Figure 1.
Co-infection of HHV6 induces persistence of C. trachomatis.
(A) Morphology of Chlamydia. Transmission electron microscopy (TEM) images of chlamydial inclusions. (a) Single Chlamydia infection, (b) co-infection with HHV6A. (B) Chlamydial inclusions of co-infected cells differ in morphology compared to single infected cells. Chlamydia was stained with an antibody against cHsp60 (red) and the host cell nuclei were visualized by Draq5 staining (blue). In the upper panel, cells were additionally transfected with Golgi-GFP fusion protein constructs. Samples were viewed under a confocal laser microscopy. (C) Co-infection with HHV6A and -6B induces chlamydial persistence. HeLa cells were infected with C. trachomatis (Ctr) alone or together with either HHV-6A, -6B, UV inactivated HHV6A (UV.HHV6A) or -HHV6B (UV.HHV6B). In parallel, latent HHV6 (lat.HHV6A and lat.HHV6B) containing HeLa cells were infected with Ctr. The bars indicate inclusion-forming units (IFU) obtained after standard infectivity assays as described in Materials and Methods. Data represent the mean ± SEM of three independent infection experiments. (D) Early co-infection with HHV6 is necessary for inducing chlamydial persistence. HeLa cells were infected with Chlamydia for 2 h prior to the addition of viral particles for different time points as indicated. In a parallel infection set up, HHV6A was added to Chlamydia-infected cells after 2 h, but subsequently HHV6 was removed from the infection media at the indicated time points. Infectivity assays were performed as described in materials and methods. NI: no infection. Data represent the mean ± SEM of three independent infection experiments. (E) TEM images of chlamydial inclusions in HSB2 cells (a, b) and in monocyte-derived macrophages from healthy blood donors (c, d). Cells were either infected with Ctr alone (a, c) or together with HHV6A (b, d). (F) Chlamydial infectivity is down regulated in monocyte-derived macrophages in presence of HHV6A co-infection. Freshly isolated macrophages were infected with C. trachomatis (Ctr) alone or together with HHV6A and chlamydial infectivity was determined. Data represent the mean ± SEM of three independent samples.
Figure 2.
HHV6-mediated chlamydial persistence differs from other forms of chlamydial persistence and depends on viral entry.
(A,B) HHV6-mediated chlamydial persistence differs from that induced by Penicillin G. HeLa cells were co-infected with Chlamydia and HHV6A (A) or HHV6B (B) for different time intervals in the absence (-PenG) or presence of 10 U/ml of Penicillin G (+PenG). Chlamydial DNA was quantified by qPCR using a primer set against chlamydial ORF LcrH/SycD and normalization against 5S rDNA. Data represent the mean ± SEM of three independent infection experiments. (C,D) CHO cells do not permit HHV6-mediated chlamydial persistence. Infectivity assays were performed in CHO cells using both HHV6A and HHV6B and analyzed by Western blot (C) or inclusion counting after staining the chlamydial inclusions with an antibody against cHsp60 and Cy2-labeled secondary antibody (D). Statistical analysis was based on the Student t-test, and p>0.05 was considered as insignificant. IFU, inclusion forming units. (E,F) Silencing of CD46 prevents HHV6A-mediated chlamydial persistence, but not that of HHV6B. Human CD46 was silenced by transfection of siRNAs in HeLa cells and the knock down efficiency was checked with an antibody against human CD46. Infectivity assays were performed and analyzed by Western blot (E) or inclusion counting (F) after staining the chlamydial inclusions with an antibody against cHsp60 and Cy2 labeled secondary antibody. Statistical analysis was based on the Student t-test, and p>0.05 was considered as insignificant whereas p<0.05 was considered as significant. IFU/ml in figure (D) and (F) represent the mean ± SEM of three independent infection experiments. (G) Productive and latent HHV6 infection affects chlamydial replication in HUVEC cells. C. trachomatis (Ctr) and HHV6A were used to infect either HUVEC cells, which were or were not pre-infected with HHV6A for 2–3 weeks (latH6A) as indicated. DNA was extracted and used for amplifying bacterial DNA as described under (A). Data represent the mean of 3 independent experiments. hpi, hours post infection; dpi, days post infection. (H) Productive HHV6 infection induces chlamydial persistence. Chlamydial infectivity assays were performed and evaluated by detecting chlamydial Hsp60 (cHsp60) by immunoblotting. Fold change values indicate the ratio of cHsp60 to actin values obtained after signal quantification using densitometric analysis. PI, primary infection; SI, secondary infection. NI: no infection.
