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Sample records for agent yersinia pestis

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. [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

  16. 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.

  17. 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...

  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. 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...

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  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. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. [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

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

    PubMed Central

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

    2015-01-01

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

  6. 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

  7. 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

  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. 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

  10. 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

  11. 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

  12. 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...

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. [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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  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. 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

  10. 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.

  11. 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

  12. 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...

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. [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

  2. 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

  3. 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

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  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. 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

  1. 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

  2. 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

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

    PubMed

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

    2016-04-01

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

  4. 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

  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. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

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

    PubMed

    Mize, Erica L; Britten, Hugh B

    2016-02-01

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

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

    SciTech Connect

    Sample, A.K.

    1987-01-01

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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. [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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

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

    PubMed Central

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

    2013-01-01

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

  15. 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

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

    PubMed

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

    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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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).

  5. 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.

  6. 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

  7. [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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

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

    PubMed Central

    Bland, David M.; Hinnebusch, B. Joseph

    2016-01-01

    Background The cat flea, Ctenocephalides felis, is prevalent worldwide, will parasitize animal reservoirs of plague, and is associated with human habitations in known plague foci. Despite its pervasiveness, limited information is available about the cat flea’s competence as a vector for Yersinia pestis. It is generally considered to be a poor vector, based on studies examining early-phase transmission during the first week after infection, but transmission potential by the biofilm-dependent proventricular-blocking mechanism has never been systematically evaluated. In this study, we assessed the vector competence of cat fleas by both mechanisms. Because the feeding behavior of cat fleas differs markedly from important rat flea vectors, we also examined the influence of feeding behavior on transmission dynamics. Methodology/Principal Findings Groups of cat fleas were infected with Y. pestis and subsequently provided access to sterile blood meals twice-weekly, 5 times per week, or daily for 4 weeks and monitored for infection, the development of proventricular biofilm and blockage, mortality, and the ability to transmit. In cat fleas allowed prolonged, daily access to blood meals, mimicking their natural feeding behavior, Y. pestis did not efficiently colonize the digestive tract and could only be transmitted during the first week after infection. In contrast, cat fleas that were fed intermittently, mimicking the feeding behavior of the efficient vector Xenopsylla cheopis, could become blocked and regularly transmitted Y. pestis for 3–4 weeks by the biofilm-mediated mechanism, but early-phase transmission was not detected. Conclusions The normal feeding behavior of C. felis, more than an intrinsic resistance to infection or blockage by Y. pestis, limits its vector competence. Rapid turnover of midgut contents results in bacterial clearance and disruption of biofilm accumulation in the proventriculus. Anatomical features of the cat flea foregut may also restrict

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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...

  1. 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...

  2. 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

  3. 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

  4. 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

  5. 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.

  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. 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

  8. 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

  9. 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

  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. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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...

  19. 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

  20. 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

  1. 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

  2. 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.

  3. [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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  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. 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

  9. 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

  10. 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

  11. 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

  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. 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

  15. 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

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

    PubMed Central

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

    2016-01-01

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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  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. 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.

  4. 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

  5. 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.

  6. 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

  7. N-hydroxybenzimidazole Inhibitors of the Transcription Factor LcrF in Yersinia: Novel Anti-Virulence Agents

    PubMed Central

    Kim, Oak K.; Garrity-Ryan, Lynne K.; Bartlett, Victoria J.; Grier, Mark C.; Verma, Atul K.; Medjanis, Gabriel; Donatelli, Janice E.; Macone, Ann B.; Tanaka, S. Ken; Levy, Stuart B.; Alekshun, Michael N.

    2009-01-01

    LcrF, a Multiple Adaptational Response (MAR) transcription factor, regulates virulence in Yersinia pestis and Yersinia pseudotuberculosis. In a search for small molecule inhibitors of LcrF, an acrylic amide series of N-hydroxybenzimidazoles was synthesized, and the SAR (structure-activity relationship) was examined. Selected test compounds demonstrated inhibitory activity in a primary cell-free LcrF-DNA binding assay as well as in a secondary whole cell assay (Type III secretion system dependent Y. pseudotuberculosis cytotoxicity assay). The inhibitors exhibited no measurable antibacterial activity in vitro, confirming that they do not target bacterial growth. These results demonstrate that N-hydroxybenzimidazole inhibitors, exemplified by 14, 22 and 36, are effective anti-virulence agents, and have the potential to prevent infections caused by Yersinia spp. PMID:19708663

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Behavior of Avirulent Yersinia pestis in Liquid Whole Egg as Affected by Antimicrobials and Thermal Pasteurization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia spp. is a psychrotrophic bacterium that can grow at temperatures as low as minus two degrees Celsius, and is known to contaminate shell eggs in the United States and shell eggs and liquid egg in South America. A study was performed to determine the thermal sensitivity of avirulent Yersinia...

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. [Biological and physico-chemical properties of Yersinia pseudotuberculosis bacterial culture having the fra-operon Yersinia pestis].

    PubMed

    Byvalov, A A; Gavrilov, K E; Krupin, V V; Chebotarev, E V; Zheludkova, E V; Drubkov, V I; Smirnov, A E; Mal'kov, V N; Dupiasheva, T Iu; Pechenkin, D V; Bondarev, V P

    2008-01-01

    The biological and physico-chemical properties of cultures of two isogenous recombinant variants of Yersinia pseudotuberculosis were studied. The cell genomes of the cultures are distinguished from one another only by the presence or by the absence of the fra-operon, which is a determined attribute of the plague microbe capsule-forming process. The expression of the attribute is amplified by rising the microbial biomass cultivation temperature and stimulates the decrease in the viability of the bacteria and adaptation potential in vitro. In the warm-blooded owner organism the microbes of the capsule-forming recombinant variant are characterized by the greater residual pathogenicity and immunogenic ability to the experimental plague of the laboratory animals as compared to the reference-variant cells. These specific features could be explained by more expressed colonizing ability of the capsule-forming microbes provided by owner cells' stability to the phagocyte process. PMID:18368776

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  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. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. [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

  5. 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

  6. 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

  7. 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.

  8. 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

  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. 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

  11. Yersinia--flea interactions and the evolution of the arthropod-borne transmission route of plague.

    PubMed

    Chouikha, Iman; Hinnebusch, B Joseph

    2012-06-01

    Yersinia pestis, the causative agent of plague, is unique among the enteric group of Gram-negative bacteria in relying on a blood-feeding insect for transmission. The Yersinia-flea interactions that enable plague transmission cycles have had profound historical consequences as manifested by human plague pandemics. The arthropod-borne transmission route was a radical ecologic change from the food-borne and water-borne transmission route of Yersinia pseudotuberculosis, from which Y. pestis diverged only within the last 20000 years. Thus, the interactions of Y. pestis with its flea vector that lead to colonization and successful transmission are the result of a recent evolutionary adaptation that required relatively few genetic changes. These changes from the Y. pseudotuberculosis progenitor included loss of insecticidal activity, increased resistance to antibacterial factors in the flea midgut, and extending Yersinia biofilm-forming ability to the flea host environment. PMID:22406208

  12. 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

  13. 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

  14. Loss of a Biofilm-Inhibiting Glycosyl Hydrolase during the Emergence of Yersinia pestis▿

    PubMed Central

    Erickson, David L.; Jarrett, Clayton O.; Callison, Julie A.; Fischer, Elizabeth R.; Hinnebusch, B. Joseph

    2008-01-01

    Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-β-1,6-N-acetyl-d-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved β-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission. PMID:18931111

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

    PubMed

    McGillick, Brian E; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency. PMID:26818694

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  17. 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

  18. 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

  19. 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

  20. [Ecological regularities of the existence of pathogenic Yersinia in soil ecosystems].

    PubMed

    Breneva, N V; Maramovich, A S; Klimov, V T

    2005-01-01

    In this review the data on the ecology of pathogenic Yersinia in soil ecosystems, based on prolonged observations, were analyzed and summarized. In contrast to saprophytic species, ubiquitously spread in nature, pathogenic representatives of the genus Yersinia occurred only in the soil of natural foci and of these, Y. pestis were found only in the soil of burrows of the main carriers. The complex of abiotic and biotic factors (temperature, humidity, chemical composition, interactions in biocenosis) which determined the possibility of the existence of Yersinia in the soil environment and the preservation of their pathogenic properties was considered. Special attention was paid to their geno-phenotypic variability as the main factor of the adaptation of the causative agents of plague, pseudotuberculosis and intestinal yersiniosis in the environment. PMID:16438385

  1. 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

  2. [Sympatric Speciation of the Plague Microbe Yersinia pestis: Monohostal Specialization in the Host-Parasite Marmot-Flea (Marmota sibirica-Oropsylla silantiewi) System].

    PubMed

    Suntsov, V V

    2016-01-01

    An ecological scenario of the origin of the plague microbe that is interpreted in the light of modern Darwinism (synthetic theory of evolution) is presented. It is shown that the plague microbe emerged from a clone of the psychrophilic saprozoonotic pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan time, 22000-15000 years ago, in the monohostal Mongolian marmot (Marmota sibirica)-flea (Oropsylla silantiewi) host-parasite system. It was noted that the evolutionary process described corresponds to the sympatric form of speciation by transition ofthe clone of migrant founders to a new, already-existing ecological niche. It was established that monohostal specialization of the plague microbe was made possible due to heterothermia (5-37 degrees C) of marmots in the hibernation period. The factors of the speciation process--isolation, the struggle for existence, and natural selection--were analyzed. PMID:27396172

  3. Coregulation of host-adapted metabolism and virulence by pathogenic yersiniae

    PubMed Central

    Heroven, Ann Kathrin; Dersch, Petra

    2014-01-01

    Deciphering the principles how pathogenic bacteria adapt their metabolism to a specific host microenvironment is critical for understanding bacterial pathogenesis. The enteric pathogenic Yersinia species Yersinia pseudotuberculosis and Yersinia enterocolitica and the causative agent of plague, Yersinia pestis, are able to survive in a large variety of environmental reservoirs (e.g., soil, plants, insects) as well as warm-blooded animals (e.g., rodents, pigs, humans) with a particular preference for lymphatic tissues. In order to manage rapidly changing environmental conditions and interbacterial competition, Yersinia senses the nutritional composition during the course of an infection by special molecular devices, integrates this information and adapts its metabolism accordingly. In addition, nutrient availability has an impact on expression of virulence genes in response to C-sources, demonstrating a tight link between the pathogenicity of yersiniae and utilization of nutrients. Recent studies revealed that global regulatory factors such as the cAMP receptor protein (Crp) and the carbon storage regulator (Csr) system are part of a large network of transcriptional and posttranscriptional control strategies adjusting metabolic changes and virulence in response to temperature, ion and nutrient availability. Gained knowledge about the specific metabolic requirements and the correlation between metabolic and virulence gene expression that enable efficient host colonization led to the identification of new potential antimicrobial targets. PMID:25368845

  4. Coregulation of host-adapted metabolism and virulence by pathogenic yersiniae.

    PubMed

    Heroven, Ann Kathrin; Dersch, Petra

    2014-01-01

    Deciphering the principles how pathogenic bacteria adapt their metabolism to a specific host microenvironment is critical for understanding bacterial pathogenesis. The enteric pathogenic Yersinia species Yersinia pseudotuberculosis and Yersinia enterocolitica and the causative agent of plague, Yersinia pestis, are able to survive in a large variety of environmental reservoirs (e.g., soil, plants, insects) as well as warm-blooded animals (e.g., rodents, pigs, humans) with a particular preference for lymphatic tissues. In order to manage rapidly changing environmental conditions and interbacterial competition, Yersinia senses the nutritional composition during the course of an infection by special molecular devices, integrates this information and adapts its metabolism accordingly. In addition, nutrient availability has an impact on expression of virulence genes in response to C-sources, demonstrating a tight link between the pathogenicity of yersiniae and utilization of nutrients. Recent studies revealed that global regulatory factors such as the cAMP receptor protein (Crp) and the carbon storage regulator (Csr) system are part of a large network of transcriptional and posttranscriptional control strategies adjusting metabolic changes and virulence in response to temperature, ion and nutrient availability. Gained knowledge about the specific metabolic requirements and the correlation between metabolic and virulence gene expression that enable efficient host colonization led to the identification of new potential antimicrobial targets. PMID:25368845

  5. [A personal view of the history of the genus Yersinia].

    PubMed

    Mollaret, H H

    1987-01-01

    The first recorded experience Australia had of the genus Yersinia was the arrival in 1889 of a French expedition led by Pasteur's nephew, Dr. Adrien Loir. At that time Australia was in the grips of an epidemic of rabbits, and Loir's purpose was to eradicate the rabbits by means of fowl plague (Pasteurella multocida). Sadly, bureaucratic and political obstacles prevailed, and Loir was never granted permission to release his biological control agent. Alexander Yersin had been tempted to join Loir's expedition, but elected in the end to travel to Hong Kong, where he discovered the plague bacillus. Had he gone to Australia, we might not now be speaking of the genus Yersinia... Historically, Yersinia pestis has affected not only world history but literature as well. In Shakespeare's Romeo and Juliet, the tragic denouement can be attributed directly to the consequences of the Great Plague. In times of plague, cities closed their gates to travellers, and houses their doors and windows. Thus Laurence's explanatory letter was prevented from reaching Romeo, who returned to take his life beside the drugged (but living) body of his beloved. Not only was the contemporary literature from which Shakespeare drew inspiration full of references to the plague, but he himself had experienced the social effects of the plague at first hand. The recent rejection of the name Y. pseudotuberculosis var. pestis in favour of Y. pestis is fitting, not simply on the grounds of preventing confusion - after all, Y. pseudotuberculosis can be an equally lethal pathogen. However, a review of the epidemiology for Y. pestis since the First Pandemic in the 6th Century AD lends support to Devignat's hypothesis that Y. pseudotuberculosis evolved from Y. pestis, rather than vice versa. This probably occurred in Europe shortly before the Second Pandemic, and the new mutant spread slowly through the European rodent population, immunising the carriers against plague. In other parts of the world which

  6. Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

    SciTech Connect

    Phan, Jason; Tropea, Joseph E.; Waugh, David S.

