The T. vaginalis T016 isolate was cultured in a glass, screw-capped tube containing Diamond's trypticase-yeast extract-maltose (TYM) medium (NAPCO, Winchester, Virginia, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St. Louis, Missouri, USA) in 5% CO₂ at 37℃ for 24 hr. Cultured parasites were monitored for motility, and the viability of T. vaginalis was determined before each experiment using trypan blue staining (>99%).

Total RNA was extracted from T. vaginalis T016 isolate using Trizol Reagent (Invitrogen, Carlsbad, California, USA), and RNA concentrations were determined spectrophotometrically. RNA (1 µg) was reverse transcribed using the ReverTra Ace RT kit (Toyobo, Osaka, Japan). Following reverse transcription, cDNA was amplified by PCR using Ex Taq (Takara, Tokyo, Japan) under the following conditions: 5 min at 94℃, followed by 30 cycles of 30 sec at 98℃, 30 sec at 57℃, and 1 min at 72℃. The final elongation step was continued for 10 min at 72℃. The primer sequences were used as follows: GP63, MG aminopeptidase P-like metallopeptidase (MP50), M41 FtsH endopeptidase-like metallopeptidase (TVAG_243780.2), oligopeptidase A-like metallopeptidase (TVAG_477180), zinc carboxypeptidase-like metallopeptidase (TVAG_075740), and aspartyl aminopeptidase-like metallopeptidase (TVAG_392410) (Table 1). PCR products were electrophoresed through a 2% agarose gel and visualized with RedSafe (iNtRON Biotechnology, Seoul, Korea).

To construct pET32a-GP63 expression plasmids, the coding sequence of the T. vaginalis GP63 (GenBank accession no. GU356538.1) were amplified by PCR using Pfu-X DNA Polymerase (Solgent, Daejeon, Korea) from cDNA of T. vaginalis T016 with a pair of oligonucleotide primers: GP63 forward, 5'-CGCGGATCCATGATATTGTCTCTACTCCTCGTTG-3' and reverse, 5'-CCCAAGCTTAATCGATAAATTTGCATCAGGTTCT-3' (recognition sites for BamHI and HindIII, respectively are underlined). The PCR products were digested with the appropriate restriction enzyme and cloned into the BamHI/HindIII sites of the pET-32a (+) expression vector with N-terminal His-tag. The resulting plasmids were named pET32a-GP63. Recombinant plasmids were confirmed by restriction analysis and PCR sequencing (Solgent, Daejeon, South Korea). His-tagged GP63 protein was induced by 1 mM IPTG at 37℃ for 3 hr in BL21 (DE3) Escherichia coli. Finally, GP63 proteins in the supernatant were separated by 10% SDS-PAGE and visualized by Coomassie blue staining or detected by western blotting using the anti-His antibody.

To prepare T. vaginalis lysates, T. vaginalis were harvested in the logarithmic phase and washed 3 times in PBS (pH 6.2). T. vaginalis pellets were resuspended in PBS and lysed using a sonicator, and then centrifuged at 10,000 g for 30 min. The supernatant was collected and used as T. vaginalis lysate. The lysate was filtered through a 0.2-µm-pore-size filter and stored at -70℃ until required. The T. vaginalis lysate concentration was determined by the Bradford assay using bovine serum albumin (BSA) as the standard and stored at -70℃ until use.

Human cervical cancer cell lines, SiHa cells, were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, New York, USA) and antibiotic-antimycotic (Gibco BRL) in a 5% CO2 at 37℃.

SiHa cell monolayers (1×10⁶) were washed with PBS (pH 7.4) and live T. vaginalis were incubated with SiHa cell monolyers in the medium mixture (DMEM/TYM=2:1) at a multiplicity of infection (MOI) of 2 for 16 hr. To investigate whether T. vaginalis ESP were critical for SiHa cell protein synthesis, we used the 6-well Transwell permeable supports system (Costar, Cambridge, Massachusetts, USA). These inserts, which have a porous (pore diameter 3 µm) membrane, serve as the upper chambers and ordinary tissue culture plate wells serve as the lower chambers. SiHa cells (1×10⁶) was added to the lower chamber and medium containing T. vaginalis (2×10⁶) was added to the upper chambers and the plates were then incubated for 16 hr. Otherwise, SiHa cells were treated with T. vaginalis lysate (100 µg/ml) for 16 hr.

To evaluate the effect of mTOR according to the incubation time and parasite load, SiHa cells were incubated with live T. vaginalis at MOI 2 for 0.5, 1, 2, 4, 8, 16 hr, or SiHa cells were infected with live T. vaginalis at MOI 0.5, 1, and 2 for 2 hr. At the indicated time points, cells were collected and washed in PBS.

To determine whether T. vaginalis metalloproteinases are involved in the cleavage host mTOR, T. vaginalis (MOI 2) were treated for 30 min with 5 mM 1,10-phenanthroline (1,10-PT; Sigma-Aldrich) and the control group T. vaginalis were mock-treated in PBS. Then, T. vaginalis were washed with PBS and infected with SiHa cells for 16 hr.

Proteins were extracted using the PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) supplemented with a complete cocktail of protease inhibitors (Roche, Basel, Switzerland) for 15 min on ice. After centrifugation at 14,000 g for 15 min at 4℃, the supernatant was collected, and equal volumes of protein from each sample were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were blocked in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST) and 5% skim milk. After being washed once in TBST, membranes were incubated overnight at 4℃ with the primary antibodies diluted in TBST supplemented with 5% BSA. The antibodies used included rabbit polyclonal anti-mTOR, rabbit polyclonal anti-AKT, rabbit polyclonal anti-p70S6K (all from Cell Signaling Technology Inc., Beverly, Massachusetts, USA), and mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology, Santa Cruz, California, USA). Following 3 consecutive washes in TBST, membranes were incubated for 90 min with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) diluted 1:10,000 with incubation buffer, as described above. After extensive washing, bound secondary antibodies were visualized using an enhanced ECL chemiluminescence detection kit (GE Healthcare, Little Chalfont, UK).

