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  1. Article ; Online: Identification of Virulence Determinants During Host-Pathogen Interaction Using Tn-Seq Technology.

    Peek, Christopher T / Ibberson, Carolyn B / Cassat, James E

    Methods in molecular biology (Clifton, N.J.)

    2019  Volume 2069, Page(s) 155–175

    Abstract: Transposon sequencing (Tn-seq) is a powerful genetic tool that enables the detection of essential genes within a given environment. The application of Tn-seq to Staphylococcus aureus has generated transposon libraries in numerous strains with ... ...

    Abstract Transposon sequencing (Tn-seq) is a powerful genetic tool that enables the detection of essential genes within a given environment. The application of Tn-seq to Staphylococcus aureus has generated transposon libraries in numerous strains with inactivation of virtually every nonessential gene in the genome. This exciting technology coupled with increasingly available computational tools has been deployed in animal models of infection to identify essential S. aureus genes within specific host environments. In this chapter, we describe the application of Tn-seq to a murine model of osteomyelitis as a paradigm for using this powerful technology to elucidate mechanisms of bacterial pathogenesis.
    MeSH term(s) Animals ; DNA Transposable Elements ; Gene Library ; High-Throughput Nucleotide Sequencing ; Mice ; Staphylococcal Infections/genetics ; Staphylococcal Infections/metabolism ; Staphylococcus aureus/genetics ; Staphylococcus aureus/metabolism ; Staphylococcus aureus/pathogenicity ; Virulence Factors/genetics ; Virulence Factors/metabolism
    Chemical Substances DNA Transposable Elements ; Virulence Factors
    Language English
    Publishing date 2019-09-12
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-9849-4_12
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Loss of

    Ford, Caleb A / Hurford, Ian M / Fulbright, Laura E / Curry, Jacob M / Peek, Christopher T / Spoonmore, Thomas J / Cruz Victorio, Virginia / Johnson, Joshua R / Peck, Sun H / Cassat, James E

    Frontiers in cellular and infection microbiology

    2022  Volume 12, Page(s) 985467

    Abstract: Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. ...

    Abstract Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system.
    MeSH term(s) Animals ; Anti-Bacterial Agents ; Cancellous Bone ; Cytokines ; Ligands ; Methicillin-Resistant Staphylococcus aureus ; Mice ; Mice, Knockout ; Osteomyelitis ; Osteoprotegerin/genetics ; Receptor Activator of Nuclear Factor-kappa B ; Staphylococcus aureus/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics
    Chemical Substances Anti-Bacterial Agents ; Cytokines ; Ligands ; Osteoprotegerin ; Receptor Activator of Nuclear Factor-kappa B ; Von Hippel-Lindau Tumor Suppressor Protein (EC 2.3.2.27)
    Language English
    Publishing date 2022-09-20
    Publishing country Switzerland
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2619676-1
    ISSN 2235-2988 ; 2235-2988
    ISSN (online) 2235-2988
    ISSN 2235-2988
    DOI 10.3389/fcimb.2022.985467
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Intestinal Inflammation Promotes MDL-1

    Peek, Christopher T / Ford, Caleb A / Eichelberger, Kara R / Jacobse, Justin / Torres, Teresa P / Maseda, Damian / Latour, Yvonne L / Piazuelo, M Blanca / Johnson, Joshua R / Byndloss, Mariana X / Wilson, Keith T / Rathmell, Jeffrey C / Goettel, Jeremy A / Cassat, James E

    Cellular and molecular gastroenterology and hepatology

    2022  Volume 14, Issue 4, Page(s) 731–750

    Abstract: Background & aims: Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation, but many patients experience extra-intestinal disease. Bone loss is one common extra-intestinal manifestation of IBD that occurs through ... ...

