Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in the Chlamydia trachomatis developmental cycle

Abstract

Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes to Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the growth of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated while early genes maintained their expression. Importantly, the growth defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes of C. trachomatis.

Keywords: CRISPRi; Chlamydia trachomatis; DNA topoisomerase; DNA topology; TopA; antibacterial mechanism; bacterial developmental cycle; dCas12; quinolone; transcription.

Figure 1.. Conditional repression of topA transcription in C. trachomatis.

(a) Schematic representation of the strategy used to make a targeted topA knockdown through dCas12 and a specific crRNA, whose targeting site is indicated by the red X. (b) Immunoblotting analysis of dCas12 expression. C. trachomatis L2/topA-kd and L2/Nt (control) infected cells were cultured in medium containing aTC (10ng/mL) for 20 hrs starting at 4 h pi and sampled for immunoblotting with rabbit anti-dCas12 antibody. Host cell α-tubulin was probed with a mouse anti-tubulin antibody and used as a protein loading control. (c) Immunofluorescence micrograph of C. trachomatis grown in the absence (-aTC) or presence of aTC (+aTC). Fixed cells at 40 h pi were immunolabeled with rabbit anti-dCas12 antibody and visualized with Alexa Fluor 568-conjugated goat anti-rabbit IgG. C. trachomatis expressing GFP (green) and dCas12 (red) are shown. Host cell and bacterial DNA were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar=100μm. (d) Fold change in relative topA transcript levels in the absence or presence of aTC. RT-qPCR was performed with C. trachomatis infected cells grown under dCas12-inducing or mock inducing conditions for 11 hrs (to 15h pi) and 20 hrs (to 24h pi) starting from 4 h pi. Chlamydial genomic DNA (gDNA) copy from respective culture was determined by qPCR using primers specific to housekeeping tufA gene. Relative quantitation of topA specific transcripts were normalized to the gDNA value. The data are presented as the ratio of relative topA transcript in the presence of aTC to that in the absence of aTC, which is set at 1 as shown by a black dashed line. The data and standard deviation (SD) of three independent biological replicates are shown. ***p < 0.005, ****p < 0.0001. Statistical significance in all panels was determined by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 2.. Targeted knockdown of topA causes intracellular growth arrest of C. trachomatis.

(a) One-step growth curve of C. trachomatis. HeLa cells were infected with C. trachomatis L2/topA-kd or L2/Nt at the dose that resulted in 40% cell infection (multiplicity of infection, MOI= 0.4) and cultured in the absence or presence of aTC (at 10ng/mL). Cells sampled at 0, 12, 24, 30, or 48h pi (x-axis) were used for determination of inclusion forming unit (IFUs; y-axis) on fresh HeLa monolayers. IFU values are expressed as the mean ± standard deviation (SD) from triplicate samples. Experiment was repeated three times. (b) Representative immunofluorescence images of C. trachomatis L2/topA-kd. Infected HeLa cells were grown under the conditions of dCas12 induction for 20 h (+aTC4–24h), transient induction from 4 to 8 h pi (+aTC 4h/−8h), or mock induction (−aTC). Fixed cells at 24 h pi were immunolabeled with monoclonal antibody to C. trachomatis major outer membrane protein (MOMP) and visualized with Alexa Fluor 568-conjugated goat anti-mouse IgG. The DAPI-stained DNA (blue), MOMP (red), and C. trachomatis expressing GFP (green) are shown. The automated images were obtained with a 20× objective using Cytation 1. Scale bar=20 μm. (c) Histogram displays frequency of the individual C. trachomatis inclusion sizes that were calculated using Gen 5 software. Graph shows measurement of one representative well with 9 different fields per condition. Three independent trials were performed. (d) Relative IFUs in C. trachomatis in the absence or presence of aTC for 20 hrs or 4 hrs. Triplicate results in a representative experiment are shown as mean ± SD. Values are presented as the percentage of IFU from dCas12 induced sample to that from respective mock induction sample, which is set at 100 as indicated by a red line. At least four independent experiments were performed. Statistical significance in all panels was determined by one-way ANOVA followed by Tukey’s post-hoc test. ****P<0.0001; ***P<0.001.

Figure 3.. Dose- and time-dependent effects of targeted topA knockdown on P_Nmen-gfp expression in C. trachomatis L2/topA-kd.

(a) Quantification of gfp expression using RT-qPCR. The sites of primers used to detect gfp from the sample cDNA are indicated. The gfp mRNA concentrations were normalized to the DNA control as determined by qPCR targeting tufA and presented as mean ± SD of three biological replicates. (b) Live-cell images of C. trachomatis. HeLa cells were infected with C. trachomatis L2/topA-kd at MOI~0.3 and cultured in aTC free medium. Increasing concentrations of aTC (0, 2.5, 5, or 10ng/mL) were added starting at 4 h pi or 16 h pi. The automated imaging acquisition was performed at 24 h pi under the same exposure conditions with Cytation 1. Scale bar=20 μm. (c) Immunoblotting analysis of dCas12 and MOMP expression. Increasing concentration of aTC was added at 4 h pi to induce dCas12 expression. Densitometry of the blot was assessed using ImageJ. Values are presented as the density of the dCas12 band (the upper panel) normalized to the MOMP band (the lower panel) from the same sample. Host cell α-tubulin was used as protein loading control. Data were collected from two independent experiments. Note: a small amount of dCas12 leaky expression was detected in the absence of aTC. (d) Measurement of GFP MFI (mean fluorescence intensity) in C. trachomatis infected cells grown in the absence or presence of aTC. Individual inclusions were analyzed using the Gen5 software. The MFI values are presented as mean ± SD from the indicated inclusion numbers (N) per condition in replicate wells. ****p < 0.0001, comparison was made using one-way ANOVA followed by Tukey’s post-hoc test.

