Identification of T3S signals in C. trachomatis proteins using Y. enterocolitica as a heterologous system. Y. enterocolitica T3S-proficient (ΔHOPEMT) (A) and T3S-defective (ΔHOPEMT ΔYscU) (B) were used to analyze secretion of hybrid proteins comprising the first 20 amino acids of selected C. trachomatis proteins or the first 20 amino acids of Y. enterocolitica SycT fused to the mature form of TEM-1 β-lactamase (TEM-1). Immunoblots show the result of T3S assays in which proteins in culture supernatants (S, secreted proteins) and in bacterial pellets (P, non-secreted proteins) from ~2.5x108 and ~5x107 bacteria, respectively, were loaded per lane. TEM-1 hybrids of the known C. trachomatis T3S substrates CT082 [26, 27] and CT694  were used as positive controls. SycT and SycO are strictly cytosolic Yersinia T3S chaperones [44, 51]. SycT20-TEM-1 was a negative control for the T3S assays. Immunodetection of SycO ensured that the presence of TEM-1 hybrid proteins in the culture supernatants was not a result of bacterial lysis or contamination. The percentage (%) of secretion of each TEM-1 hybrid was calculated by densitometry, as the ratio between the amount of secreted and total protein. The threshold to decide whether a protein was secreted was set to 5% (dashed line), based on the% of secretion of SycT20-TEM-1. Data are the mean ± SEM from at least 3 independent experiments.