Activity-based profiling of Shigella virulence factor OspF in human cells

ABSTRACT: Phosphothreonine lyases are a small family of bacterial virulence factors secreted into host cells during infection by certain Gram-negative pathogens. This family of enzymes is known for its unique mechanism of silencing host immune signaling via the irreversible β-elimination of phosphothreonine (pThr) into dehydrobutyrine (Dhb). Phosphothreonine lyases have been reported to target mitogen-activated protein kinases (MAPKs) during infection, but the role of Dhb in the mammalian proteome remains elusive. Here, we explore the substrate profile for OspF from Shigella flexneri using a nucleophilic phosphine probe that is able to covalently capture Dhb sites as a result of enzyme exposure. In this work, we use enrichment-based chemoproteomics to define OspF’s activity through streptavidin pulldown of biotinylated probe adducts, allowing us to profile purified recombinant OspF activity at a range of concentrations, while simultaneously profiling the proteomic consequence of removing OspF’s N-terminal docking domain (ΔN26) and by profiling a catalytically dead OspF mutant (K134A). Likewise, we compare our chemoproteomics findings to traditional phosphoproteomics workflows using recombinant OspF. Further, we apply our chemoproteomic workflow to profile OspF activity in HeLa lysates during Shigella infection with (WT) or without (ΔospF) the gene encoding OspF. Simultaneously, we profile protein abundance in un-enriched, untreated lysates during Shigella infection with or without the gene encoding OspF. These studies reveal a wider range of cellular targets of OspF than previously appreciated, highlighting novel areas of discovery within the context of Shigella pathogenesis while additionally revealing a broad activity for OspF’s unique catalytic mechanism.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Nicholas McCurtin  

LAB HEAD: Rebecca A. Scheck

PROVIDER: PXD073985 | Pride | 2026-02-27

REPOSITORIES: Pride