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Sample records for obligate intracellular pathogen

  1. Adaptive immunity to the obligate intracellular pathogen Coxiella burnetii.

    PubMed

    Shannon, Jeffrey G; Heinzen, Robert A

    2009-01-01

    Coxiella burnetii is an obligate intracellular bacterial pathogen that causes the zoonosis Q fever. While an effective whole-cell vaccine (WCV) against Q fever exists, the vaccine has limitations in being highly reactogenic in sensitized individuals. Thus, a safe and effective vaccine based on recombinant protein antigen (Ag) is desirable. To achieve this goal, a better understanding of the host response to primary infection and the precise mechanisms involved in protective immunity to C. burnetii are needed. This review summarizes our current understanding of adaptive immunity to C. burnetii with a focus on recent developments in the field. PMID:18813881

  2. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

    PubMed Central

    Giengkam, Suparat; Blakes, Alex; Utsahajit, Peemdej; Chaemchuen, Suwittra; Atwal, Sharanjeet; Blacksell, Stuart D.; Paris, Daniel H.; Day, Nicholas P. J.; Salje, Jeanne

    2015-01-01

    Background Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families. Methodology A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells. Key results Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing. Conclusions Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments. PMID:26317517

  3. Proteomic Profiling of the Outer Membrane Fraction of the Obligate Intracellular Bacterial Pathogen Ehrlichia ruminantium

    PubMed Central

    Moumène, Amal; Marcelino, Isabel; Ventosa, Miguel; Gros, Olivier; Lefrançois, Thierry; Vachiéry, Nathalie

    2015-01-01

    The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium. PMID:25710494

  4. Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis

    PubMed Central

    Blume, Martin; Rodriguez-Contreras, Dayana; Landfear, Scott; Fleige, Tobias; Soldati-Favre, Dominique; Lucius, Richard; Gupta, Nishith

    2009-01-01

    Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1–3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike ?tgst2, the modest impairment in ?tggt1 and ?tggt1/?tgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The ?tggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in ?tggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of ?tggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells. PMID:19617561

  5. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungalrelated parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and...

  6. Modulation of innate immune signaling pathways by the intracellular pathogen Toxoplasma gondii

    E-print Network

    Rosowski, Emily E. (Emily Elizabeth)

    2013-01-01

    Toxoplasma gondii, an obligate intracellular protozoan parasite, is one of the most successful eukaryotic pathogens. It can infect virtually any warm-blooded animal, including humans, in whom it can cause serious disease. ...

  7. Transient Transfection and Expression in the Obligate Intracellular Parasite Toxoplasma gondii

    NASA Astrophysics Data System (ADS)

    Soldati, Dominique; Boothroyd, John C.

    1993-04-01

    Toxoplasma gondii is a protozoan pathogen that produces severe disease in humans and animals. This obligate intracellular parasite provides an excellent model for the study of how such pathogens are able to invade, survive, and replicate intracellularly. DNA encoding chloramphenicol acetyltransferase was introduced into T. gondii and transiently expressed with the use of three vectors based on different Toxoplasma genes. The ability to introduce genes and have them efficiently and faithfully expressed is an essential tool for understanding the structure-function relation of genes and their products.

  8. Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

    PubMed Central

    Gillespie, Joseph J.; Williams, Kelly; Shukla, Maulik; Snyder, Eric E.; Nordberg, Eric K.; Ceraul, Shane M.; Dharmanolla, Chitti; Rainey, Daphne; Soneja, Jeetendra; Shallom, Joshua M.; Vishnubhat, Nataraj Dongre; Wattam, Rebecca; Purkayastha, Anjan; Czar, Michael; Crasta, Oswald; Setubal, Joao C.; Azad, Abdu F.; Sobral, Bruno S.

    2008-01-01

    Background Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular ?-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs). Methodology/Principal Findings We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (?1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. Conclusion/Significance Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets. PMID:19194535

  9. Metabolic Interdependence of Obligate Intracellular Bacteria and Their Insect Hosts†

    PubMed Central

    Zientz, Evelyn; Dandekar, Thomas; Gross, Roy

    2004-01-01

    Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism—that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts. PMID:15590782

  10. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    PubMed Central

    Leon-Sicairos, Nidia; Reyes-Cortes, Ruth; Guadrón-Llanos, Alma M.; Madueña-Molina, Jesús; Leon-Sicairos, Claudia; Canizalez-Román, Adrian

    2015-01-01

    Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed. PMID:26120582

  11. The role of autophagy in intracellular pathogen nutrient acquisition

    PubMed Central

    Steele, Shaun; Brunton, Jason; Kawula, Thomas

    2015-01-01

    Following entry into host cells intracellular pathogens must simultaneously evade innate host defense mechanisms and acquire energy and anabolic substrates from the nutrient-limited intracellular environment. Most of the potential intracellular nutrient sources are stored within complex macromolecules that are not immediately accessible by intracellular pathogens. To obtain nutrients for proliferation, intracellular pathogens must compete with the host cell for newly-imported simple nutrients or degrade host nutrient storage structures into their constituent components (fatty acids, carbohydrates, and amino acids). It is becoming increasingly evident that intracellular pathogens have evolved a wide variety of strategies to accomplish this task. One recurrent microbial strategy is to exploit host degradative processes that break down host macromolecules into simple nutrients that the microbe can use. Herein we focus on how a subset of bacterial, viral, and eukaryotic pathogens leverage the host process of autophagy to acquire nutrients that support their growth within infected cells. PMID:26106587

  12. The genome of obligately intracellular Ehrlichia canis revealsthemes of complex membrane structure and immune evasion strategies

    SciTech Connect

    Mavromatis, K.; Kuyler Doyle, C.; Lykidis, A.; Ivanova, N.; Francino, P.; Chain, P.; Shin, M.; Malfatti, S.; Larimer, F.; Copeland,A.; Detter, J.C.; Land, M.; Richardson, P.M.; Yu, X.J.; Walker, D.H.; McBride, J.W.; Kyrpides, N.C.

    2005-09-01

    Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, a-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7).

  13. Evolutionary Genomics of a Temperate Bacteriophage in an Obligate Intracellular Bacteria (Wolbachia)

    E-print Network

    Bordenstein, Seth

    Evolutionary Genomics of a Temperate Bacteriophage in an Obligate Intracellular Bacteria (Wolbachia, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant

  14. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms

    PubMed Central

    Mansilla Pareja, Maria Eugenia; Colombo, Maria I.

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance. PMID:24137567

  15. Bacterial pathogens commandeer Rab GTPases to establish intracellular niches.

    PubMed

    Stein, Mary-Pat; Müller, Matthias P; Wandinger-Ness, Angela

    2012-12-01

    Intracellular bacterial pathogens deploy virulence factors termed effectors to inhibit degradation by host cells and to establish intracellular niches where growth and differentiation take place. Here, we describe mechanisms by which human bacterial pathogens (including Chlamydiae; Coxiella burnetii; Helicobacter pylori; Legionella pneumophila; Listeria monocytogenes; Mycobacteria; Pseudomonas aeruginosa, Salmonella enterica) modulate endocytic and exocytic Rab GTPases in order to thrive in host cells. Host cell Rab GTPases are critical for intracellular transport following pathogen phagocytosis or endocytosis. At the molecular level bacterial effectors hijack Rab protein function to: evade degradation, direct transport to particular intracellular locations and monopolize host vesicles carrying molecules that are needed for a stable niche and/or bacterial growth and differentiation. Bacterial effectors may serve as specific receptors for Rab GTPases or as enzymes that post-translationally modify Rab proteins or endosomal membrane lipids required for Rab function. Emerging data indicate that bacterial effector expression is temporally and spatially regulated and multiple virulence factors may act concertedly to usurp Rab GTPase function, alter signaling and ensure niche establishment and intracellular bacterial growth, making this field an exciting area for further study. PMID:22901006

  16. Intracellular immunity: finding the enemy within—how cells recognize and respond to intracellular pathogens

    PubMed Central

    Tam, Jerry C. H.; Jacques, David A.

    2014-01-01

    Historically, once a cell became infected, it was considered to be beyond all help. By this stage, the invading pathogen had breached the innate defenses and was beyond the reach of the humoral arm of the adaptive immune response. The pathogen could still be removed by cell-mediated immunity (e.g., by NK cells or cytotoxic T lymphocytes), but these mechanisms necessitated the destruction of the infected cell. However, in recent years, it has become increasingly clear that many cells possess sensor and effector mechanisms for dealing with intracellular pathogens. Most of these mechanisms are not restricted to professional immune cells nor do they all necessitate the destruction of the host. In this review, we examine the strategies that cells use to detect and destroy pathogens once the cell membrane has been penetrated. PMID:24899588

  17. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila

    PubMed Central

    Fonseca, Maris V.; Swanson, Michele S.

    2014-01-01

    The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication. PMID:24575391

  18. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  19. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites.

    PubMed

    Woo, Yong H; Ansari, Hifzur; Otto, Thomas D; Klinger, Christen M; Kolisko, Martin; Michálek, Jan; Saxena, Alka; Shanmugam, Dhanasekaran; Tayyrov, Annageldi; Veluchamy, Alaguraj; Ali, Shahjahan; Bernal, Axel; del Campo, Javier; Cihlá?, Jaromír; Flegontov, Pavel; Gornik, Sebastian G; Hajdušková, Eva; Horák, Aleš; Janouškovec, Jan; Katris, Nicholas J; Mast, Fred D; Miranda-Saavedra, Diego; Mourier, Tobias; Naeem, Raeece; Nair, Mridul; Panigrahi, Aswini K; Rawlings, Neil D; Padron-Regalado, Eriko; Ramaprasad, Abhinay; Samad, Nadira; Tom?ala, Aleš; Wilkes, Jon; Neafsey, Daniel E; Doerig, Christian; Bowler, Chris; Keeling, Patrick J; Roos, David S; Dacks, Joel B; Templeton, Thomas J; Waller, Ross F; Lukeš, Julius; Oborník, Miroslav; Pain, Arnab

    2015-01-01

    The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. PMID:26175406

  20. Specific isolation of RNA from the grape powdery mildew pathogen Erysiphe necator, an epiphytic, obligate parasite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA expression profiling of obligately parasitic plant microbes is hampered by the requisite interaction of host and parasite. For superficial pathogens like grape powdery mildew as well as for epiphytic saprophytes, growth along the outside surface of the plant allows separation from the host and ...

  1. The Obligate Intracellular Parasite Toxoplasma gondii Secretes a Soluble Phosphatidylserine Decarboxylase*

    PubMed Central

    Gupta, Nishith; Hartmann, Anne; Lucius, Richard; Voelker, Dennis R.

    2012-01-01

    Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent Km of 67 ?m. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ?20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ?20% of its total PSD activity at 37 °C, and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism. PMID:22563079

  2. The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite

    PubMed Central

    2005-01-01

    We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular ?-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin–antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as ?-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria. PMID:15984913

  3. Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

    NASA Astrophysics Data System (ADS)

    Reverey, Julia F.; Jeon, Jae-Hyung; Bao, Han; Leippe, Matthias; Metzler, Ralf; Selhuber-Unkel, Christine

    2015-06-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

  4. Superdiffusion dominates intracellular particle motion in the supercrowded space of pathogenic Acanthamoeba castellanii

    E-print Network

    J. F. Reverey; J. -H. Jeon; H. Bao; M. Leippe; R. Metzler; C. Selhuber-Unkel

    2015-07-02

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

  5. Superdiffusion dominates intracellular particle motion in the supercrowded space of pathogenic Acanthamoeba castellanii

    E-print Network

    Reverey, J F; Bao, H; Leippe, M; Metzler, R; Selhuber-Unkel, C

    2015-01-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved ...

  6. Genetic analysis of host resistance to intracellular pathogens: lessons from studies of Toxoplasma gondii infection.

    PubMed

    Yap, George S; Shaw, Michael H; Ling, Yun; Sher, Alan

    2006-04-01

    Cell-mediated immunity to Toxoplasma gondii establishes and maintains a balanced host-pathogen relationship. Recent analyses using spontaneous and genetically engineered mouse mutants have yielded a clearer picture of factors positively and negatively regulating the host immune response and a better understanding of cytokine-inducible intracytoplasmic mechanisms that lead to intracellular pathogen suppression and demise. PMID:16513380

  7. Host-Directed Antimicrobial Drugs with Broad-Spectrum Efficacy against Intracellular Bacterial Pathogens

    PubMed Central

    Czy?, Daniel M.; Potluri, Lakshmi-Prasad; Jain-Gupta, Neeta; Riley, Sean P.; Martinez, Juan J.; Steck, Theodore L.; Crosson, Sean; Gabay, Joëlle E.

    2014-01-01

    ABSTRACT We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. PMID:25073644

  8. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    NASA Astrophysics Data System (ADS)

    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  9. Comparative Genomics Suggests That the Human Pathogenic Fungus Pneumocystis jirovecii Acquired Obligate Biotrophy through Gene Loss

    PubMed Central

    Cissé, Ousmane H.; Pagni, Marco; Hauser, Philippe M.

    2014-01-01

    Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses. PMID:25062922

  10. Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood.

    PubMed

    Vojvodic, S; Jensen, A B; James, R R; Boomsma, J J; Eilenberg, J

    2011-04-21

    Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic honeybee-specific disease that can persist in colonies for years, reducing both brood and honey production, whereas stonebrood is a rare facultative pathogen that also affects hosts other than honeybees and can likely survive outside insect hosts. Hive infection trials have indicated that accidental drops in comb temperature increase the prevalence of chalkbrood, but it has remained unclear whether virulence is directly temperature-dependent. We used a newly established in vitro rearing technique for honeybee larvae to test whether there are systematic temperature effects on mortality induced by controlled infections, and whether such effects differed between the two fungal pathogens. We found that increasing spore dosage at infection had a more dramatic effect on mortality from stonebrood compared to chalkbrood. In addition, a 24h cooling period after inoculation increased larval mortality from chalkbrood infection, whereas such a cooling period decreased mortality after stonebrood infection. These results raise interesting questions about honeybee defenses against obligate and facultative pathogens and about the extent to which stress factors in the host (dis)favor pathogens with lesser degrees of specialization. PMID:21050682

  11. A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

    PubMed Central

    Laubier, Aurélie; Bleves, Sophie; Blanc-Potard, Anne-Béatrice

    2015-01-01

    Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies. PMID:26080006

  12. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    E-print Network

    Higgins, Darren

    Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen H. G for review October 27, 2005) We have developed a vaccine strategy for generating an attenu- ated strain intra- cellularly and is readily killed. However, after degradation of the vaccine strain within

  13. Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes

    PubMed Central

    Odendall, Charlotte; Dixit, Evelyn; Stavru, Fabrizia; Bierne, Helene; Franz, Kate M.; Fiegen, Ann; Boulant, Steeve; Gehrke, Lee; Cossart, Pascale; Kagan, Jonathan C.

    2014-01-01

    Type I Interferon (IFN) responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of Type I IFNs. The mechanisms controlling Type I IFN-independent responses are undefined. We have found that RIG-I like Receptors (RLRs) induce Type III IFN expression in a variety of human cell types, and identified factors that differentially regulate Type I and III IFN expression. We identified peroxisomes as a primary site that initiates Type III IFN expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust Type III IFN responses in human cells. These findings highlight the interconnections between innate immunity and cell biology. PMID:24952503

  14. A new view to intracellular pathogens and host responses in the South of Spain

    PubMed Central

    Portillo, Francisco García-del; Cossart, Pascale

    2012-01-01

    A workshop on ‘The Biology of Intracellular Bacterial Pathogens’ was held last October in a venue of the International University of Andalusia (UNIA) located in the World Historic Heritage town of Baeza, in the South of Spain. This Workshop gathered leading scientists from around the world to discuss their latest findings related to the mechanisms that intracellular pathogens use to subvert and manipulate host cell functions. The workshop focused on novel aspects that imprint current research in this discipline, including the heterogeneous behaviour of the pathogen at the population level, the host determinants that modulate susceptibility to the infection, the search for new drugs to combat these particular types of infections and also cutting edge technologies based on new imaging approaches and the use of microfluidics. Discussion on these topics provided new insights into the biology of these pathogens and enriched the field with new ideas for understanding why colonization of the intracellular niche of eukaryotic cells is a preferred strategy used by important human pathogens. PMID:22323444

  15. Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts.

    PubMed

    Chacón-Díaz, Carlos; Altamirano-Silva, Pamela; González-Espinoza, Gabriela; Medina, María-Concepción; Alfaro-Alarcón, Alejandro; Bouza-Mora, Laura; Jiménez-Rojas, César; Wong, Melissa; Barquero-Calvo, Elías; Rojas, Norman; Guzmán-Verri, Caterina; Moreno, Edgardo; Chaves-Olarte, Esteban

    2015-12-01

    Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly. PMID:26438796

  16. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21.

    PubMed

    McEwan, William A; Tam, Jerry C H; Watkinson, Ruth E; Bidgood, Susanna R; Mallery, Donna L; James, Leo C

    2013-04-01

    During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-?B, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors. PMID:23455675

  17. Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California

    PubMed Central

    Straub, Mary H.; Kelly, Terra R.; Rideout, Bruce A.; Eng, Curtis; Wynne, Janna; Braun, Josephine; Johnson, Christine K.

    2015-01-01

    Throughout the world, populations of scavenger birds are declining rapidly with some populations already on the brink of extinction. Much of the current research into the factors contributing to these declines has focused on exposure to drug residues, lead, and other toxins. Despite increased monitoring of these declining populations, little is known about infectious diseases affecting scavenger bird species. To assess potential infectious disease risks to both obligate and facultative scavenger bird species, we performed a serosurvey for eleven potential pathogens in three species of scavenging birds in California: the California condor (Gymnogyps californianus), turkey vulture (Cathartes aura) and golden eagle (Aquila chrysaetos). California condors were seropositive for avian adenovirus, infectious bronchitis virus, Mycoplasma gallisepticum, avian paramyxovirus-2, West Nile virus (WNV) and Toxoplasma gondii. Golden eagles were seropositive for avian adenovirus, Chlamydophila psittaci and Toxoplasma gondii, and turkey vultures were seropositive for avian adenovirus, Chlamydophila psittaci, avian paramyxovirus-1, Toxoplasma gondii and WNV. Risk factor analyses indicated that rearing site and original release location were significantly associated with a positive serologic titer to WNV among free-flying condors. This study provides preliminary baseline data on infectious disease exposure in these populations for aiding in early disease detection and provides potentially critical information for conservation of the endangered California condor as it continues to expand its range and encounter new infectious disease threats. PMID:26606755

  18. Characterization of an obligate intracellular bacterium in the midgut epithelium of the bulrush bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae).

    PubMed

    Kuechler, Stefan Martin; Dettner, Konrad; Kehl, Siegfried

    2011-05-01

    Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This "mycetocytic belt" might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, "Candidatus Rohrkolberia cinguli," is proposed for this newly characterized clade of symbiotic bacteria. PMID:21378044

  19. Identification of a Quorum-Sensing Signal Molecule in the Facultative Intracellular Pathogen Brucella melitensis

    PubMed Central

    Taminiau, Bernard; Daykin, Mavis; Swift, Simon; Boschiroli, Maria-Laura; Tibor, Anne; Lestrate, Pascal; De Bolle, Xavier; O'Callaghan, David; Williams, Paul; Letesson, Jean-Jacques

    2002-01-01

    Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella. PMID:12010991

  20. Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice

    PubMed Central

    Collantes-Fernandez, Esther; Arrighi, Romanico B. G.; Álvarez-García, Gema; Weidner, Jessica M.; Regidor-Cerrillo, Javier; Boothroyd, John C.; Ortega-Mora, Luis M.; Barragan, Antonio

    2012-01-01

    The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites. PMID:22403627

  1. M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae

    PubMed Central

    Buchacher, Tanja; Ohradanova-Repic, Anna; Stockinger, Hannes

    2015-01-01

    Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-? and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of C. pneumoniae to evade cellular defense and to persist in human macrophages. PMID:26606059

  2. The Mutualistic Side of Wolbachia–Isopod Interactions: Wolbachia Mediated Protection Against Pathogenic Intracellular Bacteria

    PubMed Central

    Braquart-Varnier, Christine; Altinli, Mine; Pigeault, Romain; Chevalier, Frédéric D.; Grève, Pierre; Bouchon, Didier; Sicard, Mathieu

    2015-01-01

    Wolbachia is a vertically transmitted endosymbiont whose radiative success is mainly related to various host reproductive manipulations that led to consider this symbiont as a conflictual reproductive parasite. However, lately, some Wolbachia have been shown to act as beneficial symbionts by protecting hosts against a broad range of parasites. Still, this protection has been mostly demonstrated in artificial Wolbachia-host associations between partners that did not co-evolved together. Here, we tested in two terrestrial isopod species Armadillidium vulgare and Porcellio dilatatus whether resident Wolbachia (native or non-native) could confer protection during infections with Listeria ivanovii and Salmonella typhimurium and also during a transinfection with a Wolbachia strain that kills the recipient host (i.e., wVulC in P. dilatatus). Survival analyses showed that (i) A. vulgare lines hosting their native Wolbachia (wVulC) always exhibited higher survival than asymbiotic ones when infected with pathogenic bacteria (ii) P. dilatatus lines hosting their native wDil Wolbachia strain survived the S. typhimurium infection better, while lines hosting non-native wCon Wolbachia strain survived the L. ivanovii and also the transinfection with wVulC from A. vulgare better. By studying L. ivanovii and S. typhimurium loads in the hemolymph of the different host-Wolbachia systems, we showed that (i) the difference in survival between lines after L. ivanovii infections were not linked to the difference between their pathogenic bacterial loads, and (ii) the difference in survival after S. typhimurium infections corresponds to lower loads of pathogenic bacteria. Overall, our results demonstrate a beneficial effect of Wolbachia on survival of terrestrial isopods when infected with pathogenic intracellular bacteria. This protective effect may rely on different mechanisms depending on the resident symbiont and the invasive bacteria interacting together within the hosts.

  3. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    PubMed

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A; Xet-Mull, Ana M; Sisk, Dana M; Smith, Kristen L Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H; Tobin, David M; Cullen, Bryan R

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ? 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin. PMID:25184567

  4. Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana

    PubMed Central

    Kemen, Eric; Gardiner, Anastasia; Schultz-Larsen, Torsten; Kemen, Ariane C.; Balmuth, Alexi L.; Robert-Seilaniantz, Alexandre; Bailey, Kate; Holub, Eric; Studholme, David J.; MacLean, Dan; Jones, Jonathan D. G.

    2011-01-01

    Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the “CHXCs”, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata. PMID:21750662

  5. Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen Brucella melitensis 16 M

    PubMed Central

    2010-01-01

    Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A ?vjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment. PMID:20387905

  6. Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

    PubMed

    McCormack, Ryan M; de Armas, Lesley R; Shiratsuchi, Motoaki; Fiorentino, Desiree G; Olsson, Melissa L; Lichtenheld, Mathias G; Morales, Alejo; Lyapichev, Kirill; Gonzalez, Louis E; Strbo, Natasa; Sukumar, Neelima; Stojadinovic, Olivera; Plano, Gregory V; Munson, George P; Tomic-Canic, Marjana; Kirsner, Robert S; Russell, David G; Podack, Eckhard R

    2015-01-01

    Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. PMID:26402460

  7. Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria

    PubMed Central

    McCormack, Ryan M; de Armas, Lesley R; Shiratsuchi, Motoaki; Fiorentino, Desiree G; Olsson, Melissa L; Lichtenheld, Mathias G; Morales, Alejo; Lyapichev, Kirill; Gonzalez, Louis E; Strbo, Natasa; Sukumar, Neelima; Stojadinovic, Olivera; Plano, Gregory V; Munson, George P; Tomic-Canic, Marjana; Kirsner, Robert S; Russell, David G; Podack, Eckhard R

    2015-01-01

    Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. DOI: http://dx.doi.org/10.7554/eLife.06508.001 PMID:26402460

  8. Comparative Genome Analysis of Wheat Blue Dwarf Phytoplasma, an Obligate Pathogen That Causes Wheat Blue Dwarf Disease in China

    PubMed Central

    Chen, Wang; Li, Yan; Wang, Qiang; Wang, Nan; Wu, Yunfeng

    2014-01-01

    Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ?94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms. PMID:24798075

  9. Comparative genome analysis of wheat blue dwarf phytoplasma, an obligate pathogen that causes wheat blue dwarf disease in China.

    PubMed

    Chen, Wang; Li, Yan; Wang, Qiang; Wang, Nan; Wu, Yunfeng

    2014-01-01

    Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ?94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms. PMID:24798075

  10. Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs

    PubMed Central

    Hauser, Philippe M.; Burdet, Frédéric X.; Cissé, Ousmane H.; Keller, Laurent; Taffé, Patrick; Sanglard, Dominique; Pagni, Marco

    2010-01-01

    Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8’085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4’974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the “amino acid metabolism” category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans. PMID:21188143

  11. 13C-Flux Spectral Analysis of Host-Pathogen Metabolism Reveals a Mixed Diet for Intracellular Mycobacterium tuberculosis

    PubMed Central

    Beste, Dany J.V.; Nöh, Katharina; Niedenführ, Sebastian; Mendum, Tom A.; Hawkins, Nathaniel D.; Ward, Jane L.; Beale, Michael H.; Wiechert, Wolfgang; McFadden, Johnjoe

    2013-01-01

    Summary Whereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world’s population, remain mostly unknown. We used a systems-based approach—13C-flux spectral analysis (FSA) complemented with manual analysis—to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. 13C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. 13C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics. PMID:23911587

  12. Functional Characterization of the Incomplete Phosphotransferase System (PTS) of the Intracellular Pathogen Brucella melitensis

    PubMed Central

    Dozot, Marie; Poncet, Sandrine; Nicolas, Cécile; Copin, Richard; Bouraoui, Houda; Mazé, Alain; Deutscher, Josef; De Bolle, Xavier; Letesson, Jean-Jacques

    2010-01-01

    Background In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions. Methodology/Principal Findings In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among ?-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase. Conclusions/Significance The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism. PMID:20844759

  13. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

    PubMed

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells. PMID:26518878

  14. Anaplasma phagocytophilum APH_1387 is expressed throughout bacterial intracellular development and localizes to the pathogen-occupied vacuolar membrane.

