Differential Induction of Er Stress, Autophagy and Cell Death in Trichomonas Vaginalis -Infected Siha Cells

Trichomonas vaginalis is an extracellular protozoan parasite
known to cause vaginitis and urethritis...


Introduction
Trichomonas vaginalis is an extracellular protozoan parasite known to cause vaginitis and urethritis. Trichomoniasis is the most common non-viral sexually transmitted disease in the world with more than 170 million cases reported per year [1,2]. T. vaginalis colonizes at the epithelium of the human genital tract and is transported into cells by hydrolyzing organic components into glucose, leading to premature membrane rupture, preterm delivery, and low birth weight. Trichomoniasis has also been associated with atypical pelvic inflammatory disease, infertility, and increased susceptibility to HIV infection and invasive cervical cancer [3][4][5][6][7].
Trichomonas vaginalis is an understudied parasitic organism whose pathogenic mechanisms remain unclear, even though it is the cause of a major public health problem. Current literature has focused its contact-dependent mechanisms, where the parasite adheres to vaginal epithelial cells to induce cytotoxicity, as a major contributing factor towards disease development [7][8][9][10][11][12][13]. As for contact-independent mechanisms, it is well known that the secretion of virulence factors by parasitic protists plays a fundamental role in multifactorial host-parasite interactions. As such, other studies have highlighted the effector proteins and proteases that are secreted by Trichomonas vaginalis into the urogenital tract as central players in pathogenesis [14]. We, however, focus on the subsequent cellular stress events following these mechanisms in SiHa cells, a carcinoma cell line of the cervix. Cellular stress plays a key role in both of these mechanisms. It can be induced by external or internal cues and activates several well-orchestrated processes aimed at either restoring cellular homeostasis or committing to cell death [15][16][17]. One of these processes is the Endoplasmic Reticulum (ER) stress response.
The Endoplasmic Reticulum (ER) is a highly dynamic network composed of sac-like structures and tubules. The major function of the ER is to fold and mature nascent proteins that transit through the secretory pathway [18]. Perturbations at the cellular level can affect ER homeostasis and accumulate unfolded proteins in the ER lumen, a condition referred to as ER stress. To alleviate ER stress and ensure cell survival, the Unfolded Protein Response (UPR) promotes degradation of misfolded proteins and induces greater protein folding activity in the ER lumen. However, under prolonged conditions, cells that become irreversibly damaged are subjected to autophagy and ultimately eliminated by apoptosis [19][20][21][22]. In ER stress-mediated cell death, transcriptional and posttranscriptional mechanisms are activated to regulate pro-apoptotic members of the Bcl-2 family to facilitate cytochrome-c release from the mitochondria and calcium release from the ER to engage downstream apoptotic signaling events [23,24].
Notably, mitochondria-dependent apoptosis has been heavily implicated in T. vaginalis-induced pathogenesis. One study identified the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP following T. vaginalis infection [25][26][27]. T. vaginalis infection of human neutrophils also resulted in apoptosis through the activation of caspase-3 and the reduction of Mcl-1 expression [27].
vaginalis induced apoptosis through the action of Bcl-xL but not that of Bcl-2 [29]. These findings merit further investigation on the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins during T. vaginalis infection.
Furthermore, many intracellular pathogens, such as protozoan parasites belonging to genus Plasmodium, Leishmania, Toxoplasma and Cryptosporidium, are known to induce an UPR activity in infected cells [30]. However, UPR pathways in cells infected by extracellular parasites have yet to be reported. T. vaginalis-induced apoptosis may be mediated by UPR. The UPR is mediated by three ER-resident transmembrane proteins that can act as ER stress sensors, IRE1, PERK and ATF6, which are in turn regulated by BiP (also known as glucose regulated protein 78 or GRP78), a major chaperone in the ER lumen [17][18][19][20][22][23][24]. We hypothesized that T. vaginalis-infected cervical epithelial cells would demonstrate altered expression patterns of these key molecules as well as significantly affect downstream pro-apoptotic products such as JNK phosphorylation or CHOP expression and phagophore-promoting events including LC3 lipidation which then binds to the phagophore to form autophagosomes. Understanding how T. vaginalis infection regulates mechanisms such as ER stress, autophagy, and apoptosis is critical for the protection of host cells. In this study, we identify the induction of ER stress in SiHa cells by T. vaginalis infection, which ultimately sequentially resulted in the autophagy and apoptosis of the SiHa cells.

