Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Abstract

Chlamydia trachomatis is an obligate intracellular human pathogen responsible for the most prevalent sexually-transmitted infection in the world. For decades C. trachomatis has been considered an "energy parasite" that relies entirely on the uptake of ATP from the host cell. The genomic data suggest that C. trachomatis respiratory chain could produce a sodium gradient that may sustain the energetic demands required for its rapid multiplication. However, this mechanism awaits experimental confirmation. Moreover, the relationship of chlamydiae with the host cell, in particular its energy dependence, is not well understood. In this work, we are showing that C. trachomatis has an active respiratory metabolism that seems to be coupled to the sodium-dependent synthesis of ATP. Moreover, our results show that the inhibition of mitochondrial ATP synthesis at an early stage decreases the rate of infection and the chlamydial inclusion size. In contrast, the inhibition of the chlamydial respiratory chain at mid-stage of the infection cycle decreases the inclusion size but has no effect on infection rate. Remarkably, the addition of monensin, a Na⁺/H⁺ exchanger, completely halts the infection. Altogether, our data indicate that chlamydial development has a dynamic relationship with the mitochondrial metabolism of the host, in which the bacterium mostly depends on host ATP synthesis at an early stage, and at later stages it can sustain its own energy needs through the formation of a sodium gradient.

Keywords: ATP synthase; Chlamydia trachomatis; energy metabolism; host-pathogen interaction; mitochondrial metabolism; respiratory chain.

Figure 1.

Respiratory activities of non-infected and C. trachomatis–infected HeLa cells in the presence of respiratory inhibitors and ionophores. Representative traces of oxygen consumption of non-infected (A and C) and infected (B and D) intact HeLa cells in the presence of 1 μm HQNO, rotenone (Rot), oligomycin A (Oligo), or CCCP. Respiratory activities of non-infected (E) and infected (F) digitonin-permeabilized cells were in the presence of NaCl (20 mm), α-ketoglutarate (α-KG, 10 mm), and ADP (0.5 mm). G, oxygen consumption rates of non-infected (upper panel) and infected (lower panel) HeLa cells in the presence of 1 mm KCN or 1 μm of each of the following uncouplers or inhibitors: rotenone, antimycin A (AA), oligomycin A, CCCP, monensin (Mon), and HQNO. H, HQNO titration of rotenone-insensitive respiration of intact infected cells. *, p < 0.05 versus vehicle-treated control (Con).

Figure 2.

Effects of respiratory chain inhibitors or ionophores on mitochondrial membrane potential in Chlamydia-infected HeLa cells. The bright field images show non-infected cells (Control) or Chlamydia-infected cells (Infected). The mitochondrial membrane potential indicator JC-1 was used and visualized in red fluorescence (JC-1, left). The red/green fluorescent images (JC-1, middle) before and after the addition of the inhibitors (JC-1, right) are shown. A, CCCP (2 μm); B, HQNO (1 μm); and C, monensin (2 μm). Scale bar, 20 μm.

Figure 3.

Mitochondrial content and chlamydial major outer membrane protein content in Chlamydia-infected HeLa cells. A, mitochondrial content of non-infected HeLa cells (NI) and Chlamydia-infected HeLa cells (I) were analyzed by Western blot analysis, using antibodies against VDAC on mitochondrial outer membrane and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. B, protein levels of VDAC were normalized to levels of GAPDH. The plot shows the percentage of the VDAC/GAPDH ratio. Results were expressed as mean ± S.D., using three independent experiments. C and D, cell cultures were treated with respiratory chain inhibitors or ionophores at 1 hpi (C) or 12 hpi (D), and protein levels were analyzed by Western blotting using an anti-chlamydial MOMP antibody. GAPDH is shown as a loading control. E and F, graphical display of MOMP/GAPDH content expressed as ratio of MOMP/GAPDH band intensity percentage at 1 hpi (E) or 12 hpi (F) treatment with respect to the control (Con). n = 3–4. *, p < 0.05; **, p < 0.005. The vertical line between control and CCCP bands represents a splice junction (C and D).

Figure 4.

Effect of respiratory chain inhibitors on chlamydial infection in cell culture. A and B, representative images of C. trachomatis–infected HeLa cultures treated with 10 μm oligomycin A (Oligo) or 10 μm HQNO, at 1 or 12 hpi, and stained with HEMA 3 staining or immunofluorescence with anti-MOMP antibodies (green) at 36 hpi. In the fluorescent images, DNA is visualized with Hoechst 33342 (blue). Scale bars represent 20 μm. C and E, size of the chlamydial inclusion was quantified by measuring the area of the inclusions with the inhibitors added at 1 hpi (C) or 12 hpi (E). 110–340 inclusions were measured per condition per experiment using ImageJ. D and F, chlamydial infection rate was determined by quantifying the percentage of inclusion-positive cells with the inhibitors added at 1 hpi (D) or 12 hpi (F). More than 200 cells were quantified per condition per sample. Results were collected from 6 to 8 separate experiments and are expressed as mean ± S.D. Asterisks denote the significance from the vehicle-treated control (Con). *, p < 0.05; **, p < 0.005.

Figure 5.

Detail of Chlamydia-disrupted inclusion under oligomycin A or monensin treatment at 1 hpi. Representative images under ×100 objective lens of C. trachomatis–infected HeLa cells immunostained with anti-MOMP antibodies (green) at 36 hpi. A, control cells (vehicle-treated). B, 10 μm oligomycin A treatment. In type 1, small inclusions are present, and a main inclusion is still formed or it is reminiscent. In type 2, only small chlamydial inclusions are observed. C, 2 μm monensin treatment. Individual chlamydial inclusions are not fused in a single inclusion or it is disrupted. DNA is visualized with Hoechst 33342 (blue). Scale bar, 5 μm. Images are full focus images created after acquisition with BZ-X software merging 0.1 μm Z-stacks. Haze reduction was applied to optimize inclusion appearance.

Figure 6.

Effect of ionophores on chlamydial infection in cell culture. A and B, HeLa cells infected with C. trachomatis were treated with 2 μm monensin (Mon) or CCCP at 1 or 12 hpi and stained with HEMA 3 staining or immunofluorescence using anti-chlamydial antibodies (green) at 36 hpi. Hoechst was used for DNA labeling (blue in fluorescent images). Scale bars, 20 μm. C and E, area of the chlamydial inclusion was measured in >110 inclusions per condition, in six separate experiments using ImageJ. D and F, chlamydial infection rate represents the percentage of infected cells when treatment was applied at 1 hpi (D) or 12 hpi (F). >200 cells were quantified per condition of six to eight different experiments. Error bars represent standard deviation of the mean. Asterisks denote the significance from vehicle-treated control (Con). *, p < 0.05; **, p < 0.005.

Figure 7.

Scheme of Na⁺-based chlamydial energy metabolism.