    2010-11-16

    Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure of a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.

  7. Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis.

    PubMed

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Niedzialkowski, Pawel; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

    2015-07-30

    YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from -2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

  8. Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

    PubMed Central

    Kuban-Jankowska, Alicja; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

    2015-01-01

    YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

  9. 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.

  10. Whole-Genome Yersinia sp. Assemblies from 10 Diverse Strains.

    PubMed

    Daligault, H E; Davenport, K W; Minogue, T D; Bishop-Lilly, K A; Broomall, S M; Bruce, D C; Chain, P S; Coyne, S R; Frey, K G; Gibbons, H S; Jaissle, J; Koroleva, G I; Ladner, J T; Lo, C-C; Munk, C; Palacios, G F; Redden, C L; Rosenzweig, C N; Scholz, M B; Johnson, S L

    2014-01-01

    Yersinia spp. are animal pathogens, some of which cause human disease. We sequenced 10 Yersinia isolates (from six species: Yersinia enterocolitica, Y. fredericksenii, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, and Y. ruckeri) to high-quality draft or complete status. The genomes range in size from 3.77 to 4.94 Mbp. PMID:25342679

  11. Whole-Genome Yersinia sp. Assemblies from 10 Diverse Strains

    PubMed Central

    Daligault, H. E.; Davenport, K. W.; Minogue, T. D.; Bishop-Lilly, K. A.; Broomall, S. M.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Frey, K. G.; Gibbons, H. S.; Jaissle, J.; Koroleva, G. I.; Ladner, J. T.; Lo, C.-C.; Munk, C.; Palacios, G. F.; Redden, C. L.; Rosenzweig, C. N.; Scholz, M. B.

    2014-01-01

    Yersinia spp. are animal pathogens, some of which cause human disease. We sequenced 10 Yersinia isolates (from six species: Yersinia enterocolitica, Y. fredericksenii, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, and Y. ruckeri) to high-quality draft or complete status. The genomes range in size from 3.77 to 4.94 Mbp. PMID:25342679

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. The Origin of a Killer Revealed by Bronze Age Yersinia Genomes.

    PubMed

    Nelson-Sathi, Shijulal; Martin, William F

    2015-11-11

    Bubonic plaque is caused by Yersinia pestis, a deadly pathogen that left deep scars in human history. Rasmussen et al. (2015) have now retrieved Y. pestis genomes from 2,800- to 5,000-year-old human teeth, shedding new light on origins of the strain that brought Black Death to Europe 670 years ago. PMID:26567502

  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. [Yersinia uveitis].

    PubMed

    Lang, G K; Knapp, W; Völcker, H E

    1983-02-01

    A 13 year-old boy was admitted with a unilateral acute fibrinous iritis accompanied by a pauciarticular arthritis which had been preceded by a febrile lower urinary tract infection. The diagnosis of a Yersinia enterocolitica infection was established by significant titers of agglutinating antibodies vs. the serotypes O-I (=0:3). The differential diagnosis of the disease included infections with salmonella, shigella, campylobacter, chlamydiae and metastatic bacterial and mycotic infections as well as rheumatic diseases. Repeated observation of Yersinia enterocolitica in our uveitis patients during the last couple of years suggests that Yersinia enterocolitica is another pathogen causing acute uveitis. The clinical significance of Yersinia enterocolitica infections in ophthalmology will have to be clarified by further specific investigations. PMID:6843029

  19. Transmission of flea-borne zoonotic agents.

    PubMed

    Eisen, Rebecca J; Gage, Kenneth L

    2012-01-01

    Flea-borne zoonoses such as plague (Yersinia pestis) and murine typhus (Rickettsia typhi) caused significant numbers of human cases in the past and remain a public health concern. Other flea-borne human pathogens have emerged recently (e.g., Bartonella henselae, Rickettsia felis), and their mechanisms of transmission and impact on human health are not fully understood. Our review focuses on the ecology and epidemiology of the flea-borne bacterial zoonoses mentioned above with an emphasis on recent advancements in our understanding of how these organisms are transmitted by fleas, maintained in zoonotic cycles, and transmitted to humans. Emphasis is given to plague because of the considerable number of studies generated during the first decade of the twenty-first century that arose, in part, because of renewed interest in potential agents of bioterrorism, including Y. pestis. PMID:21888520

  20. 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.

  1. Yersinia enterocolitica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The detection of plasmid-bearing (pYV) human pathogenic strains of Yersinia enterocolitica depends on the expression of various pYV-associated virulence characteristics. However, diagnostic techniques based on pYV encoded phenotypes have limited reliability due to the unstable nature of pYV. Two r...

  2. Crystal structure of the Yersinia protein-tyrosine phosphatase YopH complexed with a specific small molecule inhibitor.

    PubMed

    Sun, Jin-Peng; Wu, Li; Fedorov, Alexander A; Almo, Steven C; Zhang, Zhong-Yin

    2003-08-29

    The pathogenic bacteria 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. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a Ki value of 25 microM for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-A resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors. PMID:12810712

  3. Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8.

    PubMed Central

    Miyahara, M; Maruyama, T; Wake, A; Mise, K

    1988-01-01

    The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica. Images PMID:2833162

  4. Detection of biological warfare agents using the polymerase chain reaction. Final report, June-August 1991

    SciTech Connect

    Mann, B.J.

    1992-09-01

    The detection of biological warfare agents is an important mission for the U.S. Army. This report explores the feasibility of using the polymerase chain reaction as a means of rapid detection of biological warfare agents. Two levels of detection are proposed. The first level is group specific detection, using primers derived from 16S rDNA sequences, to detect various groups of pathogenic bacteria. The second level is species-specific detection using primers derived from DNA sequences, unique to each pathogenic organism targeted for detection. Specific examples of Vibrio cholerae, Francisella tularensis, Yersinia pestis, Staphylococcus aureus, and Bacillus anthracis are described.

  5. The genus Yersinia

    SciTech Connect

    Prpic, J.K.; Davey, R.B.

    1987-01-01

    This book contains papers presented at the Fourth International symposium on Yersinia. The topics covered include: Cloning and use of Vwa plasmid DNA as gene probes for virulent Yersiniae; Studies on the role of virulence determinants of Yersinia enterocolitica in gnotobiotic piglets; and significance of specific IgA antibodies in infections due to Yersinia enterocolitica and their complications.

  6. 'Add, stir and reduce': Yersinia spp. as model bacteria for pathogen evolution.

    PubMed

    McNally, Alan; Thomson, Nicholas R; Reuter, Sandra; Wren, Brendan W

    2016-02-15

    Pathogenic species in the Yersinia genus have historically been targets for research aimed at understanding how bacteria evolve into mammalian pathogens. The advent of large-scale population genomic studies has greatly accelerated the progress in this field, and Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica have once again acted as model organisms to help shape our understanding of the evolutionary processes involved in pathogenesis. In this Review, we highlight the gene gain, gene loss and genome rearrangement events that have been identified by genomic studies in pathogenic Yersinia species, and we discuss how these findings are changing our understanding of pathogen evolution. Finally, as these traits are also found in the genomes of other species in the Enterobacteriaceae, we suggest that they provide a blueprint for the evolution of enteropathogenic bacteria. PMID:26876035

  7. Fast, sensitive point of care electrochemical molecular system for point mutation and select agent detection.

    PubMed

    MacLeod, J A; Nemeth, A C; Dicke, W C; Wang, D; Manalili Wheeler, S; Hannis, J C; Collier, G B; Drader, J J

    2016-07-01

    Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges. We describe a prototype device for performing a molecular diagnostics test for clinical and biodefense samples in 16 minutes using a prototype capable of an 8 minute PCR reaction, followed by hybridization and detection on an electrochemical microarray based on the i-STAT® system. We used human buccal swabs for hemochromatosis testing including in-device DNA extraction. Additional clinical and biodefense samples included influenza A and bacterial select agents Bacillus anthracis, Yersinia pestis and Francisella tularensis. PMID:27280174

  8. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  9. Structure and function of cytidine monophosphate kinase from Yersinia pseudotuberculosis, essential for virulence but not for survival

    PubMed Central

    Walker, Nicola J.; Clark, Elizabeth A.; Ford, Donna C.; Bullifent, Helen L.; McAlister, Erin V.; Duffield, Melanie L.; Acharya, K. Ravi; Oyston, Petra C. F.

    2012-01-01

    The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use. PMID:23271832

  10. 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

  11. Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

    PubMed Central

    Schubert, S.; Fischer, D.; Heesemann, J.

    1999-01-01

    Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae. PMID:10515929

  12. Ferric enterochelin transport in Yersinia enterocolitica: molecular and evolutionary aspects.

    PubMed

    Schubert, S; Fischer, D; Heesemann, J

    1999-10-01

    Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae. PMID:10515929

  13. 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

  14. [Molecular mechanisms of the plague pathogenic agent interaction with invertebrates].

    PubMed

    Kutyrev, V V; Eroshenko, G A; Popov, N V; Vidiaeva, N A; Konnov, N P

    2009-01-01

    Microbe Russian Anti-Plague Research Institute, Saratov, Russia The literature data and experimental results of the authors on the molecular basis of plague agent interaction with invertebrates are discussed. The details of the plague agent life cycle, its genome organization, and molecular genetic mechanisms of its survival in flea vector and on the nematode cuticule are discussed. The experimental data about the ability to form biofilms at abiotic and biotic surfaces in the Yersinia pestis strains of the main and non-main subspecies are presented. Mechanisms of horizontal and vertical transmission of plague agent are considered. The suggestion about participation of the new member in the complex parasitic biocenosis (nematode, vector parasite) is put forward. PMID:20050160

  15. 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

  16. Yersinia type III effectors perturb host innate immune responses

    PubMed Central

    Pha, Khavong; Navarro, Lorena

    2016-01-01

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  17. Yersinia type III effectors perturb host innate immune responses.

    PubMed

    Pha, Khavong; Navarro, Lorena

    2016-02-26

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  18. Complete genome sequence of Yersinia ruckeri str. CSF007-82, etiologic agent of enteric redmouth disease in salmonid fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present the complete, closed and finished chromosomal and extra-chromosomal genome sequences of Y. ruckeri strain CSF007-82, etiologic agent of enteric red mouth disease in salmonid fish. The chromosome is 3,799,036 bp with a G+C content of 47.5% and encodes 3,530 predicted CDS, 7 ribosomal opero...

  19. Crystal structure of the Yersinia type III secretion protein YscE

    SciTech Connect

    Phan, Jason; Austin, Brian P.; Waugh, David S.

    2010-12-06

    The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

  20. Understanding Virulence in the Brucellae and Francisellae: Towards Efficacious Treatments for Two Potential Biothreat Agents

    SciTech Connect

    Rasley, A; Parsons, D A; El-Etr, S; Roux, C; Tsolis, R

    2009-12-30

    Francisella tularensis, Yersinia pestis and Brucellae species are highly infectious pathogens classified as select agents by the Centers for Disease Control and Prevention (CDC) with the potential for use in bioterrorism attacks. These organisms are known to be facultative intracellular pathogens that preferentially infect human monocytes. As such, understanding how the host responds to infection with these organisms is paramount in detecting and combating human disease. We have compared the ability of fully virulent strains of each pathogen and their non-pathogenic near neighbors to enter and survive inside the human monocytic cell line THP-1 and have quantified the cellular response to infection with the goal of identifying both unique and common host response patterns. We expanded the scope of these studies to include experiments with pathogenic and non-pathogenic strains of Y. pestis, the causative agent of plague. Nonpathogenic strains of each organism were impaired in their ability to survive intracellularly compared with their pathogenic counterparts. Furthermore, infection of THP-1 cells with pathogenic strains of Y. pestis and F. tularensis resulted in marked increases in the secretion of the inflammatory chemokines IL-8, RANTES, and MIP-1{beta}. In contrast, B. melitensis infection failed to elicit any significant increases in a panel of cytokines tested. These differences may underscore distinct strategies in pathogenic mechanisms employed by these pathogens.

  1. Yersinia Type III Secretion System Master Regulator LcrF.

    PubMed

    Schwiesow, Leah; Lam, Hanh; Dersch, Petra; Auerbuch, Victoria

    2016-02-01

    Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

  2. Yersinia Type III Secretion System Master Regulator LcrF

    PubMed Central

    Schwiesow, Leah; Lam, Hanh

    2015-01-01

    Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

  3. 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

  4. Role of the Yersinia YopJ protein in suppressing interleukin-8 secretion by human polymorphonuclear leukocytes.

    PubMed

    Spinner, Justin L; Hasenkrug, Aaron M; Shannon, Jeffrey G; Kobayashi, Scott D; Hinnebusch, B Joseph

    2016-01-01

    Polymorphonuclear leukocytes, in addition to their direct bactericidal activities, produce cytokines involved in the activation and regulation of the innate and adaptive immune response to infection. In this study we evaluated the cytokine response of human PMNs following incubation with the pathogenic Yersinia species. Yersinia pestis strains with the pCD1 virulence plasmid, which encodes cytotoxic Yop proteins that are translocated into host cells, stimulated little or no cytokine production compared to pCD1-negative strains. In particular, PMNs incubated with pCD1-negative Y. pestis secreted 1000-fold higher levels of interleukin-8 (IL-8 or CXCL8), a proinflammatory chemokine important for PMN recruitment and activation. Deletion of yopE, -H, -T, -M or ypkA had no effect on pCD1-dependent inhibition, whereas deletion of yopJ resulted in significantly increased IL-8 production. Like Y. pestis, the enteropathogenic Yersinia species inhibited IL-8 secretion by PMNs, and strains lacking the virulence plasmid induced high levels of IL-8. Our results show that virulence plasmid-encoded effector Yops, particularly YopJ, prevent IL-8 secretion by human PMNs. Suppression of the chemotactic IL-8 response by Y. pestis may contribute to the delayed PMN recruitment to the infected lymph node that typifies bubonic plague. PMID:26361732

  5. Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

    PubMed Central

    Ahn, Yong-Yeol; Lee, Deok-Sun; Burd, Henry; Blank, William; Kapatral, Vinayak

    2014-01-01

    The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents. PMID:24454817

  6. Yersinia philomiragia sp. n., a new member of the Pasteurella group of bacteria, naturally pathogenic for the muskrat (Ondatra zibethica)

    USGS Publications Warehouse

    Jensen, W.I.; Owen, C.R.; Jellison, W.L.

