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Persistent and acute chlamydial infections induce different structural changes in the Golgi apparatus Huiling Zhu a,1 , Hongmei Li a,1 , Pu Wang a,1 , Mukai Chen a,1 , Zengwei Huang a,b , Kunpeng Li a , Yinyin Li a , Jian He a , Jiande Han a,∗ , Qinfen Zhang a,∗∗ a State Key Laboratory of Biocontrol, School of Life Sciences, Department of Dermatology in the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China b Guangzhou Sugarcane Industry Research Institute, Guangzhou, China
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Article history: Received 17 October 2013 Received in revised form 20 March 2014 Accepted 24 March 2014 Keywords: Chlamydia trachomatis Acute infection Persistent infection Golgi fragmentation
a b s t r a c t Chlamydia trachomatis causes a wide range of diseases that have a significant impact on public health. Acute chlamydial infections can cause fragmentation of the Golgi compartment ensuring the lipid transportation from the host cell. However, the changes that occur in the host cell Golgi apparatus after persistent infections are unclear. Here, we examined Golgi-associated gene (golga5) transcription and expression along with the structure of the Golgi apparatus in cells persistently infected with Chlamydia trachomatis. The results showed that persistent infections caused little fragmentation of the Golgi. The results also revealed that Golgi fragmentation might be associated with the suppression of transcription of the gene golga5. © 2014 Elsevier GmbH. All rights reserved.
Introduction Chlamydia trachomatis is an obligate intracellular bacterium that causes many diseases in human beings. It is the leading pathogen of preventable blindness worldwide, and the most frequently reported sexually transmitted disease (Schachter, 1999). Indeed, about 40% of sexually transmitted diseases are caused by Chlamydia trachomatis (Stephens et al., 2011). The bacterium has a biphasic developmental cycle that alternates between infectious extracellular elementary bodies (EBs) and metabolically active intracellular reticulate bodies (RBs); the latter multiply within vacuoles called inclusions (Abdelrahman and Belland, 2005; Stephens et al., 2011). The normal life cycle of the bacterium can be disturbed by antibiotics, nutrient depletion, immunological factors, phage infection, and monocytic infection, resulting in a persistent infection (Beatty et al., 1994). Persistent Chlamydiae are characterized by morphologically enlarged RBs, with a slow metabolism and weakened
∗ Corresponding author at: Build 415#, Room 101, School of Life Sciences, Sun Yat-sen University, Xingang West Road 135, Guangzhou, China, 510275. Tel.: +86 20 84112286; fax: +86 20 84110108. ∗∗ Corresponding author. E-mail addresses: hanjd [email protected] (J. Han), [email protected] (Q. Zhang). 1 These authors contributed equally.
infectivity, which are called aberrant bodies (ABs). The persistent chlamydial infection is thought to be related to many chronic diseases, including arthritis and trachoma (Gerard et al., 2010; Mpiga and Ravaoarinoro, 2006). In subfertile women with tubal pathology, serological markers of persistent Chlamydia trachomatis infections are significantly more common compared to women without tubal pathology (den Hartog et al., 2005). Therefore persistent infection of the urogenital tract in women could be an important cause of infertility. More seriously, persistent infection of Chlamydia trachomatis is difficult to diagnose and treat because it is often culture-negative and resistant to antibiotics (Patton et al., 1994). During the persistent state, Chlamydiae display a transcriptional profile that is significantly different from cells in the acute infection (Belland et al., 2003; Nicholson et al., 2003). Previous studies have undertaken genomic transcriptional profiling using microarray and differential gene expression analyses of Chlamydiae cells in the persistent and acute infections (Belland et al., 2003; Nicholson et al., 2003). Alterations in host gene expression after chlamydial infection has also been studied; however, most of these genes are involved in inflammatory response (Ren et al., 2003). Not much is known about how the expressions of the genes associated with host Golgi apparatus respond to the induction of persistent chlamydial infections. Chlamydiae interact with host cells to ensure their own survival and multiplication. Such methods include subverting the host cytoskeleton (Carabeo et al., 2002), hijacking membrane trafficking
