissão em microondas da população em precipitação é bastante fraca e portanto da nossa base de dados de 130 explosões observadas pelo Rádio Polarímetro de Nobeyama, em sete freqüências, apenas para 32 foi possível separar as duas componentes de emissão com uma boa razão sinal/ruído. A partir de estudos das escalas de tempo das emissões devidas à variação gradual da emissão no aprisionamento e da variação rápida da emissão dos elétrons em precipitação foi possível obter a separação utilizando um filtro temporal nas emissões resultantes. Em nossa análise destas explosões estudamos os espectros girossincrotrônicos da emissão gradual, a qual associamos provir do topo dos arcos magnéticos e da emissão de variação rápida associada aos elétrons em precipitação. Estes espectros foram calculados e dos quais inferimos que a indução magnética efetiva do topo e dos pés foi em média, Btopo = 236 G e Bpés = 577 G, inferidas das freqüências de pico dos espectros em ntopo = 11,8 GHz e npés = 14,6 GHz com leve anisotropia (pequeno alargamento espectral). O índice espectral da distribuição não-térmica de elétrons d, inferido do índice espectral de fótons da emissão em regime opticamente fino, foi de dtopo = 3,3 e dpés = 3,9. Estes parâmetros são típicos da maioria das análises realizadas em ambiente único de

  2. The EM SSAB Annual Work Plan Process: Focusing Board Efforts and Resources - 13667

    SciTech Connect

    Young, Ralph

    2013-07-01

    One of the most daunting tasks for any new member of a local board of the Environmental Management Site Specific Advisory Board (EM SSAB) is to try to understand the scope of the clean-up activities going on at the site. In most cases, there are at least two or three major cleanup activities in progress as well as monitoring of past projects. When planning for future projects is added to the mix, the list of projects can be long. With the clean-up activities involving all major environmental media - air, water, soils, and groundwater, new EM SSAB members can find themselves totally overwhelmed and ineffective. Helping new members get over this initial hurdle is a major objective of EM and all local boards of the EM SSAB. Even as members start to understand the size and scope of the projects at a site, they can still be frustrated at the length of time it takes to see results and get projects completed. Many project and clean-up timelines for most of the sites go beyond 10 years, so it's not unusual for an EM SSAB member to see the completion of only 1 or 2 projects over the course of their 6-year term on the board. This paper explores the annual work planning process of the EM SSAB local boards, one tool that can be used to educate EM SSAB members into seeing the broader picture for the site. EM SSAB local work plans divide the site into projects focused on a specific environmental issue or media such as groundwater and/or waste disposal options. Projects are further broken down into smaller segments by highlighting major milestones. Using these metrics, local boards of the EM SSAB can start to quantify the effectiveness of the project in achieving the ultimate goal of site clean-up. These metrics can also trigger board advice and recommendations for EM. At the beginning of each fiscal year, the EM SSAB work plan provides a road map with quantifiable checkpoints for activities throughout the year. When the work plans are integrated with site-specific, enforceable

  3. Linear array implementation of the EM algorithm for PET image reconstruction

    SciTech Connect

    Rajan, K.; Patnaik, L.M.; Ramakrishna, J.

    1995-08-01

    The PET image reconstruction based on the EM algorithm has several attractive advantages over the conventional convolution back projection algorithms. However, the PET image reconstruction based on the EM algorithm is computationally burdensome for today`s single processor systems. In addition, a large memory is required for the storage of the image, projection data, and the probability matrix. Since the computations are easily divided into tasks executable in parallel, multiprocessor configurations are the ideal choice for fast execution of the EM algorithms. In tis study, the authors attempt to overcome these two problems by parallelizing the EM algorithm on a multiprocessor systems. The parallel EM algorithm on a linear array topology using the commercially available fast floating point digital signal processor (DSP) chips as the processing elements (PE`s) has been implemented. The performance of the EM algorithm on a 386/387 machine, IBM 6000 RISC workstation, and on the linear array system is discussed and compared. The results show that the computational speed performance of a linear array using 8 DSP chips as PE`s executing the EM image reconstruction algorithm is about 15.5 times better than that of the IBM 6000 RISC workstation. The novelty of the scheme is its simplicity. The linear array topology is expandable with a larger number of PE`s. The architecture is not dependant on the DSP chip chosen, and the substitution of the latest DSP chip is straightforward and could yield better speed performance.

  4. A new method for vitrifying samples for cryoEM.

    PubMed

    Razinkov, Ivan; Dandey, Venkata P; Wei, Hui; Zhang, Zhening; Melnekoff, David; Rice, William J; Wigge, Christoph; Potter, Clinton S; Carragher, Bridget

    2016-08-01

    Almost every aspect of cryo electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. We present results from a new device for preparing vitrified samples. The successful use of the device is coupled to a new "self-blotting" grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well defined thickness containing evenly distributed single particles. We believe that these methods will in the future result in a system for vitrifying grids that is completely automated. PMID:27288865

  5. Speech articulator measurements using low power EM-wave sensors

    SciTech Connect

    Holzrichter, J.F.; Burnett, G.C.; Ng, L.C.; Lea, W.A.

