Identification of host interactors of Brucella BT4E effector proteins by Flag-tag affinity purification coupled with LC-MS/MS in HEK293T cells

ABSTRACT: This study aimed to systematically identify host cellular proteins that interact with newly discovered Brucella abortus type IV secretion system (T4SS) effector proteins, designated BT4E3, BT4E4, BT4E19, BT4E32, BT4E33, BT4E37, and BT4E43. To achieve this, we employed an affinity purification–mass spectrometry (AP-MS) strategy in a human cell context. The coding sequences of individual BT4E effectors were cloned into the p3xFLAG-CMV-7.1 vector as C-terminal fusions with 3×Flag tag. These constructs, along with a p3xFLAG-CMV-7.1-control vector (mCherry-Strep only), were transiently transfected into HEK293T cells using Lipofectamine 3000. Twenty-four hours post-transfection, cells were lysed under mild non-denaturing conditions (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40) supplemented with protease and phosphatase inhibitors. Flag-tagged effector complexes were affinity-purified using Flag Sepharose beads, followed by stringent washing and elution with 2.5 mM D-desthiobiotin. Each effector purification was performed in three independent biological replicates to ensure reproducibility. Eluted protein complexes were verified by Western blotting, then processed for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS was carried out on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled online to an EASY-nLC 1000/1200 nanoflow system. The resulting proteomics data enable the identification of high-confidence host binding partners for each BT4E effector, providing insights into the molecular mechanisms by which Brucella manipulates host cellular processes—including vesicular trafficking, innate immune signaling, autophagy, and cell death pathways—to establish intracellular infection. This dataset serves as a valuable resource for understanding Brucella pathogenesis and host–pathogen interactions mediated by T4SS effectors.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: dongjie sun  

LAB HEAD: Jiabo Ding

PROVIDER: PXD068987 | Pride | 2026-02-13

REPOSITORIES: Pride