Annexin A2 as a key interacting partner in PrsA-induced cell death pathways of Streptococcus suis serotype 2.

The virulence factor PrsA, a parvulin‑type peptidyl‑prolyl isomerase (PPIase) in Streptococcus suis serotype 2 (SS2), triggers inflammatory responses and host cell death, yet the host protein it interacts with and the downstream signaling events remain poorly defined. Here in this study, we employed an integrated strategy combining bait protein affinity purification methods by covalent coupling and co-immunoprecipitation (Co-IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, bioinformatic and molecular docking analysis to screen for the putative host proteins interacting with SS2-PrsA. Candidate interactions were further validated through pull-down, Co-IP, and molecules co-localization analysis by immunofluorescence assay. The expression of the candidate host target was knocked down using RNA interference, then the PrsA- or SS2-induced corresponding cells death were determined via LDH release and LIVE/DEAD staining, with its downstream pathway activation evaluated by Western blotting. Annexin A2 was identified as a key host interactor of SS2-PrsA. Both in vitro and cellular stimulation experiments confirmed a direct interaction and significant co-localization effect between PrsA and Annexin A2. Its knockdown markedly reduced cell death triggered by either purified PrsA or SS2 infection. At the mechanistic level, loss of Annexin A2 impaired the activation of pyroptosis markers (cleaved caspase-1, GSDMD, and IL-1β) and suppressed phosphorylation of the necroptosis mediator MLKL. These findings not only advance our understanding of SS2 pathogenesis but also highlight Annexin A2 as a critical host factor that links PrsA function and has the potential for developing anti-infective strategies based on host-pathogen interactions.