At 20 h p.i., the immunofluorescence transmission for CT143 was more intense but still restricted to the inclusion (Fig 3A). of antibodies against GSK1059865 CT142 and CT143. HeLa cells were either left untransfected (NT) or transfected using jetPEI? (Polyplus-transfection) with plasmids encoding EGFP, EGFP-CT143 or EGFP-CT142, as indicated. Whole cell lysates were analyzed with antibodies against GFP, CT142 or CT143. Bands corresponding to EGFP-CT142 and EGFP-CT143 are indicated by an arrow.(PDF) pone.0178856.s006.pdf (65K) GUID:?824754AC-0D6C-4698-B75F-CE3F5536C526 S4 Fig: Quantification of CT142, CT143, and CT144 proteins produced by recombinant strains. (A) HeLa cells were left uninfected (UI) or infected for 30 h by L2/434 or L2/434 transporting plasmids pCT142-2HA, pCT143-2HA, pCT144-2HA or pCT142-CT143-CT144-2HA. Whole cell lysates were analyzed by immunoblotting with antibodies against HA, CT142 or CT143 (as indicated), Hsp60 (bacterial loading control) and tubulin (loading control for host cells). The arrows indicate the position of CT142, CT142-2HA, CT143 and CT143-2HA bands. (B) The bands corresponding to HA-tagged proteins in each lane of the anti-HA blot in Fig A in S4 Fig (and in other replicates) were quantified by densitometry relative to the corresponding bands of Hsp60 and tubulin using Fiji software . The calculated CT142/Hsp60/tubulin GSK1059865 values in the graph indicate mean standard error of the mean (SEM) from 4 impartial experiments. P-values were calculated by a one of the ways ANOVA and Tukey post hoc analysis and the values were not significantly different (P 0.05) between the different data sets. (C and D) The bands corresponding to CT142 and CT142-2HA proteins (C), or the bands corresponding to CT143 and CT143-2HA proteins (D), in each lane of the anti-CT142 or CT143 blots, respectively, in Fig A in S4 Fig (and in other replicates) were quantified by densitometry relative to the corresponding bands of Hsp60 and tubulin using Fiji software . The calculated HA/Hsp60/tubulin values in the graph indicate mean SEM from 4 impartial experiments, relative to the values of CT142 (in C) or CT143 (in D) in cells infected by L2/434. P-values GSK1059865 were calculated by a one of the ways ANOVA and Dunett post hoc analysis (relative to the L2/434 data) and there were significant differences (P 0.05) only for data corresponding to samples GSK1059865 infected by L2/434 harbouring pCT142-2HA or pCT142-CT143-CT144-2HA (in C) or for data corresponding to samples infected by L2/434 harbouring pCT143-2HA.(PDF) pone.0178856.s007.pdf (186K) GUID:?E176DA30-B1AA-44D0-B630-058F0B3BBCB8 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract is usually a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in confirmed the prediction that and are organized in an operon and revealed that their expression is likely driven CT96 by the main factor, 66. In host cells infected by strains transporting plasmids generating CT142, CT143, or CT144 under the control of the promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the.