The Genetic Transformation of Chlamydia pneumoniae

Abstract

We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis, C. muridarum, C. caviae, C. pecorum, and C. abortus However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniaeC. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

Keywords: Chlamydia felis; Chlamydia pneumoniae; genetic manipulation; plasmid shuttle vector; plasmid tropism; transformation.

FIG 1

Map of the C. pneumoniae-derived shuttle vector pRSGFPCAT-Cpn and the RSGFP expression in C. pneumoniae LPCoLN. (A) The CDSs of pCpnE1 and pRSGFPCAT are shown in orange and light gray, respectively. The red-shifted green fluorescent protein gene (RSGFP) is shown in green. MCIP, meningococcal class I protein promoter; CAT, chloramphenicol acetyltransferase gene. (B) pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae LPCoLN. Transformed (+) C. pneumoniae was grown in HEp-2 cells with chloramphenicol, and untransformed (−) C. pneumoniae was grown without chloramphenicol for 48 h. GFP fluorescence of chlamydial inclusions was visualized in living cells without fixing and staining. Images are representative of three independent experiments. White arrows show chlamydial inclusions. Bars, 10 µm.

FIG 2

One-step growth curve and the inclusion morphology of pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae LPCoLN. (A) Recoverable C. pneumoniae at 5, 24, 48, and 72 hpi. pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae bacteria were grown in HEp-2 cells with or without chloramphenicol. The numbers of recoverable C. pneumoniae bacteria under each condition (II, III, and IV) at the indicated time were compared to those of untransformed C. pneumoniae without chloramphenicol (I) (n = 3, mean ± SEM; Sidak’s multiple comparison; *, P ≤ 0.05; ***, P ≤ 0.001). (B) Representative immunofluorescence images of pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae at 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 three independent experiments. Bars, 10 µm. (C) pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae bacteria with or without chloramphenicol treatment were analyzed with a TEM at 24, 48, and 72 hpi. Black bars, 2 µm; white bars, 10 µm.

FIG 3

The pRSGFPCAT-Cpn plasmid can be stably retained in C. pneumoniae LPCoLN and expresses RSGFP. (A) pRSGFPCAT-Cpn-transformed C. pneumoniae was subcultured in HEp-2 cells with and without chloramphenicol every 3 to 4 days over 5 passages. The ratio of live RSGFP expressed in inclusions to immunofluorescence staining (IF) of chlamydial inclusions by mouse anti-chlamydial LPS antibody was calculated. No statistically significant difference by one-way analysis of variance was found (n = 3, mean ± SEM, Sidak’s multiple comparison). (B) Representative RSGFP and immunofluorescence images of pRSGFPCAT-Cpn-transformed C. pneumoniae LPCoLN 48 hpi at passage 5. After an RSGFP signal was detected by fluorescence microscopy, the cells were fixed by methanol and 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. Bars, 20 µm.

FIG 4

RSGFP expression in pRSGFPCAT-Cpn-transformed human cardiovascular isolate C. pneumoniae CV-6. pRSGFPCAT-Cpn-transformed (+) C. pneumoniae CV-6 was grown in HEp-2 cells with chloramphenicol, and untransformed (−) C. pneumoniae was grown without chloramphenicol for 24, 48, and 72 h. GFP fluorescence of chlamydial inclusions was visualized in living cells without fixing and staining. Images are representative of three independent experiments. Bars, 10 µm.

FIG 5

One-step growth curve and the inclusion morphology of pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae CV-6. (A) Recoverable C. pneumoniae at 5, 24, 48, and 72 hpi. pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae bacteria were grown in HEp-2 cells with or without chloramphenicol. The numbers of recoverable C. pneumoniae bacteria under each condition (II, III, and IV) at the indicated time were compared to those of untransformed C. pneumoniae without chloramphenicol (I) (n = 4, mean ± SEM; Sidak’s multiple comparison, ***, P ≤ 0.001). (B) Representative immunofluorescence images of pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae at 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 four independent experiments. Bars, 10 µm. (C) pRSGFPCAT-Cpn-transformed and untransformed C. pneumoniae bacteria with or without chloramphenicol treatment were analyzed with a TEM at 24, 48, and 72 hpi. Black bars, 2 µm; white bars, 5 µm.

FIG 6

The pRSGFPCAT-Cpn plasmid can be stably retained in C. pneumoniae CV-6 and expresses RSGFP. pRSGFPCAT-Cpn-transformed C. pneumoniae was subcultured in HEp-2 cells with and without chloramphenicol every 2 to 3 days. Representative RSGFP and immunofluorescence staining (IF) images of pRSGFPCAT-Cpn-transformed C. pneumoniae CV-6 were taken 72 hpi at passage 5. After an RSGFP signal was detected by fluorescence microscopy, the cells were fixed by methanol and 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. Bars, 20 µm.

FIG 7

The RSGFP expression in different C. felis strains. pRSGFPCAT-Cpn-transformed and untransformed C. felis strains, C. felis N.I. (A), C. felis Cello (B), and C. felis 02DC26 (Cf02-23) (C). Transformed (+) C. felis strains were grown in HEp-2 cells with chloramphenicol, and untransformed (−) C. felis strains were grown without chloramphenicol for 48 h. GFP fluorescence of chlamydial inclusions was visualized in living cells without fixing and staining. Images are representative of three independent experiments. Arrows show chlamydial inclusions. Bars, 10 µm.