    PubMed

    Huang, Bernice; Troese, Matthew J; Ye, Shaojing; Sims, Jonathan T; Galloway, Nathan L; Borjesson, Dori L; Carlyon, Jason A

    2010-05-01

    Obligate vacuolar pathogens produce proteins that localize to the host cell-derived membranes of the vacuoles in which they reside, yielding unique organelles that are optimally suited for pathogen survival. Anaplasma phagocytophilum is an obligate vacuolar bacterium that infects neutrophils and causes the emerging and potentially fatal disease human granulocytic anaplasmosis. Here we identified APH_1387 as the first A. phagocytophilum-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_1387, also referred to as P100, is a 61.4-kDa acidic protein that migrates with an apparent molecular weight of 115 kDa on SDS-PAGE gels. It carries 3 tandem direct repeats that comprise 58% of the protein. Each APH_1387 repeat carries a bilobed hydrophobic alpha-helix domain, which is a structural characteristic that is consistent with the structure of chlamydia-derived proteins that traverse inclusion membranes. APH_1387 is not detectable on the surfaces of A. phagocytophilum dense core organisms bound at the HL-60 cell surface, but abundant APH_1387 is detected on the surfaces of intravacuolar reticulate cell and dense core organisms. APH_1387 accumulates on the AVM throughout infection. It associates with the AVM in human HL-60, THP-1, and HMEC-1 cells and tick ISE6 cells. APH_1387 is expressed and localizes to the AVM in neutrophils recovered from A. phagocytophilum-infected mice. This paper presents the first direct evidence that A. phagocytophilum actively modifies its host cell-derived vacuole. PMID:20212090

  15. P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance.

    PubMed

    Tang, Xiao; Basavarajappa, Devaraj; Haeggström, Jesper Z; Wan, Min

    2015-08-01

    Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence-dependent endocytotic process. Both clathrin- and caveolae/lipid raft-mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance. PMID:26116509

  16. P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

    PubMed Central

    Tang, Xiao; Basavarajappa, Devaraj

    2015-01-01

    Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence–dependent endocytotic process. Both clathrin- and caveolae/lipid raft–mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance. PMID:26116509

  17. Type II Cytokines Impair Host Defense Against Intracellular Fungal Pathogen by Amplifying Macrophage Generation of IL-33

    PubMed Central

    Verma, Akash; Kroetz, Danielle N.; Tweedle, Jamie L.; Deepe, George S.

    2014-01-01

    IL-4 subverts protective immunity to multiple intracellular pathogens including the fungus Histoplasma capsulatum. Previously, we reported that H. capsulatum-challenged CCR2?/? mice manifest elevated pulmonary fungal burden due to exaggerated IL-4. Paradoxical to our anticipation in IL-33 driving IL-4, we discovered the latter prompted IL-33 in mutant mice. In infected CCR2?/? animals, amplified IL-33 succeeded the heightened IL-4 response and inhibition of IL-4 signaling decreased IL-33. Moreover, macrophages, but not epithelial cells or dendritic cells from these mice expressed higher IL-33 in comparison to controls. Dissection of mechanisms that promulgated IL-33 revealed type-II cytokines and H. capsulatum synergistically elicited an IL-33 response in macrophages via STAT6/IRF-4 and Dectin-1 pathways respectively. Neutralizing IL-33 in CCR2?/? animals, but not controls, enhanced their resistance to histoplasmosis. Thus, we describe a previously unrecognized role for IL-4 in instigating IL-33 in macrophages. Furthermore, in presence of intracellular fungal pathogens, the type-II cytokine-driven IL-33 response impairs immunity. PMID:25118166

  18. The Equine Antimicrobial Peptide eCATH1 Is Effective against the Facultative Intracellular Pathogen Rhodococcus equi in Mice

    PubMed Central

    Schlusselhuber, Margot; Torelli, Riccardo; Martini, Cecilia; Leippe, Matthias; Cattoir, Vincent; Leclercq, Roland; Laugier, Claire; Grötzinger, Joachim; Sanguinetti, Maurizio

    2013-01-01

    Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis. PMID:23817377

  19. A Rickettsia Genome Overrun by Mobile Genetic Elements Provides Insight into the Acquisition of Genes Characteristic of an Obligate Intracellular Lifestyle

    PubMed Central

    Joardar, Vinita; Williams, Kelly P.; Driscoll, Timothy; Hostetler, Jessica B.; Nordberg, Eric; Shukla, Maulik; Walenz, Brian; Hill, Catherine A.; Nene, Vishvanath M.; Azad, Abdu F.; Sobral, Bruno W.; Caler, Elisabet

    2012-01-01

    We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ?35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity. PMID:22056929

  20. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella

    PubMed Central

    Trevijano-Contador, Nuria; Herrero-Fernández, Inés; García-Barbazán, Irene; Scorzoni, Liliana; Rueda, Cristina; Rossi, Suélen Andreia; García-Rodas, Rocío; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans. PMID:25531532

  1. Intracellular Growth Is Dependent on Tyrosine Catabolism in the Dimorphic Fungal Pathogen Penicillium marneffei

    PubMed Central

    Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex

    2015-01-01

    During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137

  2. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    SciTech Connect

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-08-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded ?-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded ?-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-?-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the ?-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other ?-barrel proteins.

  3. Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen.

    PubMed

    Willett, Jonathan W; Herrou, Julien; Briegel, Ariane; Rotskoff, Grant; Crosson, Sean

    2015-07-14

    We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi. PMID:26124143

  4. Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

    PubMed

    Tsalikis, Jessica; Tattoli, Ivan; Ling, Arthur; Sorbara, Matthew T; Croitoru, David O; Philpott, Dana J; Girardin, Stephen E

    2015-08-21

    Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-?B-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense. PMID:26134566

  5. Granulocyte Macrophage-Colony Stimulating Factor-induced Zn Sequestration Enhances Macrophage Superoxide and Limits Intracellular Pathogen Survival

    PubMed Central

    Vignesh, Kavitha Subramanian; Landero Figueroa, Julio A.; Porollo, Aleksey; Caruso, Joseph A.; Deepe, George S.

    2013-01-01

    SUMMARY Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like Zn. The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7 and the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H+ channel function and triggered reactive oxygen species (ROS) generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function. PMID:24138881

  6. AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae

    PubMed Central

    Klöckner, Anna; Otten, Christian; Derouaux, Adeline; Vollmer, Waldemar; Bühl, Henrike; De Benedetti, Stefania; Münch, Daniela; Josten, Michaele; Mölleken, Katja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-01-01

    Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation. PMID:24953137

  7. Invasion of the central nervous system by intracellular bacteria.

    PubMed

    Drevets, Douglas A; Leenen, Pieter J M; Greenfield, Ronald A

    2004-04-01

    Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens. PMID:15084504

  8. Identification of Genetic Variation between Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli Using RNA Sequencing and Genotyping-By-Sequencing

    PubMed Central

    Summers, Carly F.; Gulliford, Colwyn M.; Carlson, Craig H.; Lillis, Jacquelyn A.; Carlson, Maryn O.; Cadle-Davidson, Lance; Gent, David H.; Smart, Christine D.

    2015-01-01

    RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) were used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. Twenty isolates of P. cubensis and 19 isolates of P. humuli were genotyped using RNA-seq and GBS. Principle components analysis (PCA) of each data set showed genetic separation between the two species. Additionally, results supported previous findings that P. cubensis isolates from squash are genetically distinct from cucumber and cantaloupe isolates. A PCA-based procedure was used to identify SNPs correlated with the separation of the two species, with 994 and 4,231 PCA-correlated SNPs found within the RNA-seq and GBS data, respectively. The corresponding unigenes (n = 800) containing these potential species-specific SNPs were then annotated and 135 putative pathogenicity genes, including 3 effectors, were identified. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways. PMID:26599440

  9. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    PubMed Central

    Ortega, Álvaro D.; Quereda, Juan J.; Pucciarelli, M. Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of “intact” intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections. PMID:25429360

  10. SR-A/MARCO-mediated ligand delivery enhances intracellular TLR and NLR function, but ligand scavenging from cell surface limits TLR4 response to pathogens.

    PubMed

    Mukhopadhyay, Subhankar; Varin, Audrey; Chen, Yunying; Liu, Baoying; Tryggvason, Karl; Gordon, Siamon

    2011-01-27

    Phagocytic and pathogen sensing receptors are responsible for particle uptake and inflammation. It is unclear how these receptors' systems influence each other's function to shape an innate response. The class-A scavenger receptors SR-A (scavenger receptor A) and MARCO (macrophage receptor with collagenous structure) are 2 well-characterized phagocytic receptors that are unable to initiate inflammatory responses by themselves, yet are implicated in the pathogenesis of various inflammatory disorders. However, the mechanism for such an apparent discrepancy is still unclear. We utilized SR-A(-/-), MARCO(-/-), and SR-A(-/-)-MARCO(-/-) mice, along with microbe-derived, environmental, and synthetic polyanions to assess the inflammatory responses following combinatorial ligation of SR-A/MARCO and selected Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) by their shared ligands. In addition to ligating SR-A and MARCO, these agonists also selectively activated the cell-surface sensor TLR4, endosomal TLR3, and the cytosolic NOD2 and NALP3 (NACHT domain-, leucine-rich repeat-, and pyrin domain-containing protein 3). We show that, following recognition of common ligands, SR-A and MARCO attenuate TLR4-mediated responses while enhancing responses by the intracellular TLR3, NOD2, and NALP3. We conclude that SR-A/MARCO-mediated rapid ligand internalization prevented sensing by surface TLRs while increasing ligand availability in intracellular compartments, thus allowing sensing and robust responses by intracellular sensors. PMID:21098741

  11. Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria.

    PubMed

    Ham, Jong Hyun

    2013-04-01

    Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens. PMID:23186372

  12. Application of ?-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Fields, Kenneth A.

    2015-01-01

    Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 ?-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia. PMID:26258949

  13. Characterization of a lipopolysaccharide-targeted monoclonal antibody and its variable fragments as candidates for prophylaxis against the obligate intracellular bacterial pathogen Coxiella burnetii.

    PubMed

    Peng, Ying; Schoenlaub, Laura; Elliott, Alexandra; Mitchell, William J; Zhang, Guoquan

    2014-11-01

    Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure. PMID:25114119

  14. Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

    PubMed Central

    Peng, Ying; Schoenlaub, Laura; Elliott, Alexandra; Mitchell, William J.

    2014-01-01

    Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure. PMID:25114119

  15. Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its Adaptation to Broad Host Ranges and Intracellular Niches

    PubMed Central

    Xiao, Jingfan; Wu, Haizhen; Wang, Xin; Lv, Yuanzhi; Xu, Lili; Zheng, Huajun; Wang, Shengyue; Zhao, Guoping; Liu, Qin; Zhang, Yuanxing

    2009-01-01

    Background Edwardsiella tarda is the etiologic agent of edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries. Here we describe the complete genome of E. tarda, EIB202, a highly virulent and multi-drug resistant isolate in China. Methodology/Principal Findings E. tarda EIB202 possesses a single chromosome of 3,760,463 base pairs containing 3,486 predicted protein coding sequences, 8 ribosomal rRNA operons, and 95 tRNA genes, and a 43,703 bp conjugative plasmid harboring multi-drug resistant determinants and encoding type IV A secretion system components. We identified a full spectrum of genetic properties related to its genome plasticity such as repeated sequences, insertion sequences, phage-like proteins, integrases, recombinases and genomic islands. In addition, analysis also indicated that a substantial proportion of the E. tarda genome might be devoted to the growth and survival under diverse conditions including intracellular niches, with a large number of aerobic or anaerobic respiration-associated proteins, signal transduction proteins as well as proteins involved in various stress adaptations. A pool of genes for secretion systems, pili formation, nonfimbrial adhesions, invasions and hemagglutinins, chondroitinases, hemolysins, iron scavenging systems as well as the incomplete flagellar biogenesis might feature its surface structures and pathogenesis in a fish body. Conclusion/Significance Genomic analysis of the bacterium offered insights into the phylogeny, metabolism, drug-resistance, stress adaptation, and virulence characteristics of this versatile pathogen, which constitutes an important first step in understanding the pathogenesis of E. tarda to facilitate construction of a practical effective vaccine used for combating fish edwardsiellosis. PMID:19865481

  16. Host Jumps and Radiation, Not Co?Divergence Drives Diversification of Obligate Pathogens. A Case Study in Downy Mildews and Asteraceae

    PubMed Central

    Choi, Young-Joon; Thines, Marco

    2015-01-01

    Even though the microevolution of plant hosts and pathogens has been intensely studied, knowledge regarding macro-evolutionary patterns is limited. Having the highest species diversity and host-specificity among Oomycetes, downy mildews are a useful a model for investigating long-term host-pathogen coevolution. We show that phylogenies of Bremia and Asteraceae are significantly congruent. The accepted hypothesis is that pathogens have diverged contemporarily with their hosts. But maximum clade age estimation and sequence divergence comparison reveal that congruence is not due to long-term coevolution but rather due to host-shift driven speciation (pseudo-cospeciation). This pattern results from parasite radiation in related hosts, long after radiation and speciation of the hosts. As large host shifts free pathogens from hosts with effector triggered immunity subsequent radiation and diversification in related hosts with similar innate immunity may follow, resulting in a pattern mimicking true co-divergence, which is probably limited to the terminal nodes in many pathogen groups. PMID:26230508

  17. Contrasting host–pathogen interactions and genome evolution in two generalist and specialist microsporidian pathogens of mosquitoes

    PubMed Central

    Desjardins, Christopher A.; Sanscrainte, Neil D.; Goldberg, Jonathan M.; Heiman, David; Young, Sarah; Zeng, Qiandong; Madhani, Hiten D.; Becnel, James J.; Cuomo, Christina A

    2015-01-01

    Obligate intracellular pathogens depend on their host for growth yet must also evade detection by host defenses. Here we investigate host adaptation in two Microsporidia, the specialist Edhazardia aedis and the generalist Vavraia culicis, pathogens of disease vector mosquitoes. Genomic analysis and deep RNA-Seq across infection time courses reveal fundamental differences between these pathogens. E. aedis retains enhanced cell surface modification and signalling capacity, upregulating protein trafficking and secretion dynamically during infection. V. culicis is less dependent on its host for basic metabolites and retains a subset of spliceosomal components, with a transcriptome broadly focused on growth and replication. Transcriptional profiling of mosquito immune responses reveals that response to infection by E. aedis differs dramatically depending on the mode of infection, and that antimicrobial defensins may play a general role in mosquito defense against Microsporidia. This analysis illuminates fundamentally different evolutionary paths and host interplay of specialist and generalist pathogens. PMID:25968466

  18. Cloning and characterization of a novel invertase from the obligate biotroph Uromyces fabae and analysis of expression patterns of host and pathogen invertases in the course of infection.

    PubMed

    Voegele, Ralf T; Wirsel, Stefan; Möll, Ulla; Lechner, Melanie; Mendgen, Kurt

    2006-06-01

    Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs. PMID:16776296

  19. Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins

    PubMed Central

    Moore, Elizabeth R.; Ouellette, Scot P.

    2014-01-01

    Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches. PMID:25401095

  20. Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis.

    PubMed

    Isla, A; Haussmann, D; Vera, T; Kausel, G; Figueroa, J

    2014-10-10

    Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line. PMID:25205198

  1. Intracellular life of Coxiella burnetii in macrophages.

    PubMed

    Ghigo, Eric; Pretat, Lionel; Desnues, Benoît; Capo, Christian; Raoult, Didier; Mege, Jean-Louis

    2009-05-01

    Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that is considered a potential biological weapon of category B. C. burnetii survives within myeloid cells by subverting receptor-mediated phagocytosis and preventing phagosome maturation. The intracellular fate of C. burnetii also depends on the functional state of myeloid cells. This review describes the mechanisms used by C. burnetii to circumvent uptake and trafficking events, and the role of cytokines on C. burnetii survival in myeloid cells. PMID:19538264

  2. Exit Mechanisms of the Intracellular Bacterium Ehrlichia

    PubMed Central

    Thomas, Sunil; Popov, Vsevolod L.; Walker, David H.

    2010-01-01

    Background The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the causative agent of human monocytotropic ehrlichiosis. Ehrlichia muris and Ixodes ovatus Ehrlichia (IOE) are agents of mouse models of ehrlichiosis. The mechanism by which Ehrlichia are transported from an infected host cell to a non-infected cell has not been demonstrated. Methodology/Principal Findings Using fluorescence microscopy and transmission and scanning electron microscopy, we demonstrated that Ehrlichia was transported through the filopodia of macrophages during early stages of infection. If host cells were not present in the vicinity of an Ehrlichia-infected cell, the leading edge of the filopodium formed a fan-shaped structure filled with the pathogen. Formation of filopodia in the host macrophages was inhibited by cytochalasin D and ehrlichial transport were prevented due to the absence of filopodia formation. At late stages of infection the host cell membrane was ruptured, and the bacteria were released. Conclusions/Significance Ehrlichia are transported through the host cell filopodium during initial stages of infection, but are released by host cell membrane rupture during later stages of infection. PMID:21187937

  3. ORIGINAL ARTICLE Detection of a novel intracellular microbiome hosted

    E-print Network

    ORIGINAL ARTICLE Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria

  4. Host antioxidant enzymes and TLR-2 neutralization modulate intracellular survival of Staphylococcus aureus: Evidence of the effect of redox balance on host pathogen relationship during acute staphylococcal infection.

    PubMed

    Nandi, Ajeya; Bishayi, Biswadev

    2015-12-01

    Staphylococcus aureus is an important pathogen in bone disease and innate immune recognition receptor, TLR-2 is reported to be crucial for inflammatory bone loss. Role of TLR-2 in bacterial clearance and cytokine response to S. aureus infection in murine bone marrow macrophages has been reported but the role of host derived ROS in host-pathogen relationship still remains an obvious question. In the present study, blocking of SOD and catalase in TLR-2 neutralized fresh bone marrow cells (FBMC) with Diethyldithiocarbamic acid (DDC) and 3-Amino-1,2,4-triazole (ATZ), separately, during acute S. aureus infection, produces moderate level of ROS and limits inflammation as compared with only TLR-2 non-neutralized condition and leads to decreased bacterial count compared with only TLR-2 neutralized condition. In summary, host SOD and catalase modulates ROS generation, cytokine levels and TLR-2 expression in FBMCs during acute S. aureus infection which might be useful in the alleviation of S. aureus infection and bone loss. PMID:26416307

  5. Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during Campylobacter jejuni colonization.

    PubMed

    Awad, Wageha A; Smorodchenko, Alina; Hess, Claudia; Aschenbach, Jörg R; Molnár, Andor; Dublecz, Károly; Khayal, Basel; Pohl, Elena E; Hess, Michael

    2015-08-01

    Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca(2+)]i signaling in the intestines of broiler chickens. For this, we compared: control birds (n?=?60) and C. jejuni-infected birds (n?=?60; infected orally with 1?×?10(8) CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca(2+) indicator Fluo-4 and two-photon microscopy, we revealed that [Ca(2+)]i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca(2+)]i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca(2+)]i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca(2+) signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health. PMID:25825050

  6. NCI & Division Obligations

    Cancer.gov

    Displays obligations for grants, contracts, training fellowships, intramural research, and management and support, including the number of grant awards, funding amounts, and percent of the total NCI budget.

  7. Nitric Oxide from IFN?-Primed Macrophages Modulates the Antimicrobial Activity of ?-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella

    PubMed Central

    Jones-Carson, Jessica; Zweifel, Adrienne E.; Tapscott, Timothy; Austin, Chad; Brown, Joseph M.; Jones, Kenneth L.; Voskuil, Martin I.; Vázquez-Torres, Andrés

    2014-01-01

    Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of ?-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to ?-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the ?-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to ?-lactams. The degree of NO-induced ?-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ?H+ and ?? electrochemical gradients of the PMF prevents ?-lactam-mediated killing. According to this model, NO generated by IFN?-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFN?-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of ?-lactam antibiotics. PMID:25121731

  8. Nitric oxide from IFN?-primed macrophages modulates the antimicrobial activity of ?-lactams against the intracellular pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella.

    PubMed

    Jones-Carson, Jessica; Zweifel, Adrienne E; Tapscott, Timothy; Austin, Chad; Brown, Joseph M; Jones, Kenneth L; Voskuil, Martin I; Vázquez-Torres, Andrés

    2014-08-01

    Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of ?-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to ?-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the ?-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to ?-lactams. The degree of NO-induced ?-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ?H+ and ?? electrochemical gradients of the PMF prevents ?-lactam-mediated killing. According to this model, NO generated by IFN?-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFN?-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of ?-lactam antibiotics. PMID:25121731

  9. Intracellular bacteria and adverse pregnancy outcomes.

    PubMed

    Baud, D; Greub, G

    2011-09-01

    This review considers the role of intracellular bacteria in adverse pregnancy outcomes, such as miscarriage, stillbirths, and preterm labour. The cause of miscarriage, stillbirth and preterm labour often remains unexplained. Intracellular bacteria that grow either poorly or not at all on media used routinely to detect human pathogens could be the aetiological agents of these obstetric conditions. For example, Listeria monocytogenes and Coxiella burnetti are intracellular bacteria that have a predilection for the fetomaternal unit and may induce fatal disease in the mother and/or fetus. Both are important foodborne or zoonotic pathogens in pregnancy. Preventive measures, diagnostic tools and treatment will be reviewed. Moreover, we will also address the importance in adverse pregnancy outcomes of other intracellular bacteria, including Brucella abortus and various members of the order Chlamydiales. Indeed, there is growing evidence that Chlamydia trachomatis, Chlamydia abortus and Chlamydia pneumoniae infections may also result in adverse pregnancy outcomes in humans and/or animals. Moreover, newly discovered Chlamydia-like organisms have recently emerged as new pathogens of both animals and humans. For example, Waddlia chondrophila, a Chlamydia-related bacterium isolated from aborted bovine fetuses, has also been implicated in human miscarriages. Future research should help us to better understand the pathophysiology of adverse pregnancy outcomes caused by intracellular bacteria and to determine the precise mode of transmission of newly identified bacteria, such as Waddlia and Parachlamydia. These emerging pathogens may represent the tip of the iceberg of a large number of as yet unknown intracellular pathogenic agents. PMID:21884294

  10. Patho-epigenetics of Infectious Diseases Caused by Intracellular Bacteria.

    PubMed

    Niller, Hans Helmut; Minarovits, Janos

    2016-01-01

    In multicellular eukaryotes including plants, animals and humans, epigenetic reprogramming may play a role in the pathogenesis of a wide variety of diseases. Recent studies revealed that in addition to viruses, pathogenic bacteria are also capable to dysregulate the epigenetic machinery of their target cells. In this chapter we focus on epigenetic alterations induced by bacteria infecting humans. Most of them are obligate or facultative intracellular bacteria that produce either bacterial toxins and surface proteins targeting the host cell membrane, or synthesise effector proteins entering the host cell nucleus. These bacterial products typically elicit histone modifications, i.e. alter the "histone code". Bacterial pathogens are capable to induce alterations of host cell DNA methylation patterns, too. Such changes in the host cell epigenotype and gene expression pattern may hinder the antibacterial immune response and create favourable conditions for bacterial colonization, growth, or spread. Epigenetic dysregulation mediated by bacterial products may also facilitate the production of inflammatory cytokines and other inflammatory mediators affecting the epigenotype of their target cells. Such indirect epigenetic changes as well as direct interference with the epigenetic machinery of the host cells may contribute to the initiation and progression of malignant tumors associated with distinct bacterial infections. PMID:26659266

  11. Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.

    PubMed

    Noh, Ju Young; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Seo, Gi Won; Kim, Dong Hyun; Patnaik, Hongray Howrelia; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Han, Yeon Soo

    2014-01-25

    Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. PMID:24200961

  12. Comparison of the 'Ca Liberibacter asiaticus' genome adapted for an intracellular lifestyle with other members of the rhizobiales

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An intracellular plant pathogen ‘Ca. Liberibacter asiaticus,’ a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, Agrobacterium tumefaciens and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 52 clust...

  13. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500?pN??m-2) and its dynamic fluctuations at a sensitivity of 20?pN??m-2 (20?Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  14. Obesity and obligation.

    PubMed

    Jeppsson, Sofia

    2015-03-01

    The belief that obese people ought to lose weight and keep it off is widespread, and has a profound negative impact on the lives of the obese. I argue in this paper that most obese people have no such obligation, even if obesity is bad, and caused by calorie input exceeding output. Obese people do not have an obligation to achieve long-term weight loss if this is impossible for them, is worse than the alternative, or requires such an enormous effort in relation to what stands to be gained that this option is supererogatory rather than obligatory. It is highly plausible that most obese people fall into one of these three groups. Politicians may still have obligations to fight obesity, but they ought to do so through progressive politics rather than blaming and shaming. PMID:25843121

  15. GRANDPARENTS' ENTITLEMENTS AND OBLIGATIONS

    PubMed Central

    Draper, Heather

    2013-01-01

    In this article, it is argued that grandparents' obligations originate from parental obligations (i.e from the relationship they have with their children, the parents of their grandchildren) and not from the role of grandparent per se, and any entitlements flow from the extent to which these obligations are met. The position defended is, therefore, that grandparents qua grandparents are not entitled to form or continue relationships with their grandchildren. A continuation of grandparent-grandchildren relationships may be in the interests of children, but the grandparental nature of the relationship is not decisive. What counts is the extent to which relationships children have with any adults who are not their parents are is significant to them. Sometimes, however, grandparents become parents or co-parents of their grandchildren. They then gain parental rights, and as such are as entitled, ceteris parius, as any parent to expect their relationship with the child to continue. The issue of grandparents' entitlements can come to the fore when parents separate, and grandparents are unhappy with the access they have to their grandchildren. Grandparents' obligations may become a particular issue when parents die, struggle, or fail to care for their children. This article focuses particularly on these kinds of circumstances. PMID:23718643

  16. Human neutrophils dump Candida glabrata after intracellular killing.

    PubMed

    Essig, Fabian; Hünniger, Kerstin; Dietrich, Stefanie; Figge, Marc Thilo; Kurzai, Oliver

    2015-11-01

    Interaction between fungal pathogens and human phagocytes can lead to remarkably variable outcomes, ranging from intracellular killing to prolonged survival and replication of the pathogen in the host cell. Using live cell imaging we observed primary human neutrophils that release phagocytosed Candida glabrata yeast cells after intracellular killing. This process, for which we propose the name "dumping", adds a new outcome to phagocyte-fungus interaction which may be of potential immunological importance as it allows professional antigen presenting cells to take up and process neutrophil-inactivated pathogens that in their viable state are able to evade intracellular degradation in these cells. PMID:26385824

  17. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    EPA Science Inventory

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  18. The olive fly endosymbiont, "Candidatus Erwinia dacicola," switches from an intracellular existence to an extracellular existence during host insect development.

