Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

Abstract

Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

“…In samples treated with anhydrotetracycline at concentrations of Ն400 ng/ml for 24 h, C. trachomatis inclusions were visibly smaller in size than untreated samples, suggesting that higher concentrations of anhydrotetracycline are inhibitory to chlamydial growth (data not shown). Similar sensitivity to anhydrotetracycline was recently described by Wickstrum et al (38). Figure 3A depicts induction of mCherry under the control of the tet promoter with as little as 10 ng of anhydrotetracycline hydrochloride/ml (Fig.…”

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

“…A system based on FRT/FLP recombination would permit in vivo gene ablation by introducing flanking FRT sites into a locus of interest and promoting gene excision by inducing trans-expression of the FLP recombinase. Plasmids in which expression is controlled by the Tet-inducible operon have already been developed and can easily be coopted for such approaches (21,26). An inducible FRT/FLP system could also be harnessed for genome editing and selectable marker recycling during the engineering of strains with multiple gene knockouts.…”

“…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).…”

Using Fluorescent Proteins to Visualize and Quantitate <em>Chlamydia </em>Vacuole Growth Dynamics in Living Cells

“…A novel application of this approach is for derivation of meaningful quantitative data about the inclusion, for example inclusion area, volume and shape. Finally, this visualization method is highly adaptable and can be used in combination with other fluorescent markers to reveal additional insight into the biology of Chlamydia infected host cells, for example GFP-or mKate2-expressing Chlamydia 6,11 , fluorescent actin 12 or GFP tagged Inc proteins 13 .…”