title: Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition.
abstract: Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.
authors.forename: Julia S
authors.initials: JS
authors.lastname: Webb
authors.forename: Kiel C
authors.initials: KC
authors.lastname: Nikolakakis
authors.forename: Julia L E
authors.initials: JL
authors.lastname: Willett
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Aoki
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Hayes
authors.forename: David A
authors.initials: DA
authors.lastname: Low
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D018920:Coculture Techniques; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology* / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D057054:Molecular Imaging; D021381:Protein Transport
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; C503242:CdiA protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins