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  1. Article ; Online: Vibrio parahaemolyticus: Basic Techniques for Growth, Genetic Manipulation, and Analysis of Virulence Factors.

    Chimalapati, Suneeta / Lafrance, Alexander E / Chen, Luming / Orth, Kim

    Current protocols in microbiology

    2021  Volume 59, Issue 1, Page(s) e131

    Abstract: Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for ... ...

    Abstract Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for bacterial culture, genetic manipulation, and analysis of virulence phenotypes. Detailed protocols for growth assessment, generation of mutants, and phenotype assessment are included in this article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assessment of growth of V. parahaemolyticus Alternate Protocol 1: Assessment of growth of V. parahaemolyticus using a plate reader Basic Protocol 2: Swimming/swarming motility assay Basic Protocol 3: Genetic manipulation Alternate Protocol 2: Natural transformation Basic Protocol 4: Secretion assay and sample preparation for mass spectrometry analysis Basic Protocol 5: Invasion assay (gentamicin protection assay) Basic Protocol 6: Immunofluorescence detection of intracellular V. parahaemolyticus Basic Protocol 7: Cytotoxicity assay for T3SS2.
    MeSH term(s) Bacterial Proteins/genetics ; Bacteriological Techniques/methods ; Gentamicins/pharmacology ; HeLa Cells ; Humans ; Staining and Labeling ; Swimming ; Vibrio Infections/drug therapy ; Vibrio Infections/microbiology ; Vibrio parahaemolyticus/drug effects ; Vibrio parahaemolyticus/genetics ; Vibrio parahaemolyticus/growth & development ; Vibrio parahaemolyticus/pathogenicity ; Virulence/genetics ; Virulence Factors/genetics
    Chemical Substances Bacterial Proteins ; Gentamicins ; Virulence Factors
    Language English
    Publishing date 2021-06-09
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2213675-7
    ISSN 1934-8533 ; 1934-8525
    ISSN (online) 1934-8533
    ISSN 1934-8525
    DOI 10.1002/cpmc.131
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Enzymatic Specificity of Conserved Rho GTPase Deamidases Promotes Invasion of Vibrio parahaemolyticus at the Expense of Infection.

    Lafrance, Alexander E / Chimalapati, Suneeta / Garcia Rodriguez, Nalleli / Kinch, Lisa N / Kaval, Karan Gautam / Orth, Kim

    mBio

    2022  Volume 13, Issue 4, Page(s) e0162922

    Abstract: Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase ... ...

    Abstract Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens.
    MeSH term(s) Bacterial Infections ; Escherichia coli Proteins ; Humans ; Type III Secretion Systems/metabolism ; Uropathogenic Escherichia coli/metabolism ; Vibrio parahaemolyticus/genetics ; Vibrio parahaemolyticus/metabolism ; Virulence Factors ; rho GTP-Binding Proteins
    Chemical Substances Escherichia coli Proteins ; Type III Secretion Systems ; Virulence Factors ; rho GTP-Binding Proteins (EC 3.6.5.2)
    Language English
    Publishing date 2022-07-07
    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.01629-22
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: Manipulation of IRE1-Dependent MAPK Signaling by a Vibrio Agonist-Antagonist Effector Pair.

    De Nisco, Nicole J / Casey, Amanda K / Kanchwala, Mohammed / Lafrance, Alexander E / Coskun, Fatma S / Kinch, Lisa N / Grishin, Nick V / Xing, Chao / Orth, Kim

    mSystems

    2021  Volume 6, Issue 1

    Abstract: Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion ... ...

    Abstract Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen
    Language English
    Publishing date 2021-02-09
    Publishing country United States
    Document type Journal Article
    ISSN 2379-5077
    ISSN 2379-5077
    DOI 10.1128/mSystems.00872-20
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Vibrio deploys type 2 secreted lipase to esterify cholesterol with host fatty acids and mediate cell egress.

