The Matchmaker two-hybrid system (Clontech Laboratories, Inc.) was used to confirm the potential interaction partners of GlgA (CT798). The gene sequence of CT798 (WP-100139618) was obtain from the National Centre for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/) and amplified by PCR using the following primers, which contained SfiI sites at both ends: CT798 forward, 5′-AAGGCCATTACGGCCATGAAAATTATTCACACAGCTATCG-3′ and reverse, 5′-CCGGCCGAGGCGGCCTTGTTTATAAATTTCTAAATATTTATTGGC-3′.

PCR was performed under the following conditions: Initial denaturation at 98°C for 5 min, amplification (30 cycles) at 98°C for 30 sec, 55°C for 30 sec and 72°C for 42 sec, and afinal extension at 72°C for 5 min (Qiagen). The FastPfu DNA Polymerase was used (Beijing Transfen Biotech Co., Ltd.). The orientation and authenticity of the plasmid sequence was confirmed by sequencing (Sangon Biotech Co., Ltd.) and restriction endonuclease digestion by 8% SfiI digestion (Fermentas) at 50°C for 24 h.

The CT798 plasmid was cloned into the GAL4 binding domain of the pGBKT7 vector (Hitech Bio-Technology Co., Ltd). CT798 plasmids were treated with SfiI in a 25-µl reaction system including 20 µl CT798, 2 µl SfiI restriction enzyme, 2.5 µl 10X buffer and 0.5 µl sterile water. pGBKT7 was treated with SfiI in a 20-µl reaction system including 10 µl CT798, 1 µl SfiI restriction enzyme, 2 µl 10X buffer and 7 µl sterile water. The digested reaction mixtures were then combined and incubated at 16°C for 1 h, consisting of 5 µl Ligation High Buffer (Toyobo Life Sciences), 5 µl CT798/SfiI and 0.5 µl pGBKT7/SfiI. The recombinant plasmid wasthenused to transform TOP10 Escherichia coli. Escherichia coli was supplied by American Type Culture Collection and cultured in Luria-Bertani medium (1% NaCl; 1% Polypeptone; 0.5% Yeast extract) in a humidified incubator at 37°C with 5% CO₂. Then, it was inoculated onto Luria-Bertani medium plates (1% NaCl; 1% Polypeptone; 0.5% Yeast extract; 2% agar) containing 50 µg/ml kanamycin (LB-Kan^r+) overnight at 37°C. A total of six bacterial colonies were selected and further cultured at 16°C overnight with agitation (250 × g). The plasmids of the cultured bacteria were extracted using a Plasmid Miniprep kit (Qiagen, Inc.) and verified by sequencing.

Agarose gel electrophoresis is a standard method for identification DNA fragments (15). In the present study, 0.7% agarose gel was used. Then, 0.14 g agarose and 20 ml 1X Tris-acetate-EDTA buffer in a flask were heated in a microwave for 5 min at 100°C. Then, 2 µl ethidium bromide was added (Thermo Fisher Scientific, Inc.), and poured onto a taped plate with casting combs. Then, 2 µl DNA sample from Escherichia coli was added to the 5X agarose gel, and underwent electrophoresis at 120 mA for 40 min at 25°C until separation had been achieved. The DNA fragments were visualized using a long wave UV light box.

pGADT7, pGBKT7-p53, pGBKT7-laminC and the pGADT7-170297-LUcDNA plasmid genome were provided from Hitech Bio-Technology Co., Ltd. The pGADT7-170297-LUcDNA plasmid genome was referred to as ‘pGADT7-largeT’. As a positive control, pGBKT7-p53 and pGADT7-largeT were used to transform AH109 yeast cells. As a negative control, AH109 cells were also transformed using pGBKT7-lamin C with pGADT7-large T. AH109 were purchased from the American Type Culture Collection and cultured in yeast peptone dextrose adenine (YPDA; 1% Yeast extract; 2% Tryptone; 2% Glucose; 0.02% Adenine) with 5% CO₂. Synthetic defined (SD)/-tryptophan (Trp), SD/-leucine (Leu), SD/-Trp/-Leu, SD/-Trp/-Leu/-histidine (His) and SD/-Trp/-Leu/-His/-adenine (Ade) media were purchased from Clontech Laboratories, Inc.

To test whether the prey plasmid was able to auto-activate the reporter genes, pGADT7 and pGBKT7-CT798 were used to co-transform AH109 cells according to the lithium acetate transformation method (16). The cells were then cultured in SD medium lacking various amino acids (SD/-Trp/-Leu) at 30°C for 4 days. The isolated yeast clones were tested for the activation of the third reporter gene, including imidazoleglycerol-phosphate dehydratase (HIS3), phosphoribosylaminoimidazole carboxylase (ADE2) and LacZ.