Trichomonas vaginalis culture
The T. vaginalis T106 isolate obtained from one of the author Prof. Jae-Sook Ryu was provided by Prof. J. K. Alderete. T. vaginalis

TCA Precipitation
After cells were washed with PBS (pH 7.4), cells were suspended in ice-cold 10% (w/v) TCA, incubated on ice for 30 min, scraped into microfuge tube, and centrifuged at 13,000 rpm for 5 min at 4℃.
The resulting pellet was washed with ice-cold 5% (w/v) TCA and centrifuged. The supernatants were removed, and the pellets were

Statistical Analysis
Results were repeated at least three independent times and expressed as the mean ± Standard Deviation (SD). P values between groups were determined by a two-tails paired Student t tests. P < 0.05 was considered statistically significant.

Induction of Endoplasmic Reticulum (ER) stress in SiHa cells by T. vaginalis infection
Given that ER stress has been reported to result from pathogenic stress signals such as infections, we examined the involvement of the UPR signaling pathway in T. vaginalis infection of SiHa cells.
GRP78 (BiP) and phospho-eIF2α expression levels have been recognized as good monitors of UPR activation. Thus, to assess the involvement of ER stress, we measured the expression of phospho-eIF2α and GRP78 in T. vaginalis-infected SiHa cells.
The expression level of GRP78 was significantly increased 12 hrs after T. vaginalis MOI 1 infection and remained enhanced at 48 hrs ( Figure 1A). Phospho-eIF2α expression was also significantly  (C) Cell lysates were prepared as in (A) and analyzed with anti-Atg4B, anti-Atg5, anti-Atg7, and anti-α-tubulin anti-bodies.

Induction of autophagy in the SiHa cells infected with T. vaginalis
Since autophagy has been reported to be linked to infections and ER stress, we then investigated how the autophagy process is involved in the physiology of T. vaginalis-infected SiHa cells. As an indicator of autophagosome formation and autophagy, LC3 was detected in two forms: LC3-I and II (PE-conjugated form).
We found that the expression of the LC3-Ⅰ band was induced at 12 hrs ( Figure 1B). On the other hand, LC3-Ⅱ expression significantly increased at 18 h, peaked at 24 hrs, and decreased at later time points ( Figure 1B). ATG5 expression, which is important for phagophore formation, steadily increased in T. vaginalis-infected SiHa cells while ATG4B and ATG7 expression levels were not significantly altered ( Figure 1C). These results suggest that T.
vaginalis infection may induce autophagy in SiHa cells.
The unique LC3-II expression dynamics prompted an examination of whether autophagic flux is affected by T. vaginalis infection. The dynamics of p62 (SQSTM1/sequestosome 1) protein expression was examined, because as a ubiquitin-and LC3-interacting protein, p62 binds and transports ubiquitinated proteins into autophagosomes for degradation. Expression levels of this autophagy substrate were significantly enhanced between 18 and 24 hrs and degraded after 36 hrs ( Figure 1D). This finding suggests that clearance of ubiquitinated proteins by p62, and thus autophagic flux, was somehow blocked at 18-24 hrs following the infection and resolved later.

Induction of apoptosis the SiHa cells infected with T. vaginalis
The prolonged enhancement of GRP78 and phospho-eIF2α  Cell lysates were prepared through the procedure described in the materials and methods section and then subjected to immunoblot analysis using anti-Bcl-xL, anti-Mcl-1, and anti-α-tubulin antibodies. (B) Total cell lysates were prepared as in (A) and subjected to analysis using anti-Bim, anti-Bak, anti-Bax, and anti-α-tubulin antibodies. (C) Samples were prepared like in (A) and subjected to immunoblot analysis using anti-PARP, anti-cleaved caspase-9, anticleaved caspase-3, and anti-actin antibodies. Positions of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 are indicated. (B) Cell lysates were prepared as in (A) and subjected to analysis using anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and anti-α-tubulin antibodies.
Mitochondria-dependent apoptosis was analyzed by measuring protein expression levels of PARP and caspases 9 and 3. Cleaved caspase-9 and cleaved PARP were markedly elevated at later time points 36 and 48 hrs ( Figure 2C), indicating that apoptosis was induced in T. vaginalis infected-SiHa cells. Interestingly, cleaved caspase-3 was induced early on at 18 hrs following infection ( Figure   2C). When the activities of the MAPK signaling pathways were examined, p-ERK was dominant at early time points while p-JNK and p-p38 were induced and sustained strongly at later time points (figure 2D). These results suggest that JNK and p38 pathways are heavily involved in maintaining stress signals in SiHa cells infected with T. vaginalis.