    1969-01-01

    A bacterium experimentally pathogenic for muskrats (Ondatra zibethica), white mice, mountain voles (Microtus montanus), and deer mice (Peromyscus maniculatus) was isolated from the tissues of a sick muskrat captured on the Bear River Migratory Bird Refuge (Brigham City, Utah) and from four surface water samples collected within 15 miles of that point. In culture, the cells are chiefly coccoid, but in the tissues of muskrats and voles they resemble the bizarre forms of Yersinia pestis, except for their smaller size. The characteristics of the organism are described and the name Yersinia philomiragia sp. n. is proposed.

  7. Prevention and treatment of bacterial diseases caused by bacterial bioterrorism threat agents.

    PubMed

    Greenfield, Ronald A; Bronze, Michael S

    2003-10-01

    There is general consensus that the bacterial agents or products most likely to be used as weapons of mass destruction are Bacillus anthracis, Yersinia pestis, Francisella tularensis and the neurotoxin of Clostridium botulinum. Modern supportive and antimicrobial therapy for inhalational anthrax is associated with a 45% mortality rate, reinforcing the need for better adjunctive therapy and prevention strategies. Pneumonic plague is highly contagious, difficult to recognize and is frequently fatal. Therefore, the development of vaccines against this agent is crucial. Although tularemia is associated with low mortality, the highly infectious nature of aerosolized F. tularensis poses a substantive threat that is best met by vaccine development. Safer antitoxins and a vaccine are required to meet the threat of the use of botulinum toxin as a weapon of mass destruction. In this article, the current status of research in these areas is reviewed. PMID:14554016

  8. Genome Wide Search for Biomarkers to Diagnose Yersinia Infections.

    PubMed

    Kalia, Vipin Chandra; Kumar, Prasun

    2015-12-01

    Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4-6 base cutters). Yersinia species have 6-7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences-carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications. PMID:26543261

  9. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for....food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of several virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding...

  10. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for...food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding (dark-v...

  11. High-Throughput Screening (HTS) by NMR Guided Identification of Novel Agents Targeting the Protein Docking Domain of YopH.

    PubMed

    Bottini, Angel; Wu, Bainan; Barile, Elisa; De, Surya K; Leone, Marilisa; Pellecchia, Maurizio

    2016-04-19

    Recently we described a novel approach, named high-throughput screening (HTS) by NMR that allows the identification, from large combinatorial peptide libraries, of potent and selective peptide mimetics against a given target. Here, we deployed the "HTS by NMR" approach for the design of novel peptoid sequences targeting the N-terminal domain of Yersinia outer protein H (YopH-NT), a bacterial toxin essential for the virulence of Yersinia pestis. We aimed at disrupting the protein-protein interactions between YopH-NT and its cellular substrates, with the goal of inhibiting indirectly YopH enzymatic function. These studies resulted in a novel agent of sequence Ac-F-pY-cPG-d-P-NH2 (pY=phosphotyrosine; cPG=cyclopentyl glycine) with a Kd value against YopH-NT of 310 nm. We demonstrated that such a pharmacological inhibitor of YopH-NT results in the inhibition of the dephosphorylation by full-length YopH of a cellular substrate. Hence, potentially this agent represents a valuable stepping stone for the development of novel therapeutics against Yersinia infections. The data reported further demonstrate the utility of the HTS by NMR approach in deriving novel peptide mimetics targeting protein-protein interactions. PMID:26592695

  12. Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

    PubMed

    Souza, Roberto A; Frazão, Miliane R; Almeida, Alzira M P; Falcão, Juliana P

    2015-08-01

    The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species. PMID:25980404

  13. Yersinia lead SUMO attack.

    PubMed

    Cornelis, G R; Denecker, G

    2001-01-01

    Little is known about the mechanism by which Yops, proteins that Yersinia inject into the cytosol of macrophage, cause downregulation of the inflammatory response and diseases such as the plague. Now it appears that Yops are the first bacterial member of a new family of ubiquitin-like proteases. PMID:11135606

  14. Autonomous Detection of Aerosolized Biological Agents by Multiplexed Immunoassay with PCR Confirmation

    SciTech Connect

    Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Setlur, U S; Smith, S M; Gutierrez, D M; Metz, T R; Nasarabadi, S L; Venkateswaran, K S; Farrow, S W; Colston, Jr., B W; Dzenitis, J M

    2004-05-27

    The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii and botulinum toxoid. By coupling highly selective antibody and DNA based assays, the probability of an APDS reporting a false positive is extremely low.

  15. SUSCEPTIBILITY OF CHEMOSTAT-GROWN 'YERSINIA ENTEROCOLITICA' AND 'KLEBSIELLA PNEUMONIAE' TO CHLORINE DIOXIDE

    EPA Science Inventory

    The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a c...

  16. Yersinia virulence factors - a sophisticated arsenal for combating host defences.

    PubMed

    Atkinson, Steve; Williams, Paul

    2016-01-01

    The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six 'effector' proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen. PMID:27347390

  17. Yersinia virulence factors - a sophisticated arsenal for combating host defences

    PubMed Central

    Atkinson, Steve; Williams, Paul

    2016-01-01

    The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six ‘effector’ proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen. PMID:27347390

  18. Comparative Omics-Driven Genome Annotation Refinement: Application across Yersiniae

    SciTech Connect

    Rutledge, Alexandra C.; Jones, Marcus B.; Chauhan, Sadhana; Purvine, Samuel O.; Sanford, James; Monroe, Matthew E.; Brewer, Heather M.; Payne, Samuel H.; Ansong, Charles; Frank, Bryan C.; Smith, Richard D.; Peterson, Scott; Motin, Vladimir L.; Adkins, Joshua N.

    2012-03-27

    Genome sequencing continues to be a rapidly evolving technology, yet most downstream aspects of genome annotation pipelines remain relatively stable or are even being abandoned. To date, the perceived value of manual curation for genome annotations is not offset by the real cost and time associated with the process. In order to balance the large number of sequences generated, the annotation process is now performed almost exclusively in an automated fashion for most genome sequencing projects. One possible way to reduce errors inherent to automated computational annotations is to apply data from 'omics' measurements (i.e. transcriptional and proteomic) to the un-annotated genome with a proteogenomic-based approach. This approach does require additional experimental and bioinformatics methods to include omics technologies; however, the approach is readily automatable and can benefit from rapid developments occurring in those research domains as well. The annotation process can be improved by experimental validation of transcription and translation and aid in the discovery of annotation errors. Here the concept of annotation refinement has been extended to include a comparative assessment of genomes across closely related species, as is becoming common in sequencing efforts. Transcriptomic and proteomic data derived from three highly similar pathogenic Yersiniae (Y. pestis CO92, Y. pestis pestoides F, and Y. pseudotuberculosis PB1/+) was used to demonstrate a comprehensive comparative omic-based annotation methodology. Peptide and oligo measurements experimentally validated the expression of nearly 40% of each strain's predicted proteome and revealed the identification of 28 novel and 68 previously incorrect protein-coding sequences (e.g., observed frameshifts, extended start sites, and translated pseudogenes) within the three current Yersinia genome annotations. Gene loss is presumed to play a major role in Y. pestis acquiring its niche as a virulent pathogen, thus

  19. 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

  20. Salmonella, Shigella, and Yersinia

    PubMed Central

    Dekker, John; Frank, Karen

    2015-01-01

    Synopsis Salmonella, Shigella, and Yersinia cause a well-characterized spectrum of disease in humans, ranging from asymptomatic carriage to hemorrhagic colitis and fatal typhoidal fever. These pathogens are responsible for millions of cases of food-borne illness in the U.S. each year, with substantial costs measured in hospitalizations and lost productivity. In the developing world, illness caused by these pathogens is not only more prevalent, but is also associated with a greater case-fatality rate. Classical methods for identification rely on selective media and serology, but newer methods based on mass spectrometry and PCR show great promise for routine clinical testing. PMID:26004640

  1. Yersinia ruckeri sp. nov., the redmouth (RM) bacterium

    USGS Publications Warehouse

    Ewing, W.H.; Ross, A.J.; Brenner, Don J.; Fanning, G. R.

    1978-01-01

    Cultures of the redmouth (RM) bacterium, one of the etiological agents of redmouth disease in rainbow trout (Salmo gairdneri) and certain other fishes, were characterized by means of their biochemical reactions, by deoxyribonucleic acid (DNA) hybridization, and by determination of guanine-plus-cytosine (G+C) ratios in DNA. The DNA relatedness studies confirmed the fact that the RM bacteria are members of the family Enterobacteriaceae and that they comprise a single species that is not closely related to any other species of Enterobacteriaceae. They are about 30% related to species of both Serratia and Yersinia. A comparison of the biochemical reactions of RM bacteria and serratiae indicated that there are many differences between these organisms and that biochemically the RM bacteria are most closely related to yersiniae. The G+C ratios of RM bacteria were approximated to be between 47.5 and 48.5% These values are similar to those of yersiniae but markedly different from those of serratiae. On the basis of their biochemical reactions and their G+C ratios, the RM bacteria are considered to be a new species of Yersinia, for which the name Yersinia ruckeri is proposed. Strain 2396-61 (= ATCC 29473) is designated the type strain of the species.

  2. Comparative Genomic Hybridization Analysis of Yersinia enterocolitica and Yersinia pseudotuberculosis Identifies Genetic Traits to Elucidate Their Different Ecologies

    PubMed Central

    Jaakkola, Kaisa; Somervuo, Panu; Korkeala, Hannu

    2015-01-01

    Enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis are both etiological agents for intestinal infection known as yersiniosis, but their epidemiology and ecology bear many differences. Swine are the only known reservoir for Y. enterocolitica 4/O:3 strains, which are the most common cause of human disease, while Y. pseudotuberculosis has been isolated from a variety of sources, including vegetables and wild animals. Infections caused by Y. enterocolitica mainly originate from swine, but fresh produce has been the source for widespread Y. pseudotuberculosis outbreaks within recent decades. A comparative genomic hybridization analysis with a DNA microarray based on three Yersinia enterocolitica and four Yersinia pseudotuberculosis genomes was conducted to shed light on the genomic differences between enteropathogenic Yersinia. The hybridization results identified Y. pseudotuberculosis strains to carry operons linked with the uptake and utilization of substances not found in living animal tissues but present in soil, plants, and rotting flesh. Y. pseudotuberculosis also harbors a selection of type VI secretion systems targeting other bacteria and eukaryotic cells. These genetic traits are not found in Y. enterocolitica, and it appears that while Y. pseudotuberculosis has many tools beneficial for survival in varied environments, the Y. enterocolitica genome is more streamlined and adapted to their preferred animal reservoir. PMID:26605338

  3. 42 CFR 73.5 - Exemptions for HHS select agents and toxins.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., Botulinum neurotoxin producing species of Clostridium, Ebola viruses, Francisella tularensis, Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis. This report must be...), (3) The Act commonly known as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or (4) The...

  4. 42 CFR 73.5 - Exemptions for HHS select agents and toxins.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., Botulinum neurotoxin producing species of Clostridium, Ebola viruses, Francisella tularensis, Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis. This report must be...), (3) The Act commonly known as the Virus-Serum-Toxin Act (21 U.S.C. 151-159), or (4) The...

  5. DETECTION OF YERSINIA PESTIS BY COMPARISON OF VIRULENCE PLASMID (PYV/PCD)-ASSOCIATED PHENOTYPES IN YERSINIA SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenotypic expression of several virulence plasmid (pYV/pCD; 70-kb)-associated genetic determinants including low calcium response (Lcr; pin point colony, size= 0.36 mm), colony morphology (size= 1.13 mm), crystal violet (CV) binding (dark-violet colony), Congo Red (CR) uptake (red pin point colony;...

  6. The Virulence Plasmid of Yersinia, an Antihost Genome

    PubMed Central

    Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

    1998-01-01

    The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

  7. Temporal and pathogen-load dependent changes in rainbow trout (Oncorhynchus mykiss) immune response traits following challenge with biotype 2 Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rainbow trout infected with Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), produce a pro-inflammatory and acute-phase response attributed in part to the innate recognition of bacterial-produced flagellin. Recently, variants of Yersinia ruckeri have been identified that lac...

  8. The exoribonuclease Polynucleotide Phosphorylase influences the virulence and stress responses of yersiniae and many other pathogens.