    PubMed

    Estes, Anne M; Hearn, David J; Bronstein, Judith L; Pierson, Elizabeth A

    2009-11-01

    As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment). PMID:19767463

  19. Identification of C. burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation 

    E-print Network

    Weber, Mary

    2014-05-05

    Coxiella burnetii is a Gram-negative intracellular pathogen that encodes a specialized type IVb secretion system (T4SS) which is essential for intracellular replication, Coxiella-containing vacuole (CCV) formation, modulation ...

  20. Hijacking of Host Cellular Functions by an Intracellular Parasite, the Microsporidian Anncaliia algerae

    PubMed Central

    Panek, Johan; El Alaoui, Hicham; Mone, Anne; Urbach, Serge; Demettre, Edith; Texier, Catherine; Brun, Christine; Zanzoni, Andreas; Peyretaillade, Eric; Parisot, Nicolas; Lerat, Emmanuelle; Peyret, Pierre; Delbac, Frederic; Biron, David G.

    2014-01-01

    Intracellular pathogens including bacteria, viruses and protozoa hijack host cell functions to access nutrients and to bypass cellular defenses and immune responses. These strategies have been acquired through selective pressure and allowed pathogens to reach an appropriate cellular niche for their survival and growth. To get new insights on how parasites hijack host cellular functions, we developed a SILAC (Stable Isotope Labeling by Amino Acids in Cell culture) quantitative proteomics workflow. Our study focused on deciphering the cross-talk in a host-parasite association, involving human foreskin fibroblasts (HFF) and the microsporidia Anncaliia algerae, a fungus related parasite with an obligate intracellular lifestyle and a strong host dependency. The host-parasite cross-talk was analyzed at five post-infection times 1, 6, 12 and 24 hours post-infection (hpi) and 8 days post-infection (dpi). A significant up-regulation of four interferon-induced proteins with tetratricopeptide repeats IFIT1, IFIT2, IFIT3 and MX1 was observed at 8 dpi suggesting a type 1 interferon (IFN) host response. Quantitative alteration of host proteins involved in biological functions such as signaling (STAT1, Ras) and reduction of the translation activity (EIF3) confirmed a host type 1 IFN response. Interestingly, the SILAC approach also allowed the detection of 148 A. algerae proteins during the kinetics of infection. Among these proteins many are involved in parasite proliferation, and an over-representation of putative secreted effectors proteins was observed. Finally our survey also suggests that A. algerae could use a transposable element as a lure strategy to escape the host innate immune system. PMID:24967735

  1. Impaired stimulation of p38?-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes.

    PubMed

    Barry, Abdoulaye Oury; Boucherit, Nicolas; Mottola, Giovanna; Vadovic, Pavol; Trouplin, Virginie; Soubeyran, Philippe; Capo, Christian; Bonatti, Stefano; Nebreda, Angel; Toman, Rudolf; Lemichez, Emmanuel; Mege, Jean-Louis; Ghigo, Eric

    2012-12-13

    Variations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38?-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38?-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38?-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38?-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly. PMID:23245320

  2. Toxoplasma on the Brain: Understanding Host-Pathogen Interactions in Chronic CNS Infection

    PubMed Central

    Kamerkar, Sushrut; Davis, Paul H.

    2012-01-01

    Toxoplasma gondii is a prevalent obligate intracellular parasite which chronically infects more than a third of the world's population. Key to parasite prevalence is its ability to form chronic and nonimmunogenic bradyzoite cysts, which typically form in the brain and muscle cells of infected mammals, including humans. While acute clinical infection typically involves neurological and/or ocular damage, chronic infection has been more recently linked to behavioral changes. Establishment and maintenance of chronic infection involves a balance between the host immunity and parasite evasion of the immune response. Here, we outline the known cellular interplay between Toxoplasma gondii and cells of the central nervous system and review the reported effects of Toxoplasma gondii on behavior and neurological disease. Finally, we review new technologies which will allow us to more fully understand host-pathogen interactions. PMID:22545203

  3. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis

    PubMed Central

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D.

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses. PMID:26320027

  4. The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production

    PubMed Central

    Marques, Maria Angela M.; Berrêdo-Pinho, Marcia; Rosa, Thabatta L. S. A.; Pujari, Venugopal; Lemes, Robertha M. R.; Lery, Leticia M. S.; Silva, Carlos Adriano M.; Guimarães, Ana Carolina R.; Atella, Georgia C.; Wheat, William H.; Brennan, Patrick J.; Crick, Dean C.; Belisle, John T.

    2015-01-01

    ABSTRACT Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3?-hydroxysteroid dehydrogenase (3?-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-14C]cholesterol or [26-14C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. PMID:26391209

  5. Macrophage defense mechanisms against intracellular bacteria

    PubMed Central

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  6. Pathogen-pathogen interaction

    PubMed Central

    2010-01-01

    There is growing awareness of the health implications of the fact that infectious agents often do not act independently; rather their disease potential is mediated in diverse and significant ways by their relationships with other pathogens. Pathogen-pathogen interaction (PPI), for example, impacts various virulence factors in human infection. Although still in its infancy, the study of PPI, a form of epidemiological synergism, is emerging as an important arena of new research and new understanding in health and clinical care. The aims of this paper are to: (1) draw attention to the role of PPI in human disease patterns; (2) present the syndemics model as a biosocial approach for examining the nature, pathways, contexts, and health implications of PPI and (3) suggest the utility of this approach to PPI. Toward these ends, this paper (a) reviews three case examples of alternative PPIs, (b) describes the development and key concepts and components of the syndemics model with specific reference to interacting infectious agents, (c) contextualizes this discussion with a brief review of broader syndemics disease processes (not necessarily involving infections disease) and (d) comments on the research, treatment and prevention implications of syndemic interaction among pathogens. PMID:21178409

  7. Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics.

    PubMed

    Djulic, Alma; Schmid, Annette; Lenz, Heike; Sharma, Pia; Koch, Christin; Wirsel, Stefan G R; Voegele, Ralf T

    2011-07-01

    Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host-parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (?-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle. PMID:21724169

  8. 45 CFR 83.10 - General obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...2010-10-01 false General obligations. 83...Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION ...AND 845 OF THE PUBLIC HEALTH SERVICE ACT Discrimination...Prohibited § 83.10 General obligations....

  9. 45 CFR 83.10 - General obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...2011-10-01 false General obligations. 83...Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION ...AND 845 OF THE PUBLIC HEALTH SERVICE ACT Discrimination...Prohibited § 83.10 General obligations....

  10. 38 CFR 17.607 - Obligated service.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...AFFAIRS MEDICAL Va Health Professional Scholarship Program § 17.607 Obligated service...for which the participant received a scholarship award under these regulations...obligation. A participant who received a scholarship as a full-time student must be...

  11. 7 CFR 400.767 - Requester obligations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...2012-01-01 false Requester obligations. 400.767 Section 400.767 Agriculture Regulations of the Department of Agriculture...Interpretations of Statutory and Regulatory Provisions § 400.767 Requester obligations. (a) All requests...

  12. 7 CFR 400.767 - Requester obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...2010-01-01 false Requester obligations. 400.767 Section 400.767 Agriculture Regulations of the Department of Agriculture...Interpretations of Statutory and Regulatory Provisions § 400.767 Requester obligations. (a) All requests...

  13. 7 CFR 400.767 - Requester obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...2011-01-01 false Requester obligations. 400.767 Section 400.767 Agriculture Regulations of the Department of Agriculture...Interpretations of Statutory and Regulatory Provisions § 400.767 Requester obligations. (a) All requests...

  14. 7 CFR 400.767 - Requester obligations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...2014-01-01 false Requester obligations. 400.767 Section 400.767 Agriculture Regulations of the Department of Agriculture...Interpretations of Statutory and Regulatory Provisions § 400.767 Requester obligations. (a) All requests...

  15. 7 CFR 400.767 - Requester obligations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...2013-01-01 false Requester obligations. 400.767 Section 400.767 Agriculture Regulations of the Department of Agriculture...Interpretations of Statutory and Regulatory Provisions § 400.767 Requester obligations. (a) All requests...

  16. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...2011-10-01 2011-10-01 false Guarantee obligations. Sec. 11 Section 11 Shipping MARITIME ADMINISTRATION, DEPARTMENT...AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the...

  17. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...2010-10-01 2010-10-01 false Guarantee obligations. Sec. 11 Section 11 Shipping MARITIME ADMINISTRATION, DEPARTMENT...AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the...

  18. 7 CFR 993.56 - Reserve obligation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 2010-01-01 2010-01-01 false Reserve obligation. 993.56 Section 993.56 Agriculture...PRODUCED IN CALIFORNIA Order Regulating Handling Reserve Control § 993.56 Reserve obligation. Whenever salable and...

  19. 7 CFR 993.56 - Reserve obligation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 2011-01-01 2011-01-01 false Reserve obligation. 993.56 Section 993.56 Agriculture...PRODUCED IN CALIFORNIA Order Regulating Handling Reserve Control § 993.56 Reserve obligation. Whenever salable and...

  20. Legal obligations of NHS managers.

    PubMed

    Jacks, H J

    1988-10-01

    The loss of crown immunity resulting from section 2 of the National Health Service (Amendment) Act 1986 has understandably caused much concern relating to the application of the Health and Safety at Work Act 1974 to hospitals. The NHS managers must remember that their legal obligations can be both civil and criminal. PMID:10291890

  1. 12 CFR 987.10 - Obligations of United States with respect to consolidated obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 7 2010-01-01 2010-01-01 false Obligations of United States with respect to consolidated obligations. 987.10 Section 987.10 Banks and Banking FEDERAL HOUSING FINANCE BOARD OFFICE OF FINANCE BOOK-ENTRY PROCEDURE FOR CONSOLIDATED OBLIGATIONS § 987.10 Obligations of United States...

  2. Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

    PubMed Central

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-01-01

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes. PMID:19071962

  3. Multi-locus tree and species tree approaches toward resolving a complex clade of downy mildews (Straminipila, Oomycota), including pathogens of beet and spinach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The family Peronosporaceae (Straminipila; Oomycota) consists of obligate biotrophic pathogens that cause downy mildew disease on angiosperm...

  4. Antimicrobial susceptibility and molecular characterization of Mycobacterium intracellulare in China.

    PubMed

    Zhao, Xiuqin; Wang, Yufeng; Pang, Yu

    2014-10-01

    Mycobacterium avium complex (MAC) is the most common non-tuberculosis mycobacterial pathogen isolated from respiratory samples, mainly including two species, Mycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare). Although these two species belong to the same group, M. avium and M. intracellulare reveal significantly differences in pathogenicity and biology. Nevertheless, little is known regarding the drug resistant details profile of M. avium or M. intracellulare instead of MAC. Here, we examined the antimicrobial susceptibility profiles of 52 clinical M. intracellulare isolates against fourteen antimicrobial agents, which are widely selected for the treatment of nontuberculous mycobacteria (NTM) infection. The drug susceptibility test revealed that clarithromycin (47/52, 90.4%), rifampicin (41/52, 78.8%) and capreomycin (40/52, 76.9%) revealed highly antimicrobial activities against M. intracellulare isolates in vitro. Furthermore, all clarithromycin resistant isolates harbored mutations in the 23S rRNA gene, and the percentage of amikacin resistant ones with mutation in the rrs gene is 62.5% (10/16). The Hunter-Gaston Discriminatory Index (HGDI) value for the 16-loci Variable Number of Tandem Repeat (VNTR) typing of M. intracellulare isolates was 0.994, and M. intracellulare resistance to moxifloxacin was significantly more commonly found in clustered strains than in nonclustered strains (?(2)=5.551, P=0.040). In conclusion, our data demonstrated that clarithromycin and capreomycin revealed highly antimicrobial activities against M. intracellulare isolates, and clarithromycin and amikacin resistance could be detected more readily and rapidly using molecular scanning of corresponding drug target than conventional drug susceptibility testing. We also found that infection by clustered strains was significantly associated with resistance to moxifloxacin. PMID:25131955

  5. Real-time molecular monitoring of chemical environment in obligate anaerobes during oxygen adaptive response.

    PubMed

    Holman, Hoi-Ying N; Wozei, Eleanor; Lin, Zhang; Comolli, Luis R; Ball, David A; Borglin, Sharon; Fields, Matthew W; Hazen, Terry C; Downing, Kenneth H

    2009-08-01

    Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses. PMID:19541631

  6. Real-Time Molecular Monitoring of Chemical Environment in ObligateAnaerobes during Oxygen Adaptive Response

    SciTech Connect

    Holman, Hoi-Ying N.; Wozei, Eleanor; Lin, Zhang; Comolli, Luis R.; Ball, David. A.; Borglin, Sharon; Fields, Matthew W.; Hazen, Terry C.; Downing, Kenneth H.

    2009-02-25

    Determining the transient chemical properties of the intracellular environment canelucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms which enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier Transform Infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bonding in their cellular water. We observed a sequence of wellorchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.

  7. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

    PubMed

    Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

    2014-11-01

    TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. PMID:24920099

  8. Intracellular antibody immunity.

    PubMed

    Watkinson, Ruth E; McEwan, William A; James, Leo C

    2014-07-01

    Antibodies allow the immune system to target pathogens despite their tremendous diversity and rapid evolution. Once bound to a pathogen, antibodies induce a broad range of effector mechanisms, including phagocytosis and complement. However, these mechanisms are all initiated in the extracellular space, meaning that pathogens like viruses evade them upon infection of their target cells. Recently, it has been shown that, in addition to mediating extracellular immune responses, antibodies also activate immunity inside infected cells. Antibodies that are bound to the surface of non-enveloped viruses or bacteria are carried into the cell during pathogen entry. Once inside the cell, these pathogen-attached antibodies are recognised by a highly conserved, high affinity cytosolic antibody receptor called TRIM21. TRIM21 initiates both sensor and effector responses that reduce viral replication and induce an antiviral state. These responses are an important part of antiviral immunity and the removal of TRIM21 results in uncontrolled viraemia and death in a mouse model of infection. PMID:24722852

  9. Therapy of intracellular Staphylococcus aureus by tigecyclin

    PubMed Central

    2013-01-01

    Background In the fields of traumatology and orthopaedics staphylococci are the most frequently isolated pathogens. Staphylococcus aureus and Staphylococcus epidermidis are known to be the major causative agents of osteomyelitis. The increasing number of multiresistant Staphylococcus aureus and resistant coagulase-negative staphylococci as a trigger of complicated osteomyelitis and implant-associated infections is a major problem. Antibiotic therapy fails in 20% of cases. Therefore the development of novel antibiotics becomes necessary. Methods This study analyses tigecyclin, the first antibiotic of the glycylines, as a potential therapy for osteomyelitis caused by multiresistant Staphylococcus aureus. Therefore its intracellular activity and the potential use in polymethylmetacrylate-bone cement are examined. The intracellular activity of tigecyclin is determined by a human osteoblast infection model. The investigation of the biomechanical characteristics is conducted concerning the ISO 5833-guidelines. Results Tigecyclin shows in vitro an intracellular activity that ranges between the antimicrobial activity of gentamicin and rifampicin. A significant negative effect on the biomechanical characteristics with an impaired stability is detected after adding tigecyclin to polymethylmetacrylate-bone cement with a percentage of 1.225% per weight. Conclusions This study shows that tigecyclin might be a potent alternative for the systemic therapy of osteomyelitis and implant-associated infections whereas the local application has to be reconsidered individually. PMID:23738922

  10. 38 CFR 17.632 - Obligated service.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Impairment and Orientation and Mobility Professional Scholarship Program § 17.632 Obligated service. (a... service. Geographic relocation may be required. (e) Creditability of advanced clinical training. No...

  11. Exploring anti-bacterial compounds against intracellular Legionella.

    PubMed

    Harrison, Christopher F; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the ?-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  12. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila.

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  13. Phosphoinositides and host-pathogen interactions.

    PubMed

    Pizarro-Cerdá, Javier; Kühbacher, Andreas; Cossart, Pascale

    2015-06-01

    Phosphoinositides control key cellular processes including vesicular trafficking and actin polymerization. Intracellular bacterial pathogens manipulate phosphoinositide metabolism in order to promote their uptake by target cells and to direct in some cases the biogenesis of their replication compartments. In this chapter, we review the molecular strategies that major pathogens including Listeria, Mycobacterium, Shigella, Salmonella, Legionella and Yersinia use to hijack phosphoinositides during infection. This article is part of a Special Issue entitled Phosphoinositides. PMID:25241942

  14. Host-microbe and microbe-microbe interactions in the evolution of obligate plant parasitism.

    PubMed

    Kemen, Ariane C; Agler, Matthew T; Kemen, Eric

    2015-06-01

    Research on obligate biotrophic plant parasites, which reproduce only on living hosts, has revealed a broad diversity of filamentous microbes that have independently acquired complex morphological structures, such as haustoria. Genome studies have also demonstrated a concerted loss of genes for metabolism and lytic enzymes, and gain of diversity of genes coding for effectors involved in host defense suppression. So far, these traits converge in all known obligate biotrophic parasites, but unexpected genome plasticity remains. This plasticity is manifested as transposable element (TE)-driven increases in genome size, observed to be associated with the diversification of virulence genes under selection pressure. Genome expansion could result from the governing of the pathogen response to ecological selection pressures, such as host or nutrient availability, or to microbial interactions, such as competition, hyperparasitism and beneficial cooperations. Expansion is balanced by alternating sexual and asexual cycles, as well as selfing and outcrossing, which operate to control transposon activity in populations. In turn, the prevalence of these balancing mechanisms seems to be correlated with external biotic factors, suggesting a complex, interconnected evolutionary network in host-pathogen-microbe interactions. Therefore, the next phase of obligate biotrophic pathogen research will need to uncover how this network, including multitrophic interactions, shapes the evolution and diversity of pathogens. PMID:25622918

  15. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  16. Purification and proteomics of pathogen-modified vacuoles and membranes

    PubMed Central

    Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C.; Subbarayal, Prema; Prusty, Bhupesh K.; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E.; Rudel, Thomas; Hilbi, Hubert

    2015-01-01

    Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation. PMID:26082896

  17. Purification and proteomics of pathogen-modified vacuoles and membranes.

    PubMed

    Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C; Subbarayal, Prema; Prusty, Bhupesh K; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E; Rudel, Thomas; Hilbi, Hubert

    2015-01-01

    Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation. PMID:26082896

  18. Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication.

    PubMed

    Kaldestad, Marte; Haugland, Gyri T; Rønneseth, Anita; Wergeland, Heidrun I; Samuelsen, Ole Bent

    2014-02-01

    The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens. PMID:24492050

  19. Divine voluntarism: moral obligation supervenes on God's antecedent will 

    E-print Network

    Nam, Mi Young

    2004-11-15

    's will for human moral obligation and God's will for human moral good. After all, God's will for human moral obligation is God's willing that His own will for human moral good constitute moral obligation for humans....

  20. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...46 Shipping 8 2010-10-01 2010-10-01 false Nature and content of Obligations. 298.30 Section 298.30...ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page....

  1. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...46 Shipping 8 2011-10-01 2011-10-01 false Nature and content of Obligations. 298.30 Section 298.30...ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page....

  2. 31 CFR 225.5 - Pledge of definitive Government obligations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... false Pledge of definitive Government obligations. 225.5 Section...ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS...225.5 Pledge of definitive Government obligations. (a) Type...

  3. 31 CFR 225.5 - Pledge of definitive Government obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... false Pledge of definitive Government obligations. 225.5 Section...ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS...225.5 Pledge of definitive Government obligations. (a) Type...

  4. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  5. Proteomics of Plant Pathogenic Fungi

    PubMed Central

    González-Fernández, Raquel; Prats, Elena; Jorrín-Novo, Jesús V.

    2010-01-01

    Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. PMID:20589070

  6. Carbon based nutrition of Staphylococcus aureus and the role of sugar phosphate transporters in intracellular bacterial replication 

    E-print Network

    Bell, John Alexander

    2014-06-28

    The Gram positive bacterium Staphylococcus aureus is a major cause of human disease in industrialized countries. This multifaceted pathogen is adapted to thrive in a variety of host niches, including the intracellular ...

  7. Heat tolerance of free living and of intracellular Listeria.

    PubMed

    Ly, T M; Müller, H E

    1989-12-01

    Listeria monocytogenes, L. innocua, and L. seeligeri are ingested by Tetrahymena pyriformis. They are not killed but survive and lyse bacteriovorous protozoans after about 8 days. The question important in food processing whether intracellular L. monocytogenes are protected against pasteurization was investigated using a model of Tetrahymena containing previously ingested Listeria. The study showed that Tetrahymena containing Listeria are more susceptible against application of heat, but intracellular Listeria, phagocytized within Tetrahymena, are not more protected than free and extracellular bacteria. Therefore, a properly performed pasteurization, i.e. of milk, kills intracellular Listeria as fast as extracellular living organisms. Finally, the pathogenic L. monocytogenes showed a higher susceptibility and lower tolerance against application of heat than apathogenic L. innocua and L. seeligeri. PMID:2516720

  8. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

    PubMed

    Lehar, Sophie M; Pillow, Thomas; Xu, Min; Staben, Leanna; Kajihara, Kimberly K; Vandlen, Richard; DePalatis, Laura; Raab, Helga; Hazenbos, Wouter L; Morisaki, J Hiroshi; Kim, Janice; Park, Summer; Darwish, Martine; Lee, Byoung-Chul; Hernandez, Hilda; Loyet, Kelly M; Lupardus, Patrick; Fong, Rina; Yan, Donghong; Chalouni, Cecile; Luis, Elizabeth; Khalfin, Yana; Plise, Emile; Cheong, Jonathan; Lyssikatos, Joseph P; Strandh, Magnus; Koefoed, Klaus; Andersen, Peter S; Flygare, John A; Wah Tan, Man; Brown, Eric J; Mariathasan, Sanjeev

    2015-11-19

    Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections. PMID:26536114

  9. Intervention of Phytohormone Pathways by Pathogen Effectors[OPEN

    PubMed Central

    Kazan, Kemal; Lyons, Rebecca

    2014-01-01

    The constant struggle between plants and microbes has driven the evolution of multiple defense strategies in the host as well as offense strategies in the pathogen. To defend themselves from pathogen attack, plants often rely on elaborate signaling networks regulated by phytohormones. In turn, pathogens have adopted innovative strategies to manipulate phytohormone-regulated defenses. Tactics frequently employed by plant pathogens involve hijacking, evading, or disrupting hormone signaling pathways and/or crosstalk. As reviewed here, this is achieved mechanistically via pathogen-derived molecules known as effectors, which target phytohormone receptors, transcriptional activators and repressors, and other components of phytohormone signaling in the host plant. Herbivores and sap-sucking insects employ obligate pathogens such as viruses, phytoplasma, or symbiotic bacteria to intervene with phytohormone-regulated defenses. Overall, an improved understanding of phytohormone intervention strategies employed by pests and pathogens during their interactions with plants will ultimately lead to the development of new crop protection strategies. PMID:24920334

  10. Subversion of inflammasome activation and pyroptosis by pathogenic bacteria

    PubMed Central

    Cunha, Larissa D.; Zamboni, Dario S.

    2013-01-01

    Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila, and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis. PMID:24324933

  11. Live cell imaging of intracellular Salmonella enterica.

    PubMed

    Kehl, Alexander; Hensel, Michael

    2015-01-01

    During the intracellular phase of the pathogenic lifestyle, Salmonella enterica massively alters the endosomal system of its host cells. Two hallmarks are the remodeling of phagosomes into the Salmonella-containing vacuole (SCV) as a replicative niche, and the formation of tubular structures, such as Salmonella-induced filaments (SIFs). To study the dynamics and the fate of these Salmonella-specific compartments, live cell imaging (LCI) is a method of choice. In this chapter, we compare currently used microscopy techniques and focus on considerations and requirements specific for LCI. Detailed protocols for LCI of Salmonella infection with either confocal laser scanning microscopy (CLSM) or spinning disk confocal microscopy (SDCM) are provided. PMID:25253257

  12. Pathogen intelligence

    PubMed Central

    Steinert, Michael

    2014-01-01

    Different species inhabit different sensory worlds and thus have evolved diverse means of processing information, learning and memory. In the escalated arms race with host defense, each pathogenic bacterium not only has evolved its individual cellular sensing and behavior, but also collective sensing, interbacterial communication, distributed information processing, joint decision making, dissociative behavior, and the phenotypic and genotypic heterogeneity necessary for epidemiologic success. Moreover, pathogenic populations take advantage of dormancy strategies and rapid evolutionary speed, which allow them to save co-generated intelligent traits in a collective genomic memory. This review discusses how these mechanisms add further levels of complexity to bacterial pathogenicity and transmission, and how mining for these mechanisms could help to develop new anti-infective strategies. PMID:24551600

  13. Functional analysis of a lipolytic protein, a potential phytoplasma pathogenicity factor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wall-less bacteria known as phytoplasmas are obligate transkingdom parasites and pathogens of plants and insect vectors. These unusual bacteria possess some of the smallest genomes known among pathogenic bacteria, and have never been successfully isolated in artificial culture. Disease symptoms in...

  14. Quantitative proteomics of intracellular Porphyromonas gingivalis

    PubMed Central

    Xia, Qiangwei; Wang, Tiansong; Taub, Fred; Park, Yoonsuk; Capestany, Cindy A.; Lamont, Richard J.; Hackett, Murray

    2009-01-01

    Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 hours within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were over-expressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be under-expressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction. PMID:17979175

  15. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  16. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  17. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  18. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  19. 5 CFR 724.103 - Agency obligations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...Agency obligations. A Federal agency (or its successor agency) must reimburse the Judgment Fund for payments covered by the No FEAR Act. Such reimbursement must be made within a reasonable time as described in §...