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Please cite this article in press as: Zhu, H., et al., Persistent and acute chlamydial infections induce different structural changes in the Golgi apparatus. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.03.002
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pathways (Grieshaber et al., 2003), inducing Golgi fragmentation (Heuer et al., 2009), and inhibiting apoptosis (Fan et al., 1998). The Golgi apparatus processes and packages proteins, lipids, and other macromolecules. It receives proteins and other molecules from the endoplasmic reticulum and modifies, sorts, and packages them for storage within the cell or secretion to the cell exterior. Golgi fragmentation is a common feature of a number of physiological processes such as mitosis and apoptosis (Heuer et al., 2009; Rabouille and Jokitalo, 2003). During mitosis, fragmentation is related to Golgin-84 (Diao et al., 2008) and GM130 (Lowe et al., 1998), whereas during apoptosis, fragmentation is caused by caspase-dependent cleavage of giantin and golgin-160 (Mancini et al., 2000). Like many other intracellular pathogens, Chlamydia trachomatis requires host cell lipids, such as sphingolipids and cholesterol, for multiplication (Hatch and McClarty, 1998; van Ooij et al., 2000). Acute infection with Chlamydia trachomatis induces Golgi fragmentation. The breakdown of the Golgi structure into smaller elements around the inclusions increases lipid transport into the inclusions. However, no study has yet examined changes in Golgi structure that occur in cases of persistent infection. Golgi fragmentation is reported to be triggered by the successive cleavage of the Golgi matrix protein Golgin-84 (Heuer et al., 2009). While another research demonstrated that 11 previously reported protein cleavages in Chlamydia-infected cell are due to proteolysis during cell lysis and Chlamydia-induced Golgi reorganization occurs in the absence of detectable golgin-84 cleavage, which highlights the need to re-evaluate the Chlamydia literature on CPAF (chlamydial protease or proteasome-like activity factor) (Chen et al., 2012). However, there is currently no evidence on whether Golgin-84 is degraded in the persistent Chlamydia infection. Here, we used immunofluorescence analysis, transmission electron microscopy (TEM), electron tomography (ET), immunoblot analysis, and RT-PCR to examine the process of Golgi fragmentation during both persistent and acute chlamydial infections. The results indicated that chlamydial persistent infection of host cells caused much less Golgi fragmentation than acute infection.
organisms were then scraped from culture dishes 48 h later. The supernatant was analyzed to determine the number of infectious forming units (IFU) per ml. Data presented were the means with standard errors of three representative experiments (Fig. S1). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.03.002.
Immunofluorescence analysis Cells grown on coverslips as previously described (Huston et al., 2008) were fixed with cold 4% paraformaldehyde, permeabilized in 0.5% (v/v) Triton X-100 (Sigma–Aldrich, USA), and then blocked with 1% (w/v) BSA (Thermo Scientific, USA). The cells were then incubated for 1.5 h at room temperature with a mouse anti-CT MOMP antibody (Abcam, UK) diluted 1 in 400 of 1% BSA followed by washed three times with PBS and blocked with 1% (w/v) BSA before incubation for 1 h with goat anti-mouse IgG-FITC:sc-2010 (Santa Cruz Biotechnology, USA) diluted 1 in 500 of 1% BSA (w/v). The cells were then washed three times with PBS, and followed by incubated for a further 1.5 h with a rabbit anti-GM130, anti-Golgin-84 and anti-Giantin antibody (Abcam, UK) diluted 1 in 200 of 1% (w/v) BSA. Because of the similar results from using these three markers (Figs. S2 and S3), we finally used GM130 as the markers in all the experiments. After washing three times with PBS, the samples were incubated with goat ant-rabbit IgG-PE:sc-3739 (Santa Cruz Biotechnology, USA) diluted 1 in 250 of 1% (w/v) BSA. Finally, the samples were labeled with the nucleic acid stain, ProLong Anti-Fade DAPI (Invitrogen, USA) and observed under a ZEISS Axio Imager Z1 immunofluorescence microscope (Zeiss, Germany) fitted with a 100× oil objective lens. Images were captured using a Carl Zeiss Axio Cam, and fluorescence was quantified using the AxioVision 4.7 (Zeiss). Supplementary Figs. S2 and S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm. 2014.03.002.
