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Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota.

Ayres JS, Trinidad NJ, Vance RE - Nat. Med. (2012)

Bottom Line: The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host.In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice.These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Cell Biology, Division of Immunology & Pathogenesis, University of California, Berkeley, USA. jayres@berkeley.edu

ABSTRACT
The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host. However, the microbiota can also include potentially virulent species, termed pathobiont, which can cause disease when intestinal homeostasis is disrupted. The molecular mechanisms by which pathobionts cause disease remain poorly understood. Here we describe a sepsis-like disease that occurs upon gut injury in antibiotic-treated mice. Sepsis was associated with the systemic spread of a specific multidrug-resistant Escherichia coli pathobiont that expanded markedly in the microbiota of antibiotic-treated mice. Rapid sepsis-like death required a component of the innate immune system, the Naip5-Nlrc4 inflammasome. In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice. These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.

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E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome(a) Motility agar was inoculated with wild-type, motile S. Typhimurium LT2, flagellin-deficient S. Typhimurium ΔfliCΔfljB or E. coli-O21:H+ and incubated at 37 °C overnight. Representative images of the diffusion of motile bacteria are shown.(b) Retroviral constructs expressing fliC from E. coli-O21:H+ or flaA from L. pneumophila followed by an IRES-GFP element or GFP alone were transduced into wild-type, Naip5−/−, or Nlrc4−/− macrophages, and the percentage of GFP-positive cells was enumerated by flow cytometry 4 d post-transduction.(c) The percentage of cells expressing GFP from the retroviral lethality assay in (b) is indicated and charted.Data are representative of three independent experiments.
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Figure 5: E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome(a) Motility agar was inoculated with wild-type, motile S. Typhimurium LT2, flagellin-deficient S. Typhimurium ΔfliCΔfljB or E. coli-O21:H+ and incubated at 37 °C overnight. Representative images of the diffusion of motile bacteria are shown.(b) Retroviral constructs expressing fliC from E. coli-O21:H+ or flaA from L. pneumophila followed by an IRES-GFP element or GFP alone were transduced into wild-type, Naip5−/−, or Nlrc4−/− macrophages, and the percentage of GFP-positive cells was enumerated by flow cytometry 4 d post-transduction.(c) The percentage of cells expressing GFP from the retroviral lethality assay in (b) is indicated and charted.Data are representative of three independent experiments.

Mentions: The Naip5-Nlrc4 inflammasome generally detects flagellin translocated to the host cell cytosol via type III or IV secretion systems44. Because E. coli-O21:H+ encodes a T3SS and a functional flagellin (FliC) (Fig. 5a and Supplementary Fig. 5) similar to pathogenic E. coli species, we hypothesized that the cytosolic presence of flagellin from this pathobiont can activate the Naip5-Nlrc4 inflammasome. We tested this hypothesis using a previously described retroviral `lethality' assay44 in which flagellin is expressed from a retroviral promoter directly in host cells, allowing for analysis of the effects of flagellin in the absence of other bacterial factors. Wild-type, Nlrc4−/− and Naip5−/− bone marrow derived macrophages were retrovirally transduced with fliC-IRES-GFP, control GFP, or control L. pneumophila flagellin (flaA-IRES-GFP). Activation of Naip5-Nlrc4 by flagellin results in cell death, as indicated by the loss of GFP positive cells. As previously reported, cells expressing L. pneumophila flaA-IRES-GFP could only be recovered from Nlrc4−/− macrophages46. Detection of FlaA by Naip6 likely accounts for the residual responsiveness of Naip5−/− cells. However cells expressing E. coli-O21:H+ fliC-IRES-GFP could be recovered from both Nlrc4−/− and Naip5−/− macrophages, but not wild-type macrophages (Fig. 5b, c). These data demonstrate that cytosolic flagellin derived from E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome and triggers host cell death.


Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota.