  20. 42 CFR 408.4 - Payment obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... kidney donors. (1) No premiums are required for SMI benefits related to the donation of a kidney if the donor is not an enrollee. (2) A kidney donor who is an enrollee is not relieved of the obligation...

  1. 42 CFR 408.4 - Payment obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... kidney donors. (1) No premiums are required for SMI benefits related to the donation of a kidney if the donor is not an enrollee. (2) A kidney donor who is an enrollee is not relieved of the obligation...

  2. 42 CFR 408.4 - Payment obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... kidney donors. (1) No premiums are required for SMI benefits related to the donation of a kidney if the donor is not an enrollee. (2) A kidney donor who is an enrollee is not relieved of the obligation...

  3. 42 CFR 408.4 - Payment obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... kidney donors. (1) No premiums are required for SMI benefits related to the donation of a kidney if the donor is not an enrollee. (2) A kidney donor who is an enrollee is not relieved of the obligation...

  4. 42 CFR 408.4 - Payment obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... kidney donors. (1) No premiums are required for SMI benefits related to the donation of a kidney if the donor is not an enrollee. (2) A kidney donor who is an enrollee is not relieved of the obligation...

  5. 19 CFR 10.765 - Importer obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Morocco Free Trade Agreement Import Requirements § 10.765 Importer obligations. (a) General . An importer who...

  6. 45 CFR 2400.65 - Teaching obligation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...Teaching obligation. 2400.65 Section 2400.65 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching...

  7. 45 CFR 2400.65 - Teaching obligation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...Teaching obligation. 2400.65 Section 2400.65 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching...

  8. 45 CFR 2400.65 - Teaching obligation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...Teaching obligation. 2400.65 Section 2400.65 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching...

  9. 45 CFR 2400.65 - Teaching obligation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...Teaching obligation. 2400.65 Section 2400.65 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching...

  10. 45 CFR 2400.65 - Teaching obligation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...Teaching obligation. 2400.65 Section 2400.65 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION FELLOWSHIP PROGRAM REQUIREMENTS Special Conditions § 2400.65 Teaching...

  11. Naval Engineering A National Naval Obligation

    E-print Network

    Chryssostomidis, Chryssostomos

    2000-05-16

    As part of its national obligations, ONR must ensure US world leadership in those unique technology areas that insure naval superiority. ONR accomplishes this mission through research, recruitment and education, maintaining ...

  12. 5 CFR 724.404 - Agency obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...CONTINUED) IMPLEMENTATION OF TITLE II OF THE NOTIFICATION AND FEDERAL EMPLOYEE ANTIDISCRIMINATION AND RETALIATION ACT OF 2002 Best Practices § 724.404 Agency obligations. (a) Within 30 working days of issuance of the advisory...

  13. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  14. 7 CFR 989.37 - Obligation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN CALIFORNIA Order Regulating Handling Raisin Administrative Committee § 989.37 Obligation....

  15. Intracellular antibody-mediated immunity and the role of TRIM21.

    PubMed

    McEwan, William A; Mallery, Donna L; Rhodes, David A; Trowsdale, John; James, Leo C

    2011-11-01

    Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody-coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin-proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments. PMID:22006823

  16. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Nature and content of Obligations. 298.30 Section 298.30 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VESSEL FINANCING ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page. An Obligation, in...

  17. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Nature and content of Obligations. 298.30 Section 298.30 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VESSEL FINANCING ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page. An Obligation, in...

  18. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Nature and content of Obligations. 298.30 Section 298.30 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VESSEL FINANCING ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page. An Obligation, in...

  19. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Nature and content of Obligations. 298.30 Section 298.30 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VESSEL FINANCING ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page. An Obligation, in...

  20. 46 CFR 298.30 - Nature and content of Obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Nature and content of Obligations. 298.30 Section 298.30 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION VESSEL FINANCING ASSISTANCE OBLIGATION GUARANTEES Documentation § 298.30 Nature and content of Obligations. (a) Single page. An Obligation, in...

  1. Benefit Finding and Perceived Obligations of Victims

    E-print Network

    Warner, Ruth

    2007-12-11

    a mention of physical ilnes, mental ilnes, abuse, drug addiction, or obesity. They found that participants felt more anger and les pity for the poor copers as compared to the good copers. They also offered les social support to the poor coper. How... and target of leson were used to predict victim obligations, benefit finding significantly predicted victim obligations, ? = .51, p test was significant, Z = 2...

  2. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites

    PubMed Central

    Coutinho-Silva, Robson; Ojcius, David M.

    2014-01-01

    Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1? (IL-1?), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection. PMID:22634346

  3. Lateral phage transfer in obligate intracellular bacteria (Wolbachia): Verification from natural populations

    E-print Network

    Bordenstein, Seth

    , of Gryllus pennsylvanicus field crickets and of Neochlamisus bebbianae leaf beetles, to demonstrate WO and horizontally have dynamic genomes exhibiting phage transfer, recombination, and toxin-gene acquisitions (Oliver

  4. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  5. Metabolism of the vacuolar pathogen Legionella and implications for virulence

    PubMed Central

    Manske, Christian; Hilbi, Hubert

    2014-01-01

    Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called “Legionnaires' disease.” To establish its intracellular niche termed the “Legionella-containing vacuole” (LCV), L. pneumophila employs a type IV secretion system and translocates ~300 different “effector” proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria. Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen. PMID:25250244

  6. Intracellular Adaptation of Brucella abortus

    PubMed Central

    Lamontagne, Julie; Forest, Anik; Marazzo, Elena; Denis, François; Butler, Heather; Michaud, Jean-François; Boucher, Lyne; Pedro, Ida; Villeneuve, Annie; Sitnikov, Dmitri; Trudel, Karine; Nassif, Najib; Boudjelti, Djamila; Tomaki, Fadi; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Brunet, Sylvain; Côté-Martin, Alexandra; Hunter, Joanna; Moreno, Edgardo; Paramithiotis, Eustache

    2009-01-01

    Macrophages were infected with virulent B. abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration, adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain’s adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections. PMID:19216536

  7. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (?FTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  8. Importance of branched-chain amino acid utilization in Francisella intracellular adaptation.

    PubMed

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique; Charbit, Alain

    2015-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (?FTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  9. Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans

    PubMed Central

    Ghosh, Kaya; Cappiello, Clint D.; McBride, Sean M.; Occi, James L.; Cali, Ann; Takvorian, Peter M.; McDonald, Thomas V.; Weiss, Louis M.

    2011-01-01

    The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl2 failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections. PMID:16197948

  10. Whistleblowing and the bioethicist's public obligations.

    PubMed

    MacDougall, D Robert

    2014-10-01

    Bioethicists are sometimes thought to have heightened obligations by virtue of the fact that their professional role addresses ethics or morals. For this reason it has been argued that bioethicists ought to "whistleblow"--that is, publicly expose the wrongful or potentially harmful activities of their employer--more often than do other kinds of employees. This article argues that bioethicists do indeed have a heightened obligation to whistleblow, but not because bioethicists have heightened moral obligations in general. Rather, the special duties of bioethicists to act as whistleblowers are best understood by examining the nature of the ethical dilemma typically encountered by private employees and showing why bioethicists do not encounter this dilemma in the same way. Whistleblowing is usually understood as a moral dilemma involving conflicting duties to two parties: the public and a private employer. However, this article argues that this way of understanding whistleblowing has the implication that professions whose members identify their employer as the public-such as government employees or public servants--cannot consider whistleblowing a moral dilemma, because obligations are ultimately owed to only one party: the public. The article contends that bioethicists--even when privately employed--are similar to government employees in the sense that they do not have obligations to defer to the judgments of those with private interests. Consequently, bioethicists may be considered to have a special duty to whistleblow, although for different reasons than those usually cited. PMID:25045940

  11. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling

    PubMed Central

    Petnicki-Ocwieja, Tanja; Kern, Aurelie

    2014-01-01

    Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis, and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs). However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi. Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs). Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi. PMID:25566512

  12. Deciphering the Intracellular Fate of Propionibacterium acnes in Macrophages

    PubMed Central

    Fischer, Natalie; Mak, Tim N.; Shinohara, Debika Biswal; Sfanos, Karen S.; Meyer, Thomas F.

    2013-01-01

    Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases. PMID:23862148

  13. Iron in intracellular infection: to provide or to deprive?

    PubMed Central

    Silva-Gomes, Sandro; Vale-Costa, Sílvia; Appelberg, Rui; Gomes, Maria S.

    2013-01-01

    Due to their chemical versatility, transition metals were incorporated as cofactors for several basic metabolic pathways in living organisms. This same characteristic makes them potentially harmful, since they can be engaged in deleterious reactions like Fenton chemistry. As such, organisms have evolved highly specialized mechanisms to supply their own metal needs while keeping their toxic potential in check. This dual character comes into play in host-pathogen interactions, given that the host can either deprive the pathogen of these key nutrients or exploit them to induce toxicity toward the invading agent. Iron stands as the prototypic example of how a metal can be used to limit the growth of pathogens by nutrient deprivation, a mechanism widely studied in Mycobacterium infections. However, the host can also take advantage of iron-induced toxicity to control pathogen proliferation, as observed in infections caused by Leishmania. Whether we may harness either of the two pathways for therapeutical purposes is still ill-defined. In this review, we discuss how modulation of the host iron availability impacts the course of infections, focusing on those caused by two relevant intracellular pathogens, Mycobacterium and Leishmania. PMID:24367768

  14. 12 CFR 966.2 - Issuance of consolidated obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...Section 966.2 Banks and Banking FEDERAL HOUSING FINANCE BOARD FEDERAL HOME LOAN BANK LIABILITIES CONSOLIDATED... (a) Consolidated obligations issued by the Finance Board. The Finance Board may issue consolidated obligations...

  15. 12 CFR 966.2 - Issuance of consolidated obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Section 966.2 Banks and Banking FEDERAL HOUSING FINANCE BOARD FEDERAL HOME LOAN BANK LIABILITIES CONSOLIDATED... (a) Consolidated obligations issued by the Finance Board. The Finance Board may issue consolidated obligations...

  16. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the...

  17. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the...

  18. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the...

  19. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the...

  20. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the...

  1. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Federal Savings Associations § 560.42 State and local government obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association (“you”) may invest in obligations issued by any state,...

  2. 5 CFR 2635.101 - Basic obligation of public service.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 2011-01-01 false Basic obligation of public service. 2635.101 Section 2635.101 Administrative...Provisions § 2635.101 Basic obligation of public service. (a) Public service is a public trust. Each...

  3. 5 CFR 2635.101 - Basic obligation of public service.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 2010-01-01 false Basic obligation of public service. 2635.101 Section 2635.101 Administrative...Provisions § 2635.101 Basic obligation of public service. (a) Public service is a public trust. Each...

  4. 49 CFR 371.10 - Duties and obligations of brokers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...2010-10-01 false Duties and obligations of brokers. 371.10 Section 371.10 Transportation...FEDERAL MOTOR CARRIER SAFETY REGULATIONS BROKERS OF PROPERTY § 371.10 Duties and obligations of brokers. Where the broker acts on...

  5. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...2010-01-01 2010-01-01 false State and local government obligations. 560.42 Section 560.42 Banks...Federal Savings Associations § 560.42 State and local government obligations. (a) What limitations...

  6. 18 CFR 367.22 - Accounting for asset retirement obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 2010-04-01 false Accounting for asset retirement obligations...General Instructions § 367.22 Accounting for asset retirement obligations...retirement cost must be stated at the fair value of the asset retirement...

  7. VIEWING RESEARCH PARTICIPATION AS A MORAL OBLIGATION

    PubMed Central

    RENNIE, STUART

    2015-01-01

    A moral paradigm shift has proposed for participation in health-related research. It’s not just a praiseworthy option, some say; it’s a social obligation. Recasting research participation in this way would have global ramifications, however. Who ultimately stands to gain the most from it, and who has the most to lose? PMID:21495516

  8. 33 CFR 137.5 - Disclosure obligations.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Disclosure obligations. 137.5 Section 137.5 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OIL SPILL LIABILITY: STANDARDS FOR...

  9. 33 CFR 137.5 - Disclosure obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Disclosure obligations. 137.5 Section 137.5 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OIL SPILL LIABILITY: STANDARDS FOR...

  10. 33 CFR 137.5 - Disclosure obligations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Disclosure obligations. 137.5 Section 137.5 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OIL SPILL LIABILITY: STANDARDS FOR...

  11. 33 CFR 137.5 - Disclosure obligations.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Disclosure obligations. 137.5 Section 137.5 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OIL SPILL LIABILITY: STANDARDS FOR...

  12. 33 CFR 137.5 - Disclosure obligations.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Disclosure obligations. 137.5 Section 137.5 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE POLLUTION FINANCIAL RESPONSIBILITY AND COMPENSATION OIL SPILL LIABILITY: STANDARDS FOR...

  13. Inability and Obligation in Moral Judgment

    PubMed Central

    Buckwalter, Wesley; Turri, John

    2015-01-01

    It is often thought that judgments about what we ought to do are limited by judgments about what we can do, or that “ought implies can.” We conducted eight experiments to test the link between a range of moral requirements and abilities in ordinary moral evaluations. Moral obligations were repeatedly attributed in tandem with inability, regardless of the type (Experiments 1–3), temporal duration (Experiment 5), or scope (Experiment 6) of inability. This pattern was consistently observed using a variety of moral vocabulary to probe moral judgments and was insensitive to different levels of seriousness for the consequences of inaction (Experiment 4). Judgments about moral obligation were no different for individuals who can or cannot perform physical actions, and these judgments differed from evaluations of a non-moral obligation (Experiment 7). Together these results demonstrate that commonsense morality rejects the “ought implies can” principle for moral requirements, and that judgments about moral obligation are made independently of considerations about ability. By contrast, judgments of blame were highly sensitive to considerations about ability (Experiment 8), which suggests that commonsense morality might accept a “blame implies can” principle. PMID:26296206

  14. The author’s opportunity and obligation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peer review is a critical component of the scientific method and therefore should be an obligation for everyone who desires to publish their research results in refereed journals. This editorial is written to address a specific problem being encountered by editors of Soil & Tillage Research, but the...

  15. 47 CFR 90.1440 - Reporting obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PRIVATE LAND MOBILE RADIO SERVICES 700 MHz Public/Private Partnership § 90.1440 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly...

  16. 47 CFR 27.1340 - Reporting obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... COMMUNICATIONS SERVICES 700 MHz Public/Private Partnership § 27.1340 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly reports with... requirements of public safety are being met, detailed information on the areas where broadband service has...

  17. 47 CFR 90.1440 - Reporting obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PRIVATE LAND MOBILE RADIO SERVICES 700 MHz Public/Private Partnership § 90.1440 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly...

  18. 47 CFR 27.1340 - Reporting obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... COMMUNICATIONS SERVICES 700 MHz Public/Private Partnership § 27.1340 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly reports with... requirements of public safety are being met, detailed information on the areas where broadband service has...

  19. 47 CFR 27.1340 - Reporting obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... COMMUNICATIONS SERVICES 700 MHz Public/Private Partnership § 27.1340 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly reports with... requirements of public safety are being met, detailed information on the areas where broadband service has...

  20. 47 CFR 90.1440 - Reporting obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PRIVATE LAND MOBILE RADIO SERVICES 700 MHz Public/Private Partnership § 90.1440 Reporting obligations. (a) The Upper 700 MHz D Block licensee and the Public Safety Broadband Licensee shall jointly file quarterly...

  1. 21 CFR 26.62 - General obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 1 2011-04-01 2011-04-01 false General obligations. 26.62 Section 26.62 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL MUTUAL RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT...

  2. 21 CFR 26.62 - General obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false General obligations. 26.62 Section 26.62 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL MUTUAL RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT...

  3. 19 CFR 10.412 - Importer obligations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Importer obligations. 10.412 Section 10.412 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free...

  4. 19 CFR 10.412 - Importer obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Importer obligations. 10.412 Section 10.412 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free...

  5. 19 CFR 10.412 - Importer obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Importer obligations. 10.412 Section 10.412 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free...

  6. 19 CFR 10.412 - Importer obligations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Importer obligations. 10.412 Section 10.412 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free...

  7. 19 CFR 10.412 - Importer obligations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Importer obligations. 10.412 Section 10.412 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free...

  8. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    PubMed Central

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ?cypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ?cypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ?cypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  9. 26 CFR 1.454-1 - Obligations issued at discount.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 6 2011-04-01 2011-04-01 false Obligations issued at discount. 1.454-1 Section... Obligations issued at discount. (a) Certain non-interest-bearing obligations issued at discount—(1) Election... issued at a discount and redeemable for fixed amounts increasing at stated intervals (other than...

  10. 26 CFR 1.454-1 - Obligations issued at discount.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 6 2010-04-01 2010-04-01 false Obligations issued at discount. 1.454-1 Section... at discount. (a) Certain non-interest-bearing obligations issued at discount—(1) Election to include... discount and redeemable for fixed amounts increasing at stated intervals (other than an obligation...

  11. 49 CFR 22.17 - Compliance with child support obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Compliance with child support obligations. 22.17...) Policies Applying to STLP Loans § 22.17 Compliance with child support obligations. Any holder of 50% or... than 60 days delinquent on any obligation to pay child support arising under: (a) An...

  12. 49 CFR 22.17 - Compliance with child support obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Compliance with child support obligations. 22.17...) Policies Applying to STLP Loans § 22.17 Compliance with child support obligations. Any holder of 50% or... than 60 days delinquent on any obligation to pay child support arising under: (a) An...

  13. 49 CFR 22.17 - Compliance with child support obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Compliance with child support obligations. 22.17...) Policies Applying to STLP Loans § 22.17 Compliance with child support obligations. Any holder of 50% or... than 60 days delinquent on any obligation to pay child support arising under: (a) An...

  14. 49 CFR 22.17 - Compliance with child support obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Compliance with child support obligations. 22.17...) Policies Applying to STLP Loans § 22.17 Compliance with child support obligations. Any holder of 50% or... than 60 days delinquent on any obligation to pay child support arising under: (a) An...

  15. Obligate biotrophy features unraveled by the genomic analysis of the rust fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance...

  16. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  17. Molecular basis of host specificity in human pathogenic bacteria

    PubMed Central

    Pan, Xiaolei; Yang, Yang; Zhang, Jing-Ren

    2014-01-01

    Pathogenic bacteria display various levels of host specificity or tropism. While many bacteria can infect a wide range of hosts, certain bacteria have strict host selectivity for humans as obligate human pathogens. Understanding the genetic and molecular basis of host specificity in pathogenic bacteria is important for understanding pathogenic mechanisms, developing better animal models and designing new strategies and therapeutics for the control of microbial diseases. The molecular mechanisms of bacterial host specificity are much less understood than those of viral pathogens, in part due to the complexity of the molecular composition and cellular structure of bacterial cells. However, important progress has been made in identifying and characterizing molecular determinants of bacterial host specificity in the last two decades. It is now clear that the host specificity of bacterial pathogens is determined by multiple molecular interactions between the pathogens and their hosts. Furthermore, certain basic principles regarding the host specificity of bacterial pathogens have emerged from the existing literature. This review focuses on selected human pathogenic bacteria and our current understanding of their host specificity. PMID:26038515

  18. Intracellular Calcium Channels in Protozoa

    PubMed Central

    Docampo, Roberto; Moreno, Silvia N.J.; Plattner, Helmut

    2014-01-01

    Ca2+-signaling pathways and intracellular Ca2+ channels are present in protozoa. Ancient origin of inositol 1,4,5-trisphosphate receptors (IP3Rs) and other intracellular channels predates the divergence of animals and fungi as evidenced by their presence in the choanoflagellate Monosiga brevicollis, the closest known relative to metazoans. The first protozoan IP3R cloned, from the ciliate Paramecium, displays strong sequence similarity to the rat type 3 IP3R. This ciliate has a large number of IP3- and ryanodine(Ry)-like receptors in 6 subfamilies suggesting the evolutionary adaptation to local requirements for an expanding diversification of vesicle trafficking. IP3Rs have also been functionally characterized in trypanosomatids, where they are essential for growth, differentiation, and establishment of infection. The presence of the mitochondrial calcium uniporter (MCU) in a number of protozoa indicates that mitochondrial regulation of Ca2+ signaling is also an early appearance in evolution, and contributed to the discovery of the molecular nature of this channel in mammalian cells. There is only sequence evidence for the occurrence of two-pore channels (TPCs), transient receptor potential Ca2+ channels (TRPCs) and intracellular mechanosensitive Ca2+-channels in Paramecium and in parasitic protozoa. PMID:24291099

  19. Wholesale service obligation of electric utilities

    SciTech Connect

    Norton, F.L. IV; Spivak, M.R.

    1985-01-01

    The basic concepts of public utility status and utility regulation intertwine the obligation to provide service to the public as reasonably demanded with rate regulation and shielding from competitive interference. While a common law service obligation was not part of the Federal Power Act, the Federal Energy Regulatory Commission has taken the position that service, once commenced, may not be terminated without its approval. This view of Commission authority may not be supported by the legislative history of the Federal Power Act or by judicial precedent. The requirement to serve apart from recognition of a right to serve may result in increased rates in the near term and insufficient capacity, or both, in the long run. A review by the Commission and the courts is examining ways to introduce competition and shift risks from ratepayers to shareholders.

  20. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria.

    PubMed

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180?nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7?cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria. PMID:26728712

  1. Pathogens and polymers: Microbe–host interactions illuminate the cytoskeleton

    PubMed Central

    Haglund, Cat M.

    2011-01-01

    Intracellular pathogens subvert the host cell cytoskeleton to promote their own survival, replication, and dissemination. Study of these microbes has led to many discoveries about host cell biology, including the identification of cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Actin is a common target of bacterial pathogens, but recent work also highlights the use of microtubules, cytoskeletal motors, intermediate filaments, and septins. The study of pathogen interactions with the cytoskeleton has illuminated key cellular processes such as phagocytosis, macropinocytosis, membrane trafficking, motility, autophagy, and signal transduction. PMID:21969466

  2. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the G? subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  3. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  4. Abstract Inactivation of the sapABCDF genes results in a loss of virulence in several bacterial pathogens of ani-

    E-print Network

    McFall-Ngai, Margaret

    et al. 1994; Visick and Skoufos 2001; Millikan and Ruby 2002); (2) attain a normal level in the intracellular pathogen Salmonella typhimurium. A transposon mutant Claudia Lupp · Robert E. W. Hancock · Edward

  5. What are the limits to the obligations of the nurse?

    PubMed Central

    Edwards, S D

    1996-01-01

    This paper enquires into the nature and the extent of the obligations of nurses. It is argued that nurses appear to be obliged to undertake supererogatory acts if they take clause one of the United Kingdom Central Council for Nursing, Midwifery and Health Visiting (UKCC) Code of Professional Conduct seriously (as, indeed, they are required to do). In the first part of the paper, the nature of nursing obligations is outlined, and then the groups and individuals to whom nurses have obligations are identified. Following a brief discussion of the moral foundation of the nurse's obligations to her/his employer, a common conflict of obligations is identified. Then a distinction is drawn between ordinary and extraordinary moral standards. Appreciation of this is necessary for an understanding of the criterion of what constitutes a supererogatory act. By the definition of supererogatory acts proposed below, it is suggested that actions such as whistleblowing satisfy that definition. PMID:8731534

  6. Comparative Analysis of Chlamydia psittaci Genomes Reveals the Recent Emergence of a Pathogenic Lineage with a Broad Host Range

    PubMed Central

    Read, Timothy D.; Joseph, Sandeep J.; Didelot, Xavier; Liang, Brooke; Patel, Lisa; Dean, Deborah

    2013-01-01

    ABSTRACT Chlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots. PMID:23532978

  7. Effector-triggered defence against apoplastic fungal pathogens

    PubMed Central

    Stotz, Henrik U.; Mitrousia, Georgia K.; de Wit, Pierre J.G.M.; Fitt, Bruce D.L.

    2014-01-01

    R gene-mediated host resistance against apoplastic fungal pathogens is not adequately explained by the terms pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI). Therefore, it is proposed that this type of resistance is termed ‘effector-triggered defence’ (ETD). Unlike PTI and ETI, ETD is mediated by R genes encoding cell surface-localised receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrast to this extracellular recognition, ETI is initiated by intracellular detection of pathogen effectors. ETI is usually associated with fast, hypersensitive host cell death, whereas ETD often triggers host cell death only after an elapsed period of endophytic pathogen growth. In this opinion, we focus on ETD responses against foliar fungal pathogens of crops. PMID:24856287

  8. Nitric oxide detoxification by Fusarium verticillioides flavohemoglobin and role in pathogenicity of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium verticillioides is a non-obligate plant pathogen of maize causing a number of specific diseases, including root rot, kernel rot, seed rot, stalk rot, and seedling blight. The saprophytic nature of this fungus, its production of the mycotoxin fumonisin, and complex relationship maize puts t...

  9. 7 CFR 1450.105 - Obligations of participant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Agriculture (Continued) COMMODITY CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS BIOMASS CROP ASSISTANCE PROGRAM (BCAP) Matching Payments § 1450.105 Obligations of participant. (a) All...