Figure 4.. Secondary differentiation of RB to EB is impaired by topA knockdown in L2/topA-kd.

(a) Analysis of C. trachomatis genomic copy numbers (i.e., gDNA) in the absence or presence of aTC using real-time qPCR targeting the tufA gene. Values are presented as the ratio of chlamydial DNA copy numbers per ng DNA in +aTC sample to that of −aTC sample, which is set at 1. Triplicate results in a representative experiment are shown. At least two independent experiments were performed. (b) Quantification of transcripts of omcB or incD in C. trachomatis using RT-qPCR. The mRNA concentrations were normalized to the DNA control as determined by qPCR targeting tufA and presented as mean ± SD of four biological replicates. (c)-(d) Immunofluorescent micrographs of C. trachomatis expressing OmcB. HeLa 229 cells were infected with C. trachomatis L2/topA-kd (c) or L2/Nt (d), cultured in the absence (−aTC,) and presence of aTC (+aTC, 10 ng/mL) for 20 h pi, and fixed at 24 h pi for IFA. Cells were immunolabeled with rabbit polyclonal antibody to C. trachomatis OmcB and visualized with Alexa Fluor 568-conjugated goat anti-rabbit antibody. DAPI-counterstained DNAs (blue) and C. trachomatis organisms expressing GFP (green) and OmcB (red) were shown. Scale bar= 10 μm. Statistical significance in all panels was determined by one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5.. Complementation of the growth defect of topA knockdown in C. trachomatis by co-expressing topA-His₆.

(a) Schematic map of the expression vector containing topA-His₆ that is co-regulated with the dcas12 by Ptet. (b) RT-qPCR analysis of topA transcripts in C. trachomatis L2/topA-kdcom. Nucleic acid samples from HeLa cells infected with L2/topA-kdcom were collected at 24 h pi. The locations of primers used to detect topA from the cDNA samples are shown in (a). (c) Immunoblotting displays the inducible expression of TopA-His₆ in C. trachomatis. TopA-His₆ protein from the lysates of C. trachomatis infected cells were isolated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis for immunoblotting with antibody against His₆ or MOMP as a protein loading control. (d) Enumeration of EBs. C. trachomatis L2/Nt, L2/topA-kdcom, or L2/topA-kd infected cells were cultured for 40 hrs in the presence of increasing aTC amounts (at 0, 2.5, 5, and 10 ng/mL) and used for IFU assays. Values are presented from triplicate results in a representative experiment and are shown as mean ± SD. At least four independent experiments were performed. (e) Live-cell images of Chlamydia infected HeLa cells. Images were taken at 24 h pi with a 20× objective using Cytation 1. Scale bar=20 μm. (f) Analysis of changes in P_Nmen-GFP levels as indicated as mean fluorescence intensity (MFI). Individual chlamydial inclusions from (e) were measured and calculated using Gen 5 software. The inclusion numbers (n) measured per condition are as indicated. For all panels, comparison was performed by ANOVA. ****p<0.0001; ***p<0.001, ****p < 0.0001.

Figure 6.. The effects of CRISPRi-induced topA knockdown on expression of DNA gyrase genes and topoIV genes in C. trachomatis.

(a) Schematic map of gyrB/gyrA operons in C. trachomatis and detection of their transcript products using RT-qPCR. (b) Schematic map of parE/parC in C. trachomatis and detection of their transcript products using RT-qPCR. L2/top-kd infected HeLa cells grown in the presence or absence of aTC were harvested at 24 h pi for total RNA preparation and then cDNA synthesis. Results of a representative experiment from triplicate samples are reported as mean ± SD. Three independent experiments were performed. **p < 0.005, *** P< 0.001. Comparison was made using one-way ANOVA and Tukey’s post-hoc test. Primer pairs overlapping gene pairs used for RT-qPCR analysis are shown (arrows).

Figure 7.. Analysis of the response of C. trachomatis to the antibiotic moxifloxacin.

(a) Live-cell images of Chlamydia infected HeLa cells. C. trachomatis L2/topA-kd, L2/Nt, or L2/topA-kdcom infected cells were cultured in the absence or presence of Mox or aTC+Mox and imaged at 44 h pi. Scale bar=30μm. (b)-(c) Comparison of the chlamydial inclusion sizes (b) and the GFP MFI (c) of L2/topA-kd to those of L2/Nt and L2/topA-kdcom. Two hundred individual chlamydial inclusions per condition from images in (a) were measured using Gen 5 software. d. Enumeration of EB yields using IFU assay. C. trachomatis infected cells were harvested at 40 h pi for IFU assay. Values are presented from triplicate results in a representative experiment and are shown as mean ± SD. Three independent experiments were performed. (e) Analysis of C. trachomatis gDNA in the presence or absence of aTC using real-time qPCR. Values are presented as the ratio of chlamydial DNA copy numbers per ng DNA in treated sample to that in the untreated sample, which is set at 1. Triplicate results in a representative experiment are shown. Three independent experiments were performed. Statistical significance in all panels was determined by one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 8.. Schematic highlighting the role of TopA in C. trachomatis developmental cycle.

a. In wild-type C. trachomatis, optimal supercoiling levels during chlamydial developmental cycle progression is maintained by action of both topA that relaxes DNA supercoiling (RX) and gyrase that induces negative supercoiling (SC). b. When topA is repressed, the DNA supercoiling is predicted to increase, resulting in changes in expression of supercoiling-sensitive genes (e.g., chromosomal gyrB/gyrA and omcB, and the plasmid-encoded P_Nmen-gfp), thus, perturbing chlamydial development. Our data indicate that the carefully balanced activities of TopA and gyrase contribute to the completion of the chlamydial developmental cycle.