Ayres JS, Trinidad NJ, Vance RE - Nat. Med. (2012)

E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome(a) Motility agar was inoculated with wild-type, motile S. Typhimurium LT2, flagellin-deficient S. Typhimurium ΔfliCΔfljB or E. coli-O21:H+ and incubated at 37 °C overnight. Representative images of the diffusion of motile bacteria are shown.(b) Retroviral constructs expressing fliC from E. coli-O21:H+ or flaA from L. pneumophila followed by an IRES-GFP element or GFP alone were transduced into wild-type, Naip5−/−, or Nlrc4−/− macrophages, and the percentage of GFP-positive cells was enumerated by flow cytometry 4 d post-transduction.(c) The percentage of cells expressing GFP from the retroviral lethality assay in (b) is indicated and charted.Data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 5: E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome(a) Motility agar was inoculated with wild-type, motile S. Typhimurium LT2, flagellin-deficient S. Typhimurium ΔfliCΔfljB or E. coli-O21:H+ and incubated at 37 °C overnight. Representative images of the diffusion of motile bacteria are shown.(b) Retroviral constructs expressing fliC from E. coli-O21:H+ or flaA from L. pneumophila followed by an IRES-GFP element or GFP alone were transduced into wild-type, Naip5−/−, or Nlrc4−/− macrophages, and the percentage of GFP-positive cells was enumerated by flow cytometry 4 d post-transduction.(c) The percentage of cells expressing GFP from the retroviral lethality assay in (b) is indicated and charted.Data are representative of three independent experiments.
Mentions: The Naip5-Nlrc4 inflammasome generally detects flagellin translocated to the host cell cytosol via type III or IV secretion systems44. Because E. coli-O21:H+ encodes a T3SS and a functional flagellin (FliC) (Fig. 5a and Supplementary Fig. 5) similar to pathogenic E. coli species, we hypothesized that the cytosolic presence of flagellin from this pathobiont can activate the Naip5-Nlrc4 inflammasome. We tested this hypothesis using a previously described retroviral `lethality' assay44 in which flagellin is expressed from a retroviral promoter directly in host cells, allowing for analysis of the effects of flagellin in the absence of other bacterial factors. Wild-type, Nlrc4−/− and Naip5−/− bone marrow derived macrophages were retrovirally transduced with fliC-IRES-GFP, control GFP, or control L. pneumophila flagellin (flaA-IRES-GFP). Activation of Naip5-Nlrc4 by flagellin results in cell death, as indicated by the loss of GFP positive cells. As previously reported, cells expressing L. pneumophila flaA-IRES-GFP could only be recovered from Nlrc4−/− macrophages46. Detection of FlaA by Naip6 likely accounts for the residual responsiveness of Naip5−/− cells. However cells expressing E. coli-O21:H+ fliC-IRES-GFP could be recovered from both Nlrc4−/− and Naip5−/− macrophages, but not wild-type macrophages (Fig. 5b, c). These data demonstrate that cytosolic flagellin derived from E. coli-O21:H+ activates the Naip5-Nlrc4 inflammasome and triggers host cell death.

Bottom Line: The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host.In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice.These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Cell Biology, Division of Immunology & Pathogenesis, University of California, Berkeley, USA. jayres@berkeley.edu

ABSTRACT
The mammalian intestine harbors a complex microbial community that provides numerous benefits to its host. However, the microbiota can also include potentially virulent species, termed pathobiont, which can cause disease when intestinal homeostasis is disrupted. The molecular mechanisms by which pathobionts cause disease remain poorly understood. Here we describe a sepsis-like disease that occurs upon gut injury in antibiotic-treated mice. Sepsis was associated with the systemic spread of a specific multidrug-resistant Escherichia coli pathobiont that expanded markedly in the microbiota of antibiotic-treated mice. Rapid sepsis-like death required a component of the innate immune system, the Naip5-Nlrc4 inflammasome. In accordance with Koch's postulates, we found the E. coli pathobiont was sufficient to activate Naip5-Nlrc4 and cause disease when injected intravenously into unmanipulated mice. These findings reveal how sepsis-like disease can result from recognition of pathobionts by the innate immune system.

Show MeSH
Related in: MedlinePlus