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

Abstract

Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infection. 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 contributes to controlling 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 developmental cycle 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 developmental 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 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 developmental and transcriptional processes of C. trachomatis.IMPORTANCEWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.

Keywords: CRISPRi; Chlamydia trachomatis; DNA topoisomerase; antibacterial mechanism; bacterial developmental cycle; dCas12; fluoroquinolone; gene expression; moxifloxacin; topA.

Fig 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) Immunoblot for dCas12 in L2/topA-kd or L2/Nt-infected HeLa cells in the absence or presence of aTC (10 ng/mL) added at 0 h pi. Cells were sampled at 24 h pi for immunoblotting with rabbit anti-dCas12 primary antibody and anti-rabbit horseradish peroxidase-conjugated secondary 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 showing the relatively low levels of GFP in L2/topA-kd compared to L2/Nt. C. trachomatis-infected HeLa cells were grown in the absence or presence of aTC (10 ng/mL), fixed at 40 h pi, and subjected to IFA with rabbit anti-dCas12 antibody and then Alexa Fluor 568-conjugated goat anti-rabbit IgG to visualize. Host cell and bacterial DNA were counterstained with DAPI. The automated images were obtained using Cytation 1 and analyzed by the software Gen-5. C. trachomatis expressing GFP (green), dCas12 (red), and DAPI-stained DNA (blue) is shown. (d) The quantitative data from panel c are presented as relative MFI of GFP (upper panel) and dCas12 (lower panel); each is normalized to DAPI in the same inclusion. The number of the inclusion counts per condition is 43 ± 9. Scale bar = 100 µm. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ***P ≤0.001, ****P ≤ 0.0001. (e) Fold change in topA transcript levels. RT-qPCR was performed with C. trachomatis-infected cells grown under inducing (+aTC) or mock inducing (−aTC) conditions starting from 4 h pi for 11 h (to 15 h pi) and 20 h (to 24 h pi). Quantified topA-specific transcripts were normalized to the gDNA value from respective cultures using primers targeting topA. 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 red line. Data from three biological replicates of an experiment are shown. At least three independent experiments were performed. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test. ***P ≤ 0.001, ****P ≤ 0.0001. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; IFA, immunofluorescence assay; MFI, mean florescence intensity; ns, no significance.

Fig 2

Targeted knockdown of topA decelerates the developmental cycle 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 = 0.4) and cultured in the absence or presence of aTC (10 ng/mL) added at 0 h pi. Cells sampled at 0, 12, 24, 30, or 48 h pi (time h pi, x-axis) were used for determination of IFUs (y-axis) on fresh HeLa monolayers. IFU values are expressed as the mean ± SD from triplicate samples. Experiment was repeated at least 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 (+aTC4-24 h), transient induction from 4 to 8 h pi (+aTC 4 h-8h), or mock induction (−aTC). Fixed cells at 24 h pi were immunolabeled with mouse monoclonal antibody to C. trachomatis L2 MOMP and visualized with Alexa fluor 568-conjugated goat anti-mouse IgG. The automated images were acquired and analyzed using cytation1 and Gen5. The DAPI-stained DNA (blue) and C. trachomatis expressing GFP (green) and MOMP (red) are shown. Scale bar = 20 µm. (c) Histogram displays the distribution of individual C. trachomatis inclusion sizes y-axis, percentage of inclusion counts; x-axis, inclusion size [μm]). Graph shows inclusion measurement of one representative well with nine different fields per condition. The yellow lines indicate median inclusion size counted. Three independent trials were performed with similar results. (d) Relative IFUs in the absence or presence of aTC for 20 or 4 h. Triplicate results in a representative experiment are shown as mean ± SD. Values are presented as the ratio of IFU from dCas12 induced sample to that from respective mock induction sample, which is set at 1. At least four independent experiments were performed (also see Fig. S2, in which actual IFU values were presented). Statistical significance in all panels was determined by two-way ANOVA followed by Tukey’s post hoc test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. IFU, inclusion-forming unit; MOMP, major outer membrane protein.

