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. IMPORTANCE We 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.


Figure S1 .
Figure S1.Depiction of the general construct of the pBOMBL12CRia-derived shuttle plasmids that were used for C. trachomatis transformation.The coding sequence of the dCas12 gene is placed downstream of a tet repressor-regulated promoter in an expression vector and an ATG triplet serves as the initiation codon.Whereas pBOMBL12CRia(topA)::L2 contains a chlamydial topA-specific crRNA, the pBOMBL12CRia(NT)::L2 does not.

Figure S2 .
Figure S2.Numeration of EBs using IFU assay.C. trachomatis L2/topA-kd or L2/Nt infected HeLa cells were cultured for 20 or 4 h in the medium containing aTC starting at 4 h pi, harvested, and subjected to IFU assay.Triplicate results in a representative experiment are shown as mean ± SD.At least four independent experiments were performed.Statistical significance was determined by two-way ANOVA (simple effects within columns) followed by Tukey's post-hoc test.****P ≤ 0.0001; ***P ≤ 0.001, ns: no significance.(Also see Fig. 2d for relative IFUs).

Figure S3 .
Figure S3.(a) Live-cell images of C. trachomatis L2/Nt that has a wild type topA lacking the topA-specific crRNA.HeLa cells were infected with C. trachomatis L2/Nt at multiplicity of infection ~0.4 and cultured in aTC free medium.The increasing concentrations of aTC (0, 2.5, 5, or 10 ng/mL) were added starting at 4 h (upper panels) or at 16 h pi (lower panels).The automated imaging was taken at 24 h pi under the same exposure conditions using Cytation 1. Scale bar = 20 μm.(b) Quantifying GFP MFI of the single chlamydial inclusions.Values of GFP MFI are presented as mean ± SD from the individual inclusion numbers equal to 166 ± 50 per condition in replicate wells.****P ≤ 0.0001, comparison was made using one-way ANOVA followed by Tukey's post-hoc test.Note: varied, but no decrease in GFP was evident in the presence of aTC.

Figure S4 .
Figure S4.Quantifying transcript levels of euo and hctB in C. trachomatis strains, L2/topA-kd or L2/Nt (control) using RT-qPCR.The mRNA transcript levels were normalized to the gDNA control as determined by qPCR targeting chlamydial euo (left) or hctB (right).Values were presented as mean ± SD of four biological replicates.The data are presented as the ratio of transcript in the presence of aTC to that in the absence of aTC, 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 ≤ 0.05, **P ≤ 0.01.

Figure S9
Figure S9 Immunofluorescence micrographs of C. trachomatis L2/topA-kdcom expressing OmcB.Infected cells were grown in the absence or presence of aTC (at 5 ng/mL) and fixed at 24 h pi for IFA.The OmcB protein was immunolabelled with rabbit anti-OmcB antibody and visualized with Alexa Fluor 568-conjugated goat anti-rabbit IgG.DAPI-stained DNA (blue) and C. trachomatis expressing GFP (green) and OmcB (red) are shown.Scale bar = 10μm.Note: the enhancement of the inclusion-associated OmcB signal induced by aTC addition compared to that of mock aTC addition.

Figure S10 .Figure S11 .
Figure S10.(a) Map of expression vector pBOMBLs-topAhis6 showing the relevant elements, including Ptet-controlled topA-his6 and PNmem-GFP.This plasmid was transformed into C. trachomatis resulting in strain L2/topAH6 that was used for topA-his6 overexpression study.(b) Map of control plasmid pBOMBL-spc.This plasmid was transformed into C. trachomatis resulting in strain L2/pBOMBLs that was used in control experiment.

Figure S12 .
Figure S12.Immunofluorescence micrograph of C. trachomatis L2/topA-kdcom expressing dCas12.HeLa cells with infection of C. trachomatis were cultured in the presence of aTC (at 2.5 ng/mL) and Mox (at 1 ng/mL or 10 ng/mL) for 40 h pi and fixed for IFA.The dCas12 was immunolabelled with rabbit anti-dCas12 antibody and visualized with Alexa Fluor 568-conjugated goat anti-rabbit IgG.DAPI-stained DNA (blue) and C. trachomatis expressing GFP (green) and dCas12 (red) are shown.Scale bar=10μm.