  10. Pathogens Hijack the Epigenome

    PubMed Central

    Silmon de Monerri, Natalie C.; Kim, Kami

    2015-01-01

    Pathogens have evolved strategies to promote their survival by dramatically modifying the transcriptional profile and protein content of the host cells they infect. Modifications of the host transcriptome and proteome are mediated by pathogen-encoded effector molecules that modulate host cells through a variety of different mechanisms. Recent studies highlight the importance of the host chromatin and other epigenetic regulators as targets of pathogens. Host gene regulatory mechanisms may be targeted through cytoplasmic signaling, directly by pathogen effector proteins, and possibly by pathogen RNA. Although many of these changes are short-lived and persist only during the course of infection, several studies indicate that pathogens are able to induce long-term, heritable changes that are essential to pathogenesis of infectious diseases and persistence of pathogens within their hosts. In this review, we discuss how pathogens modulate the epigenome of host cells, a new and flourishing avenue of host-pathogen interaction studies. PMID:24525150

  11. Intracellular stresses in patterned cell assemblies.

    PubMed

    Moussus, Michel; der Loughian, Christelle; Fuard, David; Courçon, Marie; Gulino-Debrac, Danielle; Delanoë-Ayari, Hélène; Nicolas, Alice

    2014-04-14

    Confining cells on adhesive patterns allows performing robust, weakly dispersed, statistical analysis. A priori, adhesive patterns could be efficient tools to analyze intracellular cell stress fields, in particular when patterns are used to force the geometry of the cytoskeleton. This tool could then be very helpful in deciphering the relationship between the internal architecture of the cells and the mechanical, intracellular stresses. However, the quantification of the intracellular stresses is still something delicate to perform. Here we first propose a new, very simple and original method to quantify the intracellular stresses, which directly relates the strain the cells impose on the extracellular matrix to the intracellular stress field. This method is used to analyze how confinement influences the intracellular stress field. As a result, we show that the more confined the cells are, the more stressed they will be. The influence of the geometry of the adhesive patterns on the stress patterns is also discussed. PMID:24622969

  12. An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance

    PubMed Central

    Abed, Nadia; Saïd-Hassane, Fatouma; Zouhiri, Fatima; Mougin, Julie; Nicolas, Valérie; Desmaële, Didier; Gref, Ruxandra; Couvreur, Patrick

    2015-01-01

    The “Golden era” of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55–63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells. PMID:26311631

  13. Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages.

    PubMed

    Cheville, N F; Hostetter, J; Thomsen, B V; Simutis, F; Vanloubbeeck, Y; Steadham, E

    2001-06-01

    The granulomatous enteric lesions of cattle with Johne's disease are composed of infected macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall. We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation. Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis. Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles. Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens. Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation. PMID:11449907

  14. SERS nanosensors for intracellular redox potential measurements 

    E-print Network

    Auchinvole, Craig Alexander R

    2012-06-22

    Redox regulation and homeostasis are critically important in the regulation of cell function; however, there are significant challenges in quantitatively measuring and monitoring intracellular redox potentials. The work ...

  15. Obligately barophilic bacterium from the Mariana trench.

    PubMed Central

    Yayanos, A A; Dietz, A S; Van Boxtel, R

    1981-01-01

    An amphipod (Hirondellea gigas) was retrieved with decompression in an insulated trap from an ocean depth of 10,476 m. Bacterial isolates were obtained from the dead and cold animal by using silica gel medium incubated at 1000 bars (1 bar = 10(5) Pa) and 2 degrees C. The isolate designated MT41 was found to be obligately barophilic and did not grow at a pressure close to that of 380 bars found at average depths of the sea. The optimal generation time of about 25 hr was at 2 degrees C and 690 bars. The generation time at 2 degrees C and 1,035 bars, a pressure close to that at the depth of origin, was about 33 hr. Among the conclusions are: (i) pressure is an important determinant of zonation along the water column of the sea; (ii) some obligately barophilic bacteria survive decompressions; (iii) the pressure of optimal growth at 2 degrees C appears to be less than the pressure at the depth of origin and may be diagnostic for the depth of origin; (iv) rates of reproduction are slow yet significant and an order of magnitude greater than previously thought; and (v) much of deep-sea microbiology may have been done with spurious deep-sea organisms due to warming of samples. Images PMID:6946468

  16. Experimental replacement of an obligate insect symbiont.

    PubMed

    Moran, Nancy A; Yun, Yueli

    2015-02-17

    Symbiosis, the close association of unrelated organisms, has been pivotal in biological diversification. In the obligate symbioses found in many insect hosts, organisms that were once independent are permanently and intimately associated, resulting in expanded ecological capabilities. The primary model for this kind of symbiosis is the association between the bacterium Buchnera and the pea aphid (Acyrthosiphon pisum). A longstanding obstacle to efforts to illuminate genetic changes underlying obligate symbioses has been the inability to experimentally disrupt and reconstitute symbiont-host partnerships. Our experiments show that Buchnera can be experimentally transferred between aphid matrilines and, furthermore, that Buchnera replacement has a massive effect on host fitness. Using a recipient pea aphid matriline containing Buchnera that are heat sensitive because of an allele eliminating the heat shock response of a small chaperone, we reduced native Buchnera through heat exposure and introduced a genetically distinct Buchnera from another matriline, achieving complete replacement and stable inheritance. This transfer disrupted 100 million years (? 1 billion generations) of continuous maternal transmission of Buchnera in its host aphids. Furthermore, aphids with the Buchnera replacement enjoyed a dramatic increase in heat tolerance, directly demonstrating a strong effect of symbiont genotype on host ecology. PMID:25561531

  17. Obligately barophilic bacterium from the Mariana trench.

    PubMed

    Yayanos, A A; Dietz, A S; Van Boxtel, R

    1981-08-01

    An amphipod (Hirondellea gigas) was retrieved with decompression in an insulated trap from an ocean depth of 10,476 m. Bacterial isolates were obtained from the dead and cold animal by using silica gel medium incubated at 1000 bars (1 bar = 10(5) Pa) and 2 degrees C. The isolate designated MT41 was found to be obligately barophilic and did not grow at a pressure close to that of 380 bars found at average depths of the sea. The optimal generation time of about 25 hr was at 2 degrees C and 690 bars. The generation time at 2 degrees C and 1,035 bars, a pressure close to that at the depth of origin, was about 33 hr. Among the conclusions are: (i) pressure is an important determinant of zonation along the water column of the sea; (ii) some obligately barophilic bacteria survive decompressions; (iii) the pressure of optimal growth at 2 degrees C appears to be less than the pressure at the depth of origin and may be diagnostic for the depth of origin; (iv) rates of reproduction are slow yet significant and an order of magnitude greater than previously thought; and (v) much of deep-sea microbiology may have been done with spurious deep-sea organisms due to warming of samples. PMID:6946468

  18. A lack of parasitic reduction in the obligate parasitic green alga Helicosporidium.

    PubMed

    Pombert, Jean-François; Blouin, Nicolas Achille; Lane, Chris; Boucias, Drion; Keeling, Patrick J

    2014-05-01

    The evolution of an obligate parasitic lifestyle is often associated with genomic reduction, in particular with the loss of functions associated with increasing host-dependence. This is evident in many parasites, but perhaps the most extreme transitions are from free-living autotrophic algae to obligate parasites. The best-known examples of this are the apicomplexans such as Plasmodium, which evolved from algae with red secondary plastids. However, an analogous transition also took place independently in the Helicosporidia, where an obligate parasite of animals with an intracellular infection mechanism evolved from algae with green primary plastids. We characterised the nuclear genome of Helicosporidium to compare its transition to parasitism with that of apicomplexans. The Helicosporidium genome is small and compact, even by comparison with the relatively small genomes of the closely related green algae Chlorella and Coccomyxa, but at the functional level we find almost no evidence for reduction. Nearly all ancestral metabolic functions are retained, with the single major exception of photosynthesis, and even here reduction is not complete. The great majority of genes for light-harvesting complexes, photosystems, and pigment biosynthesis have been lost, but those for other photosynthesis-related functions, such as Calvin cycle, are retained. Rather than loss of whole function categories, the predominant reductive force in the Helicosporidium genome is a contraction of gene family complexity, but even here most losses affect families associated with genome maintenance and expression, not functions associated with host-dependence. Other gene families appear to have expanded in response to parasitism, in particular chitinases, including those predicted to digest the chitinous barriers of the insect host or remodel the cell wall of Helicosporidium. Overall, the Helicosporidium genome presents a fascinating picture of the early stages of a transition from free-living autotroph to parasitic heterotroph where host-independence has been unexpectedly preserved. PMID:24809511

  19. A Lack of Parasitic Reduction in the Obligate Parasitic Green Alga Helicosporidium

    PubMed Central

    Pombert, Jean-François; Blouin, Nicolas Achille; Lane, Chris; Boucias, Drion; Keeling, Patrick J.

    2014-01-01

    The evolution of an obligate parasitic lifestyle is often associated with genomic reduction, in particular with the loss of functions associated with increasing host-dependence. This is evident in many parasites, but perhaps the most extreme transitions are from free-living autotrophic algae to obligate parasites. The best-known examples of this are the apicomplexans such as Plasmodium, which evolved from algae with red secondary plastids. However, an analogous transition also took place independently in the Helicosporidia, where an obligate parasite of animals with an intracellular infection mechanism evolved from algae with green primary plastids. We characterised the nuclear genome of Helicosporidium to compare its transition to parasitism with that of apicomplexans. The Helicosporidium genome is small and compact, even by comparison with the relatively small genomes of the closely related green algae Chlorella and Coccomyxa, but at the functional level we find almost no evidence for reduction. Nearly all ancestral metabolic functions are retained, with the single major exception of photosynthesis, and even here reduction is not complete. The great majority of genes for light-harvesting complexes, photosystems, and pigment biosynthesis have been lost, but those for other photosynthesis-related functions, such as Calvin cycle, are retained. Rather than loss of whole function categories, the predominant reductive force in the Helicosporidium genome is a contraction of gene family complexity, but even here most losses affect families associated with genome maintenance and expression, not functions associated with host-dependence. Other gene families appear to have expanded in response to parasitism, in particular chitinases, including those predicted to digest the chitinous barriers of the insect host or remodel the cell wall of Helicosporidium. Overall, the Helicosporidium genome presents a fascinating picture of the early stages of a transition from free-living autotroph to parasitic heterotroph where host-independence has been unexpectedly preserved. PMID:24809511

  20. Delineation of Diverse Macrophage Activation Programs in Response to Intracellular Parasites and Cytokines

    PubMed Central

    Zhang, Shuyi; Kim, Charles C.; Batra, Sajeev; McKerrow, James H.; Loke, P'ng

    2010-01-01

    Background The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated. Methodology/Principal Findings To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. Conclusions/Significance This study provides global gene expression data for a diverse set of biologically significant pathogens and cytokines and identifies the relationships between macrophage activation states induced by these stimuli. By comparing macrophage activation programs to pathogens and cytokines under identical experimental conditions, we provide new insights into how macrophage responses to kinetoplastids correlate with the overall range of macrophage activation states. PMID:20361029

  1. The evolution of genomic instability in the obligate endosymbionts of whiteflies.

    PubMed

    Sloan, Daniel B; Moran, Nancy A

    2013-01-01

    Many insects depend on ancient associations with intracellular bacteria to perform essential metabolic functions. These endosymbionts exhibit striking examples of convergence in genome architecture, including a high degree of structural stability that is not typical of their free-living counterparts. However, the recently sequenced genome of the obligate whitefly endosymbiont Portiera revealed features that distinguish it from other ancient insect associates, such as a low gene density and the presence of perfectly duplicated sequences. Here, we report the comparative analysis of Portiera genome sequences both within and between host species. In one whitefly lineage (Bemisia tabaci), we identify large-scale structural polymorphisms in the Portiera genome that exist even within individual insects. This variation is likely mediated by recombination across identical repeats that are maintained by gene conversion. The complete Portiera genome sequence from a distantly related whitefly host (Trialeurodes vaporarium) confirms a history of extensive genome rearrangement in this ancient endosymbiont. Using gene-order-based phylogenetic analysis, we show that the majority of rearrangements have occurred in the B. tabaci lineage, coinciding with an increase in the rate of nucleotide substitutions, a proliferation of short tandem repeats (microsatellites) in intergenic regions, and the loss of many widely conserved genes involved in DNA replication, recombination, and repair. These results indicate that the loss of recombinational machinery is unlikely to be the cause of the extreme structural conservation that is generally observed in obligate endosymbiont genomes and that large, repetitive intergenic regions are an important substrate for genomic rearrangements. PMID:23542079

  2. Tick vaccines and the control of tick-borne pathogens.

    PubMed

    Merino, Octavio; Alberdi, Pilar; Pérez de la Lastra, José M; de la Fuente, José

    2013-01-01

    Ticks are obligate hematophagous ectoparasites that transmit a wide variety of pathogens to humans and animals. The incidence of tick-borne diseases has increased worldwide in both humans and domestic animals over the past years resulting in greater interest in the study of tick-host-pathogen interactions. Advances in vector and pathogen genomics and proteomics have moved forward our knowledge of the vector-pathogen interactions that take place during the colonization and transmission of arthropod-borne microbes. Tick-borne pathogens adapt from the vector to the mammalian host by differential gene expression thus modulating host processes. In recent years, studies have shown that targeting tick proteins by vaccination can not only reduce tick feeding and reproduction, but also the infection and transmission of pathogens from the tick to the vertebrate host. In this article, we review the tick-protective antigens that have been identified for the formulation of tick vaccines and the effect of these vaccines on the control of tick-borne pathogens. PMID:23847771

  3. Tick vaccines and the control of tick-borne pathogens

    PubMed Central

    Merino, Octavio; Alberdi, Pilar; Pérez de la Lastra, José M.; de la Fuente, José

    2013-01-01

    Ticks are obligate hematophagous ectoparasites that transmit a wide variety of pathogens to humans and animals. The incidence of tick-borne diseases has increased worldwide in both humans and domestic animals over the past years resulting in greater interest in the study of tick-host-pathogen interactions. Advances in vector and pathogen genomics and proteomics have moved forward our knowledge of the vector-pathogen interactions that take place during the colonization and transmission of arthropod-borne microbes. Tick-borne pathogens adapt from the vector to the mammalian host by differential gene expression thus modulating host processes. In recent years, studies have shown that targeting tick proteins by vaccination can not only reduce tick feeding and reproduction, but also the infection and transmission of pathogens from the tick to the vertebrate host. In this article, we review the tick-protective antigens that have been identified for the formulation of tick vaccines and the effect of these vaccines on the control of tick-borne pathogens. PMID:23847771

  4. 29 CFR 37.21 - How long will the recipient's obligation under the assurance last, and how broad is the obligation?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true How long will the recipient's obligation under the assurance last, and how broad is the obligation? 37.21 Section 37.21 Labor Office of the Secretary of Labor...'s obligation under the assurance last, and how broad is the obligation? (a) Where the WIA Title...

  5. Intracellular survival of Candida glabrata in macrophages: immune evasion and persistence.

    PubMed

    Kasper, Lydia; Seider, Katja; Hube, Bernhard

    2015-08-01

    Candida glabrata is a successful human opportunistic pathogen which causes superficial but also life-threatening systemic infections. During infection, C. glabrata has to cope with cells of the innate immune system such as macrophages, which belong to the first line of defense against invading pathogens. Candida glabrata is able to survive and even replicate inside macrophages while causing surprisingly low damage and cytokine release. Here, we present an overview of recent studies dealing with the interaction of C. glabrata with macrophages, from phagocytosis to intracellular growth and escape. We review the strategies of C. glabrata that permit intracellular survival and replication, including poor host cell activation, modification of phagosome maturation and phagosome pH, adaptation to antimicrobial activities, and mechanisms to overcome the nutrient limitations within the phagosome. In summary, these studies suggest that survival within macrophages may be an immune evasion and persistence strategy of C. glabrata during infection. PMID:26066553

  6. 12 CFR 614.4358 - Computation of obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Computation of obligations. 614.4358 Section 614.4358 Banks and Banking FARM CREDIT ADMINISTRATION FARM CREDIT SYSTEM LOAN POLICIES AND OPERATIONS Lending and Leasing Limits § 614.4358 Computation of obligations. (a) Inclusions. The computation of...

  7. 18 CFR 35.18 - Asset retirement obligations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Asset retirement obligations. 35.18 Section 35.18 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Other Filing Requirements § 35.18 Asset retirement obligations. (a) A public utility that files a...

  8. 18 CFR 35.18 - Asset retirement obligations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Asset retirement obligations. 35.18 Section 35.18 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Other Filing Requirements § 35.18 Asset retirement obligations. (a) A public utility that files a...

  9. 18 CFR 35.18 - Asset retirement obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Asset retirement obligations. 35.18 Section 35.18 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Other Filing Requirements § 35.18 Asset retirement obligations. (a) A public utility that files a...

  10. 18 CFR 35.18 - Asset retirement obligations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Asset retirement obligations. 35.18 Section 35.18 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Other Filing Requirements § 35.18 Asset retirement obligations. (a) A public utility that files a...

  11. 12 CFR 612.2270 - Purchase of System obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Purchase of System obligations. 612.2270... REFERRAL OF KNOWN OR SUSPECTED CRIMINAL VIOLATIONS Standards of Conduct § 612.2270 Purchase of System... Funding Corporation, may only purchase joint, consolidated, or Systemwide obligations that are: (1)...

  12. 12 CFR 612.2270 - Purchase of System obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Purchase of System obligations. 612.2270... REFERRAL OF KNOWN OR SUSPECTED CRIMINAL VIOLATIONS Standards of Conduct § 612.2270 Purchase of System... Funding Corporation, may only purchase joint, consolidated, or Systemwide obligations that are: (1)...

  13. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation. (a) Reserve obligation. Whenever... kernelweight of certified merchantable walnuts equal to a quantity derived by the application of the...

  14. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation. (a) Reserve obligation. Whenever... kernelweight of certified merchantable walnuts equal to a quantity derived by the application of the...

  15. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation. (a) Reserve obligation. Whenever... kernelweight of certified merchantable walnuts equal to a quantity derived by the application of the...

  16. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation. (a) Reserve obligation. Whenever... kernelweight of certified merchantable walnuts equal to a quantity derived by the application of the...

  17. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation. (a) Reserve obligation. Whenever... kernelweight of certified merchantable walnuts equal to a quantity derived by the application of the...

  18. 47 CFR 211.7 - Obligation of carriers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Obligation of carriers. 211.7 Section 211.7 Telecommunication OFFICE OF SCIENCE AND TECHNOLOGY POLICY AND NATIONAL SECURITY COUNCIL EMERGENCY RESTORATION PRIORITY PROCEDURES FOR TELECOMMUNICATIONS SERVICES § 211.7 Obligation of carriers. (a) During...

  19. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the Fiscal Agent to perform any of...

  20. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the Fiscal Agent to perform any of...

  1. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the Fiscal Agent to perform any of...

  2. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the Fiscal Agent to perform any of...

  3. 22 CFR 221.15 - Fiscal Agent obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Fiscal Agent obligations. 221.15 Section 221.15 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEE STANDARD TERMS AND CONDITIONS The Guarantee § 221.15 Fiscal Agent obligations. Failure of the Fiscal Agent to perform any of...

  4. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 2013-04-01 false Obligations of OASIS users. 37.8 Section 37.8 Conservation...SAME-TIME INFORMATION SYSTEMS § 37.8 Obligations of OASIS users. Each OASIS user must notify the Responsible Party one...

  5. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 2011-04-01 false Obligations of OASIS users. 37.8 Section 37.8 Conservation...SAME-TIME INFORMATION SYSTEMS § 37.8 Obligations of OASIS users. Each OASIS user must notify the Responsible Party one...

  6. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 2012-04-01 false Obligations of OASIS users. 37.8 Section 37.8 Conservation...SAME-TIME INFORMATION SYSTEMS § 37.8 Obligations of OASIS users. Each OASIS user must notify the Responsible Party one...

  7. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 2010-04-01 false Obligations of OASIS users. 37.8 Section 37.8 Conservation...SAME-TIME INFORMATION SYSTEMS § 37.8 Obligations of OASIS users. Each OASIS user must notify the Responsible Party one...

  8. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 2014-04-01 false Obligations of OASIS users. 37.8 Section 37.8 Conservation...SAME-TIME INFORMATION SYSTEMS § 37.8 Obligations of OASIS users. Each OASIS user must notify the Responsible Party one...

  9. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Obligations of OASIS... Obligations of OASIS users. Each OASIS user must notify the Responsible Party one month in advance of initiating a significant amount of automated queries. The OASIS user must also notify the Responsible...

  10. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Obligations of OASIS... Obligations of OASIS users. Each OASIS user must notify the Responsible Party one month in advance of initiating a significant amount of automated queries. The OASIS user must also notify the Responsible...

  11. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Obligations of OASIS... Obligations of OASIS users. Each OASIS user must notify the Responsible Party one month in advance of initiating a significant amount of automated queries. The OASIS user must also notify the Responsible...

  12. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Obligations of OASIS... Obligations of OASIS users. Each OASIS user must notify the Responsible Party one month in advance of initiating a significant amount of automated queries. The OASIS user must also notify the Responsible...

  13. 18 CFR 37.8 - Obligations of OASIS users.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Obligations of OASIS... Obligations of OASIS users. Each OASIS user must notify the Responsible Party one month in advance of initiating a significant amount of automated queries. The OASIS user must also notify the Responsible...

  14. 7 CFR 783.7 - Obligations of a participant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Obligations of a participant. 783.7 Section 783.7 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS TREE ASSISTANCE PROGRAM § 783.7 Obligations of a participant. (a)...

  15. 5 CFR 2635.101 - Basic obligation of public service.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Basic obligation of public service. 2635.101 Section 2635.101 Administrative Personnel OFFICE OF GOVERNMENT ETHICS GOVERNMENT ETHICS STANDARDS OF ETHICAL CONDUCT FOR EMPLOYEES OF THE EXECUTIVE BRANCH General Provisions § 2635.101 Basic obligation of public service. (a) Public service is...

  16. 34 CFR 75.707 - When obligations are made.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false When obligations are made. 75.707 Section 75.707 Education...Education DIRECT GRANT PROGRAMS What Are the Administrative Responsibilities of a...Responsibilities § 75.707 When obligations are made. The following table shows...

  17. 34 CFR 76.707 - When obligations are made.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false When obligations are made. 76.707 Section 76.707 Education...Education STATE-ADMINISTERED PROGRAMS What Are the Administrative Responsibilities of...Responsibilities § 76.707 When obligations are made. The following table shows...

  18. 47 CFR 76.309 - Customer service obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 4 2014-10-01 2014-10-01 false Customer service obligations. 76.309 Section 76.309 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE General Operating Requirements § 76.309 Customer service obligations. (a) A cable franchise authority...

  19. 29 CFR 4043.20 - Post-Event filing obligation.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 9 2012-07-01 2012-07-01 false Post-Event filing obligation. 4043.20 Section 4043.20 Labor... EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.20 Post-Event filing obligation. The plan administrator and each contributing sponsor of a plan for which...

  20. 29 CFR 4043.20 - Post-Event filing obligation.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 9 2014-07-01 2014-07-01 false Post-Event filing obligation. 4043.20 Section 4043.20 Labor... EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.20 Post-Event filing obligation. The plan administrator and each contributing sponsor of a plan for which...

  1. 29 CFR 4043.20 - Post-Event filing obligation.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 9 2013-07-01 2013-07-01 false Post-Event filing obligation. 4043.20 Section 4043.20 Labor... EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.20 Post-Event filing obligation. The plan administrator and each contributing sponsor of a plan for which...

  2. 29 CFR 4043.20 - Post-Event filing obligation.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 9 2011-07-01 2011-07-01 false Post-Event filing obligation. 4043.20 Section 4043.20 Labor... EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.20 Post-Event filing obligation. The plan administrator and each contributing sponsor of a plan for which...

  3. 29 CFR 4043.20 - Post-Event filing obligation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Post-Event filing obligation. 4043.20 Section 4043.20 Labor... EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.20 Post-Event filing obligation. The plan administrator and each contributing sponsor of a plan for which...

  4. 34 CFR 686.40 - Documenting the service obligation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (TEACH) GRANT PROGRAM Service and Repayment Obligations § 686.40 Documenting the service obligation. (a... in a program of study for which a TEACH Grant was received, the grant recipient must confirm to the... period being certified in any of the high-need fields of mathematics, science, a foreign...

  5. Parental Beliefs about Nonresident Fathers' Obligations and Rights

    ERIC Educational Resources Information Center

    Lin, I-Fen; McLanahan, Sara S.

    2007-01-01

    We examine whether parents rely on principles of equity or equality in making judgments about nonresident fathers' obligations and rights. The data are taken from the first wave of the Fragile Families and Child Wellbeing Study. The analysis sample includes 4,304 new mothers and 3,414 new fathers. Results indicate that fathers perceive obligations

  6. 43 CFR 3162.2-1 - Drilling and producing obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false Drilling and producing obligations. 3162.2... Requirements for Operating Rights Owners and Operators § 3162.2-1 Drilling and producing obligations. (a) The operator, at its election, may drill and produce other wells in conformity with any system of well...

  7. 43 CFR 3162.2-1 - Drilling and producing obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Drilling and producing obligations. 3162.2... Requirements for Operating Rights Owners and Operators § 3162.2-1 Drilling and producing obligations. (a) The operator, at its election, may drill and produce other wells in conformity with any system of well...

  8. 43 CFR 3162.2-1 - Drilling and producing obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Drilling and producing obligations. 3162.2... Requirements for Operating Rights Owners and Operators § 3162.2-1 Drilling and producing obligations. (a) The operator, at its election, may drill and produce other wells in conformity with any system of well...

  9. 43 CFR 3162.2-1 - Drilling and producing obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 2 2013-10-01 2013-10-01 false Drilling and producing obligations. 3162.2... Requirements for Operating Rights Owners and Operators § 3162.2-1 Drilling and producing obligations. (a) The operator, at its election, may drill and produce other wells in conformity with any system of well...

  10. 29 CFR 4.146 - Contract obligations after award, generally.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Contract obligations after award, generally. 4.146 Section 4.146 Labor Office of the Secretary of Labor LABOR STANDARDS FOR FEDERAL SERVICE CONTRACTS Application of the McNamara-O'Hara Service Contract Act Period of Coverage § 4.146 Contract obligations...

  11. 26 CFR 1.103-16 - Obligations of certain volunteer fire departments.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Obligations of certain volunteer fire departments. 1.103-16...103-16 Obligations of certain volunteer fire departments. (a) General rule. An obligation of a volunteer fire department issued after...

  12. 26 CFR 1.103-16 - Obligations of certain volunteer fire departments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Obligations of certain volunteer fire departments. 1.103-16...103-16 Obligations of certain volunteer fire departments. (a) General rule. An obligation of a volunteer fire department issued after...

  13. 26 CFR 1.1504-4 - Treatment of warrants, options, convertible obligations, and other similar interests.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Treatment of warrants, options, convertible obligations, and other...1.1504-4 Treatment of warrants, options, convertible obligations, and other...regarding the circumstances in which warrants, options, obligations convertible into...