Figure 3.
Co-infection of HHV6 and C. trachomatis (Ctr) favors viral entry and survival.
(A) HeLa cells were infected with HHV6A for different time intervals either in presence or absence of Ctr. Viral DNA was quantified by qPCR, using a primer set against viral the U94 gene. (B) The amount of HHV6A and Chlamydia (Ctr) DNA increases during co-infection. HeLa cells were co-infected with HHV6A and Ctr for different time intervals. DNA was extracted and used for qPCR with primers for viral U94 ORF and chlamydial LcrH/SycD. Relative DNA amount values are presented in log scale. (C) Removal of virus particles from the supernatant during co-infection prevents the increase of viral DNA. HeLa cells were infected with HHV6A either in presence or absence of Ctr. Two hours post infection (hpi), cells were washed and fresh culture medium was added. Viral DNA was quantified at different time points by qPCR, using a primer set against viral U94 gene. Data represent the mean of 3 independent experiments. (D,E) Inhibition of Chlamydia growth with 10 U/ml Penicillin G (B) or 100 ng/ml doxycyclin (C) reduces viral DNA amount. Antibiotics were added to the co-infected cells at 24 h p.i. Data represent the mean of 3 independent experiments. Relative DNA amount values are presented in log scale. hpi, hours post infection; dpi, days post infection. (F) Viral U94 transcription is induced during co-infection. HeLa cells were co-infected in duplicates with HHV6A and Chlamydia (Ctr) for 24 h. Total DNA and RNA was extracted and viral U94 DNA and RNA transcript levels were quantified by qRT-PCR and normalized against 5S rDNA or 5S rRNA respectively. **, p≤ 0.005. (G,H) HHV6A (G) or HHV6B (H) U94 transcription is increased during co-infection. HeLa cells were co-infected with HHV6A, UV-inactivated HHV6A (UV.HHV6A) and Chlamydia (Ctr) for 24 h as indicated. Total RNA was extracted and viral U94 transcript levels were quantified by qRT-PCR and normalized against 5S rRNA. hpi, hours post infection; dpi, days post infection. *, p≤ 0.05; **, p≤ 0.005. All the data represent the mean of 3 independent experiments performed on the same day.
Figure 4.
HHV6 co-infection induces mitochondrial membrane potential loss and reactive oxygen species (ROS) formation.
(A) Co-infection of HHV6 and Ctr down regulates mitochondrial membrane potential in HUVEC cells. HUVEC cells were infected with Ctr and/or HHV6A. Mitochondrial membrane potential was visualized by staining with MitoTracker (red) and fluorescence microscopy. (B) HHV6 infection increases ROS in host cells. HeLa cells were infected with Chlamydia (Ctr), HHV6A or both for three different time intervals. At the end of the infection, cell-permeable fluorogenic probe 2′, 7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) was added for 1 h at 37°C. Total cellular ROS content was measured using an Elisa reader. Data represent the mean ± SEM of three independent infection experiments performed at the same day. Statistical analysis was based on the Student t-test, and p values between different sample groups are mentioned above the respective line bars. (C) HHV6A and -6B induces Hif-1alpha expression. HeLa cells were infected with Ctr and/or HHV6A or -6B for different time intervals and Chlamydia (cHSP60) and Hif-1α were detected by immunoblotting. Actin was used as a loading control. Fold change values of Hif-1alpha were derived by normalisation to actin and are mentioned below each lane. (D) siRNA-mediated gene silencing efficiency of siNOX1 was checked by quantitative real time PCR. (E) Change in cellular ROS content was verified after 24 h of NOX1 silencing. Data represent the mean ± SEM of three independent experiments. **, p≤0.005. (F, G) siRNA-mediated gene silencing of NOX1 recovers HHV6-induced persistent Chlamydia. HeLa cells were transfected with 5 nM of NOX1 and control siRNA for 48 h and then infected with Ctr alone or together with HHV6A. Infectivity was determined by immunoblotting (F) and inclusion counting (G). Inclusion counting data represent the mean ± SEM of three independent experiments. **, p≤0.005. PI, primary infection; SI, secondary infection. IFU, inclusion forming units. Data in figure (D), (E) and (G) represent the mean of 3 independent experiments performed on the same day whereas the data in figure (F) represents one of the three biological replicates.
Figure 5.
HHV6 co-infection alters cellular glutathione balance.