    1998-01-01

    Very low power electromagnetic (EM) wave sensors are being used to measure speech articulator motions as speech is produced. Glottal tissue oscillations, jaw, tongue, soft palate, and other organs have been measured. Previously, microwave imaging (e.g., using radar sensors) appears not to have been considered for such monitoring. Glottal tissue movements detected by radar sensors correlate well with those obtained by established laboratory techniques, and have been used to estimate a voiced excitation function for speech processing applications. The noninvasive access, coupled with the small size, low power, and high resolution of these new sensors, permit promising research and development applications in speech production, communication disorders, speech recognition and related topics. {copyright} {ital 1998 Acoustical Society of America.}

  6. EM Properties of Magnetic Minerals at RADAR Frequencies

    NASA Technical Reports Server (NTRS)

    Stillman, D. E.; Olhoeft, G. R.

    2005-01-01

    Previous missions to Mars have revealed that Mars surface is magnetic at DC frequency. Does this highly magnetic surface layer attenuate RADAR energy as it does in certain locations on Earth? It has been suggested that the active magnetic mineral on Mars is titanomaghemite and/or titanomagnetite. When titanium is incorporated into a maghemite or magnetite crystal, the Curie temperature can be significantly reduced. Mars has a wide range of daily temperature fluctuations (303K - 143K), which could allow for daily passes through the Curie temperature. Hence, the global dust layer on Mars could experience widely varying magnetic properties as a function of temperature, more specifically being ferromagnetic at night and paramagnetic during the day. Measurements of EM properties of magnetic minerals were made versus frequency and temperature (300K- 180K). Magnetic minerals and Martian analog samples were gathered from a number of different locations on Earth.

  7. Balloting motion of SLEKE launch packages in EM railguns

    NASA Astrophysics Data System (ADS)

    Chu, Szu H.

    1993-01-01

    This paper reports some balloting motion computational results of the SLEKE (Sabot Launched Electric-gun Kinetic Energy) launch packages, which are in their early stage of development. The computation model considers the effects of the EM (Lorentz) propulsion force, friction, air resistance, gravity, elastic forces, and clearance between the launch package and the barrel. The axial, normal and yaw displacement, velocity and acceleration; friction; deformations and forces at the interfacing points are computed. The results of this study indicate that the balloting force for SLEKE launch packages is on the order of the air drag force and that a uniformly distributed power source would be more desirable than sharp pulse current for electromagnetic railguns.

  8. Detection of karst structures using airborne EM and VLF

    SciTech Connect

    Beard, L.P. Nyquist, J.E.; Carpenter, P.J.

    1994-12-31

    Through the combined use of multi-frequency helicopter electromagnetic and VLF data, it is possible to detect and delineate a wide variety of karst structures and possibly to assess their interconnectedness. Multi-frequency EM Can detect karst features if some element of the structure is conductive. This conductive aspect may derive from thick, moist soils in the depression commonly associated with a doline, from conductive fluids in the cavity, or from conductive sediments in the cavity if these occupy a significant portion of it. Multiple loop configurations may also increase the likelihood of detecting karst features. Preliminary evidence indicates total field VLF measurements may be able to detect interconnected karst pathways, so long as the pathways are water or sediment filled. Neither technique can effectively detect dry, resistive air-filled cavities.

  9. Integrated EM & Thermal Simulations with Upgraded VORPAL Software

    SciTech Connect

    D.N. Smithe, D. Karipides, P. Stoltz, G. Cheng, H. Wang

    2011-03-01

    Nuclear physics accelerators are powered by microwaves which must travel in waveguides between room-temperature sources and the cryogenic accelerator structures. The ohmic heat load from the microwaves is affected by the temperature-dependent surface resistance and in turn affects the cryogenic thermal conduction problem. Integrated EM & thermal analysis of this difficult non-linear problem is now possible with the VORPAL finite-difference time-domain simulation tool. We highlight thermal benchmarking work with a complex HOM feed-through geometry, done in collaboration with researchers at the Thomas Jefferson National Accelerator Laboratory, and discuss upcoming design studies with this emerging tool. This work is part of an effort to generalize the VORPAL framework to include generalized PDE capabilities, for wider multi-physics capabilities in the accelerator, vacuum electronics, plasma processing and fusion R&D fields, and we will also discuss user interface and algorithmic upgrades which facilitate this emerging multiphysics capability.

  10. Selling management on the cost benefits of EMS programs

    SciTech Connect

    Beck, W.B.

    1999-07-01

    One of the persistent misconceptions by management is that environmental programs are always a cost to the business. Management tolerates this situation because they recognize the legal and other ethical considerations related to environmental programs, however, they still regard them as a cost and a necessary evil. This paper will present several ideas that can be put into practice by the environmental professionals to counter this management perception. These include the identification and use of macro scale and micro scale cost considerations in the environmental balance sheet to provide management with a different and perhaps more realistic view of the worth of the program. This cost accounting approach should be viewed as just one of the several systems that should make up the modern day environmental management systems (EMS) program.