    PubMed

    Rosenzweig, Jason A; Chopra, Ashok K

    2013-01-01

    Microbes are incessantly challenged by both biotic and abiotic stressors threatening their existence. Therefore, bacterial pathogens must possess mechanisms to successfully subvert host immune defenses as well as overcome the stress associated with host-cell encounters. To achieve this, bacterial pathogens typically experience a genetic re-programming whereby anti-host/stress factors become expressed and eventually translated into effector proteins. In that vein, the bacterial host-cell induced stress-response is similar to any other abiotic stress to which bacteria respond by up-regulating specific stress-responsive genes. Following the stress encounter, bacteria must degrade unnecessary stress responsive transcripts through RNA decay mechanisms. The three pathogenic yersiniae (Yersinia pestis, Y. pseudo-tuberculosis, and Y. enterocolitica) are all psychrotropic bacteria capable of growth at 4°C; however, cold growth is dependent on the presence of an exoribonuclease, polynucleotide phosphorylase (PNPase). PNPase has also been implicated as a virulence factor in several notable pathogens including the salmonellae, Helicobacter pylori, and the yersiniae [where it typically influences the type three secretion system (TTSS)]. Further, PNPase has been shown to associate with ribonuclease E (endoribonuclease), RhlB (RNA helicase), and enolase (glycolytic enzyme) in several Gram-negative bacteria forming a large, multi-protein complex known as the RNA degradosome. This review will highlight studies demonstrating the influence of PNPase on the virulence potentials and stress responses of various bacterial pathogens as well as focusing on the degradosome-dependent and -independent roles played by PNPase in yersiniae stress responses. PMID:24312901

  9. Caenorhabditis elegans Mutants Resistant to Attachment of Yersinia Biofilms

    PubMed Central

    Darby, Creg; Chakraborti, Amrita; Politz, Samuel M.; Daniels, Calvin C.; Tan, Li; Drace, Kevin

    2007-01-01

    The detailed composition and structure of the Caenorhabditis elegans surface are unknown. Previous genetic studies used antibody or lectin binding to identify srf genes that play roles in surface determination. Infection by Microbacterium nematophilum identified bus (bacterially unswollen) genes that also affect surface characteristics. We report that biofilms produced by Yersinia pestis and Y. pseudotuberculosis, which bind the C. elegans surface predominantly on the head, can be used to identify additional surface-determining genes. A screen for C. elegans mutants with a biofilm absent on the head (Bah) phenotype identified three novel genes: bah-1, bah-2, and bah-3. The bah-1 and bah-2 mutants have slightly fragile cuticles but are neither Srf nor Bus, suggesting that they are specific for surface components involved in biofilm attachment. A bah-3 mutant has normal cuticle integrity, but shows a stage-specific Srf phenotype. The screen produced alleles of five known surface genes: srf-2, srf-3, bus-4, bus-12, and bus-17. For the X-linked bus-17, a paternal effect was observed in biofilm assays. PMID:17339204

  10. Spectrally-resolved fluorescence cross sections of aerosolized biological live agents and simulants using five excitation wavelengths in a BSL-3 laboratory.

    PubMed

    Pan, Yong-Le; Hill, Steven C; Santarpia, Joshua L; Brinkley, Kelly; Sickler, Todd; Coleman, Mark; Williamson, Chatt; Gurton, Kris; Felton, Melvin; Pinnick, Ronald G; Baker, Neal; Eshbaugh, Jonathan; Hahn, Jerry; Smith, Emily; Alvarez, Ben; Prugh, Amber; Gardner, Warren

    2014-04-01

    A system for measuring spectrally-resolved fluorescence cross sections of single bioaerosol particles has been developed and employed in a biological safety level 3 (BSL-3) facility at Edgewood Chemical and Biological Center (ECBC). It is used to aerosolize the slurry or solution of live agents and surrogates into dried micron-size particles, and to measure the fluorescence spectra and sizes of the particles one at a time. Spectrally-resolved fluorescence cross sections were measured for (1) bacterial spores: Bacillus anthracis Ames (BaA), B. atrophaeus var. globigii (BG) (formerly known as Bacillus globigii), B. thuringiensis israelensis (Bti), B. thuringiensis kurstaki (Btk), B. anthracis Sterne (BaS); (2) vegetative bacteria: Escherichia coli (E. coli), Pantoea agglomerans (Eh) (formerly known as Erwinia herbicola), Yersinia rohdei (Yr), Yersinia pestis CO92 (Yp); and (3) virus preparations: Venezuelan equine encephalitis TC83 (VEE) and the bacteriophage MS2. The excitation wavelengths were 266 nm, 273 nm, 280 nm, 365 nm and 405 nm. PMID:24718194

  11. Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

    PubMed Central

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools. PMID:22911756

  12. Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

    PubMed

    Cho, Minkyung; Chung, Soyi; Jung, Jae Hwan; Rhie, Gi-eun; Jeon, Jun Ho; Seo, Tae Seok

    2014-11-15

    Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (μCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification. PMID:24878840

  13. Generalized Transduction of Small Yersinia enterocolitica Plasmids

    PubMed Central

    Hertwig, Stefan; Popp, Andreas; Freytag, Barbara; Lurz, Rudi; Appel, Bernd

    1999-01-01

    To study phage-mediated gene transfer in Yersinia, the ability of Yersinia phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic Yersinia enterocolitica strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10−5 to 10−7/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic Yersinia strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes. PMID:10473387

  14. 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.

  15. Environmental Regulation of Yersinia Pathophysiology

    PubMed Central

    Chen, Shiyun; Thompson, Karl M.; Francis, Matthew S.

    2016-01-01

    Hallmarks of Yersinia pathogenesis include the ability to form biofilms on surfaces, the ability to establish close contact with eukaryotic target cells and the ability to hijack eukaryotic cell signaling and take over control of strategic cellular processes. Many of these virulence traits are already well-described. However, of equal importance is knowledge of both confined and global regulatory networks that collaborate together to dictate spatial and temporal control of virulence gene expression. This review has the purpose to incorporate historical observations with new discoveries to provide molecular insight into how some of these regulatory mechanisms respond rapidly to environmental flux to govern tight control of virulence gene expression by pathogenic Yersinia. PMID:26973818

  16. Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

    PubMed Central

    Yang, Samuel; Rothman, Richard E.; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A.

    2013-01-01

    Objectives To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. Methods The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. Results The UniProbe detected the presence of all tested Eubacteria (31 / 31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. Conclusions A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents. PMID:18370996

  17. 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

  18. Flagella biosynthesis and regulation by the Rcs pathway within the fish pathogen Yersinia ruckeri during infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gram-negative Enterobacterium Yersinia ruckeri is the etiologic agent of enteric redmouth disease (ERM) within farmed rainbow trout (Oncorhynchus mykiss, Walbaum). Over the past decade, there has been an increase in the prevalence of non-motile variants of Y. ruckeri and the appearance of these ...

  19. A role for Toll-like receptor 4 in the host response to the lung infection of Yersinia pseudotuberculosis in mice.

    PubMed

    Choi, Jin-A; Jeong, Yu-Jin; Kim, Jae-Eun; Kang, Min-Jung; Kim, Jee-Cheon; Oh, Sang-Muk; Lee, Kyung-Bok; Kim, Dong-Hyun; Kim, Dong-Jae; Park, Jong-Hwan

    2016-02-01

    Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological features induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain, IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we generated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen, liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient mice. In addition, the levels of TNF-α and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of Yptb infection for mimicking Y. pestis infection. PMID:26851596

  20. Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

    PubMed Central

    Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

    2013-01-01

    Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

  1. Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

    PubMed

    Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T; Weidmann, Manfred

    2013-04-01

    Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

  2. Inactivation of Yersinia pseudotuberculosis 197 and Francisella tularensis LVS in beverages by high pressure processing.

    PubMed

    Schlesser, Joseph E; Parisi, Brian

    2009-01-01

    In 2003, the U.S. Department of Health and Human Services announced a new research program to develop technologies and strategies to prevent and minimize potential food safety and security threats. The threat of terrorist attacks against the nation's food supplies has created the need to study microorganisms not typically associated with foodborne illness. High-pressure processing has been proposed as a treatment to reduce Yersinia pestis and Francisella tularensis LVS levels in beverages. The objectives of this work were to determine the pressure resistance of Y. pseudotuberculosis 197 (surrogate for Y. pestis) and F. tularensis LVS (vaccine strain). For each bacterium, samples of ultrahigh-temperature pasteurized skim milk and pasteurized reduced-acid orange juice (pH ca. 4.2) were inoculated at a minimum level of 5 log CFU/ml. Ten-milliliter samples of the inoculated product were vacuum sealed in polyester pouches and subjected to pressures of 300 and 500 MPa for holding times ranging from 30 s to 6 min. One set of trials was performed at an initial temperature of 10 degrees C and another at 25 degrees C. Processed samples were immediately plated and enumerated. A pressure treatment of 300 MPa at 25 degrees C for less than 6 min was not sufficient to achieve a 5-log reduction of Y. pseudotuberculosis 197 or F. tularensis LVS in milk. However, a pressure treatment of 500 MPa was effective at hold times as low as 30 s. Overall, F. tularensis LVS demonstrated less pressure resistance than Y. pseudotuberculosis 197. Based on these findings, a high-pressure process designed to inactivate 5 log CFU of Y. pseudotuberculosis 197 per ml and F. tularensis LVS in orange juice or milk should be set at or above 500 MPa with a hold time of 2 min or greater. PMID:19205479

  3. Oral administration of a recombinant attenuated Yersinia pseudotuberculosis strain elicits protective immunity against plague.

    PubMed

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

    2015-11-27

    A Yersinia pseudotuberculosis PB1+ (Yptb PB1+) mutant strain combined with chromosome insertion of the caf1R-caf1A-caf1M-caf1 operon and deletions of yopJ and yopK, χ10068 [pYV-ω2 (ΔyopJ315 ΔyopK108) ΔlacZ044::caf1R-caf1M-caf1A-caf1] was constructed. Results indicated that gene insertion and deletion did not affect the growth rate of χ10068 compared to wild-type Yptb cultured at 26 °C. In addition, the F1 antigen in χ10068 was synthesized and secreted on the surface of bacteria at 37 °C (mammalian body temperature), not at ambient culture temperature (26 °C). Immunization with χ10068 primed antibody responses and specific T-cell responses to F1 and YpL (Y. pestis whole cell lysate). Oral immunization with a single dose of χ10068 provided 70% protection against a subcutaneous (s.c.) challenge with ∼ 2.6 × 10(5) LD50 of Y. pestis KIM6+ (pCD1Ap) (KIM6+Ap) and 90% protection against an intranasal (i.n.) challenge with ∼ 500 LD50 of KIM6+Ap in mice. Our results suggest that χ10068 can be used as an effective precursor to make a safe vaccine to prevent plague in humans and to eliminate plague circulation among humans and animals. PMID:26514425

  4. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-01

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis ( YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability ( R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  5. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    SciTech Connect

    Balaev, V. V.; Lashkov, A. A. Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-15

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} = 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  6. 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

  7. Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy▿

    PubMed Central

    Schwudke, Dominik; Ergin, Asgar; Michael, Kathrin; Volkmar, Sven; Appel, Bernd; Knabner, Dorothea; Konietzny, Antje; Strauch, Eckhard

    2008-01-01

    PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2. PMID:17965162

  8. Generation of a CRISPR database for Yersinia pseudotuberculosis complex and role of CRISPR-based immunity in conjugation.

    PubMed

    Koskela, Katja A; Mattinen, Laura; Kalin-Mänttäri, Laura; Vergnaud, Gilles; Gorgé, Olivier; Nikkari, Simo; Skurnik, Mikael

    2015-11-01

    The clustered regularly interspaced short palindromic repeat - CRISPR-associated genes (CRISPR-Cas) system is used by bacteria and archaea against invading conjugative plasmids or bacteriophages. Central to this immunity system are genomic CRISPR loci that contain fragments of invading DNA. These are maintained as spacers in the CRISPR loci between direct repeats and the spacer composition in any bacterium reflects its evolutionary history. We analysed the CRISPR locus sequences of 335 Yersinia pseudotuberculosis complex strains. Altogether 1902 different spacer sequences were identified and these were used to generate a database for the spacer sequences. Only ∼10% of the spacer sequences found matching sequences. In addition, surprisingly few spacers were shared by Yersinia pestis and Y. pseudotuberculosis strains. Interestingly, 32 different protospacers were present in the conjugative plasmid pYptb32953. The corresponding spacers were identified from 35 different Y. pseudotuberculosis strains indicating that these strains had encountered pYptb32953 earlier. In conjugation experiments, pYptb32953-specific spacers generally prevented conjugation with spacer-positive and spacer-free strains. However, some strains with one to four spacers were invaded by pYptb32953 and some spacer-free strains were fully resistant. Also some spacer-positive strains were intermediate resistant to conjugation. This suggests that one or more other defence systems are determining conjugation efficiency independent of the CRISPR-Cas system. PMID:25712141

  9. 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

  10. Identification of O-Antigen biosynthetic genes specific to Serovar 01 Yersinia Ruckeri: Role in virulence and protective immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a hemorrhagic septicemia that primarily affects farmed rainbow trout (Oncorhynchus mykiss, Walbaum). Y. ruckeri strains comprise several O-serotypes; however the vast majority of outbreaks are caused by serotype O1 isolates of this...

