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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 and HA-CdiAUPEC536 are exported to the cell surface.A) Schematic of the CdiAUPEC536 exoprotein depicting the locations of the inserted N-terminal hemagglutinin (HA) epitope tag and the CdiA-CT region (residues Val3016– Ile3242) used to generate anti-CdiA-CTUPEC536 polyclonal antibodies. Regions corresponding to the secretion signal sequence and the toxic tRNase domain are indicated. The vertical VENN sequence demarcates the N-terminal margin of the variable CdiA-CT sequence. Residue numbers are shown by the scale bar below. B) Whole-cell immunoblot analysis of E. coli cells expressing CdiAUPEC536 and HA-CdiAUPEC536. Cells expressing HA-CdiAUPEC536 (HA-CdiA), CdiAUPEC536 (CdiA) or no effector protein (CDI-) were fixed without permeabilization and stained with anti-HA or anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies. Where indicated, cells were treated with proteinase K (proK) prior to fixation. Stained cells were spotted onto nitrocellulose membrane and analyzed with an Odyssey infrared imager.
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pone-0057609-g001: CdiAUPEC536 and HA-CdiAUPEC536 are exported to the cell surface.A) Schematic of the CdiAUPEC536 exoprotein depicting the locations of the inserted N-terminal hemagglutinin (HA) epitope tag and the CdiA-CT region (residues Val3016– Ile3242) used to generate anti-CdiA-CTUPEC536 polyclonal antibodies. Regions corresponding to the secretion signal sequence and the toxic tRNase domain are indicated. The vertical VENN sequence demarcates the N-terminal margin of the variable CdiA-CT sequence. Residue numbers are shown by the scale bar below. B) Whole-cell immunoblot analysis of E. coli cells expressing CdiAUPEC536 and HA-CdiAUPEC536. Cells expressing HA-CdiAUPEC536 (HA-CdiA), CdiAUPEC536 (CdiA) or no effector protein (CDI-) were fixed without permeabilization and stained with anti-HA or anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies. Where indicated, cells were treated with proteinase K (proK) prior to fixation. Stained cells were spotted onto nitrocellulose membrane and analyzed with an Odyssey infrared imager.

Mentions: We chose the CDI system from uropathogenic Escherichia coli 536 (UPEC 536) [18] to examine toxin delivery because CdiA-CTUPEC536 is a tRNase with substrates in the cytosol of target cells. To facilitate genetic manipulations, 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]. We introduced a hemagglutinin (HA) peptide epitope near the mature N-terminus of CdiAUPEC536 for detection by immunofluorescence (Fig. 1A). Additionally, we raised polyclonal antiserum against the CdiA-CTUPEC536 toxin region. Thus, the N- and C-termini of CdiAUPEC536 can be tracked simultaneously using the appropriate fluorescently labeled antibodies. Because the HA epitope was inserted adjacent to the signal sequence (Fig. 1A), we first examined surface expression of HA-CdiAUPEC536 by whole-cell immunoblot to confirm that the modified exoprotein is still secreted. Cells were incubated with fluorescent anti-HA antibodies under conditions that do not permeabilize the outer membrane based on staining for a periplasmic marker and then blotted onto a nitrocellulose membrane (Fig. S1A). 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). The HA signal was sensitive to treatment with proteinase K (Fig. 1B), consistent with epitope exposure on the cell surface. However, to conclusively determine if the immunoblot procedure only detects cell-surface antigens, we repeated the analysis using antibodies to maltose-binding protein (MBP), which is localized to the periplasm [19]. MBP was not detected under the original immunoblot conditions, but a strong signal was obtained when the cells were permeabilized with Triton X-100 (Fig. S1A). Notably, the MBP signal was insensitive to proteinase K, indicating that periplasmic and presumably cytoplasmic antigens are protected from the protease (Fig. S1A). Thus, fixation conditions can be controlled to differentiate between surface exposed and internal antigens. We repeated this analysis using polyclonal antibodies to CdiA-CTUPEC536 and found that the toxin domain was present on the surface of inhibitor cells expressing either HA-tagged or untagged CdiAUPEC536 (Fig. 1B).