  11. Making connections. Voice and data solutions for EMS.

    PubMed

    Careless, James; Erich, John

    2008-08-01

    Communications used to be so simple-1) grab the radio, 2) push and talk. Now we're besieged by a confusing assortment of technology and terms-wideband, broadband, VoIP, RoIP, ect.- and a constand thrumming imperative to achieve and improve and perfect some mystical state of full interoperability. Frankly, it can all be a bit much. With this supplement, we hope to clarify you options. We examine the importance of broadband for EMS, with its potential for larger data "pipes" between the hospital and the field; advances in the promising technologies of Voice over IP and Radio over IP; and how some systems are improving their interconnectedness and resulting operations. The jargon can be overwhelming, but the ideas are worth understanding. PMID:18814746

  12. EM modeling of RF drive in DTL tank 4

    SciTech Connect

    Kurennoy, Sergey S.

    2012-06-19

    A 3-D MicroWave Studio model for the RF drive in the LANSCE DTL tank 4 has been built. Both eigensolver and time-domain modeling are used to evaluate maximal fields in the drive module and RF coupling. The LANSCE DTL tank 4 has recently been experiencing RF problems, which may or may not be related to its replaced RF coupler. This situation stimulated a request by Dan Rees to provide EM modeling of the RF drive in the DTL tank 4 (T4). Jim O'Hara provided a CAD model that was imported into the CST Microwave Studio (MWS) and after some modifications became a part of a simplified MWS model of the T4 RF drive. This technical note describes the model and presents simulation results.

  13. TrakEM2 Software for Neural Circuit Reconstruction

    PubMed Central

    Cardona, Albert; Saalfeld, Stephan; Schindelin, Johannes; Arganda-Carreras, Ignacio; Preibisch, Stephan; Longair, Mark; Tomancak, Pavel; Hartenstein, Volker; Douglas, Rodney J.

    2012-01-01

    A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis. PMID:22723842

  14. TrakEM2 software for neural circuit reconstruction.

    PubMed

    Cardona, Albert; Saalfeld, Stephan; Schindelin, Johannes; Arganda-Carreras, Ignacio; Preibisch, Stephan; Longair, Mark; Tomancak, Pavel; Hartenstein, Volker; Douglas, Rodney J

    2012-01-01

    A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis. PMID:22723842

  15. Kaisen. EMS as theater of the streets. Part two.

    PubMed

    Dernocoeur, K; Taigman, M

    1991-03-01

    Passing an EMT or paramedic course is a considerable achievement, as you have had to acquire a huge and fascinating volume of medical knowledge. But, did the program teach you acting skills? Acting? Whoever said that EMS professionals need to know anything about acting? We submit that you do; prehospital workers can gain many unanticipated benefits by using various acting skills and tactics in the field. One such benefit is the avoidance of the "complacency trap," or the boredom that can develop if you adopt the viewpoint that one crisis is much like another. But, more importantly, you will be more effective at delivering prehospital care, and you'll have a lot more fun on the job. PMID:10110186

  16. Suspended-Patch Antenna With Inverted, EM-Coupled Feed

    NASA Technical Reports Server (NTRS)

    Simons, Rainee N.

    2004-01-01

    An improved suspended-patch antenna has been designed to operate at a frequency of about 23 GHz with linear polarization and to be one of four identical antennas in a rectangular array. The antenna includes a parasitic patch on top of a suspended dielectric superstrate, an active patch on top of a suspended dielectric substrate, a microstrip on the bottom of the dielectric substrate, and a ground plane. The microstrip, the ground plane, the airgap between them, and the dielectric substrate together constitute a transmission line that has an impedance of 50 Ohm and is electromagnetically (EM) coupled to the active patch. The parasitic patch is, in turn, excited by the active patch. The microstrip feed is characterized as inverted because the microstrip is on the bottom of the substrate, whereas microstrips are usually placed on the tops of dielectric substrates

  17. User's guide for the PWR LOCA analysis capability of the WRAP-EM system

    SciTech Connect

    Beranek, F; Gregory, M V

    1980-02-01

    The Water Reactor Analysis Package (WRAP) has been expanded to provide the capability to analyze loss-of-coolant accidents (LOCAs) in both pressurized water reactors (PWRs) and boiling water reactors (BWRs) by using evaluation models (EMs). The input specifications for modules in the WRAP-EM system are presented in this document along with the JOSHUA input templates. This document, along with the WRAP user's guide, provides a step-by-step procedure for setting up a PWR data base for the WRAP-EM system. 12 refs.