  11. 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

  12. Yersinia enterocolitica: the charisma continues.

    PubMed Central

    Bottone, E J

    1997-01-01

    Yersinia enterocolitica, a gram-negative coccobacillus, comprises a heterogeneous group of bacterial strains recovered from animal and environmental reservoirs. The majority of human pathogenic strains are found among distinct serogroups (e.g. O:3, O:5,27, O:8, O:9) and contain both chromosome- and plasmid (60 to 75 kb)-mediated virulence factors that are absent in "avirulent" strains. While Y. enterocolitica is primarily a gastrointestinal tract pathogen, it may produce extraintestinal infections in hosts with underlying predisposing factors. Postinfection sequelae include arthritis and erythema nodosum, which are seen mainly in Europe among patients with serogroups O:3 and O:9 infection and HLA-B27 antigen. Y. enterocolitica is acquired through the oral route and is epidemiologically linked to porcine sources. Bacteremia is prominent in the setting of immunosuppression or in patients with iron overload or those being treated with desferrioxamine. metastatic foci following bacteremia are common and often involve the liver and spleen. Of particular concern is blood transfusion-related bacteremia. Evidence has accumulated substantiating the role of Y. enterocolitica as a food-borne pathogen that has caused six major outbreaks in the United States. The diagnosis of Y. enterocolitica gastroenteritis is best achieved through isolation of the bacterium on routine or selective bacteriologic media. When necessary, serogrouping, biogrouping, and assessment for plasmid-encoded virulence traits may aid in distinguishing virulent from "avirulent" strains. Epidemiologically, outside of identified food-borne outbreaks, the source (reservoir) of Y. enterocolitica in sporadic cases is speculative. Therefore, prevention and control measures are difficult to institute. PMID:9105754

  13. Detection of aerosolized biological agents by immunoassay followed by autonomous PCR confirmation

    SciTech Connect

    Dzenitis, J M; Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Sathyam, U S; Smith, S M; Gutierrez, D M; Metz, T R; Venkateswaran, K S; Colston, B W; Farrow, S W

    2003-12-15

    An Autonomous Pathogen Detection System (APDS) unit is an automated, podium-sized system that monitors the air for all three biological threat agents (bacteria, viruses, and toxins). The system has been developed under the auspices of the U. S. Department of Energy and Department of Homeland Security by the University of California, Lawrence Livermore National Laboratory (LLNL) to protect people in critical or high-traffic facilities and at special events. The system performs continuous aerosol collection, sample preparation, and multiplexed biological tests using advanced immunoassays as the primary screen. Over ten agents are assayed at once, and results are reported hourly. R&D work this year focused on incorporating polymerase chain-reaction (PCR) techniques for detecting DNA as confirmation of immunoassay positives. The primary objective of the Dugway testing was to demonstrate the APDS with immunoassay identification and PCR confirmation of bacteria. A secondary objective was to demonstrate immunoassay identification of a protein toxoid (denatured toxin) aerosol release. A total of 12 agent trials were conducted over 14 days of testing, for a total of four work weeks at Dugway. Both testing objectives were achieved with multiple releases and clear identifications. The APDS was shown to be effective for identifying aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. The two areas for improvement were operational as opposed to hardware-related. The first was slowing the PCR thermal cycling to achieve stronger signals, which was demonstrated during the later phases of testing. The second area is to improve the parameters for autonomous PCR triggering; this is one of the focuses of the upcoming year's work.

  14. YERSINIA ENTEROCOLITICA: AN IMPORTANT HUMAN FOODBORNE PATHOGEN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia enterocolitica is a Gram-negative microbe of public health importance and is under national FoodNet surveillance in the United States. The majority of human yersiniosis cases are foodborne. Consumption of dairy products (milk, ice cream), water, vegetables (tofu), and pork have been linke...

  15. Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

    PubMed Central

    de la Puerta, María Luisa; Trinidad, Antonio G.; del Carmen Rodríguez, María; Bogetz, Jori; Sánchez Crespo, Mariano; Mustelin, Tomas; Alonso, Andrés; Bayón, Yolanda

    2009-01-01

    YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates. PMID:19221593

  16. [ON THE ORIGIN OF HYPERVIRULENCE OF THE CAUSATIVE AGENT OF PLAGUE].

    PubMed

    Anisimov, N V; Kislichkina, A A; Platonov, M E; Evseeva, V V; Kadnikova, L A; Lipatnikova, N A; Bogun, A G; Dentovskaya, S V; Anisimov, A P

    2016-01-01

    The attempt to combine Yersinia pseudotuberculosis and Yersinia pestis into one species has been unsupported by microbiologists due to the specific features of the epidemiology and clinical presentations of their induced diseases and to basic differences in their virulence. Pseudotuberculosis is predominantly a relatively mild human intestinal infection transmitted through contaminated food and plague is an acute generalized disease with high mortality, which is most frequently transmitted by the bites of infected fleas. Y. pestis hypervirulence, the ability of single bacteria to ensure the development of predagonal bacteriemia in rodents, which is sufficient to contaminate the fleas, is one of the main events during pathogen adaptation to a new ecological niche. By analyzing the data of molecular typing of the representative kits of naturally occurring Y. pestis isolates, the authois consider the issues of formation of intraspecies groups with universal hypervirulence, as well as biovars that are highly virulent only to their major host. A strategy for searching for selective virulence factors, the potential molecular targets for vaccination and etiotropic treatment of plague, is discussed. PMID:27029142

  17. Thermal injury of Yersinia enterocolitica.

    PubMed Central

    Restaino, L; Jeter, W S; Hill, W M

    1980-01-01

    Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI. PMID:6160814

  18. DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

    EPA Science Inventory

    The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

  19. 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

  20. Regulatory principles governing Salmonella and Yersinia virulence

    PubMed Central

    Erhardt, Marc; Dersch, Petra

    2015-01-01

    Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

  1. CHROMagar Yersinia, a New Chromogenic Agar for Screening of Potentially Pathogenic Yersinia enterocolitica Isolates in Stools

    PubMed Central

    Renaud, Nicolas; Lecci, Laetitia; Courcol, René J.; Simonet, Michel

    2013-01-01

    CHROMagar Yersinia (CAY) is a new chromogenic medium for the presumptive detection of virulent Yersinia enterocolitica in stools. Based on a comparative analysis of 1,494 consecutive stools from hospitalized patients, CAY was found to be just as sensitive as the reference medium (cefsulodin-irgasan-novobiocin agar) but was significantly more specific and had a very low false-positive rate. CAY reduces the workload (and thus costs) for stool analysis and can therefore be recommended for routine laboratory use. PMID:23363840

  2. Identification of Yersinia ruckeri from diseased salmonid fish by Fourier transform infrared spectroscopy.

    PubMed

    Wortberg, F; Nardy, E; Contzen, M; Rau, J

    2012-01-01

    Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), which mainly affects salmonid fish. Isolates of Y. ruckeri from diseased salmonid fish were obtained over a 6-year period from eight fish farms in the State of Baden-Württemberg, Southwest Germany. The strains were characterized by biochemical methods and Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. These methods were complemented by 16S rDNA sequencing for several isolates. The set of strains from these fish farms included sorbitol-positive, gelatinase-positive and non-motile Y. ruckeri. These variants were differentiated with an advanced FT-IR module, which is part of a higher-ranking method including more than 200 well-defined Yersinia strains against a background of more than 1000 other Gram-negative isolates. Validation of the newly constructed method yielded 97.4% of Y. ruckeri identified correctly on the species level. Thus, the FT-IR analysis enables distinction of all Y. ruckeri from other Yersinia species (e.g. fish-borne Y. enterocolitica) and other Enterobacteriaceae typically misidentified because of similar biochemical reaction profiles, especially Hafnia alvei. The differentiation of sorbitol-positive variants of Y. ruckeri using FT-IR was demonstrated. PMID:22103737

  3. 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

  4. Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence.

    PubMed

    Schoberle, Taylor J; Chung, Lawton K; McPhee, Joseph B; Bogin, Ben; Bliska, James B

    2016-04-01

    PathogenicYersiniaspecies utilize a type III secretion system to translocate Yop effectors into infected host cells. Yop effectors inhibit innate immune responses in infected macrophages to promoteYersiniapathogenesis. In turn,Yersinia-infected macrophages respond to translocation of Yops by activating caspase-1, but different mechanisms of caspase-1 activation occur, depending on the bacterial genotype and the state of phagocyte activation. In macrophages activated with lipopolysaccharide (LPS) prior toYersinia pseudotuberculosisinfection, caspase-1 is activated by a rapid inflammasome-dependent mechanism that is inhibited by translocated YopM. The possibility that other effectors cooperate with YopM to inhibit caspase-1 activation in LPS-activated macrophages has not been investigated. Toward this aim, epistasis analysis was carried out in which the phenotype of aY. pseudotuberculosisyopMmutant was compared to that of ayopJ yopM,yopE yopM,yopH yopM,yopT yopM, orypkA yopMmutant. Activation of caspase-1 was measured by cleavage of the enzyme, release of interleukin-1β (IL-1β), and pyroptosis in LPS-activated macrophages infected with wild-type or mutantY. pseudotuberculosisstrains. Results show enhanced activation of caspase-1 after infection with theyopJ yopMmutant relative to infection by any other single or double mutant. Similar results were obtained with theyopJ,yopM, andyopJ yopMmutants ofYersinia pestis Following intravenous infection of mice, theY. pseudotuberculosisyopJmutant was as virulent as the wild type, while theyopJ yopMmutant was significantly more attenuated than theyopMmutant. In summary, through epistasis analysis this work uncovered an important role for YopJ in inhibiting caspase-1 in activated macrophages and in promotingYersiniavirulence. PMID:26810037

  5. Antigenic relationship and functional properties of Yersinia porins.

    PubMed

    Vostrikova, P; Likhatskaya, G N; Novikova, D; Solovyeva, T F

    2001-01-01

    We have studied the molecular structure and functional properties of major pore-forming proteins isolated as peptidoglycan (PG)-protein complexes from four Yersinia species (Y. intermedia, Y. enterocolitica, Y. kristensenii and Y. frederiksenii) cultured as various temperatures. Despite the close antigenic relationship, Yersinia porins revealed different functional properties. When reconstituted in model membranes, the PG-protein complexes induced conductance which was different for the "cold" (grown at 6-8 degrees C) and "warm" (grown at 37 degrees C) variants of microbial cultures. We conclude that the functional state of Yersinia porins in the outer membrane depends on the cultivation temperature. PMID:11497105

  6. Behavior of Yersinia enterocolitica in Foods.

    PubMed

    Bari, Md Latiful; Hossain, M Anwar; Isshiki, Kenji; Ukuku, Dike

    2011-01-01

    Yersinia enterocolitica are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can survive and grow at refrigeration temperatures. The ability to propagate at refrigeration temperatures is of considerable significance in food hygiene. Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture. Two chromosomal genes, inv and ail, were identified for cell invasion of mammalian. The pathogen can cause diarrhoea, appendicitis and post-infection arthritis may occur in a small proportion of cases. The most common transmission route of pathogenic Y. enterocolitica is thought to be fecal-oral via contaminated food. Direct person-to-person contact is rare. Occasionally, pathogenic Y. enterocolitica has been detected in vegetables and environmental water; thus, vegetables and untreated water are also potential sources of human yersiniosis. However, the isolation rates of pathogenic Y. enterocolitica have been low, which may be due to the limited sensitivity of the detection methods. To identify other possible transmission vehicles, different food items should be studied more extensively. Many factors related to the epidemiology of Y. enterocolitica, such as sources, transmission routes, and predominating genotypes remain obscure because of the low sensitivity of detection methods. PMID:22567332

  7. Behavior of Yersinia enterocolitica in Foods

    PubMed Central

    Bari, Md. Latiful; Hossain, M. Anwar; Isshiki, Kenji; Ukuku, Dike

    2011-01-01

    Yersinia enterocolitica are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can survive and grow at refrigeration temperatures. The ability to propagate at refrigeration temperatures is of considerable significance in food hygiene. Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture. Two chromosomal genes, inv and ail, were identified for cell invasion of mammalian. The pathogen can cause diarrhoea, appendicitis and post-infection arthritis may occur in a small proportion of cases. The most common transmission route of pathogenic Y. enterocolitica is thought to be fecal-oral via contaminated food. Direct person-to-person contact is rare. Occasionally, pathogenic Y. enterocolitica has been detected in vegetables and environmental water; thus, vegetables and untreated water are also potential sources of human yersiniosis. However, the isolation rates of pathogenic Y. enterocolitica have been low, which may be due to the limited sensitivity of the detection methods. To identify other possible transmission vehicles, different food items should be studied more extensively. Many factors related to the epidemiology of Y. enterocolitica, such as sources, transmission routes, and predominating genotypes remain obscure because of the low sensitivity of detection methods. PMID:22567332

  8. Multiplex primer-extension assay for identification of Yersinia species.

    PubMed

    Dalmasso, Alessandra; Civera, Tiziana; Filipello, Virginia; Bottero, Maria Teresa

    2014-10-01

    A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers. PMID:24985982

  9. Identification of Yersinia spp. with the API 20E system.

    PubMed

    Archer, J R; Schell, R F; Pennell, D R; Wick, P D

    1987-12-01

    The ability of the API 20E system to identify 105 clinical isolates of Yersinia spp. was compared with those of conventional biochemical tests at 28 and 37 degrees C. Elimination of the Voges-Proskauer test (recorded as a negative result) increased the percentage of correct identifications for Yersinia spp. from 66 to 93% when the API 20E strips were incubated at 28 degrees C. PMID:3323231

  10. Current activities of the Yersinia effector protein YopM.

    PubMed

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian

    2015-05-01

    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. PMID:25865799

  11. Mesenteric lymphadenitis caused by Yersinia enterocolitica

    PubMed Central

    Wojskowicz, Piotr; Kiśluk, Joanna; Fil, Dawid; Kemona, Andrzej; Dadan, Jacek

    2015-01-01

    Yersiniosis is an acute or chronic, zoonotic disease caused by infection of Gram-negative rods Yersinia enterocolitica. It can be transmitted by the consumption of originally contaminated food products (pork, unpasteurized milk) or secondarily contaminated with animal or vegetable products. The clinical picture of infection may have a variable course is related to the age and physical condition of the patient, or pathogenic properties of microorganisms. Infection caused by Y. enterocolitica can occur in different clinical forms: food poisoning, colitis, mesentric lymphadenitis, erythema nodosum, arthritis, pharyngitis, pneumonia, meningitis, sepsis. The aim of this study was to present a rare case of infection with Y. enterocolitica mesenteric lymph nodes coexistent with appendicitis. PMID:26557944

  12. Mesenteric lymphadenitis caused by Yersinia enterocolitica.

    PubMed

    Zińczuk, Justyna; Wojskowicz, Piotr; Kiśluk, Joanna; Fil, Dawid; Kemona, Andrzej; Dadan, Jacek