The pGADT7-170297-LUcDNA plasmid genome from HeLa cells was used for the yeast two-hybrid screen (17). AH109 yeast cells containing the pGBKT7-CT798 plasmid were plated and selected using SD agar plates without tryptophan. The positive clones were inoculated in 50 ml SD/-Trp medium and cultured at 30°C (225 × g) for 18 h. The cells were then transferred to 500 ml YPDA medium and cultured at 30°C (agitation at 225 × g) for 4 h until OD₆₀₀=0.6. Next, the cultured cells were centrifuged at 4,000 × g for 5 min at room temperature; the supernatant was discarded and the pellet was resuspended in 0.1 MLiAc of 20 ml. The cells were then centrifuged once more under the same conditions and suspended in 10 ml LiAc (0.1 M). Then, 9.6 ml 50% PEG3350, 1.44 ml LiAc, 300 µl single-stranded DNA and 25 µg cDNA library plasmid were added, and the cells were cultured at 30°C for 30 min. The mixture was heated in a water bath at 42°C for 25 min, followed by resuscitation at 30°C for 1 h. The cell pellet was resuspended in 8 ml sterile water following centrifugation at 4,000 × g for 5 min at room temperature. The cells were cultured on SD/-Leu/-Trp/-His and 3-amino-1,2,4-triazole (3AT) plates with 200 µl solution/plate, for a total of 40 plates. In order to determine library transformation efficiency, 20 µl cell cultures from 8 ml solution were diluted 10-fold by gradient and cultured on SD/-Leu/-Trp plates. The cells were cultured for 3–4 days at 30°C, with observation ofthe conversion results and recording of conversion efficiency. The following calculations were used: Total number of transformants = (n₁/20+n₂/2+n₃/0.2)×1/3×8,000). Where n₁, the number of colonies growing on the plate after 10-fold dilution; n₂, the number of colonies growing on the plate after 100-fold dilution; n₃, the number of colonies growing on the plate after 1,000-fold dilution. Transformation efficiency = Total number of transformants/25 µg cDNA plasmid.

After culturing on SD/-Leu/-Trp/-His and 3AT plates, the positive clones were plated onto SD/-Leu/-Trp plates. To select for clones which potentially interacted with CT798, the clones were inoculated onto SD/-Leu/-Trp/-His+3AT and SD/-Leu/-Trp/-Ade plates to assess the transcriptional activation of the HIS3 and ADE2 reporter genes, and resuspended in sterile water to detect LacZ expression. Concurrently, positive and negative control transformation experiments were conducted, for the purpose of selecting true positive colonies. The positive clone plasmids were subsequently extracted using the Plasmid Miniprep kit. Following sequencing, a BLAST search was conducted using GenBank (National Center for Biotechnology Information) to determine the associated genes.

The selected clones were used to transform AH109 yeast cells containing pGBKT7-CT798 as aforementioned, and these cells were cultured on SD/-Leu/-Trp plates. Positive clones were transferred onto SD/-Leu/-Trp/-His/-Ade+3AT and SD/-Leu/-Trp/-Ade plates to determine the transcriptional activation of the HIS3 and ADE2 reporter genes, and resuspended in sterile water to detect LacZ expression.

HeLa cells were provided from the University of South of China and cultured in complete DMEM (Gibico; Thermo Fisher Scientific, Inc), containing 10% FBS (Gibico; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO₂. HeLa cells were infected with C. trachomatis. C. trachomatis was added into HeLa cell. At 48 h post-infection, the cells were washed twice with cold PBS, and lysed with RIPA buffer (Epizyme, Inc.) for 15 min at 4°C. The lysate was pre-clarified by rotation for 10 min, centrifuged at 14,000 × g for 15 min (both 4°C), and incubated with protein-A agarose beads. Cell-free lysates were incubated with a rabbit anti-PHB antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. ab75766.) or IgM antibody (1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Protein-A beads were added for 2 h at room temperature, and the lysates were subsequently washed three timeswith PBS. The proteins were eluted into 5X SDS-PAGE sample buffer, separated by 12% SDS-PAGE gel for immunoblotting.

