We tested the hypothesis that the B. fragilis T6SS effector-toxin Bte3 forms a pre-secretion complex with its specific VgrG and the proposed accessory proteins encoded by orf2 and orf3. To test this, we generated functional pEV1/bte1_3(orf2/3) derivatives on the N1 t6-1 t6-2 background in which we HA-tagged Bte3 and alternatively Flag-tagged VgrG, Orf2, or Orf3. Reciprocal Flag-tag and HA-tag immunoprecipitations revealed protein-protein interactions between Bte3 and both Orf2 and Orf3. Anti-Flag immunoprecipitates of VgrG, Orf2, or Orf3 were subjected to mass spectrometry (MS), protein interactions supportive of a pre-secretion complex formed by Bte3, VgrG, Orf2 and Orf3 were detected in Orf2 and Orf3-tagged samples