    2015-01-01

    Yersiniosis is an acute or chronic, zoonotic disease caused by infection of Gram-negative rods Yersinia enterocolitica. It can be transmitted by the consumption of originally contaminated food products (pork, unpasteurized milk) or secondarily contaminated with animal or vegetable products. The clinical picture of infection may have a variable course is related to the age and physical condition of the patient, or pathogenic properties of microorganisms. Infection caused by Y. enterocolitica can occur in different clinical forms: food poisoning, colitis, mesentric lymphadenitis, erythema nodosum, arthritis, pharyngitis, pneumonia, meningitis, sepsis. The aim of this study was to present a rare case of infection with Y. enterocolitica mesenteric lymph nodes coexistent with appendicitis. PMID:26557944

  13. The High-Pathogenicity Island of Yersinia enterocolitica Ye8081 Undergoes Low-Frequency Deletion but Not Precise Excision, Suggesting Recent Stabilization in the Genome

    PubMed Central

    Bach, Sandrine; Buchrieser, Carmen; Prentice, Michael; Guiyoule, Annie; Msadek, Tarek; Carniel, Elisabeth

    1999-01-01

    Highly pathogenic strains of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are characterized by the possession of a pathogenicity island designated the high-pathogenicity island (HPI). This 35- to 45-kb island carries an iron uptake system named the yersiniabactin locus. While the HPIs of Y. pestis and Y. pseudotuberculosis are subject to high-frequency spontaneous deletion from the chromosome, we were initially unable to obtain HPI-deleted Y. enterocolitica 1B isolates. In the present study, using a positive selection strategy, we identified three HPI-deleted mutants of Y. enterocolitica strain Ye8081. In these three independent clones, the chromosomal deletion was not limited to the HPI but encompassed a larger DNA fragment of approximately 140 kb. Loss of this fragment, which occurred at a frequency of approximately 5 × 10−7, resulted in the disappearance of several phenotypic traits, such as growth in a minimal medium, hydrolysis of o-nitrophenyl-β-d-thiogalactopyranoside, Tween esterase activity, and motility, and in a decreased virulence for mice. However, no precise excision of the Ye8081 HPI was observed. To gain more insight into the molecular basis for this phenomenon, the putative machinery of HPI excision in Y. enterocolitica was analyzed and compared to that in Y. pseudotuberculosis. We show that the probable reasons for failure of precise excision of the HPI of Y. enterocolitica Ye8081 are (i) the interruption of the P4-like integrase gene located close to its right-hand boundary by a premature stop codon and (ii) lack of conservation of 17-bp att-like sequences at both extremities of the HPI. These mutations may represent a process of HPI stabilization in the species Y. enterocolitica. PMID:10496882

  14. The Functions of Effector Proteins in Yersinia Virulence.

    PubMed

    Zhang, Linglin; Mei, Meng; Yu, Chan; Shen, Wenwen; Ma, Lixin; He, Jiewang; Yi, Li

    2016-01-01

    Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS. PMID:27281989

  15. Human gamma delta T-cell recognition of Yersinia enterocolitica.

    PubMed Central

    Young, J L; Goodall, J C; Beacock-Sharp, H; Gaston, J S

    1997-01-01

    We have studied the human gamma delta T-cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)-B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic gamma delta T cells is seen when Yersinia-infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat-inactivated Yersinia fail to stimulate the gamma delta T-cell response. The gamma delta T-cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T-cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA-B27 less well than infected allogeneic target cells. gamma delta T-cell clones isolated from this line were found to express V gamma 9V delta 2 T-cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)-deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that gamma delta T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis. PMID:9378487

  16. LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

    PubMed Central

    Sanapala, Shilpa; Henderson, Jeremy C.; Sam, Shandiin; Olinzock, Joseph; Trent, M. Stephen; Curtiss, Roy

    2014-01-01

    Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC PBAD crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::PmsbB msbB(EC) ΔPcrp21::TT araC PBAD crp] for use with a balanced-lethal Asd-positive (Asd+) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopENt138-LcrV) was synthesized in χ10057 harboring an Asd+ plasmid (pYA5199, yopENt138-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ∼240 median lethal doses (LD50) (2.4 × 104 CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague. PMID:25114109

  17. Atypical Yersinia enterocolitica: clinical and epidemiological parameters.

    PubMed Central

    Bottone, E J

    1978-01-01

    Infections due to biochemically typical Yersinia enterocolitica usually present as gastroenteritis, mesenteric lymphadenitis, terminal ileitis, and septicemia often with visceral abscesses. In these instances, the isolates have been biochemically typical and of well-established serotypes, namely 0:3 or 0:9 and, in the United States, 0:5 or 0:8. The recovery, recognition, and significance of biochemically and serologically atypical Y. enterocolitica in human infections has proceeded more slowly. From an analysis of the clinical histories of 20 patients infected with 21 such aberrant Y. enterocolitica, it appears that these strains are of restricted pathogenic potential, producing various clinical entities such as localized skin abscesses, conjunctivitis, self-limiting enteritis, and wound and urinary tract infections in hosts with predisposing factors. Epidemiologically, whereas episodic acquisition of atypical strains by hospitalized patients is indicative of nosocomial transmission, in the present series sporadic isolations over a 4-year period, mainly from ambulatory patients, suggest an occult reservoir in the community serviced by The Mount Sinai Hospital. In contrast to typical Y. enterocolitica, which has become well adapted in animal and human hosts, it appears that environmental strains may be in the evolutionary process of becoming adapted to humans. PMID:670380

  18. Biocidal and Sporicidal Efficacy of Pathoster(®) 0.35% and Pathoster(®) 0.50% Against Bacterial Agents in Potential Bioterrorism Use.

    PubMed

    Candeliere, Antonio; Campese, Emanuele; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster(®) 0.35% and Pathoster(®) 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster(®) 0.35% was stable until 16±1 days, while Pathoster(®) 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  19. Biocidal and Sporicidal Efficacy of Pathoster® 0.35% and Pathoster® 0.50% Against Bacterial Agents in Potential Bioterrorism Use

    PubMed Central

    Candeliere, Antonio; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster® 0.35% and Pathoster® 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster® 0.35% was stable until 16±1 days, while Pathoster® 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  20. Interferon Consensus Sequence Binding Protein Confers Resistance against Yersinia enterocolitica

    PubMed Central

    Hein, Joachim; Kempf, Volkhard A. J.; Diebold, Joachim; Bücheler, Nicole; Preger, Sonja; Horak, Ivan; Sing, Andreas; Kramer, Uwe; Autenrieth, Ingo B.

    2000-01-01

    Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-γ) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacterium Yersinia enterocolitica. ICSBP−/− mice failed to produce IL-12 and IFN-γ, but also IL-4, after Yersinia challenge. In addition, granuloma formation was highly disturbed in infected ICSBP−/− mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP−/− mice rapidly succumbed to acute Yersinia infection. In vitro treatment of spleen cells from ICSBP−/− mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-γ induction. In experimental therapy of infected ICSBP−/− mice, we observed that administration of rIL-12 induced IFN-γ production which was associated with improved resistance to Yersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-γ production but nevertheless reduced bacterial burden in ICSBP−/− mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course of Yersinia infection in ICSBP−/− mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense against Yersinia. These results are important for understanding the complex host immune responses in bacterial infections. PMID:10678954

  1. Plasmacytoid Dendritic Cells Are Crucial in Bifidobacterium adolescentis-Mediated Inhibition of Yersinia enterocolitica Infection

    PubMed Central

    Wittmann, Alexandra; Autenrieth, Ingo B.; Frick, Julia-Stefanie

    2013-01-01

    In industrialized countries bacterial intestinal infections are commonly caused by enteropathogenic Enterobacteriaceae. The interaction of the microbiota with the host immune system determines the adequacy of an appropriate response against pathogens. In this study we addressed whether the probiotic Bifidobacterium adolescentis is protective during intestinal Yersinia enterocolitica infection. Female C57BL/6 mice were fed with B. adolescentis, infected with Yersinia enterocolitica, or B. adolescentis fed and subsequently infected with Yersinia enterocolitica. B. adolescentis fed and Yersinia infected mice were protected from Yersinia infection as indicated by a significantly reduced weight loss and splenic Yersinia load when compared to Yersinia infected mice. Moreover, protection from infection was associated with increased intestinal plasmacytoid dendritic cell and regulatory T-cell frequencies. Plasmacytoid dendritic cell function was investigated using depletion experiments by injecting B. adolescentis fed, Yersinia infected C57BL/6 mice with anti-mouse PDCA-1 antibody, to deplete plasmacytoid dendritic cells, or respective isotype control. The B. adolescentis-mediated protection from Yersinia dissemination to the spleen was abrogated after plasmacytoid dendritic cell depletion indicating a crucial function for pDC in control of intestinal Yersinia infection. We suggest that feeding of B. adolescentis modulates the intestinal immune system in terms of increased plasmacytoid dendritic cell and regulatory T-cell frequencies, which might account for the B. adolescentis-mediated protection from Yersinia enterocolitica infection. PMID:23977019

  2. Mice and moles inhabiting mountainous areas of Shimane Peninsula as sources of infection with Yersinia pseudotuberculosis.

    PubMed Central

    Fukushima, H; Gomyoda, M; Kaneko, S

    1990-01-01

    A total of 1,835 Yersinia spp. were isolated from 925 (60.5%) of 1,530 wild mice and from 139 (79.9%) of 174 moles living in mountainous areas of eastern Shimane Prefecture, Japan. The Yersinia spp. included 1,106 Yersinia enterocolitica, 26 Y. enterocolitica-like, 176 Yersinia mollaretii, 149 Yersinia frederiksenii, 70 Yersinia intermedia, 231 Yersinia kristensenii, 5 Yersinia aldovae, and 72 Yersinia pseudotuberculosis. Human pathogenic Y. enterocolitica was not isolated. Y. pseudotuberculosis was divided into 10 virulent 40- to 50-MDa plasmid-positive (P+) strains (serotypes 1b, 4b, and untypeable) and 62 plasmid-negative (P-) strains (serotypes 1b, 2b, 2c, 4a, 5a, 5b, 6, 7, and untypeable). P+ strains of serotypes 1b (two strains), 4b (seven strains), and untypeable (one strain) were isolated from nine Apodemus specious and one Apodemus argenteus. The isolates of Yersinia spp. were more frequently detected in newborn mice and during the breeding season. The P+ Y. pseudotuberculosis strains were recovered at less than 10(4) cells per g of the cecal contents. Thus, the prevalence of Yersinia spp. in small wild animals depends on the newborn animals born during the cold months, and wild mice in mountainous areas are important reservoirs of Y. pseudotuberculosis. Images PMID:2254420

  3. Prevalence, characterization, and antimicrobial resistance of Yersinia species and Yersinia enterocolitica isolated from raw milk in farm bulk tanks.

    PubMed

    Jamali, Hossein; Paydar, Mohammadjavad; Radmehr, Behrad; Ismail, Salmah

    2015-02-01

    The aims of this study were to investigate the prevalence and to characterize and determine the antibiotic resistance of Yersinia spp. isolates from raw milk. From September 2008 to August 2010, 446 raw milk samples were obtained from farm bulk milk tanks in Varamin, Iran. Yersinia spp. were detected in 29 (6.5%) samples, out of which 23 (79.3%), 5 (17.2%), and 1 (3.4%) were isolated from cow, sheep, and goat raw milk, respectively. The most common species isolated was Yersinia enterocolitica (65.5%), followed by Yersinia frederiksenii (31%), and Yersinia kristensenii (3.4%). Of the 19 Y. enterocolitica isolates, 14 (73.7%) were grouped into bioserotype 1A/O:9, 4 (21.1%) belonged to bioserotype 1B:O8, 1 (5.3%) belonged to bioserotype 4/O:3, and 1 isolate (biotype 1A) was not typable. All the isolates of biotypes 1B and 4harbored both the ystA and ail genes. However, all the isolates of biotype 1A were only positive for the ystB gene. The tested Yersinia spp. showed the highest percentages of resistance to tetracycline (48.3%), followed by ciprofloxacin and cephalothin (each 17.2%), ampicillin (13.8%), streptomycin (6.9%), and amoxicillin and nalidixic acid (each 3.4%). All of the tested isolates demonstrated significant sensitivity to gentamicin and chloramphenicol. Recovery of potentially pathogenic Y. enterocolitica from raw milk indicates high risks of yersiniosis associated with consumption of raw milk. PMID:25497824

  4. Identification and characterization of flagellar biosynthetic genes in Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using transposon mutagenesis we have identified a Yersinia ruckeri serovar I mutant defective in both motility and production of secreted lipase activity. Sequence analysis of this mutant revealed a single transposon insertion in an open reading frame (ORF) with homology to flhA, a flagellar biosynt...

  5. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were ...

  6. Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 II the use of Yersinia outer proteins for the specific detection of Yersinia enterocolitica infections in ruminants.