HeLa cells were infected with C. trachomatis and harvested at 48 h post-infection. Cells were washed with cold PBS and lysed with RIPA buffer (Epizyme, Inc.) with protease inhibitor and phosphatase inhibitor. Then, 10 µl protein extracts and cell lysates (Input control) were separated by 12% SDS-PAGE gel and electrophoretically transferred to nitrocellulose membranes. After blocking in 5% non-fat dry milk with TBST (50 mM Tris-HCl; pH 7.4; 5.36 mM KCl; 274 mM NaCl; 0.1% Tween-20) for 2 h at room temperature, the membranes were probed with a mouse anti-CT798 antibody (1:1,000; The University of South China) overnight at 4°C. Then, the membrane waswashed four times for 5 min in TBST and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody (1:3,000; Servicebio, Inc.; cat. no. GB23303) for 1 h at 37°C Blots were detected by Western Blot system G: BOX Chemi XXX9 (Syngene International Ltd.).

CT798 is 53 kDa; the full length of CT798 (1,440 bp) was successfully amplified from C. trachomatis genomic DNA viaPCR, and analyzed using restriction enzyme digestion (Fig. 1A). The PCR products were subsequently cloned into the bait vector pGBKT7. The construct was transformed into E. coli and inoculated onto LB-kan^r+ plates. Following incubation, six positive transformants were randomly picked and identified by PCR (Fig. 1B), and the results of recombinant plasmid sequencing were consistent with that of pGBKT7-CT798.

The AH109 yeast strain contains various nutritional reporter genes. These include: i) ADE2, coding for phosphoribosylaminoimidazole carboxylase, an important enzyme for adenine synthesis; ii) HIS3, encoding imidazoleglycerol-phosphate dehydratase, another key enzyme for adenine synthesis; and iii) LacZ, which encodes β-galactosidase enzymes. Therefore, AHI09 allows for the control of strong nutritional selection, whilst reducing the incidence of false positives. To determine the efficiency of the system, pGBKT7-CT798 and pGADT7 were used to transform AH109 cells, and pGBKT7-p53 and pGBKT7-laminC were used as the positive and negative controls, respectively. AH109 cells were then cultured on SD/-Leu/-Trp plates, and six transformants were used to test for self-activation, including pointing-plate culturing on SD/-Leu/-Trp/-His/-Ade plates to test for the expression of HIS3 and ADE2, and a colorimetric assay to determine the expression of LacZ. Control strains both grow on the SD/-Leu/-Trp plate, but only the positive control cells are able to grow on the SD/-Leu/-Trp/-His/-Ade plate. In the present study, AH109 cells containing the pGBKT7/CT798 plasmid grew on the SD/-Leu/-Trp plate, but not on the SD/-Leu/-Trp/-His/-Ade plate. Furthermore, the results of the colorimetric assay were consistent with those of the negative control (Fig. 2). These results suggest that CT798 does not possess self-activation function in AH109 cells.

pGADT7-170297-LUcDNA library used for screening was supplied by Hitech Bio-Technology Co., Ltd. Plasmids contained only pGBKT7-CT798 as bait for screening the pGADT7-170297-LUcDNA library. Library plasmids were transformed into AH109 cells containing bait plasmid and cultured on SD/-Leu/-Trp/-His and 3AT plates. AH109 cells were also cultured on SD/-Leu/-Trp plates (Fig. 3), and transformation efficiency (which is important for the success of the yeast two-hybrid system) was determined. The results revealed a total of 3.78×10⁵ transformants, indicating a transformation efficiency of 1.51×10⁴/µg. At the same time, the quality of the yeast two-hybrid library was assessed. Agarose gel electrophoresis of the PCR products indicated the average length of the insertion product to be ~1.5 kb, and the positive success rate of the library was 100%.

In the present study, 13 positive clones were obtained from two rounds of selection, which were designated A1-13. These clones were not capable of growing on SD/-Leu/-Trp/-Ade plates, and seven grew on SD/-Leu/-Trp/-His and 3AT plates (Fig. 4). In addition, 11 of the 13 clones expressed LacZ. These results indicated that the HIS3 gene was activated by the seven positive clones, and further suggested that A3, A4, A7, A8, A9, A11 and A13 interacted with CT798 to a certain degree. The positive clones were subsequently sequenced and analyzed using BLAST software. The results indicated that the seven positive clones possessed a high degree of similarity to known genes encoding four different proteins (Table I). Finally, for rotary validation, the resulting four plasmids were used to transform AH109 cells containing the pGBKT7-CT798 plasmid (Fig. 5). The results revealed that clones A3 and A11 were able to active the HIS3 gene, and that only A3 activated the LacZ gene.

Co-immunoprecipitation was performed to validate the interaction between CT798 and PHB (Fig. 6). CT798 was co-precipitated by anti-PHB from the lysates of cells infected with C. trachomatis, but not by precipitation with IgM. This suggested that PHB interacts with CT798.