  18. Time-Lapse inversion of EM Tomography data for polymer-injected hydrocarbon reservoirs

    NASA Astrophysics Data System (ADS)

    Cheon, Seiwook; Park, Chanho; Nam, Myung Jin; Son, Jeong-Sul

    2015-04-01

    Polymer flooding is a method to increase the production of hydrocarbon reservoir by injecting polymer solution into the reservoir. For a study on the monitoring fluid variation within the reservoir, we first make analysis on seismic- and electromagnetic (EM)- tomography responses for seismic and electrical-resistivity rock physics models (RPMs) of the reservoir considering polymer fluid. Constructing RPMs are dependent on not only geologic characteristics of reservoir but also reservoir parameters such as fluid-type, fluid saturation, pressure and temperature. When making RPM for monitoring analysis, we assume the geology does not changes while reservoir parameters change to affect responses of seismic and EM tomography data. Specifically when constructing electrical-resistivity RPM, we consider three different types of hydrocarbon reservoirs, which are clean sand, shaly sand, sand-shale lamination, while considering two different types of waters (fresh water and salt water) to make 2wt% polymer solution. To compute time lapse EM and seismic tomography responses for corresponding RPMs of polymer-injected reservoirs, we used 2.5D finite element EM modeling algorithm and staggered-grid finite difference elastic modeling algorithm, respectively. Comparison between sensitivities of seismic and EM tomography to polymer injection confirms that EM tomography is more sensitivity to the polymer injection. For the evaluation of the potential of EM tomography to monitor polymer flooding, this study subsequently develops an efficient time-lapse EM tomography inversion algorithm based on the 2.5D EM tomography modeling. Using the inversion algorithm, we inverted the time-lapse EM tomography data to construct true resistivity models of polymer-injected reservoirs and analyze differences between them. From the time-lapse inversion results, we can observe the differences in time lapse responses between using fresh water and salt water have been decreased in the inverted time

  19. Integration of artificial intelligence applications in the EMS: Issues and solutions

    SciTech Connect

    Bann, J.; Irisarri, G.; Kirschen, D.; Miller, B.; Mokhtari, S.

    1996-02-01

    This paper discusses the issues which must be addressed when integrating Artificial Intelligence (AI) and, in particular, expert system applications in an Energy Management System (EMS) environment. It is argued that these issues can be resolved by creating an environment which supports all the interfaces between the Artificial Intelligence (AI) applications and the EMS. This environment should also be responsible for maintaining a model of the power system common to all the AI applications. Once this environment has been created, AI applications can be easily plugged into the EMS. The design of such an environment is described and case studies of its implementation are provided to illustrate its flexibility.

  20. Stochastic EM algorithm for nonlinear state estimation with model uncertainties

    NASA Astrophysics Data System (ADS)

    Zia, Amin; Kirubarajan, Thiagalingam; Reilly, James P.; Shirani, Shahram

    2004-01-01

    In most solutions to state estimation problems like, for example, target tracking, it is generally assumed that the state evolution and measurement models are known a priori. The model parameters include process and measurement matrices or functions as well as the corresponding noise statistics. However, there are situations where the model parameters are not known a priori or are known only partially (i.e., with some uncertainty). Moreover, there are situations where the measurement is biased. In these scenarios, standard estimation algorithms like the Kalman filter and the extended Kalman Filter (EKF), which assume perfect knowledge of the model parameters, are not accurate. In this paper, the problem with uncertain model parameters is considered as a special case of maximum likelihood estimation with incomplete-data, for which a standard solution called the expectation-maximization (EM) algorithm exists. In this paper a new extension to the EM algorithm is proposed to solve the more general problem of joint state estimation and model parameter identification for nonlinear systems with possibly non-Gaussian noise. In the expectation (E) step, it is shown that the best variational distribution over the state variables is the conditional posterior distribution of states given all the available measurements and inputs. Therefore, a particular type of particle filter is used to estimate and update the posterior distribution. In the maximization (M) step the nonlinear measurement process parameters are approximated using a nonlinear regression method for adjusting the parameters of a mixture of Gaussians (MofG). The proposed algorithm is used to solve a nonlinear bearing-only tracking problem similar to the one reported recently with uncertain measurement process. It is shown that the algorithm is capable of accurately tracking the state vector while identifying the unknown measurement dynamics. Simulation results show the advantages of the new technique over standard

  1. Stochastic EM algorithm for nonlinear state estimation with model uncertainties

    NASA Astrophysics Data System (ADS)

    Zia, Amin; Kirubarajan, Thiagalingam; Reilly, James P.; Shirani, Shahram

    2003-12-01

    In most solutions to state estimation problems like, for example, target tracking, it is generally assumed that the state evolution and measurement models are known a priori. The model parameters include process and measurement matrices or functions as well as the corresponding noise statistics. However, there are situations where the model parameters are not known a priori or are known only partially (i.e., with some uncertainty). Moreover, there are situations where the measurement is biased. In these scenarios, standard estimation algorithms like the Kalman filter and the extended Kalman Filter (EKF), which assume perfect knowledge of the model parameters, are not accurate. In this paper, the problem with uncertain model parameters is considered as a special case of maximum likelihood estimation with incomplete-data, for which a standard solution called the expectation-maximization (EM) algorithm exists. In this paper a new extension to the EM algorithm is proposed to solve the more general problem of joint state estimation and model parameter identification for nonlinear systems with possibly non-Gaussian noise. In the expectation (E) step, it is shown that the best variational distribution over the state variables is the conditional posterior distribution of states given all the available measurements and inputs. Therefore, a particular type of particle filter is used to estimate and update the posterior distribution. In the maximization (M) step the nonlinear measurement process parameters are approximated using a nonlinear regression method for adjusting the parameters of a mixture of Gaussians (MofG). The proposed algorithm is used to solve a nonlinear bearing-only tracking problem similar to the one reported recently with uncertain measurement process. It is shown that the algorithm is capable of accurately tracking the state vector while identifying the unknown measurement dynamics. Simulation results show the advantages of the new technique over standard

  2. DOE-EM-45 PACKAGING OPERATIONS AND MAINTENANCE COURSE

    SciTech Connect

    Watkins, R.; England, J.

