enterocolitica ΔHOPEMT ΔYscU. With the exception of CT583-HA, which for unknown reasons was very poorly

TSA HDAC expressed by Y. enterocolitica ΔHOPEMT ΔYscU, these assays indicated that the other 10 proteins analyzed were type III secreted (Figure 3C). Figure 3 Analysis of the T3S Selleckchem PF-4708671 of C. trachomatis full-length proteins by Y. enterocolitica . Y. enterocolitica T3S-proficient (ΔHOPEMT) (A) and T3S-defective (ΔHOPEMT ΔYscU) (B) were used to analyze secretion of full-length C. trachomatis proteins with a C-terminal HA epitope tag. 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 ~5 x 108 and ~5 x 107 bacteria, respectively, were loaded per lane. The known C. trachomatis T3S substrates CT082 [26, 27] and CT694 [14] were used as positive controls, and the C. trachomatis Selleckchem GSK1838705A ribosomal protein RplJ was used as a negative control. SycO is a strictly cytosolic Yersinia T3S chaperone [44, 51] and its immunodetection

ensured that the presence of HA-tagged proteins in the culture supernatants was not a result of bacterial lysis or contamination. (C) The percentage (%) of secretion of each protein by Y. enterocolitica ΔHOPEMT 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 2% (dashed line), based on the % of secretion of RplJ-HA. Data are the mean ± SEM from at least 3 independent experiments. Secretion of full-length CT153-HA, CT172-HA, CT203-HA, CT386-HA or CT425-HA by Y. enterocolitica could occasionally be seen by immunoblotting (Figure 3A); however,

this was not always reproducible and individual average percentage of secretion of these proteins was in all cases below 2% (Figure 3B). We did not detect significant amounts of CT273-HA, CT289-HA, CT309-HA, or CT631-HA in culture supernatants (Figure 3A and Additional file 3: Table S3), but as MycoClean Mycoplasma Removal Kit their levels of expression were either extremely low (CT273-HA, CT289-HA, and CT309-HA) or undetectable (CT631-HA) it was not possible to draw conclusions about secretion of these proteins. Furthermore, CT016-HA, and possibly CT696-HA (barely visible in Figure 3A), were immunodetected in the culture supernatant fraction in a form that migrated on SDS-PAGE at a molecular weight much lower than the one predicted from their amino acid sequence (27 kDa and 46 kDa, respectively), while in the bacterial pellet fraction their migration on SDS-PAGE corresponded roughly to their predicted molecular weight (Figure 3A). This suggests that the proteins could be cleaved during secretion, unstable in the culture supernatant, or their encoding genes possess internal Shine-Dalgarno sequences.