Screening and identification of SipC-interacting proteins inSalmonellaenteritidis using Gal4 yeast two-hybrid system in duck

Abstract

The zoonotic pathogenSalmonellanot only reduces the production performance in ducks, but also poses a serious threat to human health through eggs and pollutes water bodies through feces. SipC, an effector protein of type III secretion systems (T3SS) inSalmonella, mediates translocation of effectors into the eukaryotic host. However, the precise role of SipC effectors remains unknown in ducks. In this study, the SipC from duck granulosa cells (dGCs) was selected as bait, and the SipC-interacting proteins inSalmonellaenteritidis (SE) were screened using Gal4 yeast two-hybrid system in duck. Twelve SipC-interacting proteins were identified. Among those, the p53-effector related to PMP-22 (PERP) and TGF-β activated kinase 1-binding protein 2 (TAB2) were selected to further confirm the function by GST pull-downin vitro. Over-expression of PERP resulted in not only increasing SE adhesion and invasion but also triggering the production of IL-1β and IFN-α in SE infected dGCs, while knock-down PERP showed the opposite tendency (P < 0.01). In addition, TAB2 significantly induced the production of IL-6, IL-1β, IFN-α, and INF-γ in SE infected dGCs (P < 0.05), but did not cause obvious changes in SE adhesion and invasion. When thesipCin SE was deleted, the activities of duck PERP and TAB2 were abolished because they could not bind to SipC. Taken together, although the protein of PERP and TAB2 can interact with SipC, their mechanisms were different in duck challenged by SE. Therefore, PERP was involved in SE invasion and inflammatory response of dGC ovaries, and TAB2 only contributed to dGCs inflammatory response, which provided critical insights about the mechanism in host- bacterium protein interactions duringSalmonellainvasion in duck.