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Paradoxical activation of a type VI secretion system (T6SS) phospholipase effector by its cognate immunity protein

Steven J. Jensen, Zachary C. Ruhe, August F. Williams, Dinh Q. Nhan, Fernando Garza-Sánchez, David A. Low, View ORCID ProfileChristopher S. Hayes
doi: https://doi.org/10.1101/2023.03.28.534661
Steven J. Jensen
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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Zachary C. Ruhe
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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August F. Williams
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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Dinh Q. Nhan
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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Fernando Garza-Sánchez
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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David A. Low
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
2Biomolecular Science and Engineering Program, University of California, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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Christopher S. Hayes
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
2Biomolecular Science and Engineering Program, University of California, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA
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  • ORCID record for Christopher S. Hayes
  • For correspondence: chayes@lifesci.ucsb.edu
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Abstract

Type VI secretion systems (T6SS) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce auto-permeabilization through unopposed activity of the Tle phospholipase effector. This hyper-permeability phenotype is T6SS-dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyper-permeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyper-permeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli-dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG β-spike protein. Together, these findings indicate that Tli has distinct functions depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export.

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Posted March 29, 2023.
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Paradoxical activation of a type VI secretion system (T6SS) phospholipase effector by its cognate immunity protein
Steven J. Jensen, Zachary C. Ruhe, August F. Williams, Dinh Q. Nhan, Fernando Garza-Sánchez, David A. Low, Christopher S. Hayes
bioRxiv 2023.03.28.534661; doi: https://doi.org/10.1101/2023.03.28.534661
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Paradoxical activation of a type VI secretion system (T6SS) phospholipase effector by its cognate immunity protein
Steven J. Jensen, Zachary C. Ruhe, August F. Williams, Dinh Q. Nhan, Fernando Garza-Sánchez, David A. Low, Christopher S. Hayes
bioRxiv 2023.03.28.534661; doi: https://doi.org/10.1101/2023.03.28.534661

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