    2010-05-28

    Savannah River National Laboratory - Savannah River Packaging Technology (SRNL-SRPT) delivered the inaugural offering of the Packaging Operations and Maintenance Course for DOE-EM-45's Packaging Certification Program (PCP) at the University of South Carolina Aiken on September 1 and 2, 2009. Twenty-nine students registered, attended, and completed this training. The DOE-EM-45 Packaging Certification Program (PCP) sponsored the presentation of a new training course, Packaging Maintenance and Operations, on September 1-2, 2009 at the University of South Carolina Aiken (USC-Aiken) campus in Aiken, SC. The premier offering of the course was developed and presented by the Savannah River National Laboratory, and attended by twenty-nine students across the DOE, NNSA and private industry. This training informed package users of the requirements associated with handling shipping containers at a facility (user) level and provided a basic overview of the requirements typically outlined in Safety Analysis Report for Packaging (SARP) Chapters 1, 7, and 8. The course taught packaging personnel about the regulatory nature of SARPs to help reduce associated and often costly packaging errors. Some of the topics covered were package contents, loading, unloading, storage, torque requirements, maintaining records, how to handle abnormal conditions, lessons learned, leakage testing (including demonstration), and replacement parts. The target audience for this course was facility operations personnel, facility maintenance personnel, and field quality assurance personnel who are directly involved in the handling of shipping containers. The training also aimed at writers of SARP Chapters 1, 7, and 8, package designers, and anyone else involved in radioactive material packaging and transportation safety. Student feedback and critiques of the training were very positive. SRNL will offer the course again at USC Aiken in September 2010.

  3. Vibrational symmetry classification and torsional tunneling splitting patterns in G6(EM), G12, and G36(EM) molecules

    NASA Astrophysics Data System (ADS)

    Lattanzi, F.; di Lauro, C.

    It is shown that the torsional splitting patterns in methanol-like molecules, with the excitation of small amplitude vibrational modes in the methyl group, are determined by mechanisms that can be formulated in an almost identical fashion to that for ethane-like molecules. This is achieved by treating ethane-like molecules by the internal axis method (IAM) and methanol-like molecules by the principal axis method (PAM) or rho-axis method (RAM). Using the extended molecular groups G6(EM) or C6v(M) for methanol and G36(EM) for ethane, vibrations perpendicular to the internal rotation axis are conveniently described by modes of higher degeneracy (E for methanol and Gs for ethane) in the absence of coupling of top and frame. Head-tail coupling operators, except the cos-type barrier terms, lower the degeneracy, causing vibrational splittings. Coupled vibrational pairs with torsional splitting patterns that we call 'regular' (pure A1, A2 pairs for methanol and pure E1d, E2d pairs for ethane) or 'inverted' (pure B1, B2 pairs for methanol and pure E1s, E2s pairs for ethane) can be formed as limit cases. Actual splitting patterns occur between the above limits, and are basically determined by torsional Coriolis coupling, which can tune more or less to resonance pairs of uncoupled basis levels linked by specific head-tail coupling operators. The inversion of torsional splitting patterns, observed in perpendicular vibrational modes of the methyl group of methanol, can be predicted by these theoretical considerations. Similar considerations apply to molecules of G12 symmetry.

  4. Aquisição de Estreptococos Mutans e Desenvolvimento de Cárie Dental em Primogênitos

    PubMed Central

    NOCE, Erica; RUBIRA, Cassia Maria Fischer; da Silva ROSA, Odila Pereira; da SILVA, Salete Moura Bonifácio; BRETZ, Walter Antonio

    2011-01-01

    Objetivo Avaliar o momento de aquisição de estreptococos mutans (EM), desenvolvimento de cárie dental e as variáveis a eles associadas no decorrer de 23 meses, em primogênitos de famílias de baixo nível socioeconômico, desde os sete meses de idade. Método A amostra foi selecionada com base em mães densamente colonizadas por EM, incluindo todos os membros de 14 famílias que conviviam na mesma casa. Foram envolvidos no estudo 14 mães, pais e primogênitos e 8 parentes, na maioria avós. Exames clínicos e radiográficos iniciais determinaram os índices de cárie e condição periodontal dos adultos. Contagens de EM foram feitas em todos os adultos nas duas primeiras visitas. Nas crianças foram avaliados os níveis de EM, o número de dentes e de cáries, em quatro visitas. Resultados A prevalência de EM nos adultos foi alta, estando ausente em apenas um dos pais. EM foram detectados em 1, 2, 3 e 10 crianças, respectivamente nas visitas #1, 2, 3 e 4. A cárie dental foi detectada em apenas três crianças na última visita (aos 30 meses de idade), as quais apresentaram escores de EM significantemente maiores que as crianças sem cárie, na mesma visita. Conclusão Exclusivamente a condição social de baixa renda e mães densamente colonizadas por EM não são sinônimo de colonização precoce e alta atividade de cárie em crianças cuidadas em casa. O desenvolvimento de cárie está significantemente associado a escores elevados de EM nas crianças. PMID:22022218

