Conditional gene expression in Chlamydia trachomatis using the tet system.

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

Shuttle vector with GFP expression under Tet control.

<p>The <i>E</i>. <i>coli</i> cloning vector pASK-IBA33plus, with GFP inserted under Tet control, was ligated to the wild-type plasmid from <i>C</i>. <i>trachomatis</i> strain L2 using PCR-generated restriction endonuclease sites (see Materials and Methods for details). A variation of this shuttle vector was later constructed with a gene encoding mKate2 inserted at the indicated <i>Kas</i>I site.</p

Detection of constitutive mKate2 and inducible GFP.

<p>L929 cell cultures infected with pASK-GFP/mKate2-L2 transformed <i>C</i>. <i>trachomatis</i> were induced at 24 hpi and images were then taken at indicated time points. Representative images from an uninduced sample and a sample induced with 10 ng/mL ATc at 24 hpi are shown in (A). Representative images of a sample induced with 10 ng/mL ATc at 16 hpi (from a separate experiment) are shown in (B). White arrows highlight the relatively few <i>C</i>. <i>trachomatis</i> cells within the inclusion that did not express detectable GFP at 19 hpi and 20 hpi.</p

Induction of GFP with lower ATc concentrations.

<p>L929 cells were infected with pASK-GFP/mKate2-L2 transformed <i>C</i>. <i>trachomatis</i>. At 24 hpi, samples were induced for 4 hours with the indicated concentrations of ATc. After induction, soluble GFP and mKate2 in each sample was quantified using a microplate fluorometer. To normalize samples, GFP expression was divided by mKate2 expression and the resulting ratio was graphed (A). Increases observed in GFP fluorescence of samples induced with 0.25 ng/mL or higher were statistically significant compared to the 0 ng/mL sample (P < 0.01; Student’s <i>t</i> test). Induction from 16 to 20 hpi was also tested and the resulting GFP and mKate2 expression quantified as described above (B). All ATc concentrations tested for this induction period resulted in statistically significant increases in GFP expression. GFP fluorescence from the experiments shown in panels A and B are also graphed with mKate2 normalization (C). Error bars indicate standard deviation of three separate samples.</p

Effect of anhydrotetracycline on <i>C. trachomatis</i>.

<p>L929 cells were infected with wild-type <i>C</i>. <i>trachomatis</i> L2 and then incubated in the presence of various concentrations of ATc throughout the infection (0 to 24 hpi). Cultures were fixed at 24 hpi and stained for MOMP (green). Representative images from three different concentrations are shown (A). Additionally, the same samples (and additional samples) were analyzed using Cell Profiler to calculate the average inclusion size (B). Over 700 inclusions were analyzed at each concentration. Separately, infected L929 cell cultures were treated with different concentrations of ATc and lysed at 24 hpi. Lysates were used to infect a fresh monolayer (in the absence of ATc) and infection levels were enumerated at 24 hpi using immunofluorescence microscopy. Percent inhibition of progeny was determined relative to a lysate from an untreated control (C).</p

GFP expression after induction at 24 hpi.

<p>L929 cell cultures infected with pASK-GFP-L2 transformed <i>C</i>. <i>trachomatis</i> were induced with various concentrations of ATc at 24 hpi. Images were captured every 10 minutes for a total of 6 hours. Representative images from the culture induced with 10 ng/mL and an uninduced control are shown (A). The images were also analyzed using the Otsu segmentation algorithm to determine the mean fluorescence intensities in arbitrary units within the inclusions (B). Three microscope fields were averaged for each sample with approximately 40 infected cells per field. The mean fluorescence intensity of an uninduced sample was also calculated at each time point and the resulting values were subtracted from the induced samples at the corresponding time points.</p

Detection of GFP gene transcription by qPCR.

<p>L929 cell cultures infected with pASK-GFP-L2 transformed <i>C</i>. <i>trachomatis</i> were induced with 10 ng/mL ATc from 20-24 hpi. The culture medium was then replaced with ATc-free medium and samples were collected at the indicated time points for processing and analysis of GFP transcription by qPCR. Relative expression levels are in comparison to the uninduced sample. Error bars indicate standard deviation from two separate experiments.</p

Detection of GFP induction early in the developmental cycle.

<p>L929 cell cultures infected with transformed <i>C</i>. <i>trachomatis</i> were induced at the start of infection (0 hpi). Samples were taken and lysates prepared at the indicated time points after the start of infection. GFP fluorescence was measured using a microplate fluorometer with the results quantified in arbitrary units. Error bars indicate standard deviation from two separate experiments.</p