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 CdiAUPEC536 were used to visualize transfer of CdiA from CDIUPEC536+ inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiAUPEC536 protein is deposited onto the surface of target bacteria. CdiAUPEC536 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 CdiAUPEC536 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 CdiAUPEC536 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.
Highlights
Contact-dependent growth inhibition (CDI) is a mechanism that allows Gram-negative bacteria to inhibit the growth of neighboring bacteria upon direct cell-to-cell contact [1,2,3]
We used a cosmid-borne copy of the UPEC 536 cdiBAI gene cluster, which is sufficient to confer the inhibitor cell phenotype to CDI- strains of E. coli K-12 [8]
A strong fluorescent signal was detected from cells expressing HA-CdiAUPEC536, but only background fluorescence was observed from cells expressing untagged CdiAUPEC536 or control CDI- cells carrying an empty cosmid vector (Fig. 1B)
Summary
Contact-dependent growth inhibition (CDI) is a mechanism that allows Gram-negative bacteria to inhibit the growth of neighboring bacteria upon direct cell-to-cell contact [1,2,3]. CDI is mediated by the CdiB/CdiA family of two-partner secretion proteins, which are found in many Gram-negative bacteria including a, b, and c-proteobacteria. The current model of CDI postulates that CdiA filaments extend like quills from inhibitor cells, allowing the effector protein to engage specific receptors on the surface of target bacteria. The CdiA-CT region is highly variable between CDI systems, indicating that inhibitor bacteria deploy a variety of distinct toxins. Because CDI systems target bacteria, CDI+ inhibitor cells must produce an immunity protein (CdiI) to protect themselves from autoinhibition [2]. CdiI immunity proteins are highly variable and bind their cognate CdiA-CT to block toxin activity, but they provide no protection from the CDI toxins deployed by other bacteria [1,5]. CDI systems form a network of toxin/immunity proteins that function in bacterial growth competition
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