T3SS translocon induces pyroptosis by direct interaction with NLRC4/NAIP inflammasome
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eLife assessment
This important study shows that Type 3 secretion translocons in E. tarda and other bacteria activate the NAIP-NLRC4 inflammasome. The data from cellular and biochemical experiments showing that EseB is required for activation of the NLRC4 inflammasome are convincing, and comparing other translocons and additional cellular assays will provide further strength. This paper is broadly relevant to those investigating host-pathogen interactions in diverse organisms.
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Abstract
Type III secretion system (T3SS) is a virulence apparatus existing in many bacterial pathogens. Structurally, T3SS consists of the base, needle, tip, and translocon. The NLRC4 inflammasome is the major receptor for T3SS needle and basal rod proteins. Whether other T3SS components are recognized by NLRC4 is unclear. In this study, using Edwardsiella tarda as a model intracellular pathogen, we examined T3SS−inflammasome interaction and its effect on cell death. E. tarda induced pyroptosis in a manner that required the bacterial translocon and the host inflammasome proteins of NLRC4, NLRP3, ASC, and caspase 1/4. The translocon protein EseB triggered NLRC4/NAIP-mediated pyroptosis by binding NAIP via its C-terminal region, particularly the terminal 6 residues (T6R). EseB homologs exist widely in T3SS-positive bacteria and share high identities in T6R. Like E. tarda EseB, all of the representatives of the EseB homologs exhibited T6R-dependent NLRC4 activation ability. Together these results revealed the function and molecular mechanism of EseB to induce host cell pyroptosis and suggested a highly conserved inflammasome-activation mechanism of T3SS translocon in bacterial pathogens.
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eLife assessment
This important study shows that Type 3 secretion translocons in E. tarda and other bacteria activate the NAIP-NLRC4 inflammasome. The data from cellular and biochemical experiments showing that EseB is required for activation of the NLRC4 inflammasome are convincing, and comparing other translocons and additional cellular assays will provide further strength. This paper is broadly relevant to those investigating host-pathogen interactions in diverse organisms.
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Reviewer #1 (Public Review):
Summary:
In this study, Zhao and colleagues investigate inflammasome activation by E. tarda infections. They show that E. tarda induces the activation of the NLRC4 inflammasome as well as the non-canonical pathway in human THP1 macrophages. Further dissecting NLRC4 activation, they find that T3SS translocon components eseB, eseC and eseD are necessary for NLRC4 activation and that delivery of purified eseB is sufficient to trigger NAIP-dependent NLRC4 activation. Sequence analysis reveals that eseB shares homology within the C-terminus with T3SS needle and rod proteins, leading the authors to test if this region is necessary for inflammasome activation. They show that the eseB CT is required and that it mediates interaction with NAIP. Finally, they that homologs of eseB in other bacteria also share the same …Reviewer #1 (Public Review):
Summary:
In this study, Zhao and colleagues investigate inflammasome activation by E. tarda infections. They show that E. tarda induces the activation of the NLRC4 inflammasome as well as the non-canonical pathway in human THP1 macrophages. Further dissecting NLRC4 activation, they find that T3SS translocon components eseB, eseC and eseD are necessary for NLRC4 activation and that delivery of purified eseB is sufficient to trigger NAIP-dependent NLRC4 activation. Sequence analysis reveals that eseB shares homology within the C-terminus with T3SS needle and rod proteins, leading the authors to test if this region is necessary for inflammasome activation. They show that the eseB CT is required and that it mediates interaction with NAIP. Finally, they that homologs of eseB in other bacteria also share the same sequence and that they can activate NLRC4 in a HEK293T cell overexpression system.Strengths:
This is a very nice study that convincingly shows that eseB and its homologs can be recognized by the human NAIP/NLRC4 inflammasome. The experiments are well designed, controlled and described, and the papers is convincing as a whole.Weaknesses:
The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?
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Reviewer #2 (Public Review):
Summary:
This work by Zhao et al. demonstrates the role of the Edwardsiella tarda type 3 secretion system translocon in activating human macrophage inflammation and pyroptosis. The authors show the requirement of both the bacterial translocon proteins and particular host inflammasome components for E. tarda-induced pyroptosis. In addition, the authors show that the C-terminal region of the translocon protein, EseB, is both necessary and sufficient to induce pyroptosis when present in the cytoplasm. The most terminal region of EseB was determined to be highly conserved among other T3SS-encoding pathogenic bacteria and a subset of these exhibited functionally similar effects on inflammasome activation. Overall, the data support the conclusions and interpretations and provide interesting insights into …Reviewer #2 (Public Review):
Summary:
This work by Zhao et al. demonstrates the role of the Edwardsiella tarda type 3 secretion system translocon in activating human macrophage inflammation and pyroptosis. The authors show the requirement of both the bacterial translocon proteins and particular host inflammasome components for E. tarda-induced pyroptosis. In addition, the authors show that the C-terminal region of the translocon protein, EseB, is both necessary and sufficient to induce pyroptosis when present in the cytoplasm. The most terminal region of EseB was determined to be highly conserved among other T3SS-encoding pathogenic bacteria and a subset of these exhibited functionally similar effects on inflammasome activation. Overall, the data support the conclusions and interpretations and provide interesting insights into interactions between bacterial T3SS components and the host immune system.Strengths:
The authors use established and reliable molecular biology and bacterial genetics strategies to characterize the roles of the bacterial T3SS translocon and host inflammasome pathways to E. tarda-induced pyroptosis in human macrophages. These observations are naturally expanded upon by demonstrating the specific regions of EseB that are required for inflammasome activation and the conservation of this sequence among other pathogenic bacteria.Weaknesses:
The functional assessment of EseB homologues is limited to inflammasome activation at the protein level but does not include the effects on cell viability as shown for E. tarda EseB. Confirmation that EseB homologues have similar effects on cell death would strengthen this portion of the manuscript. -
Author response:
Public Reviews:
Reviewer #1 (Public Review):
Weaknesses:
The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.
We will add discussions on E. tarda flagellin and examine whether E. tarda flagellin or T3SS needle/rod can activate NLRC4.
The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those …
Author response:
Public Reviews:
Reviewer #1 (Public Review):
Weaknesses:
The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.
We will add discussions on E. tarda flagellin and examine whether E. tarda flagellin or T3SS needle/rod can activate NLRC4.
The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?
The reviewer raises an interesting question. We will explore this question and provide relevant discussions/hypothesis in the revised manuscript.
Reviewer #2 (Public Review):
Weaknesses:
The functional assessment of EseB homologues is limited to inflammasome activation at the protein level but does not include the effects on cell viability as shown for E. tarda EseB. Confirmation that EseB homologues have similar effects on cell death would strengthen this portion of the manuscript.
According to the reviewer’s suggestion, we plan to examine the effects of representative EseB homologs on cell death.
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