Fig 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 (also see Table S2). The gfp mRNA concentrations were normalized to the gDNA levels as determined by qPCR targeting tufA and presented as mean ± SD of three biological replicates. (b) Immunoblotting analysis of dCas12, MOMP, and GFP expression with infected cells grown at increasing concentration of aTC added at 4 h pi. Densitometry of the blot was assessed using ImageJ. Values are presented as the density of the dCas12 band or GFP band normalized to the MOMP band from the same sample. Host cell α-tubulin was used as protein loading control. Note: a small amount of dCas12 leaky expression was detected in the absence of aTC. (c) Live-cell images of C. trachomatis. HeLa cells were infected with C. trachomatis L2/topA-kd at a multiplicity of infection of ~0.4 and cultured in an aTC-free medium. Increasing concentrations of aTC (0, 2.5, 5, or 10 ng/mL) were added starting at 4 or 16 h pi. Automated imaging acquisition was performed at 24 h pi under the same exposure conditions with Cytation 1. Scale bar = 100 µm. (d) Measurement of GFP MFI in C. trachomatis-infected cells grown in the absence or presence of aTC. Individual inclusions were analyzed using Gen5 software. The values are presented as mean ± SD from the inclusion numbers equal to 213 ± 41 per condition in replicate wells. 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  ≤  0.001, ****P  ≤  0.0001. ns, no significance.

Fig 4

Secondary differentiation of RB to EB is impaired by topA knockdown in L2/topA-kd. (a) Schematic representation of the chlamydial developmental cycle and its coupled expression of RB- or EB-related markers. (b) Analysis of C. trachomatis gDNA in the absence or presence of aTC using real-time qPCR targeting the tufA gene. Values are presented as the ratio of chlamydial gDNA copy numbers per nanogram 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. (c) Quantification of transcripts of omcB or incD in C. trachomatis using RT-qPCR. The levels of transcripts were normalized to the gDNA levels as determined by qPCR with the same primer pair. The values are presented as mean ± SD of four biological replicates. (d and e) Immunofluorescent micrographs of C. trachomatis expressing OmcB. HeLa cells were infected with C. trachomatis L2/topA-kd (d) or L2/Nt (e), cultured in the absence (−aTC) or presence of aTC (+aTC, 10 ng/mL) for 20 h pi starting at 4 h pi. After 24 h pi, infected cells were fixed, processed, and used for IFA by immunolabeling with rabbit polyclonal antibody to C. trachomatis OmcB and visualized with Alexa fluor 568-conjugated goat anti-rabbit antibody. DAPI-counterstained DNA (blue) and C. trachomatis expressing GFP (green) and OmcB (red) are shown. Scale bar = 10 µm. Quantitative analysis of OmcB signal has been omitted as OmcB is a secretable protein that can be associated or not with the inclusion. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. ns, no significance.

Fig 5

Effect of aTC addition on the growth of C. trachomatis L2/topA-kdcom. (a) Schematic map of the expression vector containing topA-His6 that is co-regulated with dcas12 by aTC-inducible P_tet and topA-specific crRNA (not shown). (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 24h pi. The locations of primers used to detect topA from the cDNA samples are shown in panel a and detailed in Table S2. (c) Immunoblot displays the inducible expression of TopA-His6 in C. trachomatis. TopA-His6 protein from the lysates of C. trachomatis-infected cells was isolated by 10% SDS-PAGE for immunoblot with antibody against His6 or L2 MOMP. (d) Representative live-cell images of HeLa cells infected with C. trachomatis strains as indicated. HeLa cells with infection of C. trachomatis L2/topA-kdcom or the control strains, L2/Nt and L2/topA-kd, were cultured in RPMI-10 lacking (−aTC) or containing an optimal concentration of aTC (2.5 ng/mL). After 24 h pi, infected cells were imaged in combination with green fluorescence and bright light detection using Cytation1. Scale bar = 100 µm. (also see Fig. S8) (e) Enumeration of EBs using IFU assay. The infected cells and culture supernatants were collected at 40 h pi and used to infect a fresh HeLa cell monolayer for enumeration of recoverable IFU. Data are representative of those from an experiment performed in triplicate for each condition and presented as IFUs (mean ± SD). Three independent experiments were performed with similar results. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig 6

