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      Methods to Illuminate the Role of Salmonella Effector Proteins during Infection: A Review

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          Abstract

          Intracellular bacterial pathogens like Salmonella enterica use secretion systems, such as the Type III Secretion System, to deliver virulence factors into host cells in order to invade and colonize these cells. Salmonella virulence factors include a suite of effector proteins that remodel the host cell to facilitate bacterial internalization, replication, and evasion of host immune surveillance. A number of diverse and innovative approaches have been used to identify and characterize the role of effector proteins during infection. Recent techniques for studying infection using single cell and animal models have illuminated the contribution of individual effector proteins in infection. This review will highlight the techniques applied to study Salmonella effector proteins during infection. It will describe how different approaches have revealed mechanistic details for effectors in manipulating host cellular processes including: the dynamics of effector translocation into host cells, cytoskeleton reorganization, membrane trafficking, gene regulation, and autophagy.

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          Most cited references76

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          Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.

          Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.
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            Salmonellae interplay with host cells.

            Salmonellae are important causes of enteric diseases in all vertebrates. Characterization of the molecular mechanisms that underpin the interactions of salmonellae with their animal hosts has advanced greatly over the past decade, mainly through the study of Salmonella enterica serovar Typhimurium in tissue culture and animal models of infection. Knowledge of these bacterial processes and host responses has painted a dynamic and complex picture of the interaction between salmonellae and animal cells. This Review focuses on the molecular mechanisms of these host-pathogen interactions, in terms of their context, significance and future perspectives.
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              Simultaneous identification of bacterial virulence genes by negative selection.

              An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                10 August 2017
                2017
                : 7
                : 363
                Affiliations
                Department of Chemistry and Biochemistry, BioFrontiers Institute, University of Colorado Boulder Boulder, CO, United States
                Author notes

                Edited by: Leigh A. Knodler, Washington State University, United States

                Reviewed by: Charles Lawrence Larson, Rocky Mountain Laboratory, United States; Matteo Bonazzi, Centre National de la Recherche Scientifique (CNRS), France; Isabelle Derré, University of Virginia, United States

                *Correspondence: Amy E. Palmer amy.palmer@ 123456colorado.edu
                Article
                10.3389/fcimb.2017.00363
                5554337
                28848721
                34f6ba06-925c-40ff-8890-7e144a8681be
                Copyright © 2017 Young and Palmer.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 14 June 2017
                : 27 July 2017
                Page count
                Figures: 3, Tables: 1, Equations: 0, References: 81, Pages: 12, Words: 9401
                Funding
                Funded by: National Science Foundation 10.13039/100000001
                Award ID: MCB-0950411
                Categories
                Microbiology
                Review

                Infectious disease & Microbiology
                live cell imaging,fluorescence microscopy,salmonella effector proteins,translocation of effector proteins,localization of effector proteins

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