    Abstract Background & aims: Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation, but many patients experience extra-intestinal disease. Bone loss is one common extra-intestinal manifestation of IBD that occurs through dysregulated interactions between osteoclasts and osteoblasts. Systemic inflammation has been postulated to contribute to bone loss, but the specific pathologic mechanisms have not yet been fully elucidated. We hypothesized that intestinal inflammation leads to bone loss through increased abundance and altered function of osteoclast progenitors.
    Methods: We used chemical, T cell driven, and infectious models of intestinal inflammation to determine the impact of intestinal inflammation on bone volume, the skeletal cytokine environment, and the cellular changes to pre-osteoclast populations within bone marrow. Additionally, we evaluated the potential for monoclonal antibody treatment against an inflammation-induced osteoclast co-receptor, myeloid DNAX activation protein 12-associating lectin-1 (MDL-1) to reduce bone loss during colitis.
    Results: We observed significant bone loss across all models of intestinal inflammation. Bone loss was associated with an increase in pro-osteoclastogenic cytokines within the bone and an expansion of a specific Cd11b
    Conclusions: Collectively, these data implicate the pathologic expansion and altered function of OCPs expressing MDL-1 in bone loss during IBD.
    MeSH term(s) Animals ; Antibodies, Monoclonal/metabolism ; Bone Resorption/genetics ; Bone Resorption/metabolism ; Bone Resorption/pathology ; Cell Differentiation/physiology ; Cytokines/metabolism ; Inflammation/genetics ; Inflammation/metabolism ; Inflammation/pathology ; Inflammatory Bowel Diseases/genetics ; Inflammatory Bowel Diseases/metabolism ; Inflammatory Bowel Diseases/pathology ; Intestines/metabolism ; Lectins/metabolism ; Lectins, C-Type/genetics ; Lectins, C-Type/metabolism ; Mice ; Osteoclasts/metabolism ; Osteoclasts/pathology ; Osteogenesis/genetics ; Osteogenesis/physiology ; Receptors, Cell Surface/genetics ; Receptors, Cell Surface/metabolism
    Chemical Substances Antibodies, Monoclonal ; Clec5a protein, mouse ; Cytokines ; Lectins ; Lectins, C-Type ; Receptors, Cell Surface
    Language English
    Publishing date 2022-07-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2819778-1
    ISSN 2352-345X ; 2352-345X
    ISSN (online) 2352-345X
    ISSN 2352-345X
    DOI 10.1016/j.jcmgh.2022.07.002
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Interleukin-23 receptor signaling impairs the stability and function of colonic regulatory T cells.

    Jacobse, Justin / Brown, Rachel E / Li, Jing / Pilat, Jennifer M / Pham, Ly / Short, Sarah P / Peek, Christopher T / Rolong, Andrea / Washington, M Kay / Martinez-Barricarte, Ruben / Byndloss, Mariana X / Shelton, Catherine / Markle, Janet G / Latour, Yvonne L / Allaman, Margaret M / Cassat, James E / Wilson, Keith T / Choksi, Yash A / Williams, Christopher S /
    Lau, Ken S / Flynn, Charles R / Casanova, Jean-Laurent / Rings, Edmond H H M / Samsom, Janneke N / Goettel, Jeremy A

    Cell reports

    2023  Volume 42, Issue 2, Page(s) 112128

    Abstract: The cytokine interleukin-23 (IL-23) is involved in the pathogenesis of inflammatory and autoimmune conditions including inflammatory bowel disease (IBD). IL23R is enriched in intestinal Tregs, yet whether IL-23 modulates intestinal Tregs remains unknown. ...

    Abstract The cytokine interleukin-23 (IL-23) is involved in the pathogenesis of inflammatory and autoimmune conditions including inflammatory bowel disease (IBD). IL23R is enriched in intestinal Tregs, yet whether IL-23 modulates intestinal Tregs remains unknown. Here, investigating IL-23R signaling in Tregs specifically, we show that colonic Tregs highly express Il23r compared with Tregs from other compartments and their frequency is reduced upon IL-23 administration and impairs Treg suppressive function. Similarly, colonic Treg frequency is increased in mice lacking Il23r specifically in Tregs and exhibits a competitive advantage over IL-23R-sufficient Tregs during inflammation. Finally, IL-23 antagonizes liver X receptor pathway, cellular cholesterol transporter Abca1, and increases Treg apoptosis. Our results show that IL-23R signaling regulates intestinal Tregs by increasing cell turnover, antagonizing suppression, and decreasing cholesterol efflux. These results suggest that IL-23 negatively regulates Tregs in the intestine with potential implications for promoting chronic inflammation in patients with IBD.
    MeSH term(s) Animals ; Humans ; Mice ; Colitis/pathology ; Forkhead Transcription Factors/metabolism ; Inflammation/pathology ; Inflammatory Bowel Diseases/pathology ; Interleukin-23/metabolism ; T-Lymphocytes, Regulatory
    Chemical Substances Forkhead Transcription Factors ; Interleukin-23 ; interleukin-23 receptor, mouse ; IL23R protein, human
    Language English
    Publishing date 2023-02-17
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2649101-1
    ISSN 2211-1247 ; 2211-1247
    ISSN (online) 2211-1247
    ISSN 2211-1247
    DOI 10.1016/j.celrep.2023.112128
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Coronaviruses induce entry-independent, continuous macropinocytosis.

