The lack of virulence following the placement of a 0.22-μm filter between V52 and E. coli, which is expected to disable direct contact while allowing the diffusion of secreted substrates, suggests that V52 directly contacts E. coli to enable bacterial killing and refutes the possibility that V52 secretes a diffusible substrate with toxicity toward E. coli (Fig. 4). These findings are consistent with the current T6SS model where the substrates are directly secreted into the target cell and suggest that the T6SS apparatus punctures the target bacterial cell, requiring contact, and injects one or more substrate(s) directly into the target cell.