    Chimalapati, Suneeta / de Souza Santos, Marcela / Lafrance, Alexander E / Ray, Ann / Lee, Wan-Ru / Rivera-Cancel, Giomar / Vale, Gonçalo / Pawlowski, Krzysztof / Mitsche, Matthew A / McDonald, Jeffrey G / Liou, Jen / Orth, Kim

    eLife

    2020  Volume 9

    Abstract: Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. ...

    Abstract Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment.
    MeSH term(s) Bacterial Proteins/metabolism ; Cholesterol/metabolism ; Esterification ; Fatty Acids/metabolism ; Genomic Islands ; Lipase/metabolism ; Type III Secretion Systems ; Vibrio parahaemolyticus/enzymology ; Vibrio parahaemolyticus/metabolism
    Chemical Substances Bacterial Proteins ; Fatty Acids ; Type III Secretion Systems ; Cholesterol (97C5T2UQ7J) ; Lipase (EC 3.1.1.3)
    Language English
    Publishing date 2020-08-18
    Publishing country England
    Document type Journal Article
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.58057
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Manipulation of IRE1-dependent MAPK signaling by a Vibrio agonist-antagonist effector pair

    De Nisco, Nicole J. / Casey, Amanda K. / Kanchwala, Mohammed / Lafrance, Alexander E. / Coskun, Fatma S. / Kinch, Lisa N. / Grishin, Nick V. / Xing, Chao / Orth, Kim

    bioRxiv

    Abstract: Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (1). The type III secretion system 1 of the marine pathogen, Vibrio parahaemolyticus, utilizes the sequential action of four effectors to induce ... ...

    Abstract Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (1). The type III secretion system 1 of the marine pathogen, Vibrio parahaemolyticus, utilizes the sequential action of four effectors to induce a rapid, pro-inflammatory cell death uniquely characterized by a pro-survival host transcriptional response (2, 3). Herein, we show that this pro-survival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ causing a disruption in membrane integrity. Additionally, we observe that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR) resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveal new connections between V-ATPase function, UPR and MAPK signaling. Importance Vibrio parahaemolyticus (V. para) is a seafood-borne pathogen that encodes two Type 3 Secretion Systems (T3SS). The first system T3SS1 is thought to be maintained in all strains of V. para to to maintain survival in the environment, whereas the second sytem T3SS2 is linked to clinical isolates and disease in humans. Herein, we find that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within three hours. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host surval signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector VopS reverses activation and inhibition of these signaling systems ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.
    Keywords covid19
    Publisher BioRxiv
    Document type Article ; Online
    DOI 10.1101/2020.09.01.278937
    Database COVID19

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  6. Article ; Online: Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.

    Cookmeyer, David L / Winesett, Emily S / Kokona, Bashkim / Huff, Adam R / Aliev, Sabina / Bloch, Noah B / Bulos, Joshua A / Evans, Irene L / Fagre, Christian R / Godbe, Kerilyn N / Khromava, Maryna / Konstantinovsky, Daniel M / Lafrance, Alexander E / Lamacki, Alexandra J / Parry, Robert C / Quinn, Jeanne M / Thurston, Alana M / Tsai, Kathleen J S / Mollo, Aurelio /
    Cryle, Max J / Fairman, Robert / Charkoudian, Louise K

    PLoS biology

    2017  Volume 15, Issue 11, Page(s) e2003145

    Abstract: How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original ... ...

    Abstract How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.
    MeSH term(s) Biochemistry/education ; Curriculum ; Cytochrome P-450 Enzyme System/metabolism ; Humans ; Laboratories/standards ; Learning ; Mixed Function Oxygenases/metabolism ; Protein Interaction Maps ; Research/education ; Students ; Teaching
    Chemical Substances Cytochrome P-450 Enzyme System (9035-51-2) ; Mixed Function Oxygenases (EC 1.-) ; pentachlorophenol monooxygenase (EC 1.14.13.50)
    Language English
    Publishing date 2017-11
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2126776-5
    ISSN 1545-7885 ; 1544-9173
    ISSN (online) 1545-7885
    ISSN 1544-9173
    DOI 10.1371/journal.pbio.2003145
    Database MEDical Literature Analysis and Retrieval System OnLINE

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