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Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening

Figure 5

Characterization of CBU_1260 (OmpA), a Coxiella protein involved in host cell invasion.

(A). Schematic representation of the genomic context of CBU_1260 and sites of transposon insertion of 5 independent isolated mutants (Tn175, Tn208, Tn27, Tn907, Tn749). Black area indicates the position of the predicted N-terminal α-helix and grey areas indicate the position of predicted β-sheets. Predicted extracellular loops are indicated from L1 to L4. (B). Intracellular growth curves of Coxiella mutants containing transposon insertions in CBU_1260 measured as variation of GFP fluorescence over 7 days of infection and compared to GFP-tagged wt Coxiella (Ctrl). The growth curves of all CBU_1260 mutants were significantly different from that of GFP-Coxiella (P<0.001, 2way ANOVA) from day 2 post-infection. (C). For each CBU_1260 mutant analyzed, the total number of Coxiella colonies identified by automated image analysis was divided by the total number of cell nuclei and expressed as relative to GFP-tagged wt Coxiella (Ctrl) to derive the efficiency of host cell invasion. Values are mean ± standard deviation of triplicate samples where an average of 12000 cells were analyzed per condition (values were compared with Ctrl condition. *** = P<0.001, 2way ANOVA). (D). iTASSER-derived prediction the CBU_1260 product. The central core of the protein is composed of an 8-β-sheet transmembrane barrel domain (light blue). The putative periplasmic domain is composed of a α-helix (dark blue), whereas the predicted extracellular domain is composed of four (L1 to 4) unstructured loops. (E). Representative Western blot of bacterial lysates from wt Coxiella, the control mutant Tn1832 and the 5 mutants presenting transposon insertions in CBU_1260. (F). Representative Western blot of wt Coxiella and mutant Tn208 subjected to membrane fractionation to assess the localization of OmpA (I = insoluble fraction; SN/IM = supernatant and inner membrane fraction, OM = outer membrane fraction). Blots were revealed with an antibody raised against the first extracellular loop (L1) of OmpA (top panels) and with a polyclonal antibody against Coxiella NMII (bottom panels) used as loading control. The arrows indicate the expected size of OmpA (23 kDa).

Figure 5

doi: https://doi.org/10.1371/journal.ppat.1004013.g005