The SWIB domain-containing DNA topoisomerase I of Chlamydia trachomatis mediates DNA relaxation

Abstract

The obligate intracellular bacterial pathogen, Chlamydia trachomatis (Ct), has a distinct DNA topoisomerase I (TopA) with a C-terminal domain (CTD) homologous to eukaryotic SWIB domains. Despite the lack of sequence similarity at the CTDs between C. trachomatis TopA (CtTopA) and Escherichia coli TopA (EcTopA), full-length CtTopA removed negative DNA supercoils in vitro and complemented the growth defect of an E. coli topA mutant. We demonstrated that CtTopA is less processive in DNA relaxation than EcTopA in dose-response and time course studies. An antibody generated against the SWIB domain of CtTopA specifically recognized CtTopA but not EcTopA or Mycobacterium tuberculosis TopA (MtTopA), consistent with the sequence differences in their CTDs. The endogenous CtTopA protein is expressed at a relatively high level during the middle and late developmental stages of C. trachomatis. Conditional knockdown of topA expression using CRISPRi in C. trachomatis resulted in not only a developmental defect but also in the downregulation of genes linked to nucleotide acquisition from the host cells. Because SWIB-containing proteins are not found in prokaryotes beyond Chlamydia spp., these results imply a significant function for the SWIB-containing CtTopA in facilitating the energy metabolism of C. trachomatis for its unique intracellular growth.

Keywords: Chlamydia trachomatis; DNA relaxation; DNA topoisomerase I (TopA); SWIB domain; chlamydial developmental cycle; nucleotide metabolism; transcriptional regulation.

Figure 1.. C-terminal SWIB domain is unique in CtTopA.

(a) Domain composition of CtTopA (CTL0011) predicted by InterPro. Zf: 4C zinc fingers. (b) Alignment of amino acid residues of the CTDs of TopAs from E. coli, M. tuberculosis, H. pylori, P. aeruginosa, N. gonorrhoeae and C. trachomatis. Accession numbers are shown on the left. The conserved 4C zinc fingers are boxed. The position of SWIB-domain in CtTopA is underlined (green). ClustalW was used for alignment with Matric BLOSUM62. See Fig. S1 for entire sequence alignments of these bacterial TopAs. (c) Schematic diagram showing domains of the EcTopA (D1–D9) compared to domains found in MtTopA and CtTopA. The gray or light blue bar represents the N- and C-terminal domains. The TOPRIM (red), zinc finger (cyan), Topo_C-Rpt (black), lysine repeats (yellow), and SWIB domain (green) are as indicated. (c) Structural model of CtTopA by AlphaFold. The NTD, CTD zinc fingers, and the SWIB domain are as indicated. Model confidences are shown on right.

Figure 2.. Comparison of the in vitro DNA relaxation activity of recombinant CtTopA to EcTopA.

(a) SDS-PAGE/coomassie staining gel showing recombinant CtTopA protein purified from E. coli. (b) Concentration-dependent DNA relaxation. Serial dilutions of EcTopA and CtTopA as indicated were incubated with 0.3 μg (5.2 nM) negatively supercoiled DNA for 30 min, followed by agarose gel electrophoresis. (c) Time course of DNA relaxation. EcTopA or CtTopA (25 nM) was incubated with 0.3 μg negatively supercoiled DNA for different times (1–30 min). (d) Quantification of DNA relaxation based on time course studies. The percent of relaxation was determined by dividing the distance between the negatively supercoiled band (SC); and the weighted center of the partially relaxed band (PR); by the distance between the supercoiled band (SC); and the fully relaxed band (FR). (Formula: percent relaxation = (SC-PR)/(SC-FR)*100 (60). The values are reported as mean ± standard derivation (SD) of results obtained from three independent experiments (also see Figure S4). Statistical comparison between EcTopA and CtTopA was analyzed by Two-Way ANOVA. **P<0.01, *** P<0.001, ****P<0.0001.

Figure 3.. Complementation assay in E. coli topA mutant strains.

