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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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CdiA-CTUPEC536 is delivered into target cells.A) Anti-CdiA-CTUPEC536 immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells expressing either CdiAUPEC536 (CdiA) or HA-CdiAUPEC536 (HA-CdiA) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h. Cells were then fixed and permeabilized for fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation. The histogram quantifies the percentage of target cells (average ± SEM) with surface and internal CdiA-CTUPEC536 antigen staining. At least 150 target cells from two independent experiments were scored for the quantification of CdiA delivery. B) Anti-HA epitope immunofluorescence microscopy of CDIUPEC536 co-cultures. Co-culture conditions and sample preparation was as described in panel A except that anti-HA antibodies were used for immunofluorescence. Green fluorescence is indicative of anti-HA immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation.
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pone-0057609-g003: CdiA-CTUPEC536 is delivered into target cells.A) Anti-CdiA-CTUPEC536 immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells expressing either CdiAUPEC536 (CdiA) or HA-CdiAUPEC536 (HA-CdiA) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h. Cells were then fixed and permeabilized for fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation. The histogram quantifies the percentage of target cells (average ± SEM) with surface and internal CdiA-CTUPEC536 antigen staining. At least 150 target cells from two independent experiments were scored for the quantification of CdiA delivery. B) Anti-HA epitope immunofluorescence microscopy of CDIUPEC536 co-cultures. Co-culture conditions and sample preparation was as described in panel A except that anti-HA antibodies were used for immunofluorescence. Green fluorescence is indicative of anti-HA immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation.

Mentions: We next sought to detect CdiA-derived antigens inside target bacteria. Control experiments using antibodies to periplasmic MBP showed that internal antigens are only detectable when cells are permeabilized with EDTA and lysozyme treatment (Fig. S1B). We applied the EDTA/lysozyme procedure to CDI co-cultures and observed diffuse CdiA-CTUPEC536 staining of both inhibitor and target cells (Fig. 3A). This staining was specific with no signal detected from either CDI- cells using anti-CdiA-CT antibodies (Fig. 3A) or CdiA+ cells using anti-HA antibodies (Fig. 3B). Because there is significant CdiA-CTUPEC536 antigen on cell surfaces (see Fig. 2A), the signal from permeabilized cells likely represents the sum of both surface and internal antigens. Therefore, to specifically visualize internal CdiA-CTUPEC536, we removed surface antigen with proteinase K treatment and then permeabilized the cells for immunostaining. Using the protease/permeabilization protocol, we found that virtually all inhibitor cells and approximately 30% of the target cells contain internal CdiA-CTUPEC536 antigen (Fig. 3A). The HA-CdiAUPEC536 effector protein delivered CdiA-CTUPEC536 antigen to targets at a level similar to untagged CdiAUPEC536 (Fig. 3A), indicating that the N-terminal HA tag does not adversely affect CdiA-CTUPEC536 translocation. We used the same fixation regimen to determine whether the N-terminal HA epitope is also internalized by target cells. Although there was abundant HA epitope detected on cells in the co-culture, this signal was ablated by proteinase K treatment (Fig. 3B), strongly suggesting that all of the HA peptide is surface exposed. These data indicate that the N-terminal HA epitope remains on the target cell surface, whereas the CdiA-CTUPEC536 toxin is internalized.


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)

CdiA-CTUPEC536 is delivered into target cells.A) Anti-CdiA-CTUPEC536 immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells expressing either CdiAUPEC536 (CdiA) or HA-CdiAUPEC536 (HA-CdiA) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h. Cells were then fixed and permeabilized for fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation. The histogram quantifies the percentage of target cells (average ± SEM) with surface and internal CdiA-CTUPEC536 antigen staining. At least 150 target cells from two independent experiments were scored for the quantification of CdiA delivery. B) Anti-HA epitope immunofluorescence microscopy of CDIUPEC536 co-cultures. Co-culture conditions and sample preparation was as described in panel A except that anti-HA antibodies were used for immunofluorescence. Green fluorescence is indicative of anti-HA immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation.
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pone-0057609-g003: CdiA-CTUPEC536 is delivered into target cells.A) Anti-CdiA-CTUPEC536 immunofluorescence microscopy of CDIUPEC536 co-cultures. Non-fluorescent inhibitor cells expressing either CdiAUPEC536 (CdiA) or HA-CdiAUPEC536 (HA-CdiA) were mixed with red fluorescent target cells (2∶1 inhibitor-to-target ratio) for 1 h. Cells were then fixed and permeabilized for fluorescence microscopy using anti-CdiA-CTUPEC536 (anti-CdiA-CT) antibodies as described in Methods. Green fluorescence is indicative of anti-CdiA-CT immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation. The histogram quantifies the percentage of target cells (average ± SEM) with surface and internal CdiA-CTUPEC536 antigen staining. At least 150 target cells from two independent experiments were scored for the quantification of CdiA delivery. B) Anti-HA epitope immunofluorescence microscopy of CDIUPEC536 co-cultures. Co-culture conditions and sample preparation was as described in panel A except that anti-HA antibodies were used for immunofluorescence. Green fluorescence is indicative of anti-HA immunostaining. Where indicated (+), cells were treated with proteinase K prior to fixation.
Mentions: We next sought to detect CdiA-derived antigens inside target bacteria. Control experiments using antibodies to periplasmic MBP showed that internal antigens are only detectable when cells are permeabilized with EDTA and lysozyme treatment (Fig. S1B). We applied the EDTA/lysozyme procedure to CDI co-cultures and observed diffuse CdiA-CTUPEC536 staining of both inhibitor and target cells (Fig. 3A). This staining was specific with no signal detected from either CDI- cells using anti-CdiA-CT antibodies (Fig. 3A) or CdiA+ cells using anti-HA antibodies (Fig. 3B). Because there is significant CdiA-CTUPEC536 antigen on cell surfaces (see Fig. 2A), the signal from permeabilized cells likely represents the sum of both surface and internal antigens. Therefore, to specifically visualize internal CdiA-CTUPEC536, we removed surface antigen with proteinase K treatment and then permeabilized the cells for immunostaining. Using the protease/permeabilization protocol, we found that virtually all inhibitor cells and approximately 30% of the target cells contain internal CdiA-CTUPEC536 antigen (Fig. 3A). The HA-CdiAUPEC536 effector protein delivered CdiA-CTUPEC536 antigen to targets at a level similar to untagged CdiAUPEC536 (Fig. 3A), indicating that the N-terminal HA tag does not adversely affect CdiA-CTUPEC536 translocation. We used the same fixation regimen to determine whether the N-terminal HA epitope is also internalized by target cells. Although there was abundant HA epitope detected on cells in the co-culture, this signal was ablated by proteinase K treatment (Fig. 3B), strongly suggesting that all of the HA peptide is surface exposed. These data indicate that the N-terminal HA epitope remains on the target cell surface, whereas the CdiA-CTUPEC536 toxin is internalized.

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