(A) Effect of HHV6A and Chlamydia infection on cellular GSH and GSSG content. HeLa cells were infected with Chlamydia (Ctr) and/or HHV6A for different time intervals. Total GSH and GSSG contents were measured using commercially available kits. Data represent the mean ± SD of three biological replicates performed on the same day. (B) Effect of glutathione precursor NAC and inhibitor (BSO) on chlamydial infectivity. HeLa cells were infected with Ctr for 2 h before NAC together with HHV6A or BSO without HHV6A were added at different concentrations. Infectivity assay were performed and analyzed by quantitative immunoblotting and counting of the inclusions. Cellular GSH and GSSG content was measured in parallel sets of infected cells 24 h after addition of NAC and BSO. IFU, inclusion forming units. *, p≤0.05; **, p≤0.005. (C) Exogenous supplement of DTT rescues chlamydial infectivity. HeLa cells were infected either with Chlamydia (Ctr) or together with HHV6A. Infected cells were supplemented with DTT (1 mM). Infectivity assay were performed to check chlamydial infectivity. The cellular GSH content was measured in parallel sets of primary infected cells 20 h after addition of DTT. PI, primary infection; SI, secondary infection. (D) 2-AAPA, an inhibitor of glutathione reductase reduces chlamydial infectivity. 2-AAPA was added to HeLa cells at 3 different concentrations and time points of single (Ctr) infected cells. Co-infection with HHV6A and treatment with the solvent DMSO were used controls. NI, no infection. (E) Change in cellular ROS content was measured in HeLa after 24 h of siRNA-mediated GSR silencing. Data represent the mean ± SEM of three independent experiments. **, p≤0.005. (F) siRNA-mediated gene silencing of GSR induces Chlamydia persistence. HeLa cells were transfected with 5 nM of GSR siRNA for 48 h. In parallel, a control siRNA pool was also transfected. siRNA transfected cells were then infected with Ctr for 48 h followed by Infectivity assay. Inclusion counting was carried out to check Chlamydial infectivity. Inclusion counting data represent the mean ± SEM of three independent experiments. *, p≤0.05. NI: no infection. Data in figure (D), (E) and (F) represent the mean of 3 independent experiments performed on the same day.
Figure 6.
HHV6 co-infection alters cellular NADPH balance by inhibiting GSR activity.
(A) Chlamydia infection depletes, HHV6 infection increases cellular NADPH level. HeLa cells were infected with Chlamydia (Ctr) in the presence or absence of HHV6A or HHV6B. Total NADPH content of the cells was measured after 24 h of infection. NADPH level was also measured under similar infection conditions from cells pre-treated with 5 nM 6-ANAM for 24 h. Data represent the mean ± SEM of three independent experiments performed on the same day. *, p≤0.05 (B) 6-ANAM induces chlamydial persistency. HeLa cells were treated with 5 nM 6-ANAM for 24 h followed by infectivity assay to measure chlamydial persistency. Inclusion counting was used to check the chlamydial infectivity. Data represent the mean ± SEM of three independent experiments. (C) Electron microscopy picture of a chlamydial inclusion in HeLa cells treated with 5 mM 6-ANAM for 24 h. (D) Change in cellular ROS content was measured in HeLa cells after 24 h of siRNA-mediated G6PDH silencing. **, p≤0.005. (E) siRNA mediated silencing of G6PDH expression reduces Chlamydia infectivity. HeLa cells were transfected with 5 nM of G6PDH siRNA for 48 h. In parallel, a control siRNA pool was also transfected. siRNA transfected cells were then infected with Ctr for 48 h followed by Infectivity assay. Inclusion counting was carried out to check chlamydial infectivity. *, p≤0.05. Data in figure (D) and (E) represent the mean ± SEM of three independent experiments performed on the same day.
Figure 7.
HCMV and HSV-1 induce chlamydial persistence.
HeLa cells were infected or co-infected with Chlamydia (Ctr) and (A,B) HSV-1 or (C,D) HCMV. Infectivity was monitored by counting inclusion numbers after staining the chlamydial inclusions with an antibody against cHsp60 and Cy2 labeled secondary antibody (A and C) and immunoblotting (B and D). PI, primary infection; SI, secondary infection; NI: no infection; IFU, inclusion forming units. Data in figure (A) and (C) represent the mean ± SEM of three independent experiments performed on the same day. *, p≤0.05; **, p≤0.005. Figure (B) and (D) represent one of the three biological replicates.