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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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DNase delivery is dependent upon the BamA receptor.A) bamA101 target cells are resistant to the chimeric EC93-Dd3937 CDI system. Mock inhibitors (CDI-) or CDI3937 cells were co-cultured with bamA+ cells or bamA101 cells at a 1∶100 inhibitor to target ratio. Cultures were sampled at the indicated times and viable target cells quantified as colony forming units (CFU) per mL. Reported values are the average ± SEM for three independent experiments. B) Visualization of DNase delivery. Competitions were conducted as described for panel A, but inhibitors were labeled with GFP (green) and targets with DsRed (red) to differentiate the two cell populations by fluorescence microscopy. Cells were stained with DAPI to visualize DNA. C) Quantification of anucleate target cells. The percentage of anucleate target cells (defined as cells lacking DAPI staining) was determined by inspection of random microscopy fields. Two independent co-cultures were analyzed and between 186–260 target cells were scored for each competition. Values represent the average ± SEM.
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pone-0057609-g005: DNase delivery is dependent upon the BamA receptor.A) bamA101 target cells are resistant to the chimeric EC93-Dd3937 CDI system. Mock inhibitors (CDI-) or CDI3937 cells were co-cultured with bamA+ cells or bamA101 cells at a 1∶100 inhibitor to target ratio. Cultures were sampled at the indicated times and viable target cells quantified as colony forming units (CFU) per mL. Reported values are the average ± SEM for three independent experiments. B) Visualization of DNase delivery. Competitions were conducted as described for panel A, but inhibitors were labeled with GFP (green) and targets with DsRed (red) to differentiate the two cell populations by fluorescence microscopy. Cells were stained with DAPI to visualize DNA. C) Quantification of anucleate target cells. The percentage of anucleate target cells (defined as cells lacking DAPI staining) was determined by inspection of random microscopy fields. Two independent co-cultures were analyzed and between 186–260 target cells were scored for each competition. Values represent the average ± SEM.

Mentions: CDI systems deploy a variety of toxins with diverse sequences, all of which must be efficiently transported into target bacteria. Therefore, we sought to visualize the delivery of another distinct CDI toxin into E. coli cells. We showed previously that the CdiA-CT3937−2 toxin from Dickeya dadantii 3937 is a non-specific DNase that degrades plasmid DNA in vitro[1]. We reasoned that this toxin should alter target cell DNA, which could be visualized by DAPI staining and microscopy. The CDI system from D. dadantii 3937 does not target E. coli[1]. Therefore, we replaced the CdiA-CT of E. coli CdiAEC93 with the CdiA-CT3937−2 toxin to generate a chimeric effector protein capable of delivering the DNase into E. coli cells. We first tested the CdiAEC93-CT3937−2 chimera to determine whether it is active in CDI. CDI3937−2 inhibitor cells were mixed with target cells at a 1∶1 ratio and incubated in shaking broth cultures. After four hours of co-culture, the number of viable target cells decreased ∼1,000-fold (Fig. 4A). In contrast, target cells carrying a plasmid-borne copy of the cdiI3937−2 immunity gene grew unimpeded (Fig. 4A), indicating that the grafted CdiA-CT3937−2 toxin is responsible for growth inhibition. To visualize DNase activity in target cells, we repeated the co-culture experiments with GFP labeled inhibitor cells and DsRed labeled targets and stained the cells with DAPI to visualize nucleoids. Upon initial mixing, inhibitor and target cells both exhibited similar nucleoid staining and morphology (Fig. 4B). However, a fraction of target cells lost DAPI staining after four hours of co-culture with CDI3937−2 inhibitor cells (Fig. 4B), consistent with the degradation of genomic DNA. This loss of DAPI staining was CDI-dependent (Fig. 4C), and target cells that express the cdiI3937−2 immunity gene showed no changes in nucleoid appearance during co-culture with inhibitors (Fig. 4D). Finally, we tested whether DNase delivery was dependent upon BamA. We repeated the growth competition experiments using an inhibitor to target cell ratio of 1∶100 and found that bamA101 targets were resistant to the chimeric CDI3937−2 system under these conditions (Fig. 5A). Microscopic analysis showed that wild-type bamA+ cells were more likely to lose cellular DNA (∼45% anucleate cells) compared to bamA101 targets (∼20% anucleate cells) after co-culture with CDI3937−2 inhibitors (Figs. 5B & 5C). Together, these results indicate that CdiA-CT3937−2 is translocated into E. coli in a BamA-dependent manner and that the toxin destroys target cell genomic DNA to inhibit growth.