  5. EMDataBank.org: unified data resource for CryoEM

    PubMed Central

    Lawson, Catherine L.; Baker, Matthew L.; Best, Christoph; Bi, Chunxiao; Dougherty, Matthew; Feng, Powei; van Ginkel, Glen; Devkota, Batsal; Lagerstedt, Ingvar; Ludtke, Steven J.; Newman, Richard H.; Oldfield, Tom J.; Rees, Ian; Sahni, Gaurav; Sala, Raul; Velankar, Sameer; Warren, Joe; Westbrook, John D.; Henrick, Kim; Kleywegt, Gerard J.; Berman, Helen M.; Chiu, Wah

    2011-01-01

    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 global ‘one-stop shop’ resource for deposition and retrieval of cryoEM maps, models and associated metadata. The resource unifies public access to the two major archives containing EM-based structural data: 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:20935055

  6. Analysis of the electrochemistry of hemes with Ems spanning 800 mV

    PubMed Central

    Zheng, Zhong; Gunner, M. R.

    2009-01-01

    The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of Ems with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b-, and c-type, with bis-His, His-Met, and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental Ems range over 800 mV from −350 mV in cytochrome c3 to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated Ems are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental Ems is 0.73 (R2 = 0.90), showing the method accounts for 73% of the observed Em range. Adding a +160 mV correction to the His-Met c-type hemes yields a slope of 0.97 (R2 = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and Ems shows both the lowest and highest potential hemes are c-type, whereas the b-type hemes are found in the middle Em range. In solution, bis-His ligation lowers the Em by ≈205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His, and His-Met ligated b-type hemes all cluster about Ems which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, whereas the high potential His-Met c-type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the Em, has been suggested to be a major factor in

  7. Volta phase plate cryo-EM of the small protein complex Prx3

    PubMed Central

    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. PMID:26817416

  8. 7. PHOTOCOPY, ELEVATIONS FOR E.M. BARRACKS, N.C.O. AND OFFICERS QUARTERS. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. PHOTOCOPY, ELEVATIONS FOR E.M. BARRACKS, N.C.O. AND OFFICERS QUARTERS. - NIKE Missile Base SL-40, Barracks No. 1, North end of base, southest of Basketball Court & northwest of Barracks No. 2, Hecker, Monroe County, IL

  9. The development of cryo-EM into a mainstream structural biology technique

    PubMed Central

    Nogales, Eva

    2016-01-01

    Single-particle cryo-electron microscopy (cryo-EM) has emerged over the last two decades as a technique capable of studying challenging systems that otherwise defy structural characterization. Recent technical advances have resulted in a ‘quantum leap’ in applicability, throughput and achievable resolution that has gained this technique worldwide attention. Here I discuss some of the major historical landmarks in the development of the cryo-EM field, ultimately leading to its present success. PMID:27110629

  10. The front lines of domestic violence. Training model for rural EMS personnel.

    PubMed

    Hall, Marcia; Becker, Vanessa

    2002-09-01

    1. Domestic violence is a major public health problem requiring committed, coordinated community response. 2. Domestic violence is a significant cause of morbidity and mortality for women and children in the United States. 3. EMS personnel play a frontline role in the critical response and prevention of domestic violence. 4. EMS education and training are requisite for safe, effective responses to domestic violence in rural communities. PMID:12235968

  11. Space resolution obtained with a highly segmented SCIFI e.m. calorimeter

    NASA Astrophysics Data System (ADS)

    Bertino, M.; Bini, C.; De Pedis, D.; De Zorzi, G.; Diambrini Palazzi, G.; Di Cosimo, G.; Di Domenico, A.; Gauzzi, P.; Zanello, D.

    1992-05-01

    During the setting up of the LEP-5 experiment, we tested a longitudinal SCIFI e.m. calorimeter, having a module cross area 25 × 25 mm 2 and 12.5 × 12.5 mm 2 for large and small modules respectively. The results were obtained with 10 and 50 GeV electrons, and concern the impact point resolution and the transverse distribution of the e.m. shower energy inside the calorimeter.

  12. Estudo comparativo entre estrelas centrais de nebulosas planetárias deficientes em hidrogênio

    NASA Astrophysics Data System (ADS)

    Marcolino, W. L. F.; de Araújo, F. X.