Discussion
Trichomonas vaginalis is an anaerobic, aerotolerant pathogen known to cause trichomoniasis, the most common sexuality transmitted disease in the world. Previous studies have implicated the importance of ER stress dynamics in pathogen-induced cell death. Here, we examine various protein expression patterns in cervical epithelial cancer (SiHa) cells to argue that T. vaginalis also utilizes ER stress pathways to promote disease. Specifically, we demonstrate that there is a differential induction of UPR signaling, autophagy, and apoptosis pathways. Affirming the induction of ER stress following T. vaginalis infection, we found that both GRP78 and phospho-eIF2α expression levels were significantly enhanced early on (12 hrs) and sustained at 48 hrs ( Figure 1A). GRP78 is an important regulator of ER stress sensors. It can bind to IRE1, PERK, and ATF6 to inhibit their signaling under normal conditions. When ER stress is enhanced, GRP78 is released and these stress sensors can change conformation to their active states. Released GRP78 can then bind to misfolded proteins while activated IRE1, PERK, and ATF6 upregulate GRP78 production [16,19,21] making GRP78 a strong marker for ER stress.
The increase in phospho-eIF2α expression between 24 and 48 hrs suggests an upregulation of this PERK/p-eIF2α/ATF4 pathway. In addition to lipidation of LC3, PERK induces the apoptosis through increase of CHOP expression. ATF4 and C/EBP homologous protein (CHOP), a transcription factor induced by ATF4, are known to transcriptionally regulate more than a dozen ATG genes [16,17,19,21]. In line with these pathways, we found an early increase in expression of LC3-Ⅱ and ATG5, suggesting the induction of autophagy in SiHa cells ( Figure 1B, C). Interestingly, the expression of p62 was only briefly enhanced at 18-24 hrs and then resolved. p62 is responsible for transporting ubiquitinylated proteins into autophagosomes for degradation, suggesting that the autophagic flux was blocked for a short period following infection ( Figure 1D).
PERK induced-apoptosis is regulated via inhibition of antiapoptotic Bcl-2 members and activation of pro-apoptotic BH3 proteins [17,21,24]. We found that pro-apoptotic factor Bim, as well as key cell death executioner proteins Bax and Bak, showed a strong and sustained induction at later time points. Compared to the sustained strong expression of p-elF2a, Bim, and Bak, autophagy indicator proteins LC3-Ⅱ and ATG5 showed only transient induction, suggesting that even though ER stress-induced autophagy serves as a pro-survival mechanism, cell death-promoting branches of the ER stress pathways prevailed. Relatively higher enhancement of pro-apoptotic proteins Bim and Bak compared to pro-survival BCLXL and Mcl-1 at late time points may also contribute to cell death [24,28,31]. Expression of the cleaved forms of Caspase-9 and PARP indicate a mitochondria-dependent apoptotic mechanism [16,17,21]. IRE1 is also known to contribute to both the autophagic and apoptotic pathways, specifically by promoting Jun-N-Terminal Kinase (JNK) phosphorylation [32]. Our data show that JNK and p38 were phosphorylated early following T. vaginalis infection and continued to be phosphorylated, demonstrating particularly strong expression after 18 hrs ( Figure 2D). Phosphorylated JNK can in turn phosphorylate Bcl-2, allowing the dissociation of Bcl-2 from Beclin-1 and the initiation of autophagy [15,17]. IRE1α also stimulates activation of the Apoptotic-Signaling Kinase-1 (ASK1), which activates downstream products of stress kinases and p38 MAPK to promote apoptosis [32]. Among the apoptosisinducing substrates of JNK are Bcl-2 and Bim, which are inhibited and activated, respectively, by JNK phosphorylation [33,34]. In addition, p38 MAPK phosphorylates and activates the transcription factor CHOP, which causes changes in gene expression that favor apoptosis, including increasing expression of Bim and DR5, while decreasing expression of Bcl-2 [15,17,22,31,35]. The enhancement of JNK and p38 phosphorylation pathways at later time points may be crucial to maintaining the stressed status of the T. vaginalisinfected SiHa cells and promoting apoptosis.
Our data strongly suggest that T. vaginalis infection induced significant ER dysfunction in cervical epithelial cells and activated an autophagic response as a protective mechanism. The brief period in which autophagy seems to be blocked needs further investigation, as it may represent a potential shift in the balance of pro-apoptotic and pro-survival Bcl-2 proteins. In addition, JNK could be a potential regulation point of various signaling pathways involved in the regulation of autophagy and apoptosis in T. vaginalisinfected SiHa cells. Understanding the interaction of ER stress, autophagy, and apoptosis can provide valuable insight into host defense mechanisms and protection against formidable pathogens.

Funding
This work was supported by research fund of Chungnam National University.

Conflicts of Interests
The authors declare that they have no competing interests.