  14. 26 CFR 1.1504-4 - Treatment of warrants, options, convertible obligations, and other similar interests.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Treatment of warrants, options, convertible obligations, and other...1.1504-4 Treatment of warrants, options, convertible obligations, and other...regarding the circumstances in which warrants, options, obligations convertible into...

  15. 12 CFR 1510.5 - How does the Funding Corporation make interest payments on its obligations?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...Funding Corporation make interest payments on its obligations? 1510.5 Section 1510...Funding Corporation make interest payments on its obligations? (a) The Funding Corporation...Corporation must pay the interest due on its obligations with funds it obtains...

  16. 12 CFR 1510.5 - How does the Funding Corporation make interest payments on its obligations?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Funding Corporation make interest payments on its obligations? 1510.5 Section 1510...Funding Corporation make interest payments on its obligations? (a) The Funding Corporation...Corporation must pay the interest due on its obligations with funds it obtains...

  17. 12 CFR 1270.18 - Additional requirements; notice of attachment for Book-entry consolidated obligations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...requirements; notice of attachment for Book-entry consolidated obligations. 1270...FEDERAL HOME LOAN BANKS LIABILITIES Book-Entry Procedure for Consolidated Obligations...requirements; notice of attachment for Book-entry consolidated obligations....

  18. 12 CFR 1270.18 - Additional requirements; notice of attachment for Book-entry consolidated obligations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...requirements; notice of attachment for Book-entry consolidated obligations. 1270...FEDERAL HOME LOAN BANKS LIABILITIES Book-Entry Procedure for Consolidated Obligations...requirements; notice of attachment for Book-entry consolidated obligations....

  19. 12 CFR 1270.18 - Additional requirements; notice of attachment for Book-entry consolidated obligations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...requirements; notice of attachment for Book-entry consolidated obligations. 1270...FEDERAL HOME LOAN BANKS LIABILITIES Book-Entry Procedure for Consolidated Obligations...requirements; notice of attachment for Book-entry consolidated obligations....

  20. 31 CFR 285.8 - Offset of tax refund payments to collect state income tax obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...State income tax obligation means State income tax obligations as determined under State law. For purposes of this section, State income tax obligation includes any...the overpayment. (b) General rule. (1) FMS will collect...

  1. 31 CFR 225.9 - Return of Government obligations to obligor.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Return of Government obligations to obligor. 225... ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS WITH SURETIES § 225.9 Return of Government obligations to obligor....

  2. 31 CFR 225.9 - Return of Government obligations to obligor.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 false Return of Government obligations to obligor. 225... ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS WITH SURETIES § 225.9 Return of Government obligations to obligor....

  3. 12 CFR 987.8 - Additional requirements; notice of attachment for Book-entry consolidated obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...requirements; notice of attachment for Book-entry consolidated obligations. 987...HOUSING FINANCE BOARD OFFICE OF FINANCE BOOK-ENTRY PROCEDURE FOR CONSOLIDATED OBLIGATIONS...requirements; notice of attachment for Book-entry consolidated obligations....

  4. 12 CFR 987.8 - Additional requirements; notice of attachment for Book-entry consolidated obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...requirements; notice of attachment for Book-entry consolidated obligations. 987...HOUSING FINANCE BOARD OFFICE OF FINANCE BOOK-ENTRY PROCEDURE FOR CONSOLIDATED OBLIGATIONS...requirements; notice of attachment for Book-entry consolidated obligations....

  5. Targeting of a Chlamydial Protease Impedes Intracellular Bacterial Growth

    PubMed Central

    Paschen, Stefan A.; Vier, Juliane; Schauenburg, Linda; Rupp, Jan; Meyer, Thomas F.; Häcker, Georg; Heuer, Dagmar

    2011-01-01

    Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target. PMID:21990969

  6. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity

    PubMed Central

    Ishikawa, Hiroki; Ma, Zhe; Barber, Glen N.

    2015-01-01

    The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN)1–3. However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection4. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines. PMID:19776740

  7. Settling down: the genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life.

    PubMed

    Manzano-Marín, Alejandro; Latorre, Amparo

    2014-07-01

    Particularly interesting cases of mutualistic endosymbioses come from the establishment of co-obligate associations of more than one species of endosymbiotic bacteria. Throughout symbiotic accommodation from a free-living bacterium, passing through a facultative stage and ending as an obligate intracellular one, the symbiont experiences massive genomic losses and phenotypic adjustments. Here, we scrutinized the changes in the coevolution of Serratia symbiotica and Buchnera aphidicola endosymbionts in aphids, paying particular attention to the transformations undergone by S. symbiotica to become an obligate endosymbiont. Although it is already known that S. symbiotica is facultative in Acyrthosiphon pisum, in Cinara cedri it has established a co-obligate endosymbiotic consortium along with B. aphidicola to fulfill the aphid's nutritional requirements. The state of this association in C. tujafilina, an aphid belonging to the same subfamily (Lachninae) that C. cedri, remained unknown. Here, we report the genome of S. symbiotica strain SCt-VLC from the aphid C. tujafilina. While being phylogenetically and genomically very closely related to the facultative endosymbiont S. symbiotica from the aphid A. pisum, it shows a variety of metabolic, genetic, and architectural features, which point toward this endosymbiont being one step closer to an obligate intracellular one. We also describe in depth the process of genome rearrangements suffered by S. symbiotica and the role mobile elements play in gene inactivations. Finally, we postulate the supply to the host of the essential riboflavin (vitamin B2) as key to the establishment of S. symbiotica as a co-obligate endosymbiont in the aphids belonging to the subfamily Lachninane. PMID:24951564

  8. Intracellular signalling during neutrophil recruitment

    PubMed Central

    Mócsai, Attila; Walzog, Barbara; Lowell, Clifford A.

    2015-01-01

    Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage. PMID:25998986

  9. Review of International Experience with Renewable Energy Obligation Support Mechanisms

    SciTech Connect

    Wiser, R.

    2005-06-01

    The main policy instruments currently used in the EU Member States to achieve the targets set for electricity produced from renewable energy sources are: (1) the quota obligation system; (2) the feed-in tariff system; and (3) the tendering system. The current study aims to review the experience gained with the quota obligation system. The report provides an overview of the regions where obligation systems have been implemented and contains a detailed evaluation of the performance of the obligation systems in the USA, the UK and in Sweden. The obligation systems in these countries have been evaluated based on the following criteria: Effectiveness; Market efficiency; Certainty for the renewable energy industry; Cost effectiveness; Stakeholder support for the obligation system; and Equity. The evaluation of international experiences with the obligation system gives rise to a mixed picture. Although an obligation in theory is effective and cost effective, it seems too early to conclude that the system delivers these promises in practice. On the one hand this is due to the limited period of implementation that makes it hard to distinguish between the direct effect of the system and some teething problems that will be solved in due time. On the other hand, the conclusion can be drawn that the obligation is a complex system, which will only function well if designed carefully. It does seem worthwhile, however, to continue monitoring the experiences with the obligation system abroad, because this will further reveal whether the system is indeed effective and cost effective in practice. In the longer term, e.g. beyond 2010, the introduction of an obligation system in the Netherlands could be considered. Finally, as the design of support schemes is being improved, it appears that the basic concepts of both the obligation system and the feed in system have been refined in such a way that the two systems are gradually converging. An important difference between the two systems however remains, namely that an obligation system relies more on market forces whereas the feed-in system is based on a greater involvement of the government.

  10. Legionella pneumophila requires polyamines for optimal intracellular growth.

    PubMed

    Nasrallah, Gheyath K; Riveroll, Angela L; Chong, Audrey; Murray, Lois E; Lewis, P Jeffrey; Garduño, Rafael A

    2011-09-01

    The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  11. Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ?

    PubMed Central

    Nasrallah, Gheyath K.; Riveroll, Angela L.; Chong, Audrey; Murray, Lois E.; Lewis, P. Jeffrey; Garduño, Rafael A.

    2011-01-01

    The Gram-negative intracellular pathogen Legionella pneumophilareplicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophilaexpressing a translocation reporter consisting of the Bordetella pertussisadenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophilamay require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophilareplication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophilaproliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  12. Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

    PubMed

    Labouta, Hagar Ibrahim; Menina, Sara; Kochut, Annika; Gordon, Sarah; Geyer, Rebecca; Dersch, Petra; Lehr, Claus-Michael

    2015-12-28

    Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via ?1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments. PMID:26522071

  13. Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

    E-print Network

    Markham, Aaron P.; Jaafar, Zane A.; Kemege, Kyle Evan; Middaugh, C. Russell; Hefty, P. Scott

    2009-11-01

    Chlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Likemany Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche...

  14. Bats as 'special' reservoirs for emerging zoonotic pathogens.

    PubMed

    Brook, Cara E; Dobson, Andrew P

    2015-03-01

    The ongoing West African Ebola epidemic highlights a recurring trend in the zoonotic emergence of virulent pathogens likely to come from bat reservoirs that has caused epidemiologists to ask 'Are bats special reservoirs for emerging zoonotic pathogens?' We collate evidence from the past decade to delineate mitochondrial mechanisms of bat physiology that have evolved to mitigate oxidative stress incurred during metabolically costly activities such as flight. We further describe how such mechanisms might have generated pleiotropic effects responsible for tumor mitigation and pathogen control in bat hosts. These synergisms may enable 'special' tolerance of intracellular pathogens in bat hosts; paradoxically, this may leave them more susceptible to immunopathological morbidity when attempting to clear extracellular infections such as 'white-nose syndrome' (WNS). PMID:25572882

  15. Pathogenic adaptations to host-derived antibacterial copper

    PubMed Central

    Chaturvedi, Kaveri S.; Henderson, Jeffrey P.

    2014-01-01

    Recent findings suggest that both host and pathogen manipulate copper content in infected host niches during infections. In this review, we summarize recent developments that implicate copper resistance as an important determinant of bacterial fitness at the host-pathogen interface. An essential mammalian nutrient, copper cycles between copper (I) (Cu+) in its reduced form and copper (II) (Cu2+) in its oxidized form under physiologic conditions. Cu+ is significantly more bactericidal than Cu2+ due to its ability to freely penetrate bacterial membranes and inactivate intracellular iron-sulfur clusters. Copper ions can also catalyze reactive oxygen species (ROS) generation, which may further contribute to their toxicity. Transporters, chaperones, redox proteins, receptors and transcription factors and even siderophores affect copper accumulation and distribution in both pathogenic microbes and their human hosts. This review will briefly cover evidence for copper as a mammalian antibacterial effector, the possible reasons for this toxicity, and pathogenic resistance mechanisms directed against it. PMID:24551598

  16. Subverting Toll-Like Receptor Signaling by Bacterial Pathogens

    PubMed Central

    McGuire, Victoria A.; Arthur, J. Simon C.

    2015-01-01

    Pathogenic bacteria are detected by pattern-recognition receptors (PRRs) expressed on innate immune cells, which activate intracellular signal transduction pathways to elicit an immune response. Toll-like receptors are, perhaps, the most studied of the PRRs and can activate the mitogen-activated protein kinase (MAPK) and Nuclear Factor-?B (NF-?B) pathways. These pathways are critical for mounting an effective immune response. In order to evade detection and promote virulence, many pathogens subvert the host immune response by targeting components of these signal transduction pathways. This mini-review highlights the diverse mechanisms that bacterial pathogens have evolved to manipulate the innate immune response, with a particular focus on those that target MAPK and NF-?B signaling pathways. Understanding the elaborate strategies that pathogens employ to subvert the immune response not only highlights the importance of these proteins in mounting effective immune responses, but may also identify novel approaches for treatment or prevention of infection. PMID:26648936

  17. Stochastic resonance in an intracellular genetic perceptron

    NASA Astrophysics Data System (ADS)

    Bates, Russell; Blyuss, Oleg; Zaikin, Alexey

    2014-03-01

    Intracellular genetic networks are more intelligent than was first assumed due to their ability to learn. One of the manifestations of this intelligence is the ability to learn associations of two stimuli within gene-regulating circuitry: Hebbian-type learning within the cellular life. However, gene expression is an intrinsically noisy process; hence, we investigate the effect of intrinsic and extrinsic noise on this kind of intracellular intelligence. We report a stochastic resonance in an intracellular associative genetic perceptron, a noise-induced phenomenon, which manifests itself in noise-induced increase of response in efficiency after the learning event under the conditions of optimal stochasticity.

  18. Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic h...

  19. Role of Host Cell-Derived Amino Acids in Nutrition of Intracellular Salmonella enterica.

    PubMed

    Popp, Jasmin; Noster, Janina; Busch, Kim; Kehl, Alexander; Zur Hellen, Gero; Hensel, Michael

    2015-12-01

    The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients. PMID:26351287

  20. A detailed view of the intracellular transcriptome of Listeria monocytogenes in murine macrophages using RNA-seq

    PubMed Central

    Schultze, Tilman; Hilker, Rolf; Mannala, Gopala K.; Gentil, Katrin; Weigel, Markus; Farmani, Neda; Windhorst, Anita C.; Goesmann, Alexander; Chakraborty, Trinad; Hain, Torsten

    2015-01-01

    Listeria monocytogenes is a bacterial pathogen and causative agent for the foodborne infection listeriosis, which is mainly a threat for pregnant, elderly, or immunocompromised individuals. Due to its ability to invade and colonize diverse eukaryotic cell types including cells from invertebrates, L. monocytogenes has become a well-established model organism for intracellular growth. Almost 10 years ago, we and others presented the first whole-genome microarray-based intracellular transcriptome of L. monocytogenes. With the advent of newer technologies addressing transcriptomes in greater detail, we revisit this work, and analyze the intracellular transcriptome of L. monocytogenes during growth in murine macrophages using a deep sequencing based approach. We detected 656 differentially expressed genes of which 367 were upregulated during intracellular growth in macrophages compared to extracellular growth in Brain Heart Infusion broth. This study confirmed ?64% of all regulated genes previously identified by microarray analysis. Many of the regulated genes that were detected in the current study involve transporters for various metals, ions as well as complex sugars such as mannose. We also report changes in antisense transcription, especially upregulations during intracellular bacterial survival. A notable finding was the detection of regulatory changes for a subset of temperate A118-like prophage genes, thereby shedding light on the transcriptional profile of this bacteriophage during intracellular growth. In total, our study provides an updated genome-wide view of the transcriptional landscape of L. monocytogenes during intracellular growth and represents a rich resource for future detailed analysis. PMID:26579105

  1. Recent developments in copper and zinc homeostasis in bacterial pathogens.

    PubMed

    Braymer, Joseph J; Giedroc, David P

    2014-04-01

    Copper and zinc homeostasis systems in pathogenic bacteria are required to resist host efforts to manipulate the availability and toxicity of these metal ions. Central to this microbial adaptive response is the involvement of metal-trafficking and metal-sensing proteins that ultimately exercise control of metal speciation in the cell. Cu-specific and Zn-specific metalloregulatory proteins regulate the transcription of metal-responsive genes while metallochaperones and related proteins ensure that these metals are appropriately buffered by the intracellular milieu and delivered to correct intracellular targets. In this review, we summarize recent findings on how bacterial pathogens mount a metal-specific response to derail host efforts to win the 'fight over metals.' PMID:24463765

  2. Cellular/Mollecular Intracellular Patch Electrochemistry: Regulation of Cytosolic

    E-print Network

    Sulzer, David

    Cellular/Mollecular Intracellular Patch Electrochemistry: Regulation of Cytosolic Catecholamines intracellular patch electrochemistry (IPE), a tech- nique that for the first time provides direct measurements

  3. Obligate biotrophy features unraveled by the genomic analysis of rust fungi

    PubMed Central

    Duplessis, Sébastien; Cuomo, Christina A.; Lin, Yao-Cheng; Aerts, Andrea; Tisserant, Emilie; Veneault-Fourrey, Claire; Joly, David L.; Hacquard, Stéphane; Amselem, Joëlle; Cantarel, Brandi L.; Chiu, Readman; Coutinho, Pedro M.; Feau, Nicolas; Field, Matthew; Frey, Pascal; Gelhaye, Eric; Goldberg, Jonathan; Grabherr, Manfred G.; Kodira, Chinnappa D.; Kohler, Annegret; Kües, Ursula; Lindquist, Erika A.; Lucas, Susan M.; Mago, Rohit; Mauceli, Evan; Morin, Emmanuelle; Murat, Claude; Pangilinan, Jasmyn L.; Park, Robert; Pearson, Matthew; Quesneville, Hadi; Rouhier, Nicolas; Sakthikumar, Sharadha; Salamov, Asaf A.; Schmutz, Jeremy; Selles, Benjamin; Shapiro, Harris; Tanguay, Philippe; Tuskan, Gerald A.; Henrissat, Bernard; Van de Peer, Yves; Rouzé, Pierre; Ellis, Jeffrey G.; Dodds, Peter N.; Schein, Jacqueline E.; Zhong, Shaobin; Hamelin, Richard C.; Grigoriev, Igor V.; Szabo, Les J.; Martin, Francis

    2011-01-01

    Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells. PMID:21536894

  4. Intracellular FRET-based probes: a review

    NASA Astrophysics Data System (ADS)

    Rowland, Clare E.; Brown, Carl W., III; Medintz, Igor L.; Delehanty, James B.

    2015-12-01

    Probes that exploit Förster resonance energy transfer (FRET) in their feedback mechanism are touted for their sensitivity, robustness, and low background, and thanks to the exceptional distance dependence of the energy transfer process, they provide a means of probing lengthscales well below the resolution of light. These attributes make FRET-based probes superbly suited to an intracellular environment, and recent developments in biofunctionalization and expansion of imaging capabilities have put them at the forefront of intracellular studies. Here, we present an overview of the engineering and execution of a variety of recent intracellular FRET probes, highlighting the diversity of this class of materials and the breadth of application they have found in the intracellular environment.

  5. Polymer physics of intracellular phase transitions

    NASA Astrophysics Data System (ADS)

    Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.

    2015-11-01

    Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biological roles, by compartmentalizing the cell to enable spatiotemporal control of biological reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liquid droplets that form via phase separation. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liquid-liquid phase separation in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equilibrium features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics.

  6. Introduction Many cnidarians symbiotic with the intracellular

    E-print Network

    , peroxynitrite, oxidative stress, Symbiodinium, zooxanthellae, coral bleaching, Cnidaria, di2804 Introduction Many cnidarians symbiotic with the intracellular dinoflagellate Symbiodinium sp., such as the ecologically important reef-building corals, lose their algal partners in response to a variety of stresses

  7. Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

    PubMed Central

    Saka, Hector A.; Thompson, J. Will; Chen, Yi-Shan; Kumar, Yadunanda; Dubois, Laura G.; Moseley, M. Arthur; Valdivia, Raphael H.

    2011-01-01

    Summary Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyze the membranes of the pathogen-containing vacuole (“inclusion”). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of >54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient Type III secretion (T3S) in the RB stage at 18 hpi, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a “per organism” basis. Our results show that EB and RB proteomes are streamlined to fulfill their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs. PMID:22014092

  8. Stress adaptation in a pathogenic fungus.

    PubMed

    Brown, Alistair J P; Budge, Susan; Kaloriti, Despoina; Tillmann, Anna; Jacobsen, Mette D; Yin, Zhikang; Ene, Iuliana V; Bohovych, Iryna; Sandai, Doblin; Kastora, Stavroula; Potrykus, Joanna; Ballou, Elizabeth R; Childers, Delma S; Shahana, Shahida; Leach, Michelle D

    2014-01-01

    Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of a number of regulators in C. albicans have diverged relative to the benign model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This reflects the specific evolution of C. albicans as an opportunistic pathogen obligately associated with warm-blooded animals, compared with other yeasts that are found across diverse environmental niches. Our understanding of C. albicans stress signalling is based primarily on the in vitro responses of glucose-grown cells to individual stresses. However, in vivo this pathogen occupies complex and dynamic host niches characterised by alternative carbon sources and simultaneous exposure to combinations of stresses (rather than individual stresses). It has become apparent that changes in carbon source strongly influence stress resistance, and that some combinatorial stresses exert non-additive effects upon C. albicans. These effects, which are relevant to fungus-host interactions during disease progression, are mediated by multiple mechanisms that include signalling and chemical crosstalk, stress pathway interference and a biological transistor. PMID:24353214

  9. Obligate oil-degrading marine bacteria.

    PubMed

    Yakimov, Michail M; Timmis, Kenneth N; Golyshin, Peter N

    2007-06-01

    Over the past few years, a new and ecophysiologically unusual group of marine hydrocarbon-degrading bacteria - the obligate hydrocarbonoclastic bacteria (OHCB) - has been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters. The introduction of oil or oil constituents into seawater leads to successive blooms of a relatively limited number of indigenous marine bacterial genera--Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others (the OHCB)--which are present at low or undetectable levels before the polluting event. The types of OHCB that bloom depend on the latitude/temperature, salinity, redox and other prevailing physical-chemical factors. These blooms result in the rapid degradation of many oil constituents, a process that can be accelerated further by supplementation with limiting nutrients. Genome sequencing and functional genomic analysis of Alcanivorax borkumensis, the paradigm of OHCB, has provided significant insights into the genomic basis of the efficiency and versatility of its hydrocarbon utilization, the metabolic routes underlying its special hydrocarbon diet, and its ecological success. These and other studies have revealed the potential of OHCB for multiple biotechnological applications that include not only oil pollution mitigation, but also biopolymer production and biocatalysis. PMID:17493798

  10. Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Voth, Daniel E.; Howe, Dale; Cockrell, Diane C.; Bastidas, Robert J.; Valdivia, Raphael H.

    2014-01-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ?cvpB, ?cvpC, ?cvpD, and ?cvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ?cvpD and ?cvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ?cvpB and ?cvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages. PMID:25422265

  11. Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.

    PubMed

    Larson, Charles L; Beare, Paul A; Voth, Daniel E; Howe, Dale; Cockrell, Diane C; Bastidas, Robert J; Valdivia, Raphael H; Heinzen, Robert A

    2015-02-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ?cvpB, ?cvpC, ?cvpD, and ?cvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ?cvpD and ?cvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ?cvpB and ?cvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages. PMID:25422265

  12. 31 CFR 223.18 - Performance of agency obligations.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY FINANCIAL MANAGEMENT SERVICE SURETY COMPANIES DOING BUSINESS WITH THE UNITED STATES § 223.18 Performance of agency obligations. (a)...

  13. 31 CFR 223.18 - Performance of agency obligations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY FINANCIAL MANAGEMENT SERVICE SURETY COMPANIES DOING BUSINESS WITH THE UNITED STATES § 223.18 Performance of agency obligations. (a)...

  14. 31 CFR 223.18 - Performance of agency obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY FINANCIAL MANAGEMENT SERVICE SURETY COMPANIES DOING BUSINESS WITH THE UNITED STATES § 223.18 Performance of agency obligations. (a)...

  15. 31 CFR 223.18 - Performance of agency obligations.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Relating to Money and Finance (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY FINANCIAL MANAGEMENT SERVICE SURETY COMPANIES DOING BUSINESS WITH THE UNITED STATES § 223.18 Performance of agency obligations. (a)...

  16. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...NUTS), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation...kernelweight of certified merchantable walnuts equal to a quantity derived by the...

  17. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation...kernelweight of certified merchantable walnuts equal to a quantity derived by the...

  18. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...NUTS), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation...kernelweight of certified merchantable walnuts equal to a quantity derived by the...

  19. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation...kernelweight of certified merchantable walnuts equal to a quantity derived by the...

  20. 7 CFR 984.54 - Establishment of obligation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Nuts), DEPARTMENT OF AGRICULTURE WALNUTS GROWN IN CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.54 Establishment of obligation...kernelweight of certified merchantable walnuts equal to a quantity derived by the...

  1. 22 CFR 230.07 - Fiscal Agent obligations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...false Fiscal Agent obligations. 230.07 Section 230.07 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEES ISSUED UNDER THE EMERGENCY WARTIME SUPPLEMENTAL APPROPRIATIONS ACT OF 2003, PUB. L. 108-11-STANDARD...

  2. 22 CFR 230.07 - Fiscal Agent obligations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...false Fiscal Agent obligations. 230.07 Section 230.07 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEES ISSUED UNDER THE EMERGENCY WARTIME SUPPLEMENTAL APPROPRIATIONS ACT OF 2003, PUB. L. 108-11-STANDARD...

  3. 22 CFR 230.07 - Fiscal Agent obligations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...false Fiscal Agent obligations. 230.07 Section 230.07 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEES ISSUED UNDER THE EMERGENCY WARTIME SUPPLEMENTAL APPROPRIATIONS ACT OF 2003, PUB. L. 108-11-STANDARD...

  4. 22 CFR 230.07 - Fiscal Agent obligations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...false Fiscal Agent obligations. 230.07 Section 230.07 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEES ISSUED UNDER THE EMERGENCY WARTIME SUPPLEMENTAL APPROPRIATIONS ACT OF 2003, PUB. L. 108-11-STANDARD...

  5. 22 CFR 230.07 - Fiscal Agent obligations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...false Fiscal Agent obligations. 230.07 Section 230.07 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ISRAEL LOAN GUARANTEES ISSUED UNDER THE EMERGENCY WARTIME SUPPLEMENTAL APPROPRIATIONS ACT OF 2003, PUB. L. 108-11-STANDARD...

  6. 12 CFR 612.2270 - Purchase of System obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...of Conduct § 612.2270 Purchase of System obligations...Funding Corporation, may only purchase joint, consolidated, or...with a member of the selling group designated by the Federal...Banks Funding Corporation may purchase or otherwise acquire,...

  7. 38 CFR 17.608 - Deferment of obligated service.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (a) Request for deferment. A participant receiving a degree from a school of medicine, osteopathy, dentistry, optometry, or podiatry, may request deferment of obligated service to complete an approved program of advanced clinical...

  8. 32 CFR 220.9 - Rights and obligations of beneficiaries.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.9 Rights and obligations...be required. (b) Availability of healthcare services unaffected. The availability of healthcare services in any facility of the...

  9. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association... detailed analysis of credit quality. In doing so, you must consider, as appropriate, the interest...

  10. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association... detailed analysis of credit quality. In doing so, you must consider, as appropriate, the interest...

  11. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association... detailed analysis of credit quality. In doing so, you must consider, as appropriate, the interest...

