Chlamydia trachomatis Alters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells

Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron.


Introduction
Chlamydia trachomatis (CT) is one of the most successful and leading bacterial pathogens that cause sexually transmitted infections; it infects and grows within the genital mucosal epithelial cells [1]. Chlamydial infection is responsible for pelvic inflammatory disease, tubal infertility, ectopic pregnancy, and endometritis, having significant impact on the reproductive health of women worldwide [2]. The developmental cycle of CT includes two forms: an infectious elementary body (EB) and a reticulate body that multiplies within the inclusion by binary fission [3]. A third developmental form-the persistent form and is considered as a mechanism of survival under stressful conditions [4,5]. Persistence is induced in response to changes in the culture medium, including amino acid or iron deprivation and in the presence of antibiotics or cytokines such as gamma interferon (IFN-γ) [6][7][8][9][10].
Iron is the most essential element because of its role in the chemistry related to bioenergetics, being involved in a myriad of biochemical events that are required for growth and development [11]. Excess iron promotes the generation of reactive radicals, which channelize pathogenic cascades that damage cells and tissues. Iron, as an essential nutrient and potential toxin, poses an exquisite regulatory problem in biological systems [12]. Iron homeostasis of cells is regulated posttranscriptionally by the binding of iron-regulatory proteins (IRP)-1 and -2 with iron-responsive elements (IREs). In iron-starved cells, binding of IRP to IRE stabilizes the transferrin-receptor (TfR) mRNA and inhibits the translation of mRNAs that encode the heavy (H) and light (L) chains of ferritin, thus promoting cellular iron uptake and preventing iron sequestration [13].
Almost all cells and organisms exploit iron as a cofactor in a multitude of biochemical activities. CT EBs harvested from desferal-exposed polarized epithelial cells has a reduced infective potential, which is restored after replacement with iron-saturated transferrin medium, thus confirming the dependence of chlamydia on the host for its requirement of iron [14]. Chlamydial genomic annotation provides an idea about the iron-transporter analog of ExbB/D; however, an identifiable TonB is absent [15]. The CT divalent cationdependent regulator is a distant relative of the ferric-uptake regulator family of iron-responsive regulators [16]. The biphasic life cycle of chlamydia provides various opportunities for cross-communication with the host regulatory 2 Journal of Biomedicine and Biotechnology network. No study is available currently regarding the involvement of chlamydiae in the regulation of intracellular iron in infected HeLa cells. This study aims to provide a clue to the chlamydial modulatory effect on host cells at the interface of acquisition and commensal use of iron.

Materials and Methods
Unless otherwise stated, all the reagents were purchased from Sigma Aldrich (Saint Louis, Mo, USA) and antibodies, from Santa Cruz Biotechnology (Santa Cruz, Calif, USA). Plastic wares and glasswares for tissue culture were obtained from Greiner, Germany.
2.1. Culture of CT. CT, laboratory reference serovar D (D/UW-3/Cx), propagated in HeLa-229 cells as described previously [17], were purified and stored at −80 • C in sucrose-phosphate-glutamate medium (pH 7.0). The inocula were confirmed to be free from contamination with Mycoplasma spp. using specific kits (Mycoplasma detection kit, Takara, Madison, USA).

Infection Protocol.
HeLa-229 (1 × 10 6 cells/well) cells were seeded in 6-well tissue-culture plates (Greiner, Germany). Subsequently, the HeLa cell monolayer was washed with Hank's balanced salt solution (HBSS) and inoculated with chlamydial EBs at a multiplicity of infection (moi) of 2. Next, the tissue-culture plates were placed on a rocker (Bangalore Genei Pvt. Ltd, Bangalore, India) for 2 hours at 35 • C to obtain a homogeneous infectivity. The infected monolayer was washed twice with HBSS and supplemented with complete Dulbecco's modified eagle medium containing 10% fetal calf serum. Infected HeLa cells were incubated at 37 • C with 5% CO 2 in a humid environment. Deferoxamine (DFX) and ferric ammonium citrate (FAC) were added after 16 hours postinfection (hpi) in the respective wells; after 24 hpi, the cells were withdrawn for analysis. All the experiments were carried out in triplicate with mock-infected cells as their respective controls.

