Characterization of Chlamydia trachomatis Plasmid-Encoded Open Reading Frames

Abstract

The recent success in transformation of Chlamydia trachomatis represents a major advancement in Chlamydia research. Plasmid-free C. trachomatis serovar L2 organisms can be transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT. Deletion of plasmid genes coding for Pgp1 to Pgp8 in pBRCT led to the identification of Pgp1, -2, -6, and -8 as plasmid maintenance factors; Pgp4 as a transcriptional regulator of chlamydial virulence-associated gene expression; and Pgp3, -5, and -7 as being dispensable for chlamydial growth in vitro. Using the pGFP::SW2 vector system, we confirmed these findings in the current report. To further dissect the roles of pgp coding sequences and Pgp proteins in plasmid maintenance, we introduced premature stop codons into the pgp genes. Stable transformants were obtained with pGFP::SW2 derivatives carrying premature stop codons in pgp8 but not in pgp1, pgp2, and pgp6, suggesting that the pgp8 coding sequence but not the Pgp8 protein is required for maintaining the plasmid, while Pgp1, -2, and -6 proteins are necessary for plasmid maintenance. We also found that a minimum of 30 nucleotides in the pgp3 coding region was required for pgp4 expression. Finally, mCherry red fluorescent protein was successfully expressed when the mCherry gene was used to replace the pgp3, pgp4, or pgp5 coding region, indicating that these regions can be used to express nonchlamydial genes in chlamydial organisms. These novel observations have provided information for further use of chlamydial plasmid shuttle vectors as genetic tools to understand chlamydial biology and pathogenicity as well as to develop attenuated chlamydial vaccines.

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Transformation of exogenous DNA into Chlamydia has been achieved via several methods, including electroporation, dendrimer-based delivery, and a calcium chloride-based treatment (14)(15)(16)(17)(18)(19). Successful and stable transformation of C. trachomatis LGV L2 with an Escherichia coli-C. trachomatis L2 shuttle plasmid conferring ␤-lactam resistance led to the development of expression vectors for expressing Chlamydia open reading frames (ORFs), fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein [CFP], mCherry, and mKate2), and reporter proteins (␤-galactosidase, adenylate cyclase, and glycogen synthase kinase [GSK]-tagged proteins) (19)(20)(21)(22)(23)(24)(25)(26). A conditional expression vector was also developed using a tetracycline-inducible system as well as vectors conferring chloramphenicol and blasticidin resistance (21,(26)(27)(28)(29).…”

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

“…The L2-17 organisms (10) were transformed with the various plasmids created as described above by using the transformation protocol initially developed by Wang et al (32), but with additional modifications (26,33). Briefly, 10 l L2-17 organisms (1 ϫ 10 7 inclusion-forming units [IFU]) and 10 l plasmid DNA (ϳ7 g) were mixed in a total volume of 200 l CaCl 2 buffer for 45 min at room temperature (RT).…”

“…The three DNA fragments were linked together by using PCR (AccuPrime Pfx SuperMix; Life Technologies, Grand Island, NY). The cpaF-containing DNA fragment, functioning as an independent operon, was inserted between the Escherichia coli origin of replication and the gfp gene of the pGFP::SW2 vector (kindly provided by Ian Clarke, University of Southampton [32]) using an In-fusion HD cloning kit (Clontech Laboratories Inc., Mountain View, CA) as described previously (26,33). The fusion products were rescued by transformation into Stellar competent bacterial cells.…”

“…The various L2-17 transformants were generated as described below. All chlamydial organisms were propagated, purified, aliquoted, and stored as described previously (26). For infection, cells grown in 24-well or 6-well plates with or without coverslips were inoculated with chlamydial organisms as described previously (27).…”

Evaluation of protective and pathological immune response against chlamydial infection and re-infection in mice

“…Moreover, a sequence of 30 or more nucleotides in the pGP3 gene was required for the optimal expression of pGP4 86 . Moreover, we detected strong monomeric pGP3-specific antibody production after a single inoculation with C. muridarum in C57BL/6N mice.…”