Overexpression of topA-His6 has detrimental effects on C. trachomatis. (a) Schematic map of the expression vector pBOMBLs-topAHis6, in which topA-His₆ is under the control of aTC-inducible P_tet. (b) Immunoblot for TopA-His6. The proteins from lysates of C. trachomatis L2/topAH6-infected HeLa cells harvested at 40 h pi were separated by SDS-PAGE. Blot was stained with antibody against His6 (upper panel) or L2 MOMP (lower panel). (c) Immunofluorescent micrographs of C. trachomatis L2/topAH6 in the presence of aTC (5 ng/mL) added at 0 h pi. Cells were fixed at 24 h pi and used for IFA with mouse anti-His6 antibody and visualized with Alexa fluor 568-conjugated goat anti-mouse IgG. Scale bar = 10 µm. (d) Evaluation of P_Nmen-GFP levels. Live-cell images were acquired at 24 h pi. Individual chlamydial inclusions equal to 257 ± 52 per condition were measured. Values were obtained from triplicate results in a representative experiment and are shown as mean ± SD. At least three independent experiments were performed. (e) Enumeration of EB yields. C. trachomatis L2/topAH6 or L2/pBOMBLs-infected cells were cultured for 24 h in the absence (−aTC) or presence (+aTC, at 5 ng/mL) of aTC. Triplicate results in a representative experiment are shown as mean ± SD. The data are presented as the ratio of IFUs from the aTC-exposed sample to that from the aTC-unexposed sample, which is set at 1. At least three independent experiments were performed. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P ≤ 0.05, ****P ≤ 0.0001. ns, no significance.

Fig 7

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. HeLa cells infected with C. trachomatis L2/topA-kd, L2/Nt, or L2/topA-kdcom were grown in the presence or absence of aTC (at 5 ng/mL) and harvested at 24 h pi for total RNA preparation and then cDNA synthesis. The location of primer pairs used for RT-qPCR analysis is shown. Results were obtained from two independent experiments performed in duplicate for each condition. The graph indicates mean ± SD of the ratio of transcripts from the aTC-exposed sample to that from the aTC-unexposed sample, which is set at 1 as shown by a red line. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ***P ≤ 0.001, ****P ≤ 0.000. ns, no significance.

Fig 8

Analysis of the response of C. trachomatis to moxifloxacin. (a and b) Comparison of chlamydial inclusion sizes (a) and the GFP MFI (b) of L2/topA-kd to those of L2/Nt and L2/topA-kdcom. HeLa cells were infected by L2/topA-kd, L2/Nt, or L2/topA-kdcom, cultured in the absence or presence of Mox (5 ng/mL) or both aTC (5 ng/mL) and Mox (5 ng/mL), and imaged at 42 h pi. The individual chlamydial inclusions equal to 198 ± 14 were analyzed. (c) Enumeration of EB yields. C. trachomatis-infected cells were harvested at 40 h pi and used for IFU assay. IFU values from triplicate results in a representative experiment are presented as mean ± SD. Data are presented as the ratio of IFUs from treated samples to that from untreated sample, which is set at 1. Three independent experiments were performed. (d) Analysis of C. trachomatis gDNA in the presence or absence of aTC using real-time qPCR. Values are presented as the ratio of chlamydial gDNA copy numbers per nanogram 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 was determined by two-way ANOVA followed by Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001.

Fig 9

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 primarily maintained by the joint action of 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., gyrB/gyrA and omcB), 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.