  12. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association... detailed analysis of credit quality. In doing so, you must consider, as appropriate, the interest...

  13. 12 CFR 560.42 - State and local government obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... obligations. (a) What limitations apply? Pursuant to HOLA section 5(c)(1)(H), a Federal savings association... detailed analysis of credit quality. In doing so, you must consider, as appropriate, the interest...

  14. 47 CFR 25.701 - Public interest obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Public Interest Obligations...Entities licensed to operate satellites in the 12.2 to 12.7 GHz...

  15. 47 CFR 25.701 - Public interest obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Public Interest Obligations...Entities licensed to operate satellites in the 12.2 to 12.7 GHz...

  16. 47 CFR 25.701 - Public interest obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Public Interest Obligations...Entities licensed to operate satellites in the 12.2 to 12.7 GHz...

  17. 47 CFR 25.701 - Public interest obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Public Interest Obligations...Entities licensed to operate satellites in the 12.2 to 12.7 GHz...

  18. 47 CFR 54.405 - Carrier obligation to offer Lifeline.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... (CONTINUED) UNIVERSAL SERVICE Universal Service Support for Low-Income Consumers § 54.405 Carrier obligation... within the 60-day time period. A carrier providing Lifeline service in a state that has...

  19. 47 CFR 54.405 - Carrier obligation to offer Lifeline.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... (CONTINUED) UNIVERSAL SERVICE Universal Service Support for Low-Income Consumers § 54.405 Carrier obligation... demonstrate continued eligibility within the 60-day time period. A carrier providing Lifeline service in...

  20. 29 CFR 1980.102 - Obligations and prohibited acts.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...DISCRIMINATION COMPLAINTS UNDER SECTION 806 OF THE CORPORATE AND CRIMINAL FRAUD ACCOUNTABILITY ACT OF 2002, TITLE VIII OF THE SARBANES-OXLEY ACT OF 2002 Complaints, Investigations, Findings and Preliminary Orders § 1980.102 Obligations...

  1. 29 CFR 1981.102 - Obligations and prohibited acts.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...CONTINUED) PROCEDURES FOR THE HANDLING OF DISCRIMINATION COMPLAINTS UNDER SECTION 6 OF THE PIPELINE SAFETY IMPROVEMENT ACT OF 2002 Complaints, Investigations, Findings, and Preliminary Orders § 1981.102 Obligations and prohibited acts....

  2. 29 CFR 1981.102 - Obligations and prohibited acts.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...CONTINUED) PROCEDURES FOR THE HANDLING OF DISCRIMINATION COMPLAINTS UNDER SECTION 6 OF THE PIPELINE SAFETY IMPROVEMENT ACT OF 2002 Complaints, Investigations, Findings, and Preliminary Orders § 1981.102 Obligations and prohibited acts....

  3. 29 CFR 1980.102 - Obligations and prohibited acts.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...LABOR (CONTINUED) PROCEDURES FOR THE HANDLING OF RETALIATION COMPLAINTS UNDER SECTION 806 OF THE SARBANES-OXLEY ACT OF 2002, AS AMENDED Complaints, Investigations, Findings and Preliminary Orders § 1980.102 Obligations and prohibited...

  4. 29 CFR 1980.102 - Obligations and prohibited acts.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...DISCRIMINATION COMPLAINTS UNDER SECTION 806 OF THE CORPORATE AND CRIMINAL FRAUD ACCOUNTABILITY ACT OF 2002, TITLE VIII OF THE SARBANES-OXLEY ACT OF 2002 Complaints, Investigations, Findings and Preliminary Orders § 1980.102 Obligations...

  5. 32 CFR 220.9 - Rights and obligations of beneficiaries.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.9 Rights and obligations...be required. (b) Availability of healthcare services unaffected. The availability of healthcare services in any facility of the...

  6. 32 CFR 220.9 - Rights and obligations of beneficiaries.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.9 Rights and obligations...be required. (b) Availability of healthcare services unaffected. The availability of healthcare services in any facility of the...

  7. 32 CFR 220.9 - Rights and obligations of beneficiaries.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.9 Rights and obligations...be required. (b) Availability of healthcare services unaffected. The availability of healthcare services in any facility of the...

  8. 32 CFR 220.9 - Rights and obligations of beneficiaries.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.9 Rights and obligations...be required. (b) Availability of healthcare services unaffected. The availability of healthcare services in any facility of the...

  9. 34 CFR 686.43 - Obligation to repay the grant.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION (CONTINUED) TEACHER EDUCATION ASSISTANCE FOR COLLEGE AND HIGHER EDUCATION (TEACH) GRANT PROGRAM Service and Repayment Obligations §...

  10. 34 CFR 686.43 - Obligation to repay the grant.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION (CONTINUED) TEACHER EDUCATION ASSISTANCE FOR COLLEGE AND HIGHER EDUCATION (TEACH) GRANT PROGRAM Service and Repayment Obligations §...

  11. 34 CFR 686.43 - Obligation to repay the grant.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION TEACHER EDUCATION ASSISTANCE FOR COLLEGE AND HIGHER EDUCATION (TEACH) GRANT PROGRAM Service and Repayment Obligations §...

  12. 34 CFR 686.43 - Obligation to repay the grant.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION (CONTINUED) TEACHER EDUCATION ASSISTANCE FOR COLLEGE AND HIGHER EDUCATION (TEACH) GRANT PROGRAM Service and Repayment Obligations §...

  13. 34 CFR 686.43 - Obligation to repay the grant.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION (CONTINUED) TEACHER EDUCATION ASSISTANCE FOR COLLEGE AND HIGHER EDUCATION (TEACH) GRANT PROGRAM Service and Repayment Obligations §...

  14. 23 CFR 450.332 - Annual listing of obligated projects.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...OF TRANSPORTATION PLANNING AND RESEARCH PLANNING ASSISTANCE AND STANDARDS Metropolitan Transportation Planning and Programming § 450.332 Annual listing of obligated projects. (a) In metropolitan planning areas, on an annual...

  15. 7 CFR 760.507 - Obligations of a participant.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...PROGRAMS INDEMNITY PAYMENT PROGRAMS Tree Assistance Program § 760.507 Obligations... (a) Eligible orchardists and nursery tree growers must execute all required documents... (b) Eligible orchardist or nursery tree growers must allow representatives...

  16. 7 CFR 760.507 - Obligations of a participant.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...PROGRAMS INDEMNITY PAYMENT PROGRAMS Tree Assistance Program § 760.507 Obligations... (a) Eligible orchardists and nursery tree growers must execute all required documents... (b) Eligible orchardist or nursery tree growers must allow representatives...

  17. 7 CFR 760.507 - Obligations of a participant.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...PROGRAMS INDEMNITY PAYMENT PROGRAMS Tree Assistance Program § 760.507 Obligations... (a) Eligible orchardists and nursery tree growers must execute all required documents... (b) Eligible orchardist or nursery tree growers must allow representatives...

  18. 7 CFR 783.7 - Obligations of a participant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS TREE ASSISTANCE PROGRAM § 783.7 Obligations of a participant. (a) Eligible orchardists must execute all required...

  19. 7 CFR 760.507 - Obligations of a participant.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...PROGRAMS INDEMNITY PAYMENT PROGRAMS Tree Assistance Program § 760.507 Obligations... (a) Eligible orchardists and nursery tree growers must execute all required documents... (b) Eligible orchardist or nursery tree growers must allow representatives...

  20. 16 CFR 436.2 - Obligation to furnish documents.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...Obligation to furnish documents. In connection with the offer or sale of a franchise...current disclosure document, as described...proposed franchise sale. (b) For any...to the disclosure document without...

  1. 16 CFR 436.2 - Obligation to furnish documents.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...Obligation to furnish documents. In connection with the offer or sale of a franchise...current disclosure document, as described...proposed franchise sale. (b) For any...to the disclosure document without...

  2. 16 CFR 436.2 - Obligation to furnish documents.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...Obligation to furnish documents. In connection with the offer or sale of a franchise...current disclosure document, as described...proposed franchise sale. (b) For any...to the disclosure document without...

  3. 16 CFR 436.2 - Obligation to furnish documents.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...Obligation to furnish documents. In connection with the offer or sale of a franchise...current disclosure document, as described...proposed franchise sale. (b) For any...to the disclosure document without...

  4. 16 CFR 436.2 - Obligation to furnish documents.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...Obligation to furnish documents. In connection with the offer or sale of a franchise...current disclosure document, as described...proposed franchise sale. (b) For any...to the disclosure document without...

  5. Towards Regulatory Compliance: Extracting Rights and Obligation to

    E-print Network

    Breaux, Travis D.

    Towards Regulatory Compliance: Extracting Rights and Obligation to Align Requirements Regulations and Requirements Traceability in Legal Language Modeling Regulatory Semantics Case Study: HIPAA: From Regulations to Requirements Regulations govern the system "environment." Regulatory language

  6. MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth

    PubMed Central

    Isaac, Dervla T.; Laguna, Rita K.; Valtz, Nicole; Isberg, Ralph R.

    2015-01-01

    Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ?mavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ?mavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron. PMID:26330609

  7. MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth.

    PubMed

    Isaac, Dervla T; Laguna, Rita K; Valtz, Nicole; Isberg, Ralph R

    2015-09-15

    Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ?mavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ?mavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron. PMID:26330609

  8. Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

    PubMed Central

    El-Awady, Ahmed R.; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B.; Palani, Chithra D.; Arce, Roger M.; Waller, Jennifer L.; Genco, Caroline A.; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V.; Cutler, Christopher W.

    2015-01-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs. PMID:25679217

  9. BACTERIAL WATERBORNE PATHOGENS

    EPA Science Inventory

    Bacterial pathogens are examples of classical etiological agents of waterborne disease. While these agents no longer serve as major threats to U.S. water supplies, they are still important pathogens in areas with substandard sanitation and poor water treatment facilities. In th...

  10. Plant pathogen resistance

    SciTech Connect

    Greenberg, Jean T.; Jung, Ho Won; Tschaplinski, Timothy

    2015-10-20

    Azelaic acid or its derivatives or analogs induce a robust and a speedier defense response against pathogens in plants. Azelaic acid treatment alone does not induce many of the known defense-related genes but activates a plant's defense signaling upon pathogen exposure.

  11. Emerging foodborne pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The emergence of new foodborne pathogens is due to a number of factors. An important factor is the globalization of the food supply with the possibility of the introduction of foodborne pathogens from other countries. Animal husbandry, food production, food processing, and food distribution system...

  12. Plant pathogen resistance

    SciTech Connect

    Greenberg, Jean T; Jung, Ho Won; Tschaplinski, Timothy

    2012-11-27

    Azelaic acid or its derivatives or analogs induce a robust and a speedier defense response against pathogens in plants. Azelaic acid treatment alone does not induce many of the known defense-related genes but activates a plant's defense signaling upon pathogen exposure.

  13. Identification and characterization of persistent intracellular human immunodeficiency virus type 1 integrase strand transfer inhibitor activity.

    PubMed

    Koh, Yasuhiro; Haim, Hillel; Engelman, Alan

    2011-01-01

    Pharmacokinetic and pharmacodynamic considerations significantly impact infectious disease treatment options. One aspect of pharmacodynamics is the postantibiotic effect, classically defined as delayed bacterial growth after antibiotic removal. The same principle can apply to antiviral drugs. For example, significant delays in human immunodeficiency virus type 1 (HIV-1) replication can be observed after nucleoside/nucleotide reverse transcriptase inhibitor (N/NtRTI) removal from culture medium, because these prodrugs must be anabolized into active, phosphorylated forms once internalized into cells. A relatively new class of anti-HIV-1 drugs is the integrase strand transfer inhibitors (INSTIs), and the INSTIs raltegravir (RAL) and elvitegravir (EVG) were tested here alongside positive N/NtRTI controls tenofovir disoproxil fumarate (TDF) and azidothymidine (AZT), as well as the nonnucleoside reverse transcriptase inhibitor negative control nevirapine (NVP), to assess potential postantiviral effects. Transformed and primary CD4-positive cells pretreated with INSTIs significantly resisted subsequent challenge by HIV-1, revealing the following hierarchy of persistent intracellular drug strength: TDF > EVG ? AZT > RAL > NVP. A modified time-of-addition assay was moreover developed to assess residual drug activity levels. Approximately 0.8% of RAL and 2% of initial EVG and TDF 1-h pulse drug levels persisted during the acute phase of HIV-1 infection. EVG furthermore displayed significant virucidal activity. Although there is no reason to suspect obligate intracellular modification, this study nevertheless defines significant intracellular persistence of prototype INSTIs. Ongoing second-generation formulations should therefore consider the potential for significant postantiviral effects among this drug class. Combined intracellular persistence and virucidal activities suggest potential pre-exposure prophylaxis applications for EVG. PMID:21060108

  14. Virulence Plasmids of Nonsporulating Gram-Positive Pathogens

    PubMed Central

    Van Tyne, Daria; Gilmore, Michael S.

    2014-01-01

    SUMMARY Gram-positive bacteria are leading causes of many types of human infection, including pneumonia, skin and nasopharyngeal infections, as well as urinary tract and surgical wound infections among hospitalized patients. These infections have become particularly problematic because many of the species causing them have become highly resistant to antibiotics. The role of mobile genetic elements, such as plasmids, in the dissemination of antibiotic resistance among Gram-positive bacteria has been well studied; less well understood is the role of mobile elements in the evolution and spread of virulence traits among these pathogens. While these organisms are leading agents of infection, they are also prominent members of the human commensal ecology. It appears that these bacteria are able to take advantage of the intimate association between host and commensal, via virulence traits that exacerbate infection and cause disease. However, evolution into an obligate pathogen has not occurred, presumably because it would lead to rejection of pathogenic organisms from the host ecology. Instead, in organisms that exist as both commensal and pathogen, selection has favored the development of mechanisms for variability. As a result, many virulence traits are localized on mobile genetic elements, such as virulence plasmids and pathogenicity islands. Virulence traits may occur within a minority of isolates of a given species, but these minority populations have nonetheless emerged as a leading problem in infectious disease. This chapter reviews virulence plasmids in nonsporulating Gram-positive bacteria, and examines their contribution to disease pathogenesis. PMID:25544937

  15. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    PubMed

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics. PMID:25939798

  16. Chemical development of intracellular protein heterodimerizers.

    PubMed

    Erhart, Dominik; Zimmermann, Mirjam; Jacques, Olivier; Wittwer, Matthias B; Ernst, Beat; Constable, Edwin; Zvelebil, Marketa; Beaufils, Florent; Wymann, Matthias P

    2013-04-18

    Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system. PMID:23601644

  17. Regulation of toxin production in the pathogenic clostridia.

    PubMed

    Carter, Glen P; Cheung, Jackie K; Larcombe, Sarah; Lyras, Dena

    2014-01-01

    The genus Clostridium comprises a large, heterogeneous group of obligate anaerobic, Gram-positive spore forming bacilli. Members of this genus are ubiquitous in the environment and although most species are considered saprophytic, several are pathogenic to both humans and animals. These bacteria cause a variety of diseases including neuroparalysis, gas gangrene, necrotic enteritis, food poisoning, toxic shock syndrome and pseudomembraneous colitis, which in most cases arise as a consequence of the production of potent exotoxins. Treatment options are often limited, underscoring the need for new treatment strategies and novel therapeutics. Understanding the fundamental mechanisms and signals that control toxin production in the pathogenic clostridia may lead to the identification of novel therapeutic targets that can be exploited in the development of new antimicrobial agents. PMID:24563915

  18. ROS production, intracellular HSP70 levels and their relationship in human neutrophils: effects of age

    PubMed Central

    Kovalenko, Elena I.; Boyko, Anna A.; Semenkov, Victor F.; Lutsenko, Gennady V.; Grechikhina, Maria V.; Kanevskiy, Leonid M.; Azhikina, Tatyana L.; Telford, William G.; Sapozhnikov, Alexander M.

    2014-01-01

    ROS production and intracellular HSP70 levels were measured in human neutrophils for three age groups: young (20-59 years), elders (60-89 years) and nonagenarians (90 years and older). Elders showed higher levels of spontaneous intracellular ROS content compared with young and nonagenarian groups, which had similar intracellular ROS levels. Zymosan-induced (non-spontaneous) extracellular ROS levels were also similar for young and nonagenarians but were lower in elders. However, spontaneous extracellular ROS production increased continuously with age. Correlation analysis revealed positive relationships between HSP70 levels and zymosan-stimulated ROS production in the elder group. This was consistent with a promoting role for HSP70 in ROS-associated neutrophils response to pathogens. No positive correlation between ROS production and intracellular HSP70 levels was found for groups of young people and nonagenarians. In contrast, significant negative correlations of some ROS and HSP70 characteriscics were found for neutrophils from young people and nonagenarians. The observed difference in ROS and HSP70 correlations in elders and nonagenarians might be associated with an increased risk of mortality in older individuals less than 90 years old. PMID:25514461

  19. Salmonella Engages Host MicroRNAs To Modulate SUMOylation: a New Arsenal for Intracellular Survival.

    PubMed

    Verma, Smriti; Mohapatra, Gayatree; Ahmad, Salman Mustfa; Rana, Sarika; Jain, Swati; Khalsa, Jasneet Kaur; Srikanth, C V

    2015-09-01

    Posttranslational modifications (PTMs) can alter many fundamental properties of a protein. One or combinations of them have been known to regulate the dynamics of many cellular pathways and consequently regulate all vital processes. Understandably, pathogens have evolved sophisticated strategies to subvert these mechanisms to achieve instantaneous control over host functions. Here, we present the first report of modulation by intestinal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) of host SUMOylation, a PTM pathway central to all fundamental cellular processes. Both in cell culture and in a mouse model, we observed that S. Typhimurium infection led to a dynamic SUMO-conjugated proteome alteration. The intracellular survival of S. Typhimurium was dependent on SUMO status as revealed by reduced infection and Salmonella-induced filaments (SIFs) in SUMO-upregulated cells. S. Typhimurium-dependent SUMO modulation was seen as a result of depletion of crucial SUMO pathway enzymes Ubc-9 and PIAS1, at both the protein and the transcript levels. Mechanistically, depletion of Ubc-9 relied on upregulation of small noncoding RNAs miR30c and miR30e during S. Typhimurium infection. This was necessary and sufficient for both down-modulation of Ubc-9 and a successful infection. Thus, we demonstrate a novel strategy of pathogen-mediated perturbation of host SUMOylation, an integral mechanism underlying S. Typhimurium infection and intracellular survival. PMID:26100020

  20. The Zinc Transport Systems and Their Regulation in Pathogenic Fungi

    PubMed Central

    2015-01-01

    Zinc is an essential micronutrient required for many enzymes that play essential roles in a cell. It was estimated that approximately 3% of the total cellular proteins are required for zinc for their functions. Zinc has long been considered as one of the key players in host-pathogen interactions. The host sequesters intracellular zinc by utilizing multiple cellular zinc importers and exporters as a means of nutritional immunity. To overcome extreme zinc limitation within the host environment, pathogenic microbes have successfully evolved a number of mechanisms to secure sufficient concentrations of zinc for their survival and pathogenesis. In this review, we briefly discuss the zinc uptake systems and their regulation in the model fungus Saccharomyces cerevisiae and in major human pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus gattii. PMID:26539032

  1. The Zinc Transport Systems and Their Regulation in Pathogenic Fungi.

    PubMed

    Jung, Won Hee

    2015-09-01

    Zinc is an essential micronutrient required for many enzymes that play essential roles in a cell. It was estimated that approximately 3% of the total cellular proteins are required for zinc for their functions. Zinc has long been considered as one of the key players in host-pathogen interactions. The host sequesters intracellular zinc by utilizing multiple cellular zinc importers and exporters as a means of nutritional immunity. To overcome extreme zinc limitation within the host environment, pathogenic microbes have successfully evolved a number of mechanisms to secure sufficient concentrations of zinc for their survival and pathogenesis. In this review, we briefly discuss the zinc uptake systems and their regulation in the model fungus Saccharomyces cerevisiae and in major human pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus gattii. PMID:26539032

  2. Endosymbiosis In Statu Nascendi: Close Phylogenetic RelationshipBetween Obligately Endosymbiotic and Obligately Free-LivingPolynucleobacter Strains (Betaproteobacteria)

    SciTech Connect

    Vannini, Claudia; Pockl, Matthias; Petroni, Giulio; Wu, Qinglong; Lang, Elke; Stackebrandt, Erko; Schrallhammer, Martina; Richardson, PaulM.; Hahn, Martin W.

    2006-07-21

    Bacterial strains affiliated to the phylogenetically shallowsubcluster C (PnecC) of the 28 Polynucleobacter cluster, which ischaracterized by a minimal 16S rRNA gene sequence similarity of approx.98.5 percent, have been reported to occur as obligate endosymbionts of 30ciliates (Euplotes spp.), as well as to occur as free-living cells in thepelagic zone of freshwater habitats. We investigated if these two groupsof closely related bacteria represent 32 strains fundamentally differingin lifestyle, or if they simply represent different stages of afacultative endosymbiotic lifestyle. The phylogenetic analysis of 16SrRNA gene and 16S34 23S ITS sequences of five endosymbiont strains fromtwo different Euplotes species and 40 pure culture strains demonstratedhost-species-specific clustering of the endosymbiont 36 sequences withinthe PnecC subcluster. The sequences of the endosymbionts showedcharacteristics indicating an obligate endosymbiotic lifestyle.Cultivation experiments 38 revealed fundamental differences inphysiological adaptations, and determination of the genome sizesindicated a slight size reduction in endosymbiotic strains. We concludethat the 40 two groups of PnecC bacteria represent obligately free-livingand obligately endosymbiotic strains, respectively, and do not representdifferent stages of the same complex lifecycle. 42 These closely relatedstrains occupy completely separated ecological niches. To our bestknowledge, this is the closest phylogenetic relationship between obligateendosymbionts and 44 obligately free-living bacteria everrevealed.

  3. Transgenic, Fluorescent Leishmania mexicana Allow Direct Analysis of the Proteome of Intracellular Amastigotes*S?

    PubMed Central

    Paape, Daniel; Lippuner, Christoph; Schmid, Monika; Ackermann, Renate; Barrios-Llerena, Martin E.; Zimny-Arndt, Ursula; Brinkmann, Volker; Arndt, Benjamin; Pleissner, Klaus Peter; Jungblut, Peter R.; Aebischer, Toni

    2008-01-01

    Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to ?6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3?-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens. PMID:18474515

  4. The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly.

    PubMed

    Pais, Roshan; Lohs, Claudia; Wu, Yineng; Wang, Jingwen; Aksoy, Serap

    2008-10-01

    Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis. PMID:18689507

  5. Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection

    PubMed Central

    Steinberg, Gero

    2015-01-01

    Cell biological research in the wheat pathogen Zymoseptoria tritici (formerly Mycosphaerella graminicola) has led to a good understanding of the histology of the infection process. Expression profiling and bioinformatic approaches, combined with molecular studies on signaling pathways, effectors and potential necrosis factors provides first insight into the complex interplay between the host and the pathogen. Cell biological studies will help to further our understanding of the infection strategy of the fungus. The cellular organization and intracellular dynamics of the fungus itself is largely unexplored. Insight into essential cellular processes within the pathogen will expand our knowledge of the basic biology of Z. tritici, thereby providing putative new anti-fungal targets. PMID:26092785

  6. Diversity of extracellular proteins during the transition from the 'proto-apicomplexan' alveolates to the apicomplexan obligate parasites.

    PubMed

    Templeton, Thomas J; Pain, Arnab

    2016-01-01

    The recent completion of high-coverage draft genome sequences for several alveolate protozoans - namely, the chromerids, Chromera velia and Vitrella brassicaformis; the perkinsid Perkinsus marinus; the apicomplexan, Gregarina niphandrodes, as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell-cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate - guided by ultrastructure, life strategies and phylogenetic relationships - in an attempt to understand the evolution of obligate parasitism in apicomplexans. PMID:26585326

  7. A comprehensive Prunus pathogen array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comprehensive pathogen array was developed for the detection of pathogens of many major crops in the Prunus genus. The APS disease lists for peach, plum, apricot and cherry were combined into a single Prunus pathogen list, containing 102 pathogens (75 fungi, 18 viruses, 6 bacteria and 3 phytoplasm...

  8. Intracellular synthesis of silver nanoparticle by actinobacteria and its antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Otari, S. V.; Patil, R. M.; Ghosh, S. J.; Thorat, N. D.; Pawar, S. H.

    2015-02-01

    Intracellular synthesis of silver nanoparticles (AgNPs) using Rhodococcus spp. is demonstrated. The synthesized nanoparticles were characterized by UV-Vis spectroscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier trans-form infrared spectroscopy, and transmission electron microscopy. Transmission electron microscopy study of microorganisms' revealed synthesis of nanoparticle was occurring inside the cell, in the cytoplasm. AgNPs ranged from 5 to 50 nm. Formed nanoparticles were stable in the colloidal solution due to presence of proteins on the surface. AgNPs showed excellent bactericidal and bacteriostatic activity against pathogenic microorganisms.

  9. Intracellular synthesis of silver nanoparticle by actinobacteria and its antimicrobial activity.

    PubMed

    Otari, S V; Patil, R M; Ghosh, S J; Thorat, N D; Pawar, S H

    2015-02-01

    Intracellular synthesis of silver nanoparticles (AgNPs) using Rhodococcus spp. is demonstrated. The synthesized nanoparticles were characterized by UV-Vis spectroscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier trans-form infrared spectroscopy, and transmission electron microscopy. Transmission electron microscopy study of microorganisms' revealed synthesis of nanoparticle was occurring inside the cell, in the cytoplasm. AgNPs ranged from 5 to 50 nm. Formed nanoparticles were stable in the colloidal solution due to presence of proteins on the surface. AgNPs showed excellent bactericidal and bacteriostatic activity against pathogenic microorganisms. PMID:25456659

  10. Iron acquisition within host cells and the pathogenicity of Leishmania

    PubMed Central

    Huynh, Chau; Andrews, Norma W.