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 and HA-CdiAUPEC536 are exported to the cell surface.A) Schematic of the CdiAUPEC536 exoprotein depicting the locations of the inserted N-terminal hemagglutinin (HA) epitope tag and the CdiA-CT region (residues Val3016– Ile3242) used to generate anti-CdiA-CTUPEC536 polyclonal antibodies. Regions corresponding to the secretion signal sequence and the toxic tRNase domain are indicated. The vertical VENN sequence demarcates the N-terminal margin of the variable CdiA-CT sequence. Residue numbers are shown by the scale bar below. B) Whole-cell immunoblot analysis of E. coli cells expressing CdiAUPEC536 and HA-CdiAUPEC536. Cells expressing HA-CdiAUPEC536 (HA-CdiA), CdiAUPEC536 (CdiA) or no effector protein (CDI-) were fixed without permeabilization and stained with anti-HA or anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies. Where indicated, cells were treated with proteinase K (proK) prior to fixation. Stained cells were spotted onto nitrocellulose membrane and analyzed with an Odyssey infrared imager.
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getmorefigures.php?uid=PMC3585180&req=5

pone-0057609-g001: CdiAUPEC536 and HA-CdiAUPEC536 are exported to the cell surface.A) Schematic of the CdiAUPEC536 exoprotein depicting the locations of the inserted N-terminal hemagglutinin (HA) epitope tag and the CdiA-CT region (residues Val3016– Ile3242) used to generate anti-CdiA-CTUPEC536 polyclonal antibodies. Regions corresponding to the secretion signal sequence and the toxic tRNase domain are indicated. The vertical VENN sequence demarcates the N-terminal margin of the variable CdiA-CT sequence. Residue numbers are shown by the scale bar below. B) Whole-cell immunoblot analysis of E. coli cells expressing CdiAUPEC536 and HA-CdiAUPEC536. Cells expressing HA-CdiAUPEC536 (HA-CdiA), CdiAUPEC536 (CdiA) or no effector protein (CDI-) were fixed without permeabilization and stained with anti-HA or anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies. Where indicated, cells were treated with proteinase K (proK) prior to fixation. Stained cells were spotted onto nitrocellulose membrane and analyzed with an Odyssey infrared imager.
Mentions: We chose the CDI system from uropathogenic Escherichia coli 536 (UPEC 536) [18] to examine toxin delivery because CdiA-CTUPEC536 is a tRNase with substrates in the cytosol of target cells. To facilitate genetic manipulations, 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]. We introduced a hemagglutinin (HA) peptide epitope near the mature N-terminus of CdiAUPEC536 for detection by immunofluorescence (Fig. 1A). Additionally, we raised polyclonal antiserum against the CdiA-CTUPEC536 toxin region. Thus, the N- and C-termini of CdiAUPEC536 can be tracked simultaneously using the appropriate fluorescently labeled antibodies. Because the HA epitope was inserted adjacent to the signal sequence (Fig. 1A), we first examined surface expression of HA-CdiAUPEC536 by whole-cell immunoblot to confirm that the modified exoprotein is still secreted. Cells were incubated with fluorescent anti-HA antibodies under conditions that do not permeabilize the outer membrane based on staining for a periplasmic marker and then blotted onto a nitrocellulose membrane (Fig. S1A). 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). The HA signal was sensitive to treatment with proteinase K (Fig. 1B), consistent with epitope exposure on the cell surface. However, to conclusively determine if the immunoblot procedure only detects cell-surface antigens, we repeated the analysis using antibodies to maltose-binding protein (MBP), which is localized to the periplasm [19]. MBP was not detected under the original immunoblot conditions, but a strong signal was obtained when the cells were permeabilized with Triton X-100 (Fig. S1A). Notably, the MBP signal was insensitive to proteinase K, indicating that periplasmic and presumably cytoplasmic antigens are protected from the protease (Fig. S1A). Thus, fixation conditions can be controlled to differentiate between surface exposed and internal antigens. We repeated this analysis using polyclonal antibodies to CdiA-CTUPEC536 and found that the toxin domain was present on the surface of inhibitor cells expressing either HA-tagged or untagged CdiAUPEC536 (Fig. 1B).

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