    PubMed

    Kittelberger, R; Hilbink, F; Hansen, M F; Ross, G P; Joyce, M A; Fenwick, S; Heesemann, J; Wolf-Watz, H; Nielsen, K

    1995-12-01

    Yersinia outer protein (YOP) preparations from Y. enterocolitica and Y. pseudotuberculosis were used as antigens in immunoblots for the detection of Yersinia infections in experimentally and naturally infected ruminants. Sera from 9 groups of animals were used: (1) 51 sera from cattle which were false-positive in the standard brucellosis serological tests, (2) 52 sera from brucellosis-negative cattle, (3) 51 sera from a deer herd in which 16 animals were positive in the brucellosis tests and Yersina species were isolated from 5 animals, (4) 50 sera from a deer herd in which sera from all animals were negative in the brucellosis tests, (5) 107 sera from brucellosis-negative cattle which were received from throughout New Zealand, (6) 30 sera from cattle naturally infected with B. abortus and from which B. abortus was isolated, (7) 55 sera from cattle naturally infected with B. abortus, (8) 26 sera from cattle experimentally infected with B. abortus, with mostly high titres in the conventional brucellosis tests, and (9) sera taken weekly from 3 cattle experimentally infected with Y. enterocolitica 0:9. In all 3 Y. enterocolitica 0:9 experimentally infected animals the antibody reactivity against major YOPs in the Y. enterocolitica and in the Y. pseudotuberculosis YOP preparation correlated well with the strength in the classical brucellosis tests and with the staining of smooth lipopolysaccharides (SLPS) in blots, thus confirming the usefulness of YOPs for the detection of Yersinia infections. Sera from naturally infected cattle and deer herds, regardless of whether they were false positive or negative in the brucellosis tests, showed high frequencies of staining in YOP blots (53-58% in cattle and 80-100% in deer), indicating a high prevalence of field infections with Yersinia species in New Zealand. In two of the three sera groups from B. abortus infected animals, antibodies against YOPs were detected with high frequency, showing that dual infections may be common

  7. Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

    PubMed Central

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

    2014-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  8. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    PubMed

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  9. NIAID Biodefense Image Library

    MedlinePlus

    ... Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a cover slip. Credit: NIAID Yersinia pestis Scanning electron micrograph depicting a mass of Yersinia pestis bacteria (the cause of bubonic ...

  10. Structure of a pectin methylesterase from Yersinia enterocolitica

    PubMed Central

    Boraston, Alisdair B.; Abbott, D. Wade

    2012-01-01

    Pectin methylesterases (PMEs) are family 8 carbohydrate esterases (CE8s) which remove the methyl group from methylesterified galacturonic acid (GalA) residues within pectin. Although the role of pectinases such as PMEs within dedicated phytopathogens has been well established, the significance of homologous enzymes found within the genomes of human enteropathogens remains to be determined. Presented here is the low-resolution (3.5 Å) structure of the CE8 from Yersinia enterocolitica (YeCE8). The high degree of structural conservation in the topology of the active-site cleft and catalytic apparatus that is shared with a characterized PME from a bacterial phytopathogen (i) indicates that YeCE8 is active on methylated pectin and (ii) highlights a more prominent role for pectin utilization in Yersinia than in other enteropathogenic species. PMID:22297983

  11. Isolation of pathogenic yersiniae from wild animals in Bulgaria.

    PubMed

    Nikolova, S; Tzvetkov, Y; Najdenski, H; Vesselinova, A

    2001-04-01

    Pathogenic Yersinia strains were isolated between December 1998 and April 1999 from 37 wild animals: rabbit (Lepus europeus), boar (Sus scrofa scrofa), asiatic jackal (Canis aureus), red fox (Vulpes vulpes), mouflon (Ovis musimon), european river otter (Lutra lutra), beech marten (Martes foina), polecat (Musleta putorius) and wild cat (Felis silvestris). It was established that among the wild animals Y. enterocolitica strains of serotype 0:3 predominated, accompanied by Y. pseudotuberculosis strains of serotype 0:3. In one sample from asiatic jackal and one sample from rabbit, Y. enterocolitica serotype 0:8 was isolated. Yersinia enterocolitica and Y. pseudotuberculosis strains were isolated from tonsils and tongues as well as from the viscera--lung, liver, heart, spleen, kidney and lymph nodes, mainly in young animals (1-2 years of age). The results showed that wild animals are a possible natural reservoir for pathogenic Y. enterocolitica and Y. pseudotuberculosis and are included in the epidemiological chain of yersinioses. PMID:11393816

  12. Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 Provides Insights into Niche Adaptation and Pathogenic Mechanism.

    PubMed

    Liu, Tao; Wang, Kai-Yu; Wang, Jun; Chen, De-Fang; Huang, Xiao-Li; Ouyang, Ping; Geng, Yi; He, Yang; Zhou, Yi; Min, Jie

    2016-01-01

    Yersinia ruckeri is the etiologic agent of enteric red mouth disease (ERM), a severe fish disease prevailing in worldwide aquaculture industries. Here we report for the first time the complete genome of Y. ruckeri (Yersinia ruckeri) SC09, a highly virulent strain isolated from Ictalurus punctatus with severe septicemia. SC09 possesses a single chromosome of 3,923,491 base pairs, which contains 3651 predicted protein coding sequences (CDS), 19 rRNA genes, and 79 tRNA genes. Among the CDS, we have identified a Ysa locus containing genes encoding all the components of a type III secretion system (T3SS). Comparative analysis suggest that SC09-Ysa share extensive similarity in sequence, gene content, and gene arrangement with Salmonella enterica pathogenicity island 1 (SPI1) and chromosome-encoded T3SS from Yersinia enterocolitica biotype 1B. Furthermore, phylogenetic analysis shown that SC09-Ysa and SPI1-T3SS belong on the same branch of the phylogenetic tree. These results suggest that SC09-Ysa and SPI1-T3SS appear to mediate biological function to adapt to specific hosts with a similar niche, and both of them are likely to facilitate the development of an intracellular niche. In addition, our analysis also indicated that a substantial part of the SC09 genome might contribute to adaption in the intestinal microenvironment, including a number of proteins associated with aerobic or anaerobic respiration, signal transduction, and various stress reactions. Genomic analysis of the bacterium offered insights into the pathogenic mechanism associated with intracellular infection and intestinal survivability, which constitutes an important first step in understanding the pathogenesis of Y. ruckeri. PMID:27089334

  13. Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 Provides Insights into Niche Adaptation and Pathogenic Mechanism

    PubMed Central

    Liu, Tao; Wang, Kai-Yu; Wang, Jun; Chen, De-Fang; Huang, Xiao-Li; Ouyang, Ping; Geng, Yi; He, Yang; Zhou, Yi; Min, Jie

    2016-01-01

    Yersinia ruckeri is the etiologic agent of enteric red mouth disease (ERM), a severe fish disease prevailing in worldwide aquaculture industries. Here we report for the first time the complete genome of Y. ruckeri (Yersinia ruckeri) SC09, a highly virulent strain isolated from Ictalurus punctatus with severe septicemia. SC09 possesses a single chromosome of 3,923,491 base pairs, which contains 3651 predicted protein coding sequences (CDS), 19 rRNA genes, and 79 tRNA genes. Among the CDS, we have identified a Ysa locus containing genes encoding all the components of a type III secretion system (T3SS). Comparative analysis suggest that SC09-Ysa share extensive similarity in sequence, gene content, and gene arrangement with Salmonella enterica pathogenicity island 1 (SPI1) and chromosome-encoded T3SS from Yersinia enterocolitica biotype 1B. Furthermore, phylogenetic analysis shown that SC09-Ysa and SPI1-T3SS belong on the same branch of the phylogenetic tree. These results suggest that SC09-Ysa and SPI1-T3SS appear to mediate biological function to adapt to specific hosts with a similar niche, and both of them are likely to facilitate the development of an intracellular niche. In addition, our analysis also indicated that a substantial part of the SC09 genome might contribute to adaption in the intestinal microenvironment, including a number of proteins associated with aerobic or anaerobic respiration, signal transduction, and various stress reactions. Genomic analysis of the bacterium offered insights into the pathogenic mechanism associated with intracellular infection and intestinal survivability, which constitutes an important first step in understanding the pathogenesis of Y. ruckeri. PMID:27089334

  14. Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins.

    PubMed

    Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P; Zhao, Zhendong; Wang, Yejun

    2016-08-01

    Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

  15. Enteric infections due to Campylobacter, Yersinia, Salmonella, and Shigella*

    PubMed Central

    1980-01-01

    This report reviews the available information on the clinical features, pathogenesis, bacteriology, and epidemiology of Campylobacter jejuni and Yersinia enterocolitica, both of which have recently been recognized as important causes of enteric infection. In the fields of salmonellosis and shigellosis, important new epidemiological and related findings that have implications for the control of these infections are described. Priority research activities in each of these areas are outlined. PMID:6969131

  16. Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

    PubMed

    Möllerherm, Helene; Neumann, Ariane; Schilcher, Katrin; Blodkamp, Stefanie; Zeitouni, Nathalie E; Dersch, Petra; Lüthje, Petra; Naim, Hassan Y; Zinkernagel, Annelies S; von Köckritz-Blickwede, Maren

    2015-12-01

    Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens. PMID:26459885

  17. Using Comparative Genomics for Inquiry-Based Learning to Dissect Virulence of "Escherichia coli" O157:H7 and "Yersinia pestis"

    ERIC Educational Resources Information Center

    Baumler, David J.; Banta, Lois M.; Hung, Kai F.; Schwarz, Jodi A.; Cabot, Eric L.; Glasner, Jeremy D.; Perna, Nicole T.

    2012-01-01

    Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples…

  18. Three-dimensional structures of unligated uridine phosphorylase from Yersinia pseudotuberculosis at 1.4 Å resolution and its complex with an antibacterial drug

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Gabdulkhakov, A. G.; Dontsova, M. V.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2015-07-01

    Uridine phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of uridine phosphorylase from the bacterium Yersinia pseudotuberculosis ( YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 Å resolution (DPI = 0.062 Å; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.

  19. Divergence among Genes Encoding the Elongation Factor Tu of Yersinia Species▿

    PubMed Central

    Isabel, Sandra; Leblanc, Éric; Boissinot, Maurice; Boudreau, Dominique K.; Grondin, Myrian; Picard, François J.; Martel, Eric A.; Parham, Nicholas J.; Chain, Patrick S. G.; Bader, Douglas E.; Mulvey, Michael R.; Bryden, Louis; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    2008-01-01

    Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species. PMID:18790860

  20. Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri.

    PubMed

    M Monte, Milena; Urquhart, Katy; Secombes, Christopher J; Collet, Bertrand

    2016-08-01

    Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1β1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and β-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment. PMID:27245868

  1. Pheno- and genotypic analysis of antimicrobial resistance properties of Yersinia ruckeri from fish.

    PubMed

    Huang, Yidan; Michael, Geovana Brenner; Becker, Roswitha; Kaspar, Heike; Mankertz, Joachim; Schwarz, Stefan; Runge, Martin; Steinhagen, Dieter

    2014-07-16

    Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 ± 2°C for 24, 28 and 48 h. Incubation for 24h at 22 ± 2°C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 μg/mL) and nalidixic acid (0.25-0.5 μg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 μg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ∼ 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes. PMID:24331744

  2. Yersinia enterocolitica Affects Intestinal Barrier Function in the Colon.

    PubMed

    Hering, Nina A; Fromm, Anja; Kikhney, Judith; Lee, In-Fah M; Moter, Annette; Schulzke, Jörg D; Bücker, Roland

    2016-04-01

    Infection with Yersinia enterocolitica causes acute diarrhea in early childhood. A mouse infection model presents new findings on pathological mechanisms in the colon. Symptoms involve diarrhea with watery feces and weight loss that have their functional correlates in decreased transepithelial electrical resistance and increased fluorescein permeability. Y. enterocolitica was present within the murine mucosa of both ileum and colon. Here, the bacterial insult was of focal nature and led to changes in tight junction protein expression and architecture. These findings are in concordance with observations from former cell culture studies and suggest a leak flux mechanism of diarrhea. PMID:26621910

  3. Difficulties in diagnosing terminal ileitis due to Yersinia pseudotuberculosis.

    PubMed

    Wunderink, H F; Oostvogel, P M; Frénay, I H M E; Notermans, D W; Fruth, A; Kuijper, E J

    2014-02-01

    We report three patients with terminal ileitis and positive fecal cultures with Yersinia pseudotuberculosis. From one patient, a virulence plasmid (pYV)-negative Y. pseudotuberculosis was isolated, which represents the second finding of a pYV-negative isolate associated with human disease. All patients were treated with ciprofloxacin and fully recovered. Since conventional culture methods for yersiniosis are gradually replaced with molecular tests not recognizing Y. pseudotuberculosis, we recommend to include a specific culture medium or to apply a specific polymerase chain reaction (PCR) assay on fecal samples from patients suspected of terminal ileitis. PMID:23925588

  4. Yersinia enterocolitica strains isolated from beavers (Castor fiber).

    PubMed

    Platt-Samoraj, A; Syczyło, K; Bancerz-Kisiel, A; Szczerba-Turek, A; Giżejewska, A; Szweda, W

    2015-01-01

    Pseudocloacal swabs and palatine tonsils from beavers have been examined for the Yersinia enterocolitica presence. Thirty-six samples from 24 beavers were collected and subjected to bacteriological examinations including sero- and biotypisation. Amplicons confirmed by PCR as Y. enterocolitica were sequenced. Positive samples originated from 4 out of the 24 beavers (16.7 %) and all the strains belonged to biotype 1A. The study suggested that Y. enterocolitica could be isolated from beavers, which may therefore be treated as a reservoir, a significant factor of water contamination and a vector of the Y. enterocolitica. PMID:26172198

  5. Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

    PubMed Central

    Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

    2007-01-01

    Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can

  6. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

    PubMed Central

    Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  7. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  8. Prevalence of Yersinia Species in Traditional and Commercial Dairy Products in Isfahan Province, Iran

    PubMed Central

    Rahimi, Ebrahim; Sepehri, Sara; Safarpoor Dehkordi, Farhad; Shaygan, Shima; Momtaz, Hassan

    2014-01-01

    Background: Yersinia species, especially Yersinia enterocolitica, are considered as the most prevalent milk-borne pathogens. Several serological and molecular techniques have been developed for rapid and safe diagnosis of yersiniosis. Objectives: This study was carried out to assess the prevalence rate of Yersinia species, especially Y. enterocolitica, in milk and dairy products in Isfahan province, Iran. Materials and Methods: A total of 285 commercial and traditional dairy products as well as 267 pasteurized and raw milk samples were collected during one year. The samples were studied by culturing and the positive-culture samples were investigated using PCR techniques. Results: The results of culture showed that 52 (9.42%) and 28 (5.07%) of the total 552 milk and dairy samples were positive for presences of Yersinia species and Y. enterocolitica, respectively. Totally, 24 of 28 Y. enterocolitica isolates by culture were positive in PCR test (4.59%). Raw cow milk and traditional cheese had the highest prevalence of Yersinia species and Y. enterocolitica, respectively. There were no positive results for pasteurized cow milk, raw camel milk, commercial ice cream, commercial cheese, yoghurt, Doogh, butter and curd. Yersinia species and Y. enterocolitica had the highest prevalence in autumn (15.15% and 10.6%, respectively). Significant differences regarding P < 0.05 were observed between the presences of Yersinia species and Y. enterocolitica in various samples and seasons. Conclusions: Sanitation and pasteurization are the best ways to increase the microbial quality and particularly decrease the load of Yersinia species. The ability of Yersinia species to growth in Doogh, yoghurt, curd and butter is very low. PMID:25147698

  9. Phenotypic and molecular characterization of Yersinia ruckeri isolates from rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey.

