Pufferfish gasdermin Ea is a significant player in the defense against bacterial pathogens

Abstract

Gasdermins (GSDMs) are proteins cleaved by caspase (CASP) to trigger pyroptosis. In teleosts, pyroptosis is mediated by gasdermin E (GSDME). The Pufferfish, Takifugu rubripes, possesses two GSDME orthologs: named TrGSDMEa and TrGSDMEb. TrGSDMEa is cleaved by CASP3/7 to liberate the N-terminal (NT) domain that can trigger pyroptosis in mammalian cells. However, the biological function of TrGSDMEa in pufferfish is unknown, and TrGSDMEb is poorly studied. We found that TrGSDMEb was cleaved by CASP1/3/6/7/8, but the resulting NT domain, despite its similarity to TrGSDMEa-NT domain in sequence and structure, failed to induce pyroptosis. TrGSDMEa and TrGSDMEb exhibited similar expression patterns in pufferfish under normal physiological conditions but were up- and downregulated, respectively, in expression during Vibrio harveyi and Edwardsiella tarda infection. Bacterial infection induced the activation of TrGSDMEa and CASP3/7 in pufferfish cells, resulting in pyroptosis accompanied with IL-1β production and maturation. Inhibition of TrGSDMEa-mediated pyroptosis via TrCASP3/7 reduced the death of pufferfish cells and augmented bacterial dissemination in fish tissues. Structure-oriented mutagenesis identified 16 conserved residues in teleost GSDMEa that were required for the pore formation or auto-inhibition of GSDMEa. This study illustrates the role of GSDMEa-mediated pyroptosis in teleost defense against bacterial pathogens and provides new insights into the structure-based function of vertebrate GSDME.

Supplementary information: The online version contains supplementary material available at 10.1007/s42995-024-00237-x.

Keywords: Bacterial infection; Caspase; GSDME; Immune defense; Takifugu rubripes.

Fig. 1

Cleavage of TrGSDMEb by caspase. TrGSDMEb was incubated with HsCASP1, 2, 3, 6, 7, 8, and 9 for 1 h and then subjected to SDS-PAGE (A) and immunoblotting with anti-His tag antibody (B). C The proteolytic specificities of TrCASP1, 6, and 8 were determined by treatment with different colorimetric substrates and measuring the released ρNA. The values are the means ± SD of triplicate experiments. TrGSDME (D) and TrGSDME-D248R (E) were treated with TrCASPs, and the cleavage was analyzed with SDS-PAGE. F Schematic diagram of TrGSDMEb cleavage by TrCASPs. The arrow indicates cleavage site. For all panels, FL, full length; NT, N-terminal fragment; CT, C-terminal fragment

Fig. 2

The cytotoxic potential of TrGSDMEb. TrGSDMEb-FL, -NT, and -CT were tagged with mCherry and expressed in HEK293T cells for 24 h. The cells were analyzed for TrGSDME expression (A), morphological change (B), and LDH release (C). Scale bar, 50 μm. D HEK293T cells were transfected with mCherry-tagged TrGSDMEa/b–FL/NT/CT for 24 h. The cells were stained with Sytox green and analyzed with microscopy. Representative pyroptotic cells were indicated with black arrows. Scale bar, 30 μm. E The LDH release from the above transfected cells of D was measured. For panels C and E, values are the means ± SD of triplicate experiments. ***P < 0.001

Fig. 3

TrGSDME, TrCASP3/7, and inflammatory cytokine expression in pufferfish in response to bacterial infection. Pufferfish were infected with or without (control) Edwardsiella tarda for different hours, and the expression of TrGSDMEa/b (A) and TrCASP3/7 (B) in kidney was determined by qRT-PCR. Pufferfish were infected with Vibrio harveyi, and the expression of TrGSDMEa/b (C) and TrCASP3/7 (D) was determined as above. Pufferfish were infected with or without (control) E. tarda (E) or V. harveyi (F), and TrIL-1β, TrIL-6, TrIL-8, and TrIL-18 expression in kidney were determined by qRT-PCR at various hours. For all panels, values are the means ± SD. n = 3. **P < 0.01; *P < 0.05

Fig. 4

The effect of bacterial infection on the death of pufferfish cells. A Pufferfish macrophages were infected with or without (control) Edwardsiella tarda or Vibrio harveyi for 1 h and then treated with PI. Scale bar, 50 μm. B Pufferfish macrophages were incubated with or without (control) E. tarda for 3 h and then stained with Sytox green and CM-Dil. Scale bar, 5 μm

Fig. 5

TrGSDMEa and TrCASP3/7 activation in pathogen-infected pufferfish cells. Pufferfish macrophages were incubated with Edwardsiella tarda or Vibrio harveyi for various hours. The cells and culture supernatant were immunoblotted with antibodies against TrGSDMEa, TrIL-1β, or β-actin (A) and assayed for TrCASP3/7 activity (B, C). Pufferfish peripheral blood leucocytes were infected with E. tarda or V. harveyi and then immunoblotted as above (D) and assayed for TrCASP3/7 activity (E, F). For panels (B, C, E, F), values are shown as means ± SD of three experimental replicates. ***P < 0.001; **P < 0.01

Fig. 6

The effect of TrCASP3/7 inhibitor on bacterial infection in pufferfish. A Pufferfish macrophages were infected with or without (control) Vibrio harveyi in the presence or absence of AC-DAVD-CHO for 1 h. The cells were stained with PI and observed with a microscope. Bar size, 50 μm. Pufferfish were infected with V. harveyi (B) or Edwardsiella tarda (C) in the presence or absence (control) of AC-DAVD-CHO, and bacterial loads (shown as colony-forming unit, CFU) in kidney and spleen were determined at different hours. Values are the means ± SD. n = 6. ***P < 0.001; **P < 0.01

Fig. 7

The pyroptosis activity of TrGSDMEa variants. HEK293T cells were transfected with mCherry-tagged full-length (FL) TrGSDMEa or its mutants for 24 h and then observed with microscope (A) and measured for LDH release (B). HEK293T cells transfected with TrGSDMEa-NT was used as a positive control. HEK293T cells were transfected with mCherry-tagged TrGSDMEa NT domain or mutated NT domain for 24 h and then observed with a microscope (C) and measured for LDH release (D). Values in B and D are the means ± SD of triplicate experiments. ***P < 0.001; **P < 0.01; *P < 0.05. Scale bar in A and C, 100 μm

Fig. 8

A proposed model of the anti-bacterial effect of TrGSDMEa-mediated pyroptosis in pufferfish. Bacterial infection causes the production of TrGSDMEa and the activation of TrCASP3/7. Activated TrCASP3/7 then cleave TrGSDMEa to trigger pyroptosis, which leads to intracellular content release and effective bacterial clearance