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)

DNase delivery is dependent upon the BamA receptor.A) bamA101 target cells are resistant to the chimeric EC93-Dd3937 CDI system. Mock inhibitors (CDI-) or CDI3937 cells were co-cultured with bamA+ cells or bamA101 cells at a 1∶100 inhibitor to target ratio. Cultures were sampled at the indicated times and viable target cells quantified as colony forming units (CFU) per mL. Reported values are the average ± SEM for three independent experiments. B) Visualization of DNase delivery. Competitions were conducted as described for panel A, but inhibitors were labeled with GFP (green) and targets with DsRed (red) to differentiate the two cell populations by fluorescence microscopy. Cells were stained with DAPI to visualize DNA. C) Quantification of anucleate target cells. The percentage of anucleate target cells (defined as cells lacking DAPI staining) was determined by inspection of random microscopy fields. Two independent co-cultures were analyzed and between 186–260 target cells were scored for each competition. Values represent the average ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585180&req=5

pone-0057609-g005: DNase delivery is dependent upon the BamA receptor.A) bamA101 target cells are resistant to the chimeric EC93-Dd3937 CDI system. Mock inhibitors (CDI-) or CDI3937 cells were co-cultured with bamA+ cells or bamA101 cells at a 1∶100 inhibitor to target ratio. Cultures were sampled at the indicated times and viable target cells quantified as colony forming units (CFU) per mL. Reported values are the average ± SEM for three independent experiments. B) Visualization of DNase delivery. Competitions were conducted as described for panel A, but inhibitors were labeled with GFP (green) and targets with DsRed (red) to differentiate the two cell populations by fluorescence microscopy. Cells were stained with DAPI to visualize DNA. C) Quantification of anucleate target cells. The percentage of anucleate target cells (defined as cells lacking DAPI staining) was determined by inspection of random microscopy fields. Two independent co-cultures were analyzed and between 186–260 target cells were scored for each competition. Values represent the average ± SEM.
Mentions: CDI systems deploy a variety of toxins with diverse sequences, all of which must be efficiently transported into target bacteria. Therefore, we sought to visualize the delivery of another distinct CDI toxin into E. coli cells. We showed previously that the CdiA-CT3937−2 toxin from Dickeya dadantii 3937 is a non-specific DNase that degrades plasmid DNA in vitro[1]. We reasoned that this toxin should alter target cell DNA, which could be visualized by DAPI staining and microscopy. The CDI system from D. dadantii 3937 does not target E. coli[1]. Therefore, we replaced the CdiA-CT of E. coli CdiAEC93 with the CdiA-CT3937−2 toxin to generate a chimeric effector protein capable of delivering the DNase into E. coli cells. We first tested the CdiAEC93-CT3937−2 chimera to determine whether it is active in CDI. CDI3937−2 inhibitor cells were mixed with target cells at a 1∶1 ratio and incubated in shaking broth cultures. After four hours of co-culture, the number of viable target cells decreased ∼1,000-fold (Fig. 4A). In contrast, target cells carrying a plasmid-borne copy of the cdiI3937−2 immunity gene grew unimpeded (Fig. 4A), indicating that the grafted CdiA-CT3937−2 toxin is responsible for growth inhibition. To visualize DNase activity in target cells, we repeated the co-culture experiments with GFP labeled inhibitor cells and DsRed labeled targets and stained the cells with DAPI to visualize nucleoids. Upon initial mixing, inhibitor and target cells both exhibited similar nucleoid staining and morphology (Fig. 4B). However, a fraction of target cells lost DAPI staining after four hours of co-culture with CDI3937−2 inhibitor cells (Fig. 4B), consistent with the degradation of genomic DNA. This loss of DAPI staining was CDI-dependent (Fig. 4C), and target cells that express the cdiI3937−2 immunity gene showed no changes in nucleoid appearance during co-culture with inhibitors (Fig. 4D). Finally, we tested whether DNase delivery was dependent upon BamA. We repeated the growth competition experiments using an inhibitor to target cell ratio of 1∶100 and found that bamA101 targets were resistant to the chimeric CDI3937−2 system under these conditions (Fig. 5A). Microscopic analysis showed that wild-type bamA+ cells were more likely to lose cellular DNA (∼45% anucleate cells) compared to bamA101 targets (∼20% anucleate cells) after co-culture with CDI3937−2 inhibitors (Figs. 5B & 5C). Together, these results indicate that CdiA-CT3937−2 is translocated into E. coli in a BamA-dependent manner and that the toxin destroys target cell genomic DNA to inhibit growth.

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