Electrophoretic Mobility-Shift Assay.
A radiolabeled probe of the IRE was transcribed by the in vitro transcription method. The IRE sequence of 5 GAGTTCCGT-CCAAGCACUTGAAGCAGGAAACTCTCTCCCTATAGT-GAGTCGTATTA3 was used as the template, and the T7 sequence 5 TATAGTGAGTCGTATTA3 as the primer for the amplification step carried out using an in vitro transcription kit (Roche Applied Science, Penzberg, Germany). The probe was treated with RNase-free DNase I to remove DNA contamination and subsequently extracted by the phenol-chloroform (1 : 1, v/v) method. The cytoplasmic extract (20 μg) was mixed with 10 ng of [ 32 P] CTP-labeled IRE in binding buffer (10 mM HEPES, pH 7.6, 40 mM KCl, 3 mM MgCl 2 , and 5% glycerol) and incubated at room temperature for 20 minutes. After incubation, the complex was resolved on a 6% nondenaturing acrylamide gel. In a parallel experiment, samples were treated with 2-mercaptoethanol (2-ME) at a final concentration of 2% before addition of the IRE probe, to allow the complete expression of IRE-binding activity. Amersham phosphoscreen was exposed to the gels for autoradiography and scanned using a PhosphorImager (Typhoon 9700, Amersham, Uppasala, Sweden). Band-shift experiments were carried out at least three times, and one representative experiment is shown.

Immunoblotting of Proteins. CT-infected
HeLa cells were washed with phosphate buffered saline (PBS) and subsequently treated with lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 50 mM NaF, 1 mM Na 3 VO 4 , and 1 mM phenyl methyl sulfonyl fluoride) containing the complete protease-inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined by the Bradford protein assay (BioRad Laboratories) with BSA as standard. Extracted proteins (40 μg) were electrophoresed on 8-12% SDSpolyacrylamide gels and transferred to polyvinyl difluoride membranes (BioRad Laboratories); the membranes were reversibly stained with Ponceau S (Sigma Aldrich) to confirm complete transfer. Membranes were blocked with 5% nonfat dry milk in PBS-Tween-20 and incubated with rabbit anti-IgGs against TfR-1, ferritin heavy chain (FHC), IRP-1, and IRP-2; they were further incubated with the goat anti-rabbit IgG conjugated with horseradish peroxidase. Subsequently, they were developed using diaminobenzamide as the detection agent and analyzed using the Image J software.

Statistical Analysis.
Statistical analysis was carried out with the GraphPad Prism software (version 5.0). Differences were tested for statistical significance by one-way analysis of variance, followed by the Bonferroni posttest. Each experiment was carried out in triplicate.

Expression of TfR in CT-Infected HeLa Cells.
As visualized by the Western blot assay, expression of TfR decreased in CT-infected HeLa cells in comparison to mock-infected cells. After addition of the intracellular iron chelator DFX, there was no change in the expression of TfR, whereas mock infection showed a significant increase. However, iron supplementation with FAC to CT-infected HeLa cells did not lead to a change in the TfR level, in comparison to the respective controls (Figure 1(a)).

Expression of FHC in CT-Infected HeLa Cells.
Chlamydia-infected HeLa cells showed higher expression of FHC in comparison to the mock-infected cells. Following the addition of DFX, FHC expression remained above the basal (mock) level. Moreover, subsequent to addition of FAC, upregulation of FHC was observed in both infected and mock-infected HeLa cells, as visualized by the Western blot assay (Figure 1(b)).

Levels of IRP-1 and IRP-2.
In chlamydia-infected HeLa cells, the level of IRP-1 was higher in comparison to mockinfected cells (Figure 2(a)), whereas IRP-2 did not show any significant difference (Figure 2(b)). Consequent to the addition of the intracellular iron chelator DFX, the level of IRP-1 increased significantly, whereas IRP-2 did not show such change in CT-infected cells in comparison to mockinfected cells (Figure 2(b)). Addition of iron supplement led to significant changes in both infected and mock-infected HeLa cells (Figure 2(a)).

Attenuated Binding Activity of IRP-1/IRE (RNA Electrophoretic Mobility-Shift Assay).
The binding activity was determined using the electrophoretic mobility-shift assay by comparison of the maximum activity (2 ME-treated cytoplasmic extract showed maximum activity) with the actual observed activity. The cytoplasmic extract of CTinfected HeLa cells showed a significant decrease in the binding activity relative to mock-infected cells. In the presence of DFX, increased binding activity was observed in both the conditions although the signal was relatively low in CT-infected cells (Figure 3(a)). When the cytosolic extract was preincubated with anti-IRP1 and anti-IRP2 antibodies, it showed loss of binding only in the case of IRP-1 (Figure 3(b)). Binding activity resumed in CT-infected HeLa cells after chloramphenicol treatment (Figure 3(c)).

