After having determined optimal conditions, we confirmed the capability of the assay to detect disruption of Pa virulence by testing a T3SS-deficient strain of Pa. Infection of A549 cells with the mutant strain PAO1F∆pscD, which lacks the ability to produce pscD, an essential inner membrane T3SS component [26], led to a 5-fold increase of viability compared to cells infected with the PAO1F wildtype strain (Figure 1C). The gene discussed is DHRS4; the disease is infection.