Real-time PCR Materials and methods Acute and persistent chlamydial infection HeLa cells (HeLa S3 (ATCC® CCL-2.2TM ), USA) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Gibco, USA) using a six-well plate at 37 ◦ C in 5% CO2 . When the population reached 3–6 × 105 cells per well, Chlamydia trachomatis serovar D at an MOI of 8 was added into the wells and incubated with the cells by centrifugation at 3400 × g at 37 ◦ C for 1 hour (h). Samples were then incubated for another hour at 37 ◦ C in 5% CO2 . Extracellular bacteria were removed by aspirating the supernatant followed by the addition of fresh RPMI 1640 medium supplemented with 10% fetal calf serum, 0.5% glucose, and the cells were maintained under the same conditions until they were harvested at different time points respectively. For the persistent infection group, all the procedures were the same except for the addition of 100 U/ml penicillin G (Sigma Life Science, USA) in the medium after centrifugation (Skilton et al., 2009). Control HeLa cells (not infected by Chlamydia trachomatis) were also treated with the above medium with/without 100 U/ml penicillin G. The persistent infection was confirmed by the reactivation assay of treated samples (Goellner et al., 2006). The detailed steps are as follows. At 24 h post infection (p.i.) and 48 h p.i., the medium was replaced with fresh RPMI 1640 medium supplemented with 10% fetal calf serum, 0.5% glucose, with no penicillin. Chlamydial
Total RNA was extracted and purified using an RNeasy mini Kit (QIAGEN), and 31–62 g of RNA was eluted in 50 l of RNase-free H2 O. RNA (2 g) was added to RNase-free H2 O to a final volume of 15.5 l and then denatured at 70 ◦ C for 5 min. The RNA samples were then incubated with 2 l of random hexamers (Promega), 5 l of 5× reaction buffer, 1 l of 10 mM dNTP mix (Promega), 20 units of RNase inhibitor, and 200 units of Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Invitrogen) (final reaction volume, 25 l). Reverse transcription reactions were performed at 37 ◦ C for 60 min (with a final step at 72 ◦ C for 10 min). cDNA was analyzed by agarose gel electrophoresis. The primers for real-time PCR (see the supplementary table) were synthesized by Beijing Genomics Institute (BGI) and stored at a concentration of 5 M in ddH2 O. A Roche LightCycler 480 was used to measure the levels of golga5 and gapdh cDNA. Each reaction contained 1 l of gene-specific forward and reverse primers, 5 l of 2× SYBR Green Master Mix (Roche), 2 l of cDNA template, and 1 l of ddH2 O (final volume, 10 l). The conditions for real-time PCR were as follows: pre-denaturation at 95 ◦ C for 5 min, followed by 40 cycles at 95 ◦ C for 10 s, 54 ◦ C for 15 s, and 72 ◦ C for 6 s. All reverse transcription reactions and real-time PCR reactions were performed in triplicate. Roche LightCycler 480 software (version 1.5) was used for sample quantification. All experiments were repeated three times. Supplementary table related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.03.002.
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Immunoblot analysis HeLa cells (controls and infected cells) were lysed in RIPA buffer (Thermo Scientific, USA) at the indicated time points, and the protein concentrations were determined using the Pierce BCA kit (Thermo Scientific, USA). Total proteins (20 g/lane) were separated in a reducing SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was incubated with rabbit anti-GM130 (Abcam, UK) diluted 1 in 10,000 of 5% (w/v) BSA, mouse anti-Golgin-84 (BD Transduction Laboratories, USA) diluted 1 in 200 of 5% (w/v) BSA, or mouse anti-GAPDH (Bioworld, USA) diluted 1 in 15,000 of 5% (w/v) BSA to detect the respective proteins. The membranes were then washed and incubated with peroxidase conjugated goat anti-rabbit IgG (Thermo Scientific, USA) diluted 1 in 150,000 of 5% (w/v) BSA or peroxidase conjugated goat anti-mouse IgG (Thermo Scientific) diluted 1 in 20,000 of 5% (w/v) BSA and developed using the ECL reagent (Pierce, USA), as described previously (Huston et al., 2008). Sample preparation for transmission electron microscopy Cells were harvested by centrifugation at about 1000 g, fixed in 3% glutaraldehyde (SPI Supplies, USA) for 1 h, and then with 1% osmic acid (Electron Microscopy Sciences, EMS, USA) for 90 min. The cells were dehydrated in an acetone gradient and embedded in Epon812 resin (TED PELLA INC., USA), as described previously (Huang et al., 2010; Qinfen et al., 2004). Thin (∼100 nm) sections were cut with a LKB2188 microtome (LKB BROMMA, Sweden), transferred to 200 mesh carbon-coated copper grids, stained with 2% uranyl acetate for 30 min followed by Reynold’s lead citrate for 10 min, and observed under a JEM 100CX II transmission