    2003-08-01

    Apresentamos neste trabalho o resultado de um estudo das principais características espectrais das estrelas centrais de nebulosas planetárias (ECNP) deficientes em hidrogênio. A origem e a evolução dessas estrelas ainda constitui um problema em aberto na evolução estelar. Geralmente esses objetos são divididos em [WCE], [WCL] e [WELS]. Os tipos [WCE] e [WCL] apresentam um espectro típico de uma estrela Wolf-Rayet carbonada de população I e as [WELS] apresentam linhas fracas de carbono e oxigênio em emissão. Existem evidências que apontam a seguinte sequência evolutiva : [WCL] = > [WCE] = > [WELS] = > PG 1159 (pré anã-branca). No entanto, tal cenário apresenta falhas como por exemplo a falta de ECNP entre os tipos [WCL] e [WCE]. Baseados em uma amostra de 24 objetos obtida no telescópio de 1.52m em La Silla, Chile (acordo ESO/ON), ao longo do ano 2000, apresentamos os resultados da comparação das larguras equivalentes de diversas linhas relevantes entre os tipos [WCL], [WCE] e [WELS]. Verificamos que nossos dados estão de acordo com a sequência evolutiva. Baseado nas linhas de C IV, conseguimos dividir pela primeira vez as [WELS] em dois grupos principais. Além disso, os dados reforçam a afirmação de que as [WCE] são as estrelas que possuem a maior temperatura entre as ECNP deficientes em hidrogênio. Discutimos ainda, a escassez de dados disponíveis na literatura e a necessidade da obtenção de parametros físicos para estes objetos.

  13. Effects of Loaded Squat Exercise with and without Application of Superimposed EMS on Physical Performance.

    PubMed

    Wirtz, Nicolas; Zinner, Christoph; Doermann, Ulrike; Kleinoeder, Heinz; Mester, Joachim

    2016-03-01

    The aim of the present study was to investigate the effects of a multiple set squat exercise training intervention with superimposed electromyostimulation (EMS) on strength and power, sprint and jump performance. Twenty athletes from different disciplines participated and were divided into two groups: strength training (S) or strength training with superimposed EMS (S+E). Both groups completed the same training program twice a week over a six week period consisting of four sets of the 10 repetition maximum of back squats. Additionally, the S+E group had EMS superimposed to the squat exercise with simultaneous stimulation of leg and trunk muscles. EMS intensity was adjusted to 70% of individual pain threshold to ensure dynamic movement. Strength and power of different muscle groups, sprint, and vertical jump performance were assessed one week before (pre), one week after (post) and three weeks (re) following the training period. Both groups showed improvements in leg press strength and power, countermovement and squat jump performance and pendulum sprint (p < 0.05), with no changes for linear sprint. Differences between groups were only evident at the leg curl machine with greater improvements for the S+E group (p < 0.05). Common squat exercise training and squat exercise with superimposed EMS improves maximum strength and power, as well as jumping abilities in athletes from different disciplines. The greater improvements in strength performance of leg curl muscles caused by superimposed EMS with improvements in strength of antagonistic hamstrings in the S+E group are suggesting the potential of EMS to unloaded (antagonistic) muscle groups. Key pointsSimilar strength adaptations occurred after a 6 week 10 RM back squat exercise program with superimposed EMS (S+E) and 10 RM back squat exercise (S) alone.Specific adaptations for S+E at the leg curl muscles were evident.S and S+E improved SJ, CMJ and pendulum sprint performance.No improvement occurred in linear sprint

  14. Effects of Loaded Squat Exercise with and without Application of Superimposed EMS on Physical Performance

    PubMed Central

    Wirtz, Nicolas; Zinner, Christoph; Doermann, Ulrike; Kleinoeder, Heinz; Mester, Joachim

    2016-01-01

    The aim of the present study was to investigate the effects of a multiple set squat exercise training intervention with superimposed electromyostimulation (EMS) on strength and power, sprint and jump performance. Twenty athletes from different disciplines participated and were divided into two groups: strength training (S) or strength training with superimposed EMS (S+E). Both groups completed the same training program twice a week over a six week period consisting of four sets of the 10 repetition maximum of back squats. Additionally, the S+E group had EMS superimposed to the squat exercise with simultaneous stimulation of leg and trunk muscles. EMS intensity was adjusted to 70% of individual pain threshold to ensure dynamic movement. Strength and power of different muscle groups, sprint, and vertical jump performance were assessed one week before (pre), one week after (post) and three weeks (re) following the training period. Both groups showed improvements in leg press strength and power, countermovement and squat jump performance and pendulum sprint (p < 0.05), with no changes for linear sprint. Differences between groups were only evident at the leg curl machine with greater improvements for the S+E group (p < 0.05). Common squat exercise training and squat exercise with superimposed EMS improves maximum strength and power, as well as jumping abilities in athletes from different disciplines. The greater improvements in strength performance of leg curl muscles caused by superimposed EMS with improvements in strength of antagonistic hamstrings in the S+E group are suggesting the potential of EMS to unloaded (antagonistic) muscle groups. Key points Similar strength adaptations occurred after a 6 week 10 RM back squat exercise program with superimposed EMS (S+E) and 10 RM back squat exercise (S) alone. Specific adaptations for S+E at the leg curl muscles were evident. S and S+E improved SJ, CMJ and pendulum sprint performance. No improvement occurred in linear

  15. ModEM: A modular system for inversion of elecgtromagnetic geophysical data

    NASA Astrophysics Data System (ADS)

    Egbert, G. D.; Kelbert, A.; Meqbel, N.; Weng, A.

