Figure 4. Effect of the expression of Slc1 or CT584 on Yersinia type III secretion (T3S).

(A) Y. enterocolitica ΔHOPEMT carrying plasmids encoding Slc1-HA, CT584-HA or CT790-HA, and YopE-Myc (as indicated) were incubated in T3S-inducing conditions [42]. Proteins in culture supernatants (S - secreted proteins), and in bacterial pellets (P - non-secreted proteins) were analyzed by SDS-PAGE followed by Coomassie staining and immunoblotting with the indicated antibodies. Immunodetection of TEM-1 β-lactamase (encoded by the plasmid expressing YopE-Myc, a pBAD-Myc-His–derivative) ensured that presence of proteins in culture supernatants was not a result of bacterial lysis or contamination. Proteins from culture supernatants corresponding to ∼5×10⁸ or ∼2.5×10⁸ bacteria and from bacterial pellets corresponding to ∼5×10⁷ or ∼2.5×10⁷ bacteria were analyzed by Coomassie staining or by immunoblotting, respectively. (B) The amount of YopE-Myc in the bacterial pellets and in the secreted fractions was analyzed by densitometry from images of immunoblots. (C) The amount of YscP (graph on the left) and of YopB, YopD, LcrV and YopN (graph on the right) in secreted fractions was analyzed by densitometry from images of Coomassie-stained gels. YopB, YopD, LcrV, and YopN were analyzed all together because single bands corresponding to these proteins could not be easily distinguished in Coomassie-stained gels. The bands corresponding to YopB, YopD, LcrV, YopN, or YscP were deduced from the well known separation on SDS-PAGE of Yersinia secreted proteins after a T3S assay [42]. In (B) and (C), we calculated the ratio between the amounts of YopE-Myc, YscP, or YopB, YopD, LcrV, and YopN when they were expressed in the presence of chlamydial proteins (Slc1-HA, CT584-HA, or CT790-HA) relative to when they were expressed alone (+ chlamydial proteins/− chlamydial proteins). All data are the mean ± SEM from 3 independent experiments.