electron microscope (JEOL Ltd, Japan) at 100 kV to determine specimen quality and to select cells suitable for subsequent study. Blocks containing suitable cells were returned to the microtome and semi-ultrathin (∼250 nm) sections were cut and transferred to carbon-coated slot grids. The sections were post-stained with 2% uranyl acetate for 30 min followed by Reynold’s lead citrate for 10 min. Additional colloidal gold particles (15 nm) were deposited on both surfaces of the semi-ultrathin sections and used as fiducial markers during subsequent image alignment. Electron tomography data collection and reconstruction Semi-ultrathin sections (∼250 nm) were prepared for tomography as described previously (Ladinsky et al., 1999) and examined using a JEM 2010 TEM (JOEL, Japan). When a suitable Golgi region was selected, a single axis tilt series was digitally recorded using a 2k × 4k CCD (Gatan CA, USA); the samples were manually tilted at 2◦ increments over a range of 120◦ (±60◦ ). The nominal magnifica˚ tion was 10k and the final A/pixel was 6.55. Images were aligned using the IMOD software package (Kremer et al., 1996), using the colloidal gold particles as fiducial markers. The reconstructed 3D subcellular structures were then segmented and rendered using Chimera (Pettersen et al., 2004). Statistical analysis Statistical analyses were performed using the SAS program (version 8.1, 1999–2000, SAS Institute Inc., Cary, NC). Method was carried out by analysis of t-test. Data were expressed as mean ± standard error (SE). For the length of Golgi apparatus, the measuring tools in CCD (Megaview G2, OSIS, Germany) were used to determine the length of the Golgi stacks in all groups of HeLa cells at 48 h p.i. In total, we measured the lengths of 79, 77, 31 and 51 Golgi apparatus from
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the Hela cells without and with penicillin treatment, the acutely infected and persistently infected cells respectively. For the number of the different types of vesicles from the Golgi apparatus, we in all classified and calculated the vesicles from 15, 9 and 11 tomograms for the control (include the cells with and without penicillin treated), acutely infected and persistently infected cells respectively. In each tomogram, there are usually more than 10 vesicles. Results There is little Golgi fragmentation in cells harboring a persistent Chlamydia infection In order to confirm the persistent infection, reactivation assays of the samples were determined where the penicillin was removed from the culture medium at 24 h and 48 h p.i. The results (Fig. S1) showed that Chlamydiae in persistency hardly had any infecting ability, while those Chlamydiae rescued at two different time points p.i. from persistently infected cells still could regain their infectivity. These results confirmed the persistent chlamydial infection induced by penicillin. To determine whether the Golgi undergoes fragmentation in response to persistent infection, we examined its morphology by immunofluorescence microscopy. At all time points p.i., control cells treated either without (Fig. 1A, E, I, M, Q and U) or with penicillin (Fig. 1D, H, L, P, T and X) contained a normal condensed Golgi apparatus within the cytoplasm and close to the nucleus, indicating that penicillin did not affect the structure of the Golgi apparatus. At 20 h p.i., most of the cells acutely infected with Chlamydia showed a condensed Golgi apparatus that was closely associated with the chlamydial inclusions and the nucleus (Figs. 1B, S2 and S3). As the acute infection progressed, the Golgi apparatus showed significant morphological changes, resulting in the dispersed distribution of the Golgi stacks around the inclusions (Fig. 1F, S2 and S3). At 40 h p.i., not only the Golgi elements were further dispersed (Figs. 1J, S2 and S3), but also the amount of the Golgi apparatus decreased. In 18.9% of acutely infected cells, the Golgi apparatus had completely disappeared by 40 h p.i., while there was no disappeared in the persistently infected cells. This finding indicated that the lysis of the host cells occurred since 40 h p.i. in the acutely infected cells. The Golgi apparatus dispersing and lysis of the host cells became much more serious at 72 h and 96 h p.i., and it was difficult to find the intact cells with intact inclusion or Chlamydia at 120 h p.i. in acutely infected cells (Fig. 1V). These results indicate that acute chlamydial infection induces the Golgi apparatus to break down into small-dispersed fragments and finally can induce the lysis of the host cells. Surprisingly, at 30 h p.i. the Golgi apparatus in most persistently infected cells retained the intact condensed structure and remained closely associated