SpaO^S is not essential for T3SS function but is required for the stability of SpaO^L.

(A) Western blot analyzing SipB, SipC, and InvJ secretion in the presence or absence of SpaOS (WCL = whole cell lysate; Sup. = supernatant) (B) Gentamycin protection assay to test the ability of the indicated S. Typhimurium strains in the presence or absence of SpaOS to invade cultured epithelial cells, a measure of the functionality of the SPI-1 T3SS. Numbers represent the percentage of the inoculum that survived antibiotic treatment due to internalization and are the mean ± standard deviation of three independent experiments. The difference between the levels of invasion of the two strains was not statistically significant (p = 0.6728, unpaired Student’s t test). (C) Western blot analyzing the stability of SpaOL in the absence of SpaOS, OrgB, or both in whole cell lysates from the indicated S. Typhimurium strains.