Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

Joel Selkrig,Jeroen Krijgsveld,Herman S Overkleeft,Athanasios Typas,Matylda Zietek,Bogdan I Florea,Boris Turk,Jacob Bobonis,Gianluca Sigismondo,Nataša Kopitar-Jerala,André Mateus,Bachir El Debs,Mikhail M Savitski,Anna Sueki,Nan Li,Haruna Imamura,Eicke Latz,Annika Hausmann,Pedro Beltrão ,Matthew Mangan ,Wolf‐Dietrich Hardt

doi:10.1038/s41564-020-0736-7

Abstract

SummaryThe interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined click-chemistry with pulsed stable isotope labeling of amino acids in cell culture (pSILAC-AHA) to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen, Salmonella enterica Typhimurium (STm). We monitored newly synthesised proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified active cathepsins re-traffic to the nucleus and are linked to cell death. Pharmacological cathepsin inhibition and nuclear-targeting of a cellular cathepsin inhibitor (Stefin B) suppressed STm-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that LPS transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced Gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolving host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Highlights

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction
We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death
Because the SPI-2 secretion system of STm has been implicated in re-trafficking of lysosomal contents to the external milieu[9], and nuclear cathepsin activity coincides with SPI-2-dependent proliferation (Extended Data Fig. 3a), we examined whether SPI-2 secretion is required for nuclear cathepsin activity

Summary

Introduction

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. Following detection, activated cytoplasmic pattern-recognition receptors trigger the assembly of a large cytoplasmic protein signalling complex called the inflammasome, which drives an inflammatory form of cell death termed pyroptosis and the release of pro-inflammatory cytokines. When SCV integrity is compromised, bacterial LPS is released to the host cytoplasm where it triggers robust activation of the non-canonical inflammasome and cell death[5]. To shed light onto this complex interface, we leveraged a method recently developed in our laboratory designed to enrich, identify and quantify newly synthesized host secretory proteins in serum-containing media[6] We extended this approach to include sampling of three host cell compartments from STm-infected macrophages spanning three distinct stages of the infection cycle. We detected aberrant subcellular distribution of lysosomal proteases and demonstrated their active role during STm-induced cell death via the non-canonical inflammasome.

Methods

Results

Conclusion