with the inclusions and cell nucleus (Fig. 1G, S2 and S3). This was still the case at 40 h (Figs. 1K, S2 and S3), 72 h, 96 h, and 120 h p.i., although in some areas, there were a few dispersed fragments at 120 h p.i. in the persistently infected cells. And in all the time points, the chlamydial inclusions remained intact. These results suggest that persistent infection with Chlamydia trachomatis induces less fragmentation of the Golgi than acute infection. Electron microscopy analyses confirms that persistent chlamydial infection causes little Golgi fragmentation We next confirmed that the structure of the Golgi apparatus was still intact after persistent infection using TEM. We used a combination of conventional TEM and ET to examine the
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Fig. 1. Immunofluorescence analysis marked with antibody again GM130. Comparison of Golgi morphology in non-infected (control) (A, E, I, M, Q, and U), acutely infected (B, F, J, N, R and V), persistently infected (C, G, K, O, S and W), and penicillin-treated (D, H, L, P, T and X) cells at the indicated time points post-infection. Chlamydiae were stained with FITC-labeled anti-chlamydia MOMP (green channel). The Golgi was stained with rabbit monoclonal anti-GM130 antibody labeled with Cy5 (red) and then analyzed under an immunofluorescence microscope. DNA was stained with Hoechst (blue). Scale bar = 5 m.
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Fig. 2. Electron micrographs of acutely and persistently infected cell. A–C show acutely infected cells at 36 h, 48 h and 68 h p.i., respectively, whereas D–F show persistently infected cells at the same time points. (A) In the acutely infected cells at 36 h p.i., about half of the Chlamydiae are RBs. (B) In the acutely infected cells at 48 h p.i., most of the Chlamydiae are EBs and the inclusion predominately occupied the cytoplasm of the host cell. (C) In the acutely infected cells at 68 h p.i., there are few Chlamydiae left in the inclusion which still predominately occupied the cytoplasm of the host cell. (D) Fewer Chlamydiae are present in the persistently infected cells at 36 h p.i., and three of them in the image are enlarged to be at least 2 times the size of a normal RB with extra empty vesicle attached, and usually they are named as ABs. In (E) and (F), the Chlamydiae are similar at 48 h and 68 h p.i. respectively, some of them contained multiple electron-dense granules (arrows). The host cells remain intact at all time points in persistent infection group. Bar = 500 nm. N: nuclear, EB: elementary body, RB: reticulate body, AB: aberrant body
Chlamydiae, the host HeLa cells, and the Golgi apparatus. At 36 h p.i., half of the Chlamydiae in acutely infected cells had developed into EBs (Fig. 2). At 48 h p.i., most had developed into EBs, and at 68 h p.i. the majority of HeLa cells were disrupted and the Chlamydia had been released. Most Chlamydiae in penicillin-treated infected cells enlarged to be 2–5 times the size of a normal RB, and some of them contained multiple electron-dense granules or with extra empty vesicles attached. Usually this type of Chlamydiae in EM was named as AB. They also showed a near-consistent morphology at all time points tested. Also, the number of Chlamydiae was much lower than that in acutely infected cells at all time points tested.
The majority of HeLa cells were intact at 68 h p.i. These EM results indicated that persistent chlamydial infection was successfully induced by penicillin (Fig. 2). There was no obvious difference in the size of the Golgi apparatus in persistently infected cells (∼908 nm in length), control cells (∼898 nm in length), or cells treated with penicillin (∼796 nm in length) (Fig. 3). The P-values between the control cells and the persistently infected cells or cells treated with pencillin were 0.470 and 0.194 respectively, which were both larger than 0.05. However, the Golgi apparatus in acutely infected cells was clearly shorter (∼510 nm in length) (Fig. 3). And the P-value was 0.0147, smaller
Fig. 3. Structure of the Golgi in infected cells. The Golgi is indicated by the orange contour. (A) Golgi in an acutely infected cell (length, ∼0.5 m). (B) Golgi in a persistently infected cell (length, ∼2 m). M: mitochondria, EB: elementary body, AB: aberrant body. Bar = 500 nm. (C) Lengths of the Golgi in control cells, acutely infected cells, and persistently infected cells. Columns show the average length of the Golgi in each group. The bars represent the standard error of the mean. Control: Normal HeLa cells. Control P: HeLa cells treated with Penicillin. Acute: Cells acutely infected by Chlamydia trachomatis. Persistent: Cells persistently infected by Chlamydia trachomatis.
