Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

GFP expression and mCherry induction in pCps-Tet-mCherry-transformed C. psittaci strain 02DC15. Transformed C. psittaci 02DC15 bacteria were grown in epithelial cells with 1U/ml PEN for 24 and 48 h. (A) GFP fluorescence of chlamydial inclusions was visualized in living cells without fixing and staining. aTC (10 and 100 ng/ml) was added at 1 hpi to induce mCherry expression. Images are representative of three independent experiments. White arrows show chlamydial inclusions. White scale bars represent 10 μm. (B) mCherry was analyzed by Western blotting and densitometric analyses at 24 and 48 hpi. mCherry protein amounts were normalized to chlamydial HSP60. GAPDH was used as a loading control. (n = 4; mean ± SEM; Sidak’s multiple comparison: ***, P ≤ 0.001).

One-step growth curve and inclusion morphology of pCps-Tet-mCherry-transformed and untransformed C. psittaci strain 02DC15. (A) C. psittaci strain 02DC15 transformed or untransformed with pCps-Tet-mCherry was grown in epithelial cells with or without 10 U/ml PEN and 0, 10, or 100 ng/ml of aTC. Recoverable C. psittaci 02DC15 IFUs were determined at 5, 24, 48, and 72 hpi. The numbers of recoverable C. psittaci 02DC15 under each condition (II to VIII) at the indicated time were compared to those of untransformed C. psittaci 02DC15 without PEN (I). (n = 3 to 6; mean ± SEM; Sidak’s multiple comparison: **, P ≤ 0.01; ***, P ≤ 0.001). (B) Representative immunofluorescence images of pCps-Tet-mCherry-transformed or untransformed C. psittaci 02DC15 at 5, 24, 48, and 72 hpi. Chlamydial inclusions were stained by FITC-labeled monoclonal chlamydial-LPS antibodies. Evans blue counterstaining of host cells was used for better characterization of intracellular inclusions. Images are representative of 3 to 6 independent experiments. White scale bars represent 10 μm.

The pCps-Tet-mCherry plasmid can be stably maintained by C. psittaci strain 02DC15 and expresses GFP. (A) pCps-Tet-mCherry-transformed C. psittaci 02DC15 bacteria were subcultured in epithelial cells in the presence or absence of 10 U/ml PEN every 2 days over 5 passages. Quantitative PCR was performed using primers specific to genomic DNA or to the pCps-Tet-mCherry plasmid. Copy numbers of pCps-Tet-mCherry were normalized to genomic DNA at each passage in the presence or absence of PEN, and were compared to passage 0 of C. psittaci 02DC15 in the presence of PEN. (n = 3; mean ± SEM, Sidak’s multiple comparison: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Representative GFP and immunofluorescence images of pCps-Tet-mCherry-transformed C. psittaci 02DC15 cells 48 hpi at passage 5. After a GFP signal was detected by fluorescence microscopy, the cells were fixed by methanol. Then, chlamydial inclusions were stained by FITC-labeled monoclonal chlamydial-LPS antibodies. Evans blue counterstaining of host cells was used for better characterization of intracellular inclusions. IF, immunofluorescence; LPS, lipopolysaccharide. White arrows show C. psittaci 02DC15 that lost pCps-Tet-mCherry during passages. White scale bars represent 20 μm.