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Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition.

Webb JS, Nikolakakis KC, Willett JL, Aoki SK, Hayes CS, Low DA - PLoS ONE (2013)

Bottom Line: CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.

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.

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CdiAUPEC536 is transferred to the surface of target cells.A) Immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells (expressing either CdiAUPEC536 or HA-CdiAUPEC536) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h, then analyzed by fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) or anti-HA antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT and anti-HA staining of cell surfaces. Inhibitor cells in the CDI- panel carry an empty vector without a CDI system. Where indicated (+), samples were treated with proteinase K prior to fixation, but cells were not permeabilized. B) Transfer of CdiA-CTUPEC536 antigen to target cells. The percentage of bamA+ and bamA101 target cells with CdiA-CTUPEC536 antigen was quantified after one and two hours of co-culture with CdiAUPEC536 inhibitor cells. The bamA101 allele was complemented with plasmid pBamA (corresponding to pDAL950 in Table 1). C) Transfer of HA antigen to target cells. Quantifications in panels B and C were determined by analysis of 150 target cells from two independent experiments. The reported values represent the average ± SEM.
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pone-0057609-g002: CdiAUPEC536 is transferred to the surface of target cells.A) Immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells (expressing either CdiAUPEC536 or HA-CdiAUPEC536) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h, then analyzed by fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) or anti-HA antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT and anti-HA staining of cell surfaces. Inhibitor cells in the CDI- panel carry an empty vector without a CDI system. Where indicated (+), samples were treated with proteinase K prior to fixation, but cells were not permeabilized. B) Transfer of CdiA-CTUPEC536 antigen to target cells. The percentage of bamA+ and bamA101 target cells with CdiA-CTUPEC536 antigen was quantified after one and two hours of co-culture with CdiAUPEC536 inhibitor cells. The bamA101 allele was complemented with plasmid pBamA (corresponding to pDAL950 in Table 1). C) Transfer of HA antigen to target cells. Quantifications in panels B and C were determined by analysis of 150 target cells from two independent experiments. The reported values represent the average ± SEM.

Mentions: Having established that HA-CdiAUPEC536 is secreted, we next tested its inhibition activity in growth competition assays. Inhibitor cells expressing HA-CdiAUPEC536 were incubated with target E. coli cells in shaking broth cultures, and the number of viable target cells were quantified. After four hours of co-culture with HA-CdiAUPEC536 inhibitors, target cell counts decreased ∼100-fold, whereas CDI- mock inhibitor cells carrying the cosmid vector did not inhibit target cell growth (Fig. S2). Moreover, HA-CdiAUPEC536 inhibited growth to the same extent as wild-type CdiAUPEC536 (Fig. S2), demonstrating that the HA-tagged effector protein is fully functional in CDI. We examined the co-cultures with fluorescence microscopy to determine if CdiA-associated antigens are transferred to target cells during CDI. In these experiments, target cells were labeled with the red fluorescent protein DsRed to differentiate them from unlabeled CDI+ inhibitor cells. Cell surfaces were stained with fluorescein-labeled antibodies to CdiA-CTUPEC536, which revealed toxin antigen on many cells in the co-culture including a large proportion of target cells (Fig. 2A). CdiA-CTUPEC36 immunostaining was specific with virtually no signal detected in co-cultures containing CDI- mock inhibitor cells (Fig. 2A). All of the CdiA-CTUPEC536 antigen could be removed with proteinase K treatment prior to antibody staining (Fig. 2A), indicating that the toxin domain is exposed on the surface of target cells. Co-cultures containing HA-CdiAUPEC536 inhibitor cells also showed similar transfer of CdiA-CTUPEC536 toxin antigen onto target bacteria (Fig. 2A). Somewhat unexpectedly, immunostaining for the N-terminal HA epitope revealed that this antigen is also delivered to the surface of target cells (Fig. 2A). HA epitope staining was specific with no signal detected in co-cultures containing cells that express untagged CdiAUPEC536 (Fig. 2A). Together, these experiments demonstrate that both the N-terminal and C-terminal regions of CdiAUPEC535 are transferred onto the surface of target cells.


Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition.

Webb JS, Nikolakakis KC, Willett JL, Aoki SK, Hayes CS, Low DA - PLoS ONE (2013)

CdiAUPEC536 is transferred to the surface of target cells.A) Immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells (expressing either CdiAUPEC536 or HA-CdiAUPEC536) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h, then analyzed by fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) or anti-HA antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT and anti-HA staining of cell surfaces. Inhibitor cells in the CDI- panel carry an empty vector without a CDI system. Where indicated (+), samples were treated with proteinase K prior to fixation, but cells were not permeabilized. B) Transfer of CdiA-CTUPEC536 antigen to target cells. The percentage of bamA+ and bamA101 target cells with CdiA-CTUPEC536 antigen was quantified after one and two hours of co-culture with CdiAUPEC536 inhibitor cells. The bamA101 allele was complemented with plasmid pBamA (corresponding to pDAL950 in Table 1). C) Transfer of HA antigen to target cells. Quantifications in panels B and C were determined by analysis of 150 target cells from two independent experiments. The reported values represent the average ± SEM.
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pone-0057609-g002: CdiAUPEC536 is transferred to the surface of target cells.A) Immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells (expressing either CdiAUPEC536 or HA-CdiAUPEC536) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h, then analyzed by fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) or anti-HA antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT and anti-HA staining of cell surfaces. Inhibitor cells in the CDI- panel carry an empty vector without a CDI system. Where indicated (+), samples were treated with proteinase K prior to fixation, but cells were not permeabilized. B) Transfer of CdiA-CTUPEC536 antigen to target cells. The percentage of bamA+ and bamA101 target cells with CdiA-CTUPEC536 antigen was quantified after one and two hours of co-culture with CdiAUPEC536 inhibitor cells. The bamA101 allele was complemented with plasmid pBamA (corresponding to pDAL950 in Table 1). C) Transfer of HA antigen to target cells. Quantifications in panels B and C were determined by analysis of 150 target cells from two independent experiments. The reported values represent the average ± SEM.
Mentions: Having established that HA-CdiAUPEC536 is secreted, we next tested its inhibition activity in growth competition assays. Inhibitor cells expressing HA-CdiAUPEC536 were incubated with target E. coli cells in shaking broth cultures, and the number of viable target cells were quantified. After four hours of co-culture with HA-CdiAUPEC536 inhibitors, target cell counts decreased ∼100-fold, whereas CDI- mock inhibitor cells carrying the cosmid vector did not inhibit target cell growth (Fig. S2). Moreover, HA-CdiAUPEC536 inhibited growth to the same extent as wild-type CdiAUPEC536 (Fig. S2), demonstrating that the HA-tagged effector protein is fully functional in CDI. We examined the co-cultures with fluorescence microscopy to determine if CdiA-associated antigens are transferred to target cells during CDI. In these experiments, target cells were labeled with the red fluorescent protein DsRed to differentiate them from unlabeled CDI+ inhibitor cells. Cell surfaces were stained with fluorescein-labeled antibodies to CdiA-CTUPEC536, which revealed toxin antigen on many cells in the co-culture including a large proportion of target cells (Fig. 2A). CdiA-CTUPEC36 immunostaining was specific with virtually no signal detected in co-cultures containing CDI- mock inhibitor cells (Fig. 2A). All of the CdiA-CTUPEC536 antigen could be removed with proteinase K treatment prior to antibody staining (Fig. 2A), indicating that the toxin domain is exposed on the surface of target cells. Co-cultures containing HA-CdiAUPEC536 inhibitor cells also showed similar transfer of CdiA-CTUPEC536 toxin antigen onto target bacteria (Fig. 2A). Somewhat unexpectedly, immunostaining for the N-terminal HA epitope revealed that this antigen is also delivered to the surface of target cells (Fig. 2A). HA epitope staining was specific with no signal detected in co-cultures containing cells that express untagged CdiAUPEC536 (Fig. 2A). Together, these experiments demonstrate that both the N-terminal and C-terminal regions of CdiAUPEC535 are transferred onto the surface of target cells.

Bottom Line: CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus