Virulence study on putative effector proteins. To compare the viability of deletion strains lacking putative effector proteins with that of wild-type Salmonella, competitive infection was performed in five 129SvJ mice for each mutant. ΔsrfN, ΔpagK2, and ΔpagJ/pagK1/pagK2 were transformed with pSrfN or pPagK2 to test virulence complementation, whereas wild-type Salmonella was transformed with an empty plasmid, pWKS30. A reference strain (MA6054) harboring arabinose-inducible β-galactosidase activity and each test (wild-type or mutant) strain were mixed equivalently and 10,000 CFU of the mixed cells was used to infect a mouse. Mice were sacrificed at 7 days post-infection and the spleen lysates were spread on plates containing X-gal and arabinose to compare the numbers between the wild-type strain and the mutant strain. The competitive index (CI) was determined by the formula described in Methods. Each circle represents the CI for a single mouse in each group. A CI of less than 1 indicates that a test strain is outcompeted by the reference strain in mouse. The averaged CI for each group is shown as a solid line and statistically significant p-value is indicated with an asterisk (p-value < 0.001, Student's t-test).