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Fig. 4. Golgi secretory vesicles in infected cells. (A) Golgi vesicle gallery. Top row shows the non-coated vesicles with no projecting proteins on the membrane. Middle row demonstrates the non-clathrin-coated vesicles with short protruding proteins, which make the membrane appear thicker. Bottom row displays the clathrin-coated vesicles, with a regular arrangement of clathrins projecting from the vesicle membrane (arrow). All maps were slices in 26.2 nm thickness taken from the tomograms. Bar = 30 nm. (B) The average numbers of the non-coated vesicles and (C) The average numbers of the coated vesicles. The columns show the average numbers of different types of the vesicles in one Golgi tomogram of the normal HeLa cells (Control), acutely infected cells (Acute), and persistently infected cells (Persistent). The bars represented the standard error of the mean.
than 0.05. TEM analyses of persistently infected cells suggested the absence of any obvious Golgi fragmentation, whereas the length of the Golgi apparatus in acutely infected cells was about half that in persistently-infected and control cells. We next used ET to obtain 3D images of the Golgi structure. ET enables researchers to measure the length of the Golgi apparatus and makes the vesicles in the trans-Golgi network easier to be recognized (Ladinsky et al., 1999). We classified the Golgi secretory vesicles into three different types as previously (Ladinsky et al., 1999) described: vesicles without a coating, clathrin-coated vesicles, and vesicles with a non-clathrin coating (Fig. 4A). There were no obvious differences of the average numbers of the non-coated vesicles in the Golgi apparatus among the acutely infected, persistently-infected cells and the
control group (The P-values were 0.353 and 0.710 respectively, P > 0.05) (Fig. 4B). However, the numbers of the clathrin-coated and non-clathrin-coated vesicles in the Golgi apparatus of acutely infected cells obviously increased (The P-values were 0.026 and 0.023 respectively, P < 0.05) (Fig. 4C), whereas in the Golgi apparatus of persistently infected cells, they only showed a slight increase (The P-values were 0.375 and 0.345 respectively, P > 0.05) (Fig. 4C). Taken together, the immunofluorescence and TEM results showed that persistent chlamydial infection had little effect on the structure of the Golgi. Many studies showed that Golgin-84 and GM130 are associated with Golgi fragmentation. Therefore, we then used immunoblotting and real-time PCR to examine whether persistent infection induced changes of these proteins.
Fig. 5. Immunoblot analysis of Golgin-84 and GM130 in the lysate of acutely and persistently infected cells. (A) Immunoblot analysis of cell lysates at 20 h, 30 h, 40 h, 72 h, 96 h and 120 h p.i. demonstrate the degradation of Golgin-84 and GM130 in the lysates of acutely (A) and persistently (P) infected cells. In acutely infected cell lysates, Golgin-84 was degraded into a 78 kDa fragment at 20 h post-infection and further degraded into a 65 kDa fragment by 30 h p.i. and later. In persistently infected cells lysates, two separate fragments were still visible even at 120 h p.i. and the ratio of the 78 kDa fragment and 68 kDa fragment almost remain constant after 72 h p.i. There was no degradation of Golgin-84 in the lysates of non-infected cells not treated (C) or treated (Cp) with penicillin. There was a clear reduction in GM130 in the lysates of the acutely infected cells (A), whereas levels remained relatively constant in the lysates of the persistently infected (P) and control (C and Cp) cells. GAPDH levels remained almost constant in all groups. (B) Quantification ratio of Golgin-84/GAPDH demonstrated in (A). Black: the intact Golgin-84 84 kDa/GAPDH ratio, Red: cleaved Golgin-84 78 kDa/GAPDH ratio and Blue: the cleaved Golgin-84 65 kDa/GAPDH ratio; (C) Quantification ratio of GM130/GAPDH demonstrated in (A).
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Fig. 6. Real-time PCR to examine the transcript levels of golga5. Samples of acutely infected (A) and persistently infected (B) cells were taken at 16 h, 30 h and 48 h p.i. and subjected to real-time PCR analysis. The mean cDNA copy number for golga5 was normalized to the level of host gapdh. Data are expressed as the mean from three independent experiments. The error bars represent the standard error of the