Primers (available upon request) were designed using Primer Express (Applied Biosystems). The RT-qPCR assays were done using the ABI 7000 SDS, SYBR green chemistry, and optical plates (Applied

Biosystems), as previously described [52]. At each time point, raw RT-qPCR data for each gene were normalized against the data obtained for the 16S rRNA transcript, as it was previously demonstrated that this is an adequate endogenous control [52]. The final results were based on three independent experiments. Results Selection of C. trachomatis proteins analyzed in this work To search for previously unidentified T3S substrates of C. trachomatis, we first surveyed the INK1197 genome of strain L2/434 for genes encoding mostly uncharacterized proteins, or with a putative biochemical activity compatible with the function of a T3S effector (e.g., proteases). Among these genes, we selected those encoding proteins not predicted to have a signal sequence characteristic of the general secretory pathway (according to Psortb v3.0) and that had not been previously analyzed experimentally for the presence of a T3S signal. This singled out 32 proteins (CT016, CT017, CT031, CT051, CT053, CT080, CT105, CT142,

CT143, CT144, CT153, CT161, CT172, CT273, CT277, CT289, CT309, CT330, CT338, CT386, CT425, CT568, CT583, CT590, CT631, CT635, CT656, CT696, CT702, CT837, CT845, and CT849; we used the nomenclature of the annotated C. trachomatis D/UW3 strain [53]; the names of the corresponding genes as annotated A1155463 for strain L2/434 [54] can be found in Additional file 3: Table S3). Furthermore, for comparison purposes, we considered proteins that had been tested for the presence of a T3S signal using Shigella flexneri as a heterologous bacteria: eight proteins whose first ~40 amino acids of the corresponding C. pneumoniae homologs did not drive secretion of an adenylate cyclase Glutathione peroxidase (Cya) reporter protein by S. flexneri (CT066, CT429, GrgA/CT504, CT538, CT584, CT768, CT779, CT814), and three

proteins whose N-terminal region of the C. pneumoniae homologs drove secretion of a Cya reporter protein by S. flexneri (CT203, CT577, CT863) [21]. Please note that at the time this work was initiated GrgA/CT504 was an uncharacterized protein; however, it was recently described as a transcriptional ICG-001 clinical trial activator [55]. Finally, throughout this study we used as positive controls a C. trachomatis bona-fide T3S effector (CT694) [14] and a C. trachomatis likely T3S substrate (CT082) that we had previously identified [26], and which was recently independently confirmed [27], and as negative control a predicted ribosomal protein (RplJ/CT317). In summary, in experiments that will be described below, we analyzed T3S signals in 46 C. trachomatis proteins (~5% of all proteins encoded by the L2/434 strain): 32 hypothetical proteins previously not analyzed experimentally for T3S signals, 11 proteins whose C. pneumoniae homologs were previously analyzed for T3S signals using S.