Discussion
CT is one of the most successful bacterial pathogen in terms of intracellular survival due to its ability to modulate the host resources toward its own development. Both host and the pathogens have higher affinities for iron, the most essential element required for survival.
In accordance with previous studies, the current results showed a decline in the expression of TfR in CT-infected HeLa cells. In contrast to other intracellular pathogens, CT showed upregulation of TfR expression [18]. TfR-mediated iron acquisition has been reported as an important mode of iron uptake, contributing the major part of the iron requirement in various cell types [19]. Regulation of TfR is sensitive to various intracellular physiological conditions; secretory factors, such as IFN-γ, tumor necrosis factor-α, interleukin-1, and interleukin-6, downregulate the TfR level in various cell types [20]. Experimental models of chlamydial infection have shown upregulation in the levels of IFN-γ [21]. Therefore, these results may suggest the involvement of secretory factors in the downregulation of TfR in chlamydiainfected cell. Furthermore, the present study showed that TfR expression did not change significantly after addition of DFX and FAC. This result deciphers the modulation of the iron-regulatory machinery of cells infected with CT.
In this study, upregulation of FHC was observed in CTinfected cells, which did not alter significantly after addition of the iron chelator DFX. Physiological responses reported Journal of Biomedicine and Biotechnology  for TNF-α, IL-1, IL-6, and IFN-γ include stimulation of Hferritin expression and inhibition of TfR1 expression [19]. These results further explain the involvement of cytokines of the innate immune system in the upregulation of FHC. However, nonresponsiveness of the CT-infected cells to the iron chelator DFX might indicate modulation of the ironregulatory system.
Chlamydia-infected HeLa cells showed the predominant expression of IRP-1 rather than IRP-2, in contrast to mockinfected cells. In iron-depleted condition, IRP-1/2 binding to IRE blocks ferritin-mRNA translation and simultaneously stabilizes the TfR mRNA, thus regulating the iron uptake and storage [22]. Because of the additional 73 amino-acid peptide sequence, IRP-2 is prone to proteosomal degradation in the presence of nitric oxide and iron [23]. Chlamydial infection induces the production of both nitric oxide and IFN-γ/α in different cell types [24]. In response to the same stimulus, IRP-1 and -2 are regulated conversely [25]. Hence, it may suggest that lower levels of IRP-2 might be due to the proteosomal degradation induced by nitric oxide or converse regulation by IFN-γ.
In this study, Chlamydia-infected HeLa cells showed attenuation in IRP-IRE binding activity; however, the binding activity resumed after addition of chloramphenicol. Levels of TfR and ferritin are principally regulated through the binding of IRPs with IREs, and the binding activity of IRP-IRE is dependent on both the free reactive iron (labile iron pool) and the intracellular redox state [13]. The labile iron pool regulates the binding of IRP-1/2 to IREs through distinct mechanisms. In the presence of high levels of iron, IRP-1 assembles in a cubane [4Fe-4S] cluster, inhibiting the IRE-binding activity, and converting IRP-1 to an aconitase. When cellular iron stress is low, IRP-1 binds to IRE targets as an apoprotein (see Figure 4) [26,27]. IRPs are also controlled by other effectors that include reactive oxygen species, which induce the disassembly of the Fe-S cluster of IRP-1 [28], and nitric oxide and hypoxia, which affect both IRPs [23].
Serine phosphorylation is an iron-independent mechanism of IRP-1 regulation, which destabilizes the Fe-S cluster [29,30]. The attenuated binding activity of IRP-1 leads to upregulation of FHC in Chlamydia-infected cells, in comparison to mock-infected cells, which is inhibited subsequent to DFX treatment. These results indicate that the level of intracellular free iron (Labile Iron Pool) is maintained at a critically functional state in chlamydia-infected cells; however, further study is required to establish this assumption. Iron restriction-induced chlamydial persistence is only achieved by DFX and not by exporter-dependent iron deprivation [31]. In a recent study, Nupur et al. have shown that Leishmania donovanii uses labile iron pool (LIP) as a dynamic source of iron inside the cells [32]. These observations further extend the possible involvement of LIP as a source of iron for intracellular development of CT. Hence, this study showed that chlamydial infection has a modulatory effect on the activity of IRPs which leads to decreased TfR and increased FHC expressions thus limiting the intracellular availability of catalytic-free iron and finally reactive oxygen species. Furthermore, chlamydial infection may accelerate the recycling of TfR and transferrin to the surface, thus recharge the required iron in LIP by faster shuttling. Transiting iron in LIP may be sequestered in FHC as stored available iron for CT in HeLa infected cells. Thus, the current findings suggest that attenuation in the binding activity of IRP-1/IRE may be one of the mechanisms involved in modulation of iron homeostasis in CT-infected cells.