(a-b) Results with VS111-K2 transformed with pBOMLs-topAHis6 expressing CtTopA or empty vector pBOMBLs. Ten-fold serial dilutions of the bacterial cultures were spotted on LB agar plates containing chloramphenicol and spectinomycin. Images were taken 18 h after incubation at 30°C or 37 °C (a). Growth curve of E. coli strains as indicated at 37°C during 8 h incubation in the presence or absence of aTC at 200 μg/mL(b). Y-axis:OD600, x-axis: hours of incubation. Data are presented as mean ± SEM. Statistical comparisons of OD600 between induced and uninduced samples of the same strain were performed by Two-Way ANOVA. ***P<0.001, ****P<0.0001. Lower panel: immunoblotting showing CtTopA expression in E. coli with anti-His antibody. Note: leaky expression of CtTopA in the absence of aTC. (c) Results with AS17 transformed with plasmid expressing EcTopA or CtTopA as indicated. Ten-fold serial dilutions of the cultures of the transformants were spotted on LB agar plates with kanamycin and incubated at 30°C or 42°C. Images were taken after 18 h for 42°C incubation and 36 h for 30°C incubation. For all strains, two different isolates of E. coli transformants were used as biological replicates.

Figure 4.. Reaction of anti-CtTopA or anti-CtTopA_CTD with the purified recombinant CtTopA.

(a) Depiction of the scheme for the antigen sources (either full length CtTopA or synthesized peptides) used for antibody production. The location and sequence of peptides are shown. (b) Serial dilutions of recombinant EcTopA, MtTopA and CtTopA proteins on SDS-PAGE/coomassie stained gel (upper panel) and immunoblots showing their reactions to anti-CtTopA (middle panel) or anti-CtTopA_CTD (lower panel). Arrows show protein bands of interest.

Figure 5.. C. trachomatis naturally produces SWIB-containing CtTopA.

(a) Immunofluorescence micrographs of HeLa cells infected with L2/Nt or L2/topA-kd at 45 hpi. GFP-expressing chlamydial organisms (green) were stained for CtTopA (red; anti-CtTopA antibody). Cellular and bacterial DNA was counterstained with DAPI (blue). Arrows indicate the location of chlamydial inclusions. Left panels show merged images. Image adjustments of C. trachomatis and DNA were applied equally for both bacterial strains and cells. Scale bars=20 μm. (b)-(c) Immunoblotting of endogenous chlamydial Hsp60 and CtTopA levels in lysates of infected HeLa cells sampled at 16, 20, 24, and 42 h pi. GAPDH was used as a loading control. *: band corresponding to ~98kDa CtTopA. Arrow: a larger band. Densitometry of the protein band of interest was assessed using ImageJ and presented in (c). The full-length blots with the same results were shown in Fig. S3. (d) Immunoblotting of CtTopA and CtHsp60 in cell infected with different C. trachomatis strains as indicated. Lysates of cells cultured in aTC-containing medium for 40h (4–44 hpi) were used. Values are presented as the density of the CtTopA band normalized to the CtHsp60 band from the same sample using ImageJ.

Figure 6.. Repression of topA induces growth retardation and the decrease in transcription of npt1 and npt2.

(a) Live-cell images of C. trachomatis infected HeLa cells. C. trachomatis L2/Nt, L2/topA-kd, or L2/topA-kdcom at a multiplicity of infection of ~0.4 were used for infection. Cells were cultured in the absence (-aTC) or presence (+aTC) of aTC from 4 to 24 hpi. Automated imaging acquisition was performed at 24 hpi under the same exposure conditions with Cytation 1. Scale bar = 20 µm. (b) Numeration of EB yield using infection assay. The values are presented as mean ± SD from two independent experiments each with three technique repeats. (c)-(d) Fold change in npt1 or npt2 transcript levels. RT-qPCR was conducted with C. trachomatis-infected cells grown under inducing (+aTC) or mock inducing (−aTC) conditions starting from 4 hpi for 11 h (to 15 hpi) (c) and 20 h (to 24 hpi) (d). Quantified gene-specific transcripts were normalized to the gDNA levels as determined by qPCR with the same primer pair. The data are presented as the ratio of relative transcript in the presence of aTC to that in the absence of aTC, which is set at 1 as shown by a red line. The values are presented as mean ± SD of two independent experiments each with triplicates. For all panels, statistical significance was determined by One-way or Two-Way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001.