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

Abstract

Chlamydia trachomatis has a DNA topoisomerase I with a unique C-terminal domain (CTD) homologous to eukaryotic SWIB domains. This study focused on determining the function of the SWIB domain-containing TopA from C. trachomatis (CtTopA). We demonstrated that, despite the lack of sequence similarity at the CTDs between CtTopA and TopA from Escherichia coli (EcTopA), full-length CtTopA removed negative DNA supercoils in vitro and complemented the growth defect of a topA mutant of E. coli. 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, 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. Overexpressing a topA mutant allele lacking the SWIB domain in C. trachomatis resulted in slow growth when host protein synthesis was inhibited. These data suggest that productive infection of C. trachomatis requires functional SWIB domain-containing CtTopA and de novo host protein synthesis. Because SWIB domain-containing CtTopAs are not found in prokaryotes beyond Chlamydia spp., our work suggests an important function of the SWIB domain on CtTopA activity during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is a medically important bacterial pathogen that is responsible for the most prevalent bacterial sexually transmitted infection. Bioinformatics, genetics, and biochemical analyses have established that the presence of a SWIB domain in CtTopA is relevant to chlamydial physiology. Our findings also underline the mechanistic diversity among the family of TopAs that are likely driven by pathogen-specific adaptations.

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

Fig 1

C-terminal SWIB domain is unique in CtTopA. (a) Domain composition of CtTopA (CTL0011) predicted by InterPro. Zf: 4C zinc fingers. The 8.5 kDa SWIB-like domain consists of 79 amino acids (green). (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 the SWIB domain in CtTopA is underlined (green). ClustalW was used for alignment with Matrix 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. (d) Structural model of CtTopA by AlphaFold. The NTD, CTD zinc fingers, and the SWIB domain are as indicated. Model confidences are shown on the right.

Fig 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 of negatively supercoiled DNA for different times (1–30 min). (d) Quantification of DNA relaxation based on time-course studies. The percentage 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 SC and the fully relaxed band (FR). Formula: percent relaxation = (SC − PR)/(SC − FR) × 100 (30). The values are reported as mean ± standard deviation of results obtained from three independent experiments (also see Fig. S4). Statistical comparison between EcTopA and CtTopA at a given time was analyzed by t test. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3

Complementation assay in E. coli topA mutant strains. (a and b) Results with strain VS111-K2 transformed with pBOMLs-topAHis6 expressing CtTopA or vector pBOMBLs expressing mcherry. Tenfold serial dilutions of the bacterial cultures were spotted on LB agar plates containing chloramphenicol and spectinomycin. Images were taken at 18 h after incubation at 30°C or 37°C (a). Growth curve of E. coli strains as indicated during 8-h incubation at 37°C 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. (n=3) Statistical comparisons of OD₆₀₀ between induced and uninduced samples of the same strain were performed by two-way ANOVA. ***P < 0.001 and ****P < 0.0001. Lower panel: immunoblotting showing expression of His6-tagged CtTopA in E. coli with anti-His antibody. Note: leaky expression of CtTopA in the absence of aTC. (c) Results with AS17 transformed with pLIC-EcTOP expressing EcTopA or pET-CtTopA expressing CtTopA as indicated. Tenfold 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.

Fig 4

Reaction of anti-CtTopA or anti-CtTopA_CTD with the purified recombinant CtTopA. Serial dilutions of 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.

Fig 5

Overexpression of mutant TopAΔC complements the growth defect of E. coli AS17 at 42°C. (a) Schematic map of the construct encoding P_tet/riboswitch-controlled mutant topAΔC (containing nucleotides 1–2,331 of chlamydial topA and 6xHis). (b) Complementation of E. coli AS17 with topA^ts chromosomal mutation for growth at 42°C by background levels of mutant TopAΔC. Images were taken 18 h after incubation at 30°C or 37°C. (c) Immunoblot displays inducible expression of TopAΔC in AS17 grown in LB broth containing spectinomycin (50 µg/mL) at 30°C after the addition of aTC (200 µg/mL) and Theo (15 µg/mL) for 4 h. Anti-CtTopA was used. (d) Comparison of the growth of strains with CtTopA, TopAΔC, or vector control on LB agar at 30°C and 42°C under non-induction or induction conditions. Tenfold serial dilutions of the cultures of the transformants as indicated were spotted on LB agar plates with spectinomycin (50 µg/mL) and incubated at 30°C for 36 h or 42°C for 18 h. The representative plate images from three experiments are shown.

Fig 6

C. trachomatis naturally produces SWIB domain-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 4′,6-diamidino-2-phenylindole dihydrochloride (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 and c) Immunoblotting of endogenous chlamydial Hsp60 and CtTopA levels in lysates of infected HeLa cells sampled at 16, 20, 24, and 42 hpi. Host GAPDH was used as a loading control. *: band corresponding to ~97 kDa CtTopA. Arrow: a larger band. Densitometry of the protein band of interest was assessed using ImageJ and presented in (C. A representative blot is shown with the quantification of the amount relative to that of CtHsp60. Results shown are the mean ± SD (n = 2). Note: the level of CtTopA was significantly increased from 24 to 42 h (t test, P < 0.05). The full-length blots with the same results are shown in Fig. S3. (d) Immunoblotting of CtTopA and CtHsp60 in cells infected with different C. trachomatis strains as indicated. Lysates of cells cultured in aTC-containing medium for 40 h (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.

Fig 7

The influence of mutant TopAΔC overexpression on chlamydial growth. C. trachomatis L2/topAΔC and L2/e.v. at a multiplicity of infection of ~0.4 were used to infect HeLa cells individually. Cells were cultured in the absence or presence of aTC (5 ng/mL)/Theo (15 µg/mL) and/or CHX (1.5 µg/mL) starting at 4 hpi to various times as indicated in each result. (a) Live-cell images of C. trachomatis-infected cells. Image was taken at 30 hpi under the same exposure conditions. Scale bar = 100 µm. (b) Analyzing chlamydial inclusion areas. Inclusion counts were as indicated. Statistical significance was determined by Student’s t test, ****P ≤ 0.0001; ns, no significance. (c) Numeration of EB yield using infection assay. The infected cells and culture supernatants were collected and used to infect a fresh HeLa cell monolayer to numerate recoverable EBs. The data are presented as the ratio of relative EB yields in the induced cells to those in uninduced cells, which is set at 1 as shown by a red line. The values are presented as the mean ± SD of two independent experiments, each with triplicate. Statistical significance was determined by Student’s t test, *P ≤ 0.05. (d) Examining inducible expression of TopAΔC in C. trachomatis at 24 hpi using IFA. GFP-expressing chlamydial organisms (green) were stained for CtTopA (red; anti-6xHis antibody) and major outer membrane protein (MOMP) (gray; anti-L2 MOMP). Cellular and bacterial DNA was counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (blue). Scale bar = 10 µm. (e) Immunoblot analysis of TopAΔC expression in C. trachomatis at 44 hpi with anti- 6xHis and anti-L2 MOMP antibodies. (f) Immunoblot analysis of endogenous CtTopA (red arrow) and ectopically expressed TopAΔC (green arrow) in C. trachomatis with anti-CtTopA. The protein band density was obtained using ImageJ. The ratios of CtTopA or TopAΔC to those of the MOMP in a representative blot were as indicated. The experiments were repeated three times (Fig. S6).