Fig 1. H2-T6SS is required for Azu secretion.

(A) Deletion of retS enables expression and secretion of Hcp2. A mini-CTX plasmid directing the expression of Hcp2-Flag chimera was integrated into the P. aeruginosa derivative strains, respectively. Western blot analysis of Hcp2-Flag in the cell-associated (Cell) and concentrated supernatant (Sup) protein fractions from the indicated strains grown in M9 minimal medium. For the pellet fraction, an antibody against RNA polymerase α (α-RNAP) was used as a loading control in this and subsequent blots. (B) Azu is secreted by H2-T6SS. The cultured condition is the same as described in A. EV represents the empty vector pAK1900. (C) Total cell (Total), Cytoplasmic (Cyto) and periplasmic (Peri) fractions of P. aeruginosa expressing Azu, PA0943, XcpP or CtpA were examined by Western blot. The tagged proteins were detected using a Flag antibody. RNA polymerase (RNAP) and β-lactamase (β-lac) were used as cytoplasmic or periplasmic fraction controls, respectively. Data are representative of three independent replicates. (D) Interactions of Azu and VgrG2b. Cell lysates of P. aeruginosa containing pMMB67H-VgrG2a-Flag or pMMB67H-VgrG2b-Flag were incubated with GST or GST-Azu protein individually, and protein complex were captured by glutathione beads. The single and double asterisks represent GST and GST-Azu, respectively. GST-SiaD was a negative control. (E) Azu secretion is VgrG2b dependent but not VgrG2a. The cultured condition is the same as described in A.