Citation
- Authors: Chai Q. et al.
- Year: 2022
- Journal: Science 378 eabq0132
- Applications: in vitro / mRNA / jetMESSENGER
- Cell type: immortalized BMDMs (iBMMs)
Method
The indicated mRNAs were then generated in vitro using the HiScribe T7 ARCAmRNA Kit (New England BioLabs, Cat# E2060), with incorporation of themodified nucleosides 5-methyl-CTP and pseudo-UTP (APExBIO, Cat# B7972 and B7967, respectively) to minimize the immunogenicity and enhance the stability (44). The transcribed mRNA was then dephosphorylated by Antarctic phosphatase (New England BioLabs, Cat# M0289) to avoid recognition by the cytoplasmic RNA sensors inmacrophages (45, 46). Finally, the transcribed mRNA was purified using MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific, Cat# AM1908). The purified mRNAwas then transfected into BMDMs cultured in 6-well plates (500 ng for a total of 2.5 × 105 cells per well) using jetMESSENGER (Polyplus-transfection, Cat# 101000056) in accordance with the manufacturer’s instructions. After transfection for 8 hours, the expression of the indicated proteins was verified by immunoblotting with anti-PtpB or anti-GFP antibodies or was examined by confocal microscopy.
Abstract
The inflammasome-mediated cleavage of gasdermin D (GSDMD) causes pyroptosis and inflammatory cytokine release to control pathogen infection, but how pathogens evade this immune response remains largely unexplored. Here we identify the known protein phosphatase PtpB from Mycobacterium tuberculosis as a phospholipid phosphatase inhibiting the host inflammasome-pyroptosis pathway. Mechanistically, PtpB dephosphorylated phosphatidylinositol-4-monophosphate and phosphatidylinositol-(4,5)-bisphosphate in host cell membrane, thus disrupting the membrane localization of the cleaved GSDMD to inhibit cytokine release and pyroptosis of macrophages. Notably, this phosphatase activity requires PtpB binding to ubiquitin. Disrupting phospholipid phosphatase activity or the ubiquitin-interacting motif of PtpB enhanced host GSDMD-dependent immune responses and reduced intracellular pathogen survival. Thus, pathogens inhibit pyroptosis and counteract host immunity by altering host membrane composition.