    2008-01-01

    Summary Iron is an essential cofactor for several enzymes and metabolic pathways, in both microbes and in their eukaryotic hosts. To avoid toxicity, iron acquisition is tightly regulated. This represents a particular challenge for pathogens that reside within the endocytic pathway of mammalian cells, because endosomes and lysosomes are gradually depleted in iron by host transporters. An important player in this process is Nramp1 (Slc11a1), a proton efflux pump that translocates Fe2+ and Mn2+ ions from macrophage lysosomes/phagolysosomes into the cytosol. Mutations in Nramp1 cause susceptibility to infection with the bacteria Salmonella and Mycobacteria and the protozoan Leishmania, indicating that an available pool of intraphagosomal iron is critical for the intracellular survival and replication of these pathogens. Salmonella and Mycobacteria are known to express iron transporter systems that effectively compete with host transporters for iron. Until recently, however, very little was known about the molecular strategy used by Leishmania for survival in the iron-poor environment of macrophage phagolysosomes. It is now clear that intracellular residence induces Leishmania amazonensis to express LIT1, a ZIP family membrane Fe2+ transporter that is required for intracellular growth and virulence. PMID:18070118

  11. Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

    PubMed Central

    Pieper, Rembert; Fisher, C. R.; Suh, Moo-Jin; Huang, S.-T.; Parmar, P.

    2013-01-01

    Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu. PMID:24101689

  12. 76 FR 16707 - Rule 17Ad-17; Transfer Agents', Brokers', and Dealers' Obligation To Search for Lost...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-25

    ...Brokers', and Dealers' Obligation To Search for Lost Securityholders; Paying Agents' Obligation To Search for Missing Securityholders AGENCY: Securities...Transfer Agents' Obligation to Search for Lost Securityholders'' to:...

  13. 31 CFR 225.3 - Pledge of Government obligations in lieu of a bond with surety or sureties.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 false Pledge of Government obligations in lieu of a bond with... ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS WITH SURETIES § 225.3 Pledge of Government obligations in lieu of a bond...

  14. 31 CFR 225.3 - Pledge of Government obligations in lieu of a bond with surety or sureties.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Pledge of Government obligations in lieu of a bond with... ACCEPTANCE OF BONDS SECURED BY GOVERNMENT OBLIGATIONS IN LIEU OF BONDS WITH SURETIES § 225.3 Pledge of Government obligations in lieu of a bond...

  15. 26 CFR 1.165-12 - Denial of deduction for losses on registration-required obligations not in registered form.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...or obligation (including conversion privilege) was issued after...customer, custodial or nominee relationship and such institution agrees...such paragraph. (4) Conversion of obligations into registered...transfer agent or the issuer for conversion of the obligation into...

  16. Stomata and pathogens

    PubMed Central

    Gudesblat, Gustavo E; Torres, Pablo S

    2009-01-01

    Bacteria and fungi are capable of triggering stomatal closure through pathogen-associated molecular patterns (PAMPs), which prevents penetration through these pores. Therefore, the stomata can be considered part of the plant innate immune response. Some pathogens have evolved mechanisms to evade stomatal defense. The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), which infects plants of the Brassicaceae family mainly through hydathodes, has also been reported to infect plants through stomata. A recent report shows that penetration of Xcc in Arabidopsis leaves through stomata depends on a secreted small molecule whose synthesis is under control of the rpf/diffusible signal factor (DSF) cell-to-cell signaling system, which also controls genes involved in biofilm formation and pathogenesis. The same reports shows that Arabidopsis ROS- and PAMP-activated MAP kinase 3 (MPK3) is essential for stomatal innate response. Other recent and past findings about modulation of stomatal behaviour by pathogens are also discussed. In all, these findings support the idea that PAMP-triggered stomatal closure might be a more effective and widespread barrier against phytopathogens than previously thought, which has in turn led to the evolution in pathogens of several mechanisms to evade stomatal defense. PMID:20514224

  17. Neutrophil cell surface receptors and their intracellular signal transduction pathways?

    PubMed Central

    Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

    2013-01-01

    Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

  18. Physical Features of Intracellular Proteins that Moonlight on the Cell Surface

    PubMed Central

    Amblee, Vaishak; Jeffery, Constance J.

    2015-01-01

    Moonlighting proteins comprise a subset of multifunctional proteins that perform two or more biochemical functions that are not due to gene fusions, multiple splice variants, proteolytic fragments, or promiscuous enzyme activities. The project described herein focuses on a sub-set of moonlighting proteins that have a canonical biochemical function inside the cell and perform a second biochemical function on the cell surface in at least one species. The goal of this project is to consider the biophysical features of these moonlighting proteins to determine whether they have shared characteristics or defining features that might suggest why these particular proteins were adopted for a second function on the cell surface, or if these proteins resemble typical intracellular proteins. The latter might suggest that many other normally intracellular proteins found on the cell surface might also be moonlighting in this fashion. We have identified 30 types of proteins that have different functions inside the cell and on the cell surface. Some of these proteins are found to moonlight on the surface of multiple species, sometimes with different extracellular functions in different species, so there are a total of 98 proteins in the study set. Although a variety of intracellular proteins (enzymes, chaperones, etc.) are observed to be re-used on the cell surface, for the most part, these proteins were found to have physical characteristics typical of intracellular proteins. Many other intracellular proteins have also been found on the surface of bacterial pathogens and other organisms in proteomics experiments. It is quite possible that many of those proteins also have a moonlighting function on the cell surface. The increasing number and variety of known moonlighting proteins suggest that there may be more moonlighting proteins than previously thought, and moonlighting might be a common feature of many more proteins. PMID:26110848

  19. Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens.

    PubMed

    Knodler, Leigh A; Crowley, Shauna M; Sham, Ho Pan; Yang, Hyungjun; Wrande, Marie; Ma, Caixia; Ernst, Robert K; Steele-Mortimer, Olivia; Celli, Jean; Vallance, Bruce A

    2014-08-13

    Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface. PMID:25121752

  20. [Can Chlamydia trachomatis human biovars cause abortion in cattle? An immunohistochemical study on a new host-pathogen relationship].

    PubMed

    Ozbek, Ahmet; Ozbek, Elvan; Kalkan, Yildiray; Temur, Ahmet; Küçükkalem, Omer Faruk

    2008-10-01

    Chlamydiae, which are obligate intracellular bacterial pathogens, have been radiated from single-celled eukaryotes into multi-celled hosts during their evolution. Chlamydia trachomatis one of the important species in this group, is classified into three biovars as a result of their evolution. Two of those biovars, Trachoma and LGV, are pathogens only in humans. Initially, the presence of a high specificity between the host and chlamydiae has been recognized and this relation has been considered as an adaptation mechanism. However, some studies have indicated that chlamydiae can also grow in laboratory animals, yolk sacs of embryonated eggs and in vitro cell cultures. The aim of this study was to investigate if C. trachomatis human specific biovars are possible infectious agents in the aborted bovine fetuses. Ninety aborted bovine fetuses were included in the study, and the bacteria which could be the causative agents for abortion were searched by conventional microbiological methods. Twenty-three (25.6%) abortion materials which have yielded negative results with these methods for the presence of bacterial agents other than chlamydiae, were further evaluated in terms of the presence of C. trachomatis. For this purpose the samples were inoculated into the yolk sac of embryonated eggs and the slides prepared from the yolk sac membranes of embryons died after 24 hours of inoculation, were examined for the presence of inclusion bodies by staining with Giemsa method. The presence of C. trachomatis specific antigens and glycogen inclusions in those 23 samples were also investigated by immunohistochemical and Lugol's iodine staining methods, in the fetal tissue samples which were embedded in paraffin. Immunohistochemical method was performed with immunoperoxidase staining by the use of specific antibodies against C. trachomatis major outer membrane proteins. As a result, 5 (21.7%) of the 23 samples were found positive for C. trachomatis with three of the methods (Giemsa, immunoperoxidase and lugol stainings). Although the data of our study have supported that chlamydiae can adapt to new host species other than humans, further advanced studies are needed on this subject. Our results have also emphasized that novel routes of transmission should be considered for C. trachomatis infections. PMID:19149081

  1. 24 CFR 811.110 - Refunding of obligations issued to finance Section 8 projects.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...false Refunding of obligations issued to finance Section 8 projects. 811.110 Section...110 Refunding of obligations issued to finance Section 8 projects. (a) This...savings and, if necessary, HUD will finance in refunding bond debt service...

  2. 24 CFR 811.110 - Refunding of obligations issued to finance Section 8 projects.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...false Refunding of obligations issued to finance Section 8 projects. 811.110 Section...110 Refunding of obligations issued to finance Section 8 projects. (a) This...savings and, if necessary, HUD will finance in refunding bond debt service...

  3. 24 CFR 811.110 - Refunding of obligations issued to finance Section 8 projects.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...false Refunding of obligations issued to finance Section 8 projects. 811.110 Section...110 Refunding of obligations issued to finance Section 8 projects. (a) This...savings and, if necessary, HUD will finance in refunding bond debt service...

  4. 24 CFR 811.110 - Refunding of obligations issued to finance Section 8 projects.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...false Refunding of obligations issued to finance Section 8 projects. 811.110 Section...110 Refunding of obligations issued to finance Section 8 projects. (a) This...savings and, if necessary, HUD will finance in refunding bond debt service...

  5. 24 CFR 811.110 - Refunding of obligations issued to finance Section 8 projects.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...false Refunding of obligations issued to finance Section 8 projects. 811.110 Section...110 Refunding of obligations issued to finance Section 8 projects. (a) This...savings and, if necessary, HUD will finance in refunding bond debt service...

  6. 31 CFR 103.52 - Photographic or other reproductions of Government obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Photographic or other reproductions of Government obligations. 103... § 103.52 Photographic or other reproductions of Government obligations. Nothing...authorize the microfilming or other reproduction of (a) Currency or other...

  7. 31 CFR 1010.940 - Photographic or other reproductions of Government obligations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 false Photographic or other reproductions of Government obligations. 1010...1010.940 Photographic or other reproductions of Government obligations. Nothing...authorize the microfilming or other reproduction of: (a) Currency or other...

  8. 47 CFR 14.61 - Obligations with respect to internet browsers built into mobile phones.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... false Obligations with respect to internet browsers built into mobile phones...EQUIPMENT BY PEOPLE WITH DISABILITIES Internet Browsers Built Into Telephones Used With...14.61 Obligations with respect to internet browsers built into mobile phones....

  9. 47 CFR 14.61 - Obligations with respect to internet browsers built into mobile phones.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... false Obligations with respect to internet browsers built into mobile phones...EQUIPMENT BY PEOPLE WITH DISABILITIES Internet Browsers Built Into Telephones Used With...14.61 Obligations with respect to internet browsers built into mobile phones....

  10. 42 CFR 64a.105 - What are the conditions of obligated service?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...105 Section 64a.105 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING OBLIGATED SERVICE FOR MENTAL HEALTH TRAINEESHIPS § 64a.105 What are the conditions of obligated...

  11. 76 FR 36482 - Affirmative Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-22

    ...Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding Protected Veterans AGENCY: Office of Federal...Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding Protected Veterans'' (76 FR 23358)....

  12. 76 FR 23357 - Affirmative Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-26

    ...Nondiscrimination Obligations of Contractors and Subcontractors Regarding Protected Veterans; Proposed...Nondiscrimination Obligations of Contractors and Subcontractors Regarding Protected Veterans AGENCY...requires covered Federal contractors and subcontractors to take affirmative action in...

  13. 77 FR 7108 - Affirmative Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-10

    ...Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding Individuals With Disabilities AGENCY: Office...Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding Individuals with Disabilities'' (76 FR...

  14. 76 FR 77055 - Affirmative Action and Nondiscrimination Obligations of Contractors and Subcontractors Regarding...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-09

    ...Non-discrimination Obligations of Contractors and Subcontractors Regarding Individuals With Disabilities...Nondiscrimination Obligations of Contractors and Subcontractors Regarding Individuals With Disabilities...by covered Federal contractors and subcontractors against individuals on the basis...

  15. 12 CFR 1511.5 - Obligations of Funding Corporation; no adverse claims.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...2010-01-01 false Obligations of Funding Corporation; no adverse claims. ...DEPARTMENT OF THE TREASURY RESOLUTION FUNDING CORPORATION BOOK-ENTRY PROCEDURE § 1511.5 Obligations of Funding Corporation; no adverse...

  16. 40 CFR 80.1107 - How is the Renewable Volume Obligation calculated?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 2011-07-01 false How is the Renewable Volume Obligation calculated? 80.1107...REGULATION OF FUELS AND FUEL ADDITIVES Renewable Fuel Standard § 80.1107 How is the Renewable Volume Obligation calculated?...

  17. 40 CFR 80.1106 - To whom does the Renewable Volume Obligation apply?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 false To whom does the Renewable Volume Obligation apply? 80.1106...REGULATION OF FUELS AND FUEL ADDITIVES Renewable Fuel Standard § 80.1106 To whom does the Renewable Volume Obligation apply?...

  18. 40 CFR 80.1107 - How is the Renewable Volume Obligation calculated?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 2010-07-01 false How is the Renewable Volume Obligation calculated? 80.1107...REGULATION OF FUELS AND FUEL ADDITIVES Renewable Fuel Standard § 80.1107 How is the Renewable Volume Obligation calculated?...

  19. 40 CFR 80.1106 - To whom does the Renewable Volume Obligation apply?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false To whom does the Renewable Volume Obligation apply? 80.1106...REGULATION OF FUELS AND FUEL ADDITIVES Renewable Fuel Standard § 80.1106 To whom does the Renewable Volume Obligation apply?...

  20. 32 CFR 220.2 - Statutory obligation of third party payer to pay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...COLLECTION FROM THIRD PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.2 Statutory obligation of third party...obligation to pay the United States the reasonable charges for healthcare services provided in or through any facility of...

  1. 40 CFR 152.97 - Rights and obligations of data submitters.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Rights and obligations of data submitters. 152.97 Section 152...PROCEDURES Procedures To Ensure Protection of Data Submitters' Rights § 152.97 Rights and obligations of data submitters. (a) Right to be...

  2. 78 FR 63276 - Interim Policy, FAA Review of Solar Energy System Projects on Federally Obligated Airports

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ...Interim Policy, FAA Review of Solar Energy System Projects on Federally Obligated...obligated airports to construct solar energy systems on airport property. FAA...measuring ocular impact of proposed solar energy systems which are effective...

  3. Sixth Northwest Conservation and Electric Power Plan Chapter 13: Bonneville's Obligations

    E-print Network

    's balancing area. Its obligations to provide flexibility for wind-power balancing also are driven by its obligations under NERC standards as the host balancing authority for wind-power resources that are meeting

  4. 31 CFR 1010.311 - Filing obligations for reports of transactions in currency.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...obligations for reports of transactions in currency. 1010.311 Section 1010.311...obligations for reports of transactions in currency. Each financial institution other...each deposit, withdrawal, exchange of currency or other payment or transfer,...

  5. 31 CFR 1010.311 - Filing obligations for reports of transactions in currency.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...obligations for reports of transactions in currency. 1010.311 Section 1010.311...obligations for reports of transactions in currency. Each financial institution other...each deposit, withdrawal, exchange of currency or other payment or transfer,...

  6. 31 CFR 1010.311 - Filing obligations for reports of transactions in currency.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...obligations for reports of transactions in currency. 1010.311 Section 1010.311...obligations for reports of transactions in currency. Each financial institution other...each deposit, withdrawal, exchange of currency or other payment or transfer,...

  7. 31 CFR 1010.311 - Filing obligations for reports of transactions in currency.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...obligations for reports of transactions in currency. 1010.311 Section 1010.311...obligations for reports of transactions in currency. Each financial institution other...each deposit, withdrawal, exchange of currency or other payment or transfer,...

  8. 40 CFR 80.1407 - How are the Renewable Volume Obligations calculated?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...following formulas: (1) Cellulosic biofuel. RVOCB,i = (RFStdCB,i ...Renewable Volume Obligation for cellulosic biofuel for an obligated party for calendar year...RFStdCB,i = The standard for cellulosic biofuel for calendar year i, determined by...

  9. 40 CFR 80.1407 - How are the Renewable Volume Obligations calculated?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...following formulas: (1) Cellulosic biofuel. RVOCB,i = (RFStdCB,i ...Renewable Volume Obligation for cellulosic biofuel for an obligated party for calendar year...RFStdCB,i = The standard for cellulosic biofuel for calendar year i, determined by...

  10. 32 CFR 220.2 - Statutory obligation of third party payer to pay.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...COLLECTION FROM THIRD PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.2 Statutory obligation of third party...obligation to pay the United States the reasonable charges for healthcare services provided in or through any facility of...

  11. 32 CFR 220.2 - Statutory obligation of third party payer to pay.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...COLLECTION FROM THIRD PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.2 Statutory obligation of third party...obligation to pay the United States the reasonable charges for healthcare services provided in or through any facility of...

  12. 32 CFR 220.2 - Statutory obligation of third party payer to pay.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...COLLECTION FROM THIRD PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.2 Statutory obligation of third party...obligation to pay the United States the reasonable charges for healthcare services provided in or through any facility of...

  13. 32 CFR 220.2 - Statutory obligation of third party payer to pay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...COLLECTION FROM THIRD PARTY PAYERS OF REASONABLE CHARGES FOR HEALTHCARE SERVICES § 220.2 Statutory obligation of third party...obligation to pay the United States the reasonable charges for healthcare services provided in or through any facility of...

  14. 7 CFR 984.66 - Assistance of the Board in meeting reserve obligation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...false Assistance of the Board in meeting reserve obligation. 984.66 Section 984...CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.66 Assistance of the Board in meeting reserve obligation. The Board may assist...

  15. 7 CFR 984.66 - Assistance of the Board in meeting reserve obligation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...false Assistance of the Board in meeting reserve obligation. 984.66 Section 984...CALIFORNIA Order Regulating Handling Reserve Walnuts § 984.66 Assistance of the Board in meeting reserve obligation. The Board may assist...

  16. 40 CFR 80.1407 - How are the Renewable Volume Obligations calculated?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...following formulas: (1) Cellulosic biofuel. RVOCB,i = (RFStdCB,i ...Renewable Volume Obligation for cellulosic biofuel for an obligated party for calendar year...RFStdCB,i = The standard for cellulosic biofuel for calendar year i, determined by...

  17. 40 CFR 80.1407 - How are the Renewable Volume Obligations calculated?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...following formulas: (1) Cellulosic biofuel. RVOCB,i = (RFStdCB,i ...Renewable Volume Obligation for cellulosic biofuel for an obligated party for calendar year...RFStdCB,i = The standard for cellulosic biofuel for calendar year i, determined by...

  18. 40 CFR 80.1407 - How are the Renewable Volume Obligations calculated?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...following formulas: (1) Cellulosic biofuel. RVOCB,i = (RFStdCB,i ...Renewable Volume Obligation for cellulosic biofuel for an obligated party for calendar year...RFStdCB,i = The standard for cellulosic biofuel for calendar year i, determined by...

  19. 76 FR 7975 - Commodity Pool Operators and Commodity Trading Advisors: Amendments to Compliance Obligations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-11

    ...Pool Operators and Commodity Trading Advisors: Amendments to Compliance Obligations...Pool Operators and Commodity Trading Advisors: Amendments to Compliance Obligations...Pool Operators and Commodity Trading Advisors. The Commission is proposing a new...

  20. 77 FR 11251 - Commodity Pool Operators and Commodity Trading Advisors: Compliance Obligations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-24

    ...Pool Operators and Commodity Trading Advisors: Compliance Obligations; Harmonization...Pool Operators and Commodity Trading Advisors: Compliance Obligations AGENCY: Commodity...Pool Operators and Commodity Trading Advisors. The Commission is also adopting...

  1. 28 CFR 45.10 - Procedures to promote compliance with crime victims' rights obligations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Procedures to promote compliance with crime victims' rights obligations. 45.10... Procedures to promote compliance with crime victims' rights obligations. (a...that relate to protection of the rights of crime victims. See 18 U.S.C. 3771....

  2. 28 CFR 45.10 - Procedures to promote compliance with crime victims' rights obligations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Procedures to promote compliance with crime victims' rights obligations. 45.10... Procedures to promote compliance with crime victims' rights obligations. (a...that relate to protection of the rights of crime victims. See 18 U.S.C. 3771....

  3. 16 CFR 437.2 - The obligation to furnish written documents.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...false The obligation to furnish written documents. 437.2 Section 437.2 Commercial...437.2 The obligation to furnish written documents. In connection with the offer for sale, sale, or promotion of a business...

  4. 16 CFR 437.2 - The obligation to furnish written documents.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...false The obligation to furnish written documents. 437.2 Section 437.2 Commercial...437.2 The obligation to furnish written documents. In connection with the offer for sale, sale, or promotion of a business...

  5. Origins, Diversity, and Diversification of the Native Hawaiian Leafhoppers (Hemiptera: Cicadellidae: Nesophrosyne) and Their Obligate Endosymbionts

    E-print Network

    O'Grady, Patrick M.

    Origins, Diversity, and Diversification of the Native Hawaiian Leafhoppers (Hemiptera: Cicadellidae Leafhoppers (Hemiptera: Cicadellidae: Nesophrosyne) and Their Obligate Endosymbionts Copyright 2012 By Gordon Leafhoppers (Hemiptera: Cicadellidae: Nesophrosyne) and Their Obligate Endosymbionts by Gordon Morse Bennett

  6. The Complete Genome of Teredinibacter turnerae T7901: An Intracellular Endosymbiont of Marine Wood-Boring Bivalves (Shipworms)

    PubMed Central

    Yang, Joyce C.; Madupu, Ramana; Durkin, A. Scott; Ekborg, Nathan A.; Pedamallu, Chandra S.; Hostetler, Jessica B.; Radune, Diana; Toms, Bradley S.; Henrissat, Bernard; Coutinho, Pedro M.; Schwarz, Sandra; Field, Lauren; Trindade-Silva, Amaro E.; Soares, Carlos A. G.; Elshahawi, Sherif; Hanora, Amro; Schmidt, Eric W.; Haygood, Margo G.; Posfai, Janos; Benner, Jack; Madinger, Catherine; Nove, John; Anton, Brian; Chaudhary, Kshitiz; Foster, Jeremy; Holman, Alex; Kumar, Sanjay; Lessard, Philip A.; Luyten, Yvette A.; Slatko, Barton; Wood, Nicole; Wu, Bo; Teplitski, Max; Mougous, Joseph D.; Ward, Naomi; Eisen, Jonathan A.; Badger, Jonathan H.; Distel, Daniel L.

    2009-01-01

    Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2–40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels. PMID:19568419

  7. 48 CFR 252.232-7007 - Limitation of Government's obligation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... accordingly. (e) If, solely by reason of failure of the Government to allot additional funds, by the dates... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false Limitation of Government's... of Provisions And Clauses 252.232-7007 Limitation of Government's obligation. As prescribed in...

  8. 48 CFR 252.232-7007 - Limitation of Government's obligation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... accordingly. (e) If, solely by reason of failure of the Government to allot additional funds, by the dates... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Limitation of Government's... of Provisions And Clauses 252.232-7007 Limitation of Government's obligation. As prescribed in...

  9. Asset retirement obligations: a reporting concern for healthcare facilities.

    PubMed

    Berg, Gary G; Bayes, Paul E; Morgan, Robert G

    2008-11-01

    FASB statements and SEC guidelines give direction as to how healthcare organizations should account for their asset retirement obligations (AROs) where environmental issues are concerned. A key consideration is that current costs associated with environmental problems, such as encapsulating asbestos, are to be accounted for as part of an asset's cost and depreciated over the asset's remaining life. PMID:18990844

  10. 7 CFR 1488.12 - Coverage of bank obligations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Coverage of bank obligations. 1488.12 Section 1488.12 Agriculture Regulations of the Department of Agriculture (Continued) COMMODITY CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS FINANCING OF SALES OF AGRICULTURAL COMMODITIES Financing of Export Sales...

  11. 18 CFR 154.1 - Application; Obligation to file.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... to file. 154.1 Section 154.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Provisions and Conditions § 154.1 Application; Obligation to file. (a) The provisions of this part apply to filings pursuant to section 4 of the Natural Gas Act. (b) Every natural gas company must file with...

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    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... to file. 154.1 Section 154.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Provisions and Conditions § 154.1 Application; Obligation to file. (a) The provisions of this part apply to filings pursuant to section 4 of the Natural Gas Act. (b) Every natural gas company must file with...

  13. 18 CFR 154.1 - Application; Obligation to file.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... to file. 154.1 Section 154.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... Provisions and Conditions § 154.1 Application; Obligation to file. (a) The provisions of this part apply to filings pursuant to section 4 of the Natural Gas Act. (b) Every natural gas company must file with...

  14. 47 CFR 76.309 - Customer service obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 4 2013-10-01 2013-10-01 false Customer service obligations. 76.309 Section 76.309 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE General Operating Requirements § 76.309 Customer...

  15. 47 CFR 76.309 - Customer service obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 4 2011-10-01 2011-10-01 false Customer service obligations. 76.309 Section 76.309 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE General Operating Requirements § 76.309 Customer...

  16. 47 CFR 76.309 - Customer service obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 4 2012-10-01 2012-10-01 false Customer service obligations. 76.309 Section 76.309 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE General Operating Requirements § 76.309 Customer...

  17. 47 CFR 76.309 - Customer service obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Customer service obligations. 76.309 Section 76.309 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE General Operating Requirements § 76.309 Customer...

  18. 7 CFR 1948.92 - Grant approval and fund obligation.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 13 2014-01-01 2013-01-01 true Grant approval and fund obligation. 1948.92 Section 1948.92 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS-COOPERATIVE SERVICE, RURAL UTILITIES SERVICE, AND FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE (CONTINUED) PROGRAM REGULATIONS...

  19. 7 CFR 1416.705 - Obligations of a participant.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... ASSISTANCE PROGRAMS 2005 Hurricane Tree Assistance Program § 1416.705 Obligations of a participant. (a) Eligible producers must execute all required documents and complete the 2005 Hurricane TAP funded practice... becomes ineligible for all or part of a 2005 Hurricane TAP benefit, the person and successor shall...

  20. 24 CFR 291.565 - Continuing obligations after purchase.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... purchase. 291.565 Section 291.565 Housing and Urban Development Regulations Relating to Housing and Urban... Neighbor Next Door Sales Program § 291.565 Continuing obligations after purchase. To remain in compliance.../her sole residence, the home purchased through the GNND Sales Program; and (b) Certify initially...