    Freeman, Megan Culler / Peek, Christopher T / Becker, Michelle M / Smith, Everett Clinton / Denison, Mark R

    mBio

    2014  Volume 5, Issue 4, Page(s) e01340–14

    Abstract: Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV ... ...

    Abstract Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.
    MeSH term(s) Animals ; Cell Line, Tumor ; Coronavirus/physiology ; Mice ; Pinocytosis/physiology ; Pseudopodia/physiology ; SARS Virus/physiology ; Virus Replication/physiology
    Keywords covid19
    Language English
    Publishing date 2014-08-05
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2557172-2
    ISSN 2150-7511 ; 2161-2129
    ISSN (online) 2150-7511
    ISSN 2161-2129
    DOI 10.1128/mBio.01340-14
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Coronavirus replicase-reporter fusions provide quantitative analysis of replication and replication complex formation.

    Freeman, Megan Culler / Graham, Rachel L / Lu, Xiaotao / Peek, Christopher T / Denison, Mark R

    Journal of virology

    2014  Volume 88, Issue 10, Page(s) 5319–5327

    Abstract: Unlabelled: The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of ... ...

    Abstract Unlabelled: The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication.
    Importance: Coronaviruses are a family of enveloped, positive-sense RNA viruses that are important agents of disease, including severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Replication is associated with multiple virus-induced membrane structures that evolve during infection; however, the dynamics of this process remain poorly understood. In this study, we tested whether reporter molecules expressed from native locations within the replicase polyprotein of murine hepatitis virus as fusions with nonstructural proteins could define the expression and targeting of replicase proteins during infection in live cells. We demonstrate that the replicase gene tolerates the introduction of green fluorescent protein or firefly luciferase as fusions with replicase proteins. These viruses allow early quantitation of virus replication as well as real-time measurement of replication complexes.
    MeSH term(s) Animals ; Artificial Gene Fusion ; Cell Line ; Genes, Reporter/genetics ; Green Fluorescent Proteins/analysis ; Green Fluorescent Proteins/genetics ; Murine hepatitis virus/genetics ; Murine hepatitis virus/physiology ; RNA-Dependent RNA Polymerase/genetics ; RNA-Dependent RNA Polymerase/metabolism ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/genetics ; Staining and Labeling/methods ; Virus Replication
    Chemical Substances Recombinant Fusion Proteins ; Green Fluorescent Proteins (147336-22-9) ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
    Keywords covid19
    Language English
    Publishing date 2014-03-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80174-4
    ISSN 1098-5514 ; 0022-538X
    ISSN (online) 1098-5514
    ISSN 0022-538X
    DOI 10.1128/JVI.00021-14
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article: Coronaviruses Induce Entry-Independent, Continuous Macropinocytosis

    Freeman, Megan Culler / Peek, Christopher T / Becker, Michelle M / Smith, Everett Clinton / Denison, Mark R

    mBio. 2014 Aug. 29, v. 5, no. 4

    2014  

    Abstract: Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV ... ...

    Abstract Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. IMPORTANCE Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.
    Keywords Murine hepatitis virus ; cell fusion ; epidermal growth factor receptors ; giant cells ; humans ; pathogenesis ; pathogens ; pseudopodia ; surface proteins ; viral load ; virus replication ; viruses ; Middle East ; covid19
    Language English
    Dates of publication 2014-0829
    Size p. e01340-14.
    Publishing place American Society for Microbiology
    Document type Article
    ZDB-ID 2557172-2
    ISSN 2150-7511 ; 2161-2129
    ISSN (online) 2150-7511
    ISSN 2161-2129
    DOI 10.1128/mBio.01340-14
    Database NAL-Catalogue (AGRICOLA)

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