    PubMed

    Onuk, Ertan Emek; Ciftci, Alper; Findik, Arzu; Ciftci, Gülay; Altun, Soner; Balta, Fikri; Ozer, Selmin; Coban, Ahmet Yilmaz

    2011-01-01

    The aim of the study was the phenotypic and molecular characterization of Yersinia (Y) ruckeri strains, the causative agent of Enteric Redmouth Disease (ERM), by antibiotyping, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of whole cell proteins. For this aim, a total of 97 Y ruckeri isolates were analyzed. The isolates were distinguished into ten antibiotypes and six phenotypes according to their resistance properties and whole cell protein profiles, respectively. Also, a glycoprotein band of approximately 25.5 kDa was observed in all Y ruckeri strains tested. In all strains, six different RAPD types were observed. In conclusion, Y ruckeri strains isolated from rainbow trout of fish farms in Turkey showed variation according to their phenotypic and genotypic characteristics, and the use of these three typing techniques in double and triple combinations could be more useful for discriminating the strains. PMID:21848040

  10. High prevalence of pathogenic Yersinia enterocolitica in pig cheeks.

    PubMed

    Laukkanen-Ninios, Riikka; Fredriksson-Ahomaa, Maria; Maijala, Riitta; Korkeala, Hannu

    2014-10-01

    Samples from pork cuts for minced meat and cheeks from processing plants and a slaughterhouse, and modified atmosphere (MA) packaged pork from retail were studied to estimate the prevalence of pathogenic, i.e. virulence plasmid bearing, Yersinia enterocolitica and Yersinia pseudotuberculosis in pork, as well as to quantify pathogenic Y. enterocolitica in pork cuts. Pathogenic (virF-positive) Y. enterocolitica was isolated from 17 pig cheeks (23%) but not from any of the MA-packaged 54 retail pork samples and only from one of the 155 pork cut (0.6%). Most (16/17) of the cheek samples were contaminated with pathogenic Y. enterocolitica 4/O:3 and one with bioserotype 2/O:9. No Y. pseudotuberculosis was isolated. The prevalence of pathogenic Y. enterocolitica was clearly higher (39%) in 155 pork cuts when studied with nested PCR targeting yadA on the virulence plasmid pYV although the contamination level was low varying between 0.1 and 1.6 MPN/g. Raw pork cuts and especially pig cheeks may serve as possible sources for yersiniosis caused by pathogenic Y. enterocolitica. PMID:24929882

  11. In situ structural analysis of the Yersinia enterocolitica injectisome

    PubMed Central

    Kudryashev, Mikhail; Stenta, Marco; Schmelz, Stefan; Amstutz, Marlise; Wiesand, Ulrich; Castaño-Díez, Daniel; Degiacomi, Matteo T; Münnich, Stefan; Bleck, Christopher KE; Kowal, Julia; Diepold, Andreas; Heinz, Dirk W; Dal Peraro, Matteo; Cornelis, Guy R; Stahlberg, Henning

    2013-01-01

    Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI: http://dx.doi.org/10.7554/eLife.00792.001 PMID:23908767

  12. Yersinia pseudotuberculosis infection intractable by antibiotics: A rare case report

    PubMed Central

    Kimura, Jiro; Sasaki, Kiyoshi

    2016-01-01

    Introduction Yersinia pseudotuberculosis infection is usually cured spontaneously or with administration of antibiotics. Presentation of case The patient is a twelve-year-old boy with right lower quadrant pain who had enterocolitis one month previously. Contrast-enhanced abdominal computed tomography showed a distended and edematous ileum and an intra-abdominal abscess adjacent to the mesentery with a normal appendix. The patient’s general condition did not improve with antibiotics, so an ileocecectomy was performed. Discussion Yersinia pseudotuberculosis infection requiring an operation is rare. In our case, antibiotics were not effective in treating the abscess therefore surgery was required. An early diagnosis using serological studies, ultrasound of the abdomen, and fecal culture, with appropriate administration of antibiotics, may have avoided the need for surgery. Considering YP infection as a differential diagnosis is therefore important when encountering patients with enterocolitis, especially with right lower quadrant pain. Early diagnosis may assist in avoiding unnecessary operations. Conclusion Diagnosis of YP infection may be missed or delayed because it is rare and difficult to detect, and must be distinguished from appendicitis. Although most YP infections are self-limiting, some rare cases will require surgery, therefore early diagnosis is essential. PMID:27002288

  13. Growth inhibition of Aeromonas salmonicida and Yersinia ruckeri by disinfectants containing peracetic acid.

    PubMed

    Meinelt, Thomas; Phan, Thy-My; Behrens, Sascha; Wienke, Andreas; Pedersen, Lars-Flemming; Liu, Dibo; Straus, David L

    2015-04-01

    Peracetic acid (PAA) is a therapeutic agent used for disinfection in aquaculture, but it must be investigated thoroughly in order to mitigate diseases without harming the fish. Successful disinfectants (like PAA) should not leave dangerous residues in the environment in order to successfully contribute to sustainable aquaculture. The aim of our study was to compare the effectiveness of 6 commercial PAA products with different molecular PAA:H2O2 ratios to reduce bacterial growth of Aeromonas salmonicida and Yersinia ruckeri and to determine effective concentrations and exposure times. All products reduced colony-forming units (CFUs) of A. salmonicida and Y. ruckeri. Products with higher molecular PAA:H2O2 ratios inhibited growth better than products with lower molecular PAA:H2O2 ratios at the same PAA concentration; this indicates that H2O2 is not the driving force in the reduction of A. salmonicida and Y. ruckeri growth by PAA in vitro. The practical application of the products with high molecular PAA:H2O2 ratios should be prioritized if these pathogens are diagnosed. PMID:25850398

  14. Unique Activity Spectrum of Colicin FY: All 110 Characterized Yersinia enterocolitica Isolates Were Colicin FY Susceptible

    PubMed Central

    Bosák, Juraj; Micenková, Lenka; Vrba, Martin; Ševčíková, Alena; Dědičová, Daniela; Garzetti, Debora; Šmajs, David

    2013-01-01

    Colicin FY is a plasmid encoded toxin that recognizes a yersinia-specific outer membrane protein (YiuR) as a receptor molecule. We have previously shown that the activity spectrum of colicin FY comprises strains of the genus Yersinia. In this study, we analyzed the activity of colicin FY against 110 Yersinia enterocolitica isolates differing in geographical origin and source. All isolates were characterized through analysis of 16S rRNA genes, serotyping, biotyping, restriction profiling of genomic DNA, detection of virulence markers and susceptibility to antibiotics. This confirmed the broad variability of the collection, in which all 110 Y. enterocolitica isolates, representing 77 various strains, were inhibited by colicin FY. Although isolates showed variable levels of susceptibility to colicin FY, it was not associated with any strain characteristic. The universal susceptibility of Y. enterocolitica strains to colicin FY together with the absence of activity towards strains outside the Yersinia genus suggests potential therapeutic applications for colicin FY. PMID:24339971

  15. Differentiation of Yersinia enterocolitica biotype 1A from pathogenic Yersinia enterocolitica biotypes by detection of β-glucosidase activity: comparison of two chromogenic culture media and Vitek2.

    PubMed

    Karhukorpi, Jari; Päivänurmi, Marjut

    2014-01-01

    Aesculin hydrolysis (ESC) is one of the key reactions in differentiating pathogenic Yersinia enterocolitica biotypes 1B, 2, 3, 4 and 5 from the less-pathogenic biotype 1A. Because the ESC reaction is caused by β-glucosidase (βGLU) activity of the bacteria, we studied whether two commonly used methods (BBL CHROMagar Orientation and Vitek2 Gram-negative identification card) could be used in assessing βGLU activity of 74 Yersinia strains. Both methods were sensitive (100 % and 97 %) and specific (100 % and 100 %) in differentiating βGLU-positive YE BT1A from βGLU-negative Y. enterocolitica biotypes. For a subset of strains (n = 69), a new selective CHROMagar Yersinia showed excellent agreement with the strains' βGLU activity. Thus all the methods evaluated in this study may be used to differentiate between YE BT1A and other Y. enterocolitica biotypes. PMID:24072767

  16. Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein*

    PubMed Central

    Wolters, Manuel; Boyle, Erin C.; Lardong, Kerstin; Trülzsch, Konrad; Steffen, Anika; Rottner, Klemens; Ruckdeschel, Klaus; Aepfelbacher, Martin

    2013-01-01

    Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac. PMID:23803609

  17. Trends of the Major Porin Gene (ompF) Evolution: Insight from the Genus Yersinia

    PubMed Central

    Stenkova, Anna M.; Isaeva, Marina P.; Shubin, Felix N.; Rasskazov, Valeri A.; Rakin, Alexander V.

    2011-01-01

    OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species. PMID:21655186

  18. Enzymatic Degradation of Polygalacturonic Acid by Yersinia and Klebsiella Species in Relation to Clinical Laboratory Procedures

    PubMed Central

    Starr, Mortimer P.; Chatterjee, Arun K.; Starr, Phoebe B.; Buchanan, Gordon E.

    1977-01-01

    As scored by several specified plating procedures, clinical and environmental strains of Yersinia enterocolitica, Yersinia pseudotuberculosis, and Klebsiella pneumoniae “Oxytocum” showed detectable, albeit generally weak, ability to digest polygalacturonic (pectic) acid. None of these bacterial strains had the vigorous and rapid pectolytic activity on these polygalacturonic acid-containing media that is typical of soft-rot Erwinia species, although some of the Oxytocum strains came fairly close. Analyses of the pectolytic enzyme contents of the cells and culture supernatants of the Yersinia and Klebsiella species revealed that readily detectable quantities of cell-bound polygalacturonic acid trans-eliminase and hydrolytic polygalacturonase were formed by the Yersinia and Klebsiella species; however, the total units of enzyme activity produced by these bacteria were, in general, lower than were produced by soft-rot Erwinia species. Furthermore, unlike the situation in soft-rot Erwinia cultures, these pectolytic enzymes of Yersinia and Klebsiella species were not excreted rapidly and massively into the growth medium. Cultures of other enterobacteria (Citrobacter species, Enterobacter species, Erwinia amylovora, Erwinia herbicola, Escherichia coli, Proteus species, Salmonella typhimurium, and Serratia marcescens) showed no pectolytic ability whatsoever by any of the plating procedures used and (to the extent they were so examined) produced no pectolytic enzymes detectable either in their cells or culture supernatants. This slow or weak release of pectolytic enzymes by Yersinia and Klebsiella species has a bearing on clinical laboratory procedures suitable for detecting their pectolytic activity; methods adequate for this purpose are detailed. PMID:334794

  19. Yersinia enterocolitica isolates from humans in California, 1968-1975.

    PubMed Central

    Bissett, M L

    1976-01-01

    This paper reports on the serological and biochemical characteristics of 24 human isolates of Yersinia enterocolitica submitted to the California Department of Health from 1968 through 1975. Nine different serotypes were represented. The majority of strains were serotype O:8 (six strains) and serotype O:5 (five strains). Sources of the isolates included feces (12 cases), blood (3), sputum or throat (3), bile or bowel drainage (2), wounds (2), breast abscess (1), and skin abscess (1). Clinical histories indicated a number of different syndromes. Underlying medical conditions existed in 13 cases. Results of selected biochemical tests and antimicrobial susceptibility tests on the strains indicated grouping compatible with the O serotypes of the organisms. PMID:965477

  20. Presence of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis and Escherichia coli O157:H7 in wild boars.

    PubMed

    Sannö, A; Aspán, A; Hestvik, G; Jacobson, M

    2014-12-01

    The European wild boar populations are growing and spreading to new areas, which might constitute a threat to public health, since wild boar can harbour pathogens with the potential to cause serious illness in humans. Tonsils, ileocaecal lymph nodes and faecal samples were collected from 88 Swedish wild boars and analysed for the presence of the zoonotic pathogens Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). A combination of cultivation and polymerase chain reaction (PCR) analysis was used and overall, 20% of sampled individuals tested positive for Y. enterocolitica, 20% for Y. pseudotuberculosis and 10% for Salmonella spp. A total of 41% of sampled individuals tested positive for one or more of these three pathogens. No EHEC were detected. Samples PCR-positive for Salmonella spp. were cultivated further and six isolates were obtained, belonging to Salmonella enterica subspecies enterica and subspecies diarizone. The pathogens were most commonly detected in tonsil samples. PMID:24512817

  1. Comparison of virulence plasmid (pYV/pCD)-associated phenotypes in Yersinia species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenotypic expression of several virulence plasmid (pYV/pCD; 70-kb)-associated genetic determinants were compared among Yesinia species; Yesinia pestis, (an attenuated strain which does not cause plague designated to be used in BSL2 facility), Y. pseudotuberculosis and Y. enterocolitica including lo...

  2. Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria.

    PubMed

    Okwori, Ameh E J; Martínez, Pilar Ortiz; Fredriksson-Ahomaa, Maria; Agina, Samuel E; Korkeala, Hannu

    2009-12-01

    Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enter