On the basis of the current model for T6SS, substrates pass directly from the secreting cell into the cytoplasm of the target cell. To test whether T6SS-mediated antimicrobial activity requires contact between V. cholerae and E. coli, survival was measured with a 0.22-μm filter between V52 predator and E. coli prey to allow the passage of molecules but not bacterial cells (Fig. 4). The presence of the filter abrogated killing, made apparent by an increase in E. coli survival and cfu per milliliter values that were indistinguishable from those with no predator, V52ΔvasK, or E. coli as predator (Fig. 4).