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      A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact

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          Abstract

          Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.

          Author summary

          Many bacterial pathogens sense contact to host cells and respond by inducing expression of crucial virulence factors. This includes the type III secretion systems (T3SSs) and their substrates, which manipulate different host cell functions to promote colonization and survival of the pathogen within its host. In this study, we used enteropathogenic Yersinia pseudotuberculosis to elucidate the molecular mechanism of how cell contact is transmitted and translated to trigger this process. We found that multiple global riboregulators control the decay and/or translation of the major transcriptional activator of the T3SS. In the absence of cell contact, these important RNA regulators are coopted by one of the substrate proteins of the T3SS to repress expression of the secretion machinery. Translocation of the substrate protein upon cell contact relieves riboregulator-mediated repression. This leads to a strong induction of the master regulator of T3SS/effector gene expression promoting an increase of the virulence potential and provokes a fast adaptation of the pathogen's fitness, e.g. to compensate for the imposed energetic burden.

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          Most cited references66

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          Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

          Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.
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            Assembly, structure, function and regulation of type III secretion systems

            Type III secretion systems (T3SSs) are protein transport nanomachines that resemble molecular syringes and are found in numerous Gram-negative bacterial species. This Review summarizes our current understanding of the structure and function of these important protein secretion systems, incorporating new advances from cryo-electron microscopy and integrative imaging studies.
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              Flagellin A is essential for the virulence of Vibrio anguillarum.

              A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Visualization
                Role: InvestigationRole: MethodologyRole: Visualization
                Role: InvestigationRole: MethodologyRole: Visualization
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: ConceptualizationRole: MethodologyRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                7 June 2019
                June 2019
                : 15
                : 6
                : e1007813
                Affiliations
                [1 ] Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
                [2 ] Institute for Infectiology, University of Münster, Germany
                [3 ] Department of Molecular Biology, Umea University, Sweden
                Tufts University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-5194-754X
                http://orcid.org/0000-0002-2040-1747
                http://orcid.org/0000-0001-8177-3280
                Article
                PPATHOGENS-D-18-02368
                10.1371/journal.ppat.1007813
                6583979
                31173606
                d1aa06d3-fbd5-4d6a-bfdf-aaf7fc22f139
                © 2019 Kusmierek et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 December 2018
                : 2 May 2019
                Page count
                Figures: 8, Tables: 0, Pages: 30
                Funding
                Funded by: Swedish Research Council
                Award ID: grant 2015-02874
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award Recipient :
                Funded by: HZI Graduate School
                Award Recipient :
                Funded by: HZI Graduate School
                Award Recipient :
                Funded by: HZI Graduate School
                Award Recipient :
                The authors received funding from the Helmholtz Gemeinschaft for this work. PD is supported by the German Research Center for Infection Research (DZIF). HWW is supported by the Swedish Research Council (grant 2015-02874). RW, WO and MK received fellowships from the Helmholtz Centre for Infection Research Graduate School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Transmission and Infection
                Host Cells
                Research and analysis methods
                Chemical synthesis
                Biosynthetic techniques
                Nucleic acid synthesis
                RNA synthesis
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA synthesis
                Biology and Life Sciences
                Organisms
                Bacteria
                Yersinia
                Yersinia Pseudotuberculosis
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Yersinia
                Yersinia Pseudotuberculosis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Yersinia
                Yersinia Pseudotuberculosis
                Biology and Life Sciences
                Physiology
                Physiological Processes
                Secretion
                Medicine and Health Sciences
                Physiology
                Physiological Processes
                Secretion
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Nucleases
                Ribonucleases
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Hydrolases
                Nucleases
                Ribonucleases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Hydrolases
                Nucleases
                Ribonucleases
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-06-19
                All relevant data are within the manuscript and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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