Previous studies demonstrated that metalloproteinase is the largest surface protease family and the second largest surface protein family in T. vaginalis [5], and some of these molecules may be involved in the damage to the host [16]. To better define the T. vaginalis T016 isolate metalloproteinases, GP63, MP50, TVAG_243780.2, TVAG_477180, TVAG_075740, and TVAG_392410 expression levels were detected by RT-PCR. Interestingly, GP63 (501 bp), MP50 (304 bp), TVAG_075740 (400 bp), and TVAG_392410 (249 bp) mRNA expression were detected. However, we could not detect any TVAG_243780.2 and TVAG_477180 mRNA (Fig. 1).

We constructed the recombinant plasmid pET32a-GP63, and GP63 fragment was sequenced along both strands and compared to GenBank sequences (accession no. GU356538.1). As shown in Fig. 2, when the complete GP63 region nucleotide sequences were compared with the GU356538.1 sequences, they were 99.6% base pare identities. In the 1,893 bp of open reading frame, there were only 7 nucleotide differences.

To identify the expression level of GP63 protein, IPTG-induced recombinant His-tagged GP63 protein bands were separated by 10% SDS-PAGE and visualized by Coomassie blue staining (Fig. 3A). The lysate of IPTG-induced pET32a-GP63 BL21(DE3) E. coli showed the specific high expression of 89 kDa His-tagged GP63 protein. However, uninduced pET32a-GP63 transfomed E. coli lysate did not expresse 89 kDa GP63 protein. Further, His-tagged GP63 protein band was also confirmed by western blotting using the anti-His antibody (Fig. 3B). These results clearly indicate that T. vaginalis metalloproteinase GP63 recombinant protein can highly expressed in vitro using a bacterial expression system.

The AKT/mTOR/p70S6K pathway regulates cell growth and proliferation via the regulation of protein synthesis [17]. To investigate the effect of T. vaginalis infection in the AKT/mTOR/p70S6K pathway, SiHa cells were incubated with live T. vaginalis, T. vaginalis ESP (transwell inside the live T. vaginalis), or T. vaginalis lysate for 16 hr, and then the posttranslational modification of mTOR, AKT, and p70S6K were observed by western blot. As shown in Fig. 4, live T. vaginalis and T. vaginalis ESP induced cleavage of host cell mTOR to generate approximately 85 kDa smaller protein band and the live T. vaginalis induced higher levels of mTOR cleavage than T. vaginalis ESP that even smaller band approximately 80 kDa appeared right beneath the 85 kDa band. Based on the reciprocal band intensity change between original larger mTOR band and cleaved smaller band, comparison of the mTOR-immunoreactive protein band sizes and the epitope (Ser2481 containing polypeptide) information for the antibody what we used, it is presumed that the cleavage of mTOR occurs at the N-terminal region of mTOR by T. vaginalis. Interestingly, AKT protein level was not affected by T. vaginalis but mTOR downstream target p70S6K had suffered degradation or cleavage by live T. vaginalis infection.

In order to examine how fast T. vaginalis infection makes effect on host cells, we performed time-course studies of mTOR cleavage after incubation of SiHa cells with live T. vaginalis for 0.5, 1, 2, 4, 8, and 16 hr. As shown in Fig. 5, T. vaginalis induced the cleavage of SiHa cell mTOR from 30 min after infection, and these cleavage phenomena were time-dependent, and prominently high levels of cleaved mTOR were detected after infection with T. vaginalis at 8 hr and 16 hr (Fig. 5A). Similarly, when the SiHa cells were infected with T. vaginalis at MOI 0.5, 1, and 2 for 2 hr, T. vaginalis induced mTOR cleavage in parasite load-dependent manner. A high level of cleaved mTOR was also detected in SiHa cells at T. vaginalis MOI 2 for 2 hr (Fig. 5B). Interestingly, Fig. 5A and 5B shows that, based on the infection time and infection strength, mTOR cleavage occurs in more than 2 phases, and in early infection, first N-terminal cleavage occurs to generate rather intermediate form of 170 kDa mTOR fragment. Then, as infection time elongates, this 170 kDa fragments get further cleaved to generate 85 kDa fragment of mTOR, and after 16 hr, most of 170 kDa intermediate mTOR fragment disappeared, and the band intensity of 85 kDa mTOR fragment become stronger.

To determine whether the T. vaginalis metallopeptidase plays a vital role in cleavage of SiHa cell mTOR, live T. vaginalis (MOI 2) were treated for 30 min with 5 mM metallopeptidase inhibitor, 1,10-PT. After incubation finished, T. vaginalis was washed with PBS to remove the inhibitor from the surface of parasites, and then infected with SiHa cells for 16 hr, and cell morphology was observed by phase-contrast light microscopy. Some of live T. vaginalis-infected SiHa cells detached from the culture dish and were slightly darker than the control group (Fig. 6B, C). In addition, cells infected with 1,10-PT-pretreated T. vaginalis lost their lethal effect on cell survival, so that the infected host cells were morphologically similar with the control group (Fig. 6C). Our experimental results showed that the metallopeptidase inhibitor, 1,10-PT, was found to inhibit the mTOR cleavage significantly in SiHa cells (Fig. 6D). The 170 kDa and 85 kDa mTOR fragment bands completely disappeared by 1,10-PT pretreatment and regained the correct immune positive band size of intact mTOR protein. This is the first report about that the T. vaginalis metallopeptidase plays a vital role in cleavage of host cell mTOR.