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      The Second Type VI Secretion System of Pseudomonas aeruginosa Strain PAO1 Is Regulated by Quorum Sensing and Fur and Modulates Internalization in Epithelial Cells*

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          Abstract

          Background: Three T6SSs are present in P. aeruginosa. H1-T6SS secretes bacteriolytic toxins.

          Results: H2-T6SS is regulated by quorum sensing and Fur and modulates internalization in epithelial cells through PI3K-Akt host pathway activation.

          Conclusion: H2-T6SS plays a role in virulence.

          Significance: In contrast to the anti-prokaryotic H1-T6SS, H2-T6SS targets human cells. Those T6SSs can carry out different functions important in establishing infection.

          Abstract

          The genome of Pseudomonas aeruginosa PAO1 contains three type VI secretion systems (T6SSs) called H1-, H2-, and H3-T6SS. The H1-T6SS secretes three identified toxins that target other bacteria, providing a fitness advantage for P. aeruginosa, and likely contributes to bacterial pathogenesis in chronic infections. However, no specific substrates or defined roles have been described for the two other systems. Here, we demonstrate that the expression of H2-T6SS genes of strain PAO1 is up-regulated during the transition from exponential to stationary phase growth and regulated by the Las and Rhl quorum sensing systems. In addition, we identify two putative Fur boxes in the promoter region and find that H2-T6SS transcription is negatively regulated by iron. We also show that the H2-T6SS system enhances bacterial uptake into HeLa cells (75% decrease in internalization with a H2-T6SS mutant) and into lung epithelial cells through a phosphatidylinositol 3-kinase-dependent pathway that induces Akt activation in the host cell (50% decrease in Akt phosphorylation). Finally, we show that H2-T6SS plays a role in P. aeruginosa virulence in the worm model. Thus, in contrast to H1-T6SS, H2-T6SS modulates interaction with eukaryotic host cells. Together, T6SS can carry out different functions that may be important in establishing chronic P. aeruginosa infections in the human host.

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          Most cited references 60

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          Experiments in Molecular Genetics

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            Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

            pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.
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              Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis.

              There are two interrelated acyl-homoserine lactone quorum-sensing-signaling systems in Pseudomonas aeruginosa. These systems, the LasR-LasI system and the RhlR-RhlI system, are global regulators of gene expression. We performed a transcriptome analysis to identify quorum-sensing-controlled genes and to better understand quorum-sensing control of P. aeruginosa gene expression. We compared gene expression in a LasI-RhlI signal mutant grown with added signals to gene expression without added signals, and we compared a LasR-RhlR signal receptor mutant to its parent. In all, we identified 315 quorum-induced and 38 quorum-repressed genes, representing about 6% of the P. aeruginosa genome. The quorum-repressed genes were activated in the stationary phase in quorum-sensing mutants but were not activated in the parent strain. The analysis of quorum-induced genes suggests that the signal specificities are on a continuum and that the timing of gene expression is on a continuum (some genes are induced early in growth, most genes are induced at the transition from the logarithmic phase to the stationary phase, and some genes are induced during the stationary phase). In general, timing was not related to signal concentration. We suggest that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression. Acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes during P. aeruginosa growth in culture.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                3 August 2012
                4 June 2012
                4 June 2012
                : 287
                : 32
                : 27095-27105
                Affiliations
                From the []Aix-Marseille Université et CNRS, Laboratoire d'Ingénierie des Systèmes Macromoléculaires, UMR7255, 13402 Marseille cedex 20, France and the University of California San Francisco,
                Departments of [§ ]Medicine and
                []Microbiology and Immunology, San Francisco, California 94143
                Author notes
                [3 ] To whom correspondence should be addressed: Aix-Marseille Université et CNRS, Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Tel.: 33491164126; Fax: 33491712124; E-mail: bleves@ 123456imm.cnrs.fr .
                [1]

                Present address: Imperial College London, Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, South Kensington Campus, Flowers Bldg., SW7 2AZ London, UK.

                [2]

                Present address: INSERM U624, Parc Scientifique de Luminy, 163 Ave. de Luminy, Case 915 13288, Marseille Cedex 09, France.

                Article
                M112.376368
                10.1074/jbc.M112.376368
                3411052
                22665491
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                Product
                Funding
                Funded by: National Institutes of Health
                Award ID: P01 AI053194
                Award ID: R01 AI065902
                Categories
                Microbiology

                Biochemistry

                internalization, fur, iron, invasion, protein secretion, pseudomonas aeruginosa, quorum sensing, t6ss

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