    2010-12-01

    We have developed a modular system of computer codes for inversion of electromagnetic (EM) geophysical data (ModEM). ModEM allows for rapid adaptation of inversion algorithms developed for one purpose (e.g., three-dimensional magnetotellurics (MT)) to other EM problems (e.g., controlled source EM). The modular approach can also simplify maintenance of the inversion code, as well as development of new capabilities -- e.g., allowing for new data types such as the inter-site transfer functions in MT, or modifying model regularization. Basic data objects (model parameters, solution vectors, data vectors) are treated as abstract data types, with a standard set of methods developed for each class, including creation and destruction, and, as appropriate, linear algebra or other vector space methods. Operators required for gradient computations are developed as mappings between these basic object classes. Only these abstract data objects and mappings are manipulated by higher level Jacobian and inversion routines, with no reference to the problem specific details required for a specific EM method, or for the numerical implementation of the forward solver. Required problem-specific components are instantiated at the lowest levels of the system, with details hidden from generic top-level routines by an interface layer. Parallelization using MPI has been implemented at the top level, and is thus applicable to any problem embedded in ModEM. To illustrate the flexibility of the system, we consider applications to two- and three-dimensional MT inversion, as well as simple controlled source EM problems.

  16. The Soils and Groundwater – EM-20 S&T Roadmap Quality Assurance Project Plan

    SciTech Connect

    Fix, N. J.

    2008-02-11

    The Soils and Groundwater – EM-20 Science and Technology Roadmap Project is a U.S. Department of Energy, Office of Environmental Management-funded initiative designed to develop new methods, strategies and technology for characterizing, modeling, remediating, and monitoring soils and groundwater contaminated with metals, radionuclides, and chlorinated organics. This Quality Assurance Project Plan provides the quality assurance requirements and processes that will be followed by EM-20 Roadmap Project staff.

  17. Pre-flight risk assessment in emergency medical service (EMS) helicopters

    NASA Technical Reports Server (NTRS)

    Shively, Robert J.

    1990-01-01

    A preflight risk assessment system (SAFE) was developed at NASA-Ames Research Center for civil EMS operations to assist pilots in making a decision objectively to accept or decline a mission. The ability of the SAFE system to predict risk profiles was examined at an EMS operator. Results of this field study showed that the usefulness of SAFE was largely dependent on the type of mission flown.

  18. Single-particle cryo-EM data acquisition by using direct electron detection camera.

    PubMed

    Wu, Shenping; Armache, Jean-Paul; Cheng, Yifan

    2016-02-01

    Recent advances in single-particle electron cryo-microscopy (cryo-EM) were largely facilitated by the application of direct electron detection cameras. These cameras feature not only a significant improvement in detective quantum efficiency but also a high frame rate that enables images to be acquired as 'movies' made of stacks of many frames. In this review, we discuss how the applications of direct electron detection cameras in cryo-EM have changed the way the data are acquired. PMID:26546989

  19. Using Molecular Simulation to Model High-Resolution Cryo-EM Reconstructions.

    PubMed

    Kirmizialtin, Serdal; Loerke, Justus; Behrmann, Elmar; Spahn, Christian M T; Sanbonmatsu, Karissa Y

    2015-01-01

    An explosion of new data from high-resolution cryo-electron microscopy (cryo-EM) studies has produced a large number of data sets for many species of ribosomes in various functional states over the past few years. While many methods exist to produce structural models for lower resolution cryo-EM reconstructions, high-resolution reconstructions are often modeled using crystallographic techniques and extensive manual intervention. Here, we present an automated fitting technique for high-resolution cryo-EM data sets that produces all-atom models highly consistent with the EM density. Using a molecular dynamics approach, atomic positions are optimized with a potential that includes the cross-correlation coefficient between the structural model and the cryo-EM electron density, as well as a biasing potential preserving the stereochemistry and secondary structure of the biomolecule. Specifically, we use a hybrid structure-based/ab initio molecular dynamics potential to extend molecular dynamics fitting. In addition, we find that simulated annealing integration, as opposed to straightforward molecular dynamics integration, significantly improves performance. We obtain atomistic models of the human ribosome consistent with high-resolution cryo-EM reconstructions of the human ribosome. Automated methods such as these have the potential to produce atomistic models for a large number of ribosome complexes simultaneously that can be subsequently refined manually. PMID:26068751

  20. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

    PubMed

    Coscia, Francesca; Estrozi, Leandro F; Hans, Fabienne; Malet, Hélène; Noirclerc-Savoye, Marjolaine; Schoehn, Guy; Petosa, Carlo

    2016-01-01

    Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis. PMID:27485862