Figure 3

Identification of type III secretion signals in putative Inc proteins. A. Secretion assay on colonies. The ipaB (left) or mxiD (right) strains of S. flexneri were transformed with different Chlamydia/Cya constructs, isolated, and one colony for each construct was grown overnight in contact with a PVDF membrane, which served the following day to reveal the localization of the reporter protein using anti-Cya antibodies. All chimera shown in this figure carry a functional TTS assay, which allow the chimera to diffuse in a halo in the ipaB strain but not in the mxiD strain. B. Secretion assay in liquide cultures. Exponential cultures of ipaB or mxiD strains expressing the indicated chimeras were fractionated. The supernatant (S) and pellet (P) fractions were run on SDS-PAGE and western blot was performed using anti-Cya antibody. Membranes were later probed again using anti IpaD and anti CRP antibodies, to check that there was no bacterial lysis and that TTS was functional in the ipaB strain. These controls were systematically performed and are only shown for the first row of constructs tested. The supernatant fractions is concentrated 25-fold compared to the pellet fraction. Note that CPn0169/Cya is detectable in the culture supernatant, but in very low proportion relative to its very high expression level. This is unlike other secreted chimera, we therefore concluded that this protein does not carry a functional TTS signal.