Chlamydia trachomatis alters iron-regulatory protein-1 binding capacity and modulates cellular iron homeostasis in HeLa-229 cells

Abstract

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.

Figure 1

Effect of CT infection on TfR expression in HeLa cells: HeLa cells were infected at 2 moi and treated with the iron chelator DFX mesylate and ferric ammonium citrate (FAC). Mock-infected cells were considered as the control. Treated and untreated cells were harvested at 24 hpi, and the cell lysates (40 μg) were electrophoresed on 8–12% SDS-PAGE gel, followed by Western blot analysis using anti-TfR and anti-FHC antibodies, with beta-actin as the loading control. (a) Expression of the TfR was downregulated in CT-infected HeLa cells and did not change after addition of DFX and FAC. (b) Expression of FHC was studied in CT-infected HeLa cells in comparison to control. FHC level remained above the basal level (mock) in CT-infected cells after addition of DFX. Conversely, addition of FAC led to a higher degree of induction in the FHC level of CT-infected cells than that in the control. Bars represent standard error. *P < .05 and **P < .01.

Figure 2

Effect of CT infection on IRP-1 expression in HeLa cells: 40 μg cell lysates were electrophoresed on 8% SDS-PAGE gels, followed by Western blotting using anti-IRP-1 and anti-IRP-2 antibodies, with beta-actin as control. (a) CT-infected HeLa cells showing higher expression of IRP-1, which declined after addition of DFX. (b) Decline in the level of IRP-2 expression was observed in CT-infected HeLa cells, and there was further decline consequent to addition of DFX. No significant change was observed in FAC-treated control and infected cells. Bars represent standard error. *P < .05 and ***P < .001.

Figure 3

Effect of CT infection on the interaction of IRP-IRE: cytoplasmic extracts were prepared from CT-infected HeLa cells treated with DFX and FAC, along with their respective controls, at 24 hpi. Furthermore, the extracts were incubated with ³²P-labeled RNA probe, which contained the IRE sequence, in binding buffer. For the supershift assay, the corresponding antibodies (IRP-1 and IRP-2) were added. In parallel experiments, samples were treated with 2-mercaptoethanol) for determination of the maximum binding activity. (a) Binding activity of IRP-1/IRE was attenuated in CT-infected HeLa cells as observed in electrophoretic mobility-shift assay. (b) Inhibition of binding was observed with IRP-1 antibodies in CT-infected HeLa cells. (c) Attenuation of binding activity reverted to normal after treatment with chloramphenicol.

Figure 4

Translational initiation of ferritin and TfR mRNA degradation are regulated by binding of IRP to IRE in response to availability of iron. (1) In the excess iron, IRP forms an open conformation and is not able to bind IRE, subsequently translation of ferritin gets initiated and degradation of exposed TfR mRNA takes place. (2) In iron starved cells, IRP attains a closed conformation and binds to IRE present on 5′ UTR of ferritin mRNA thus interferes with translation initiation and simultaneously stabilize TfR mRNA by binding 3′ UTR. (3) Binding affinity of IRP-IRE is attenuated in presence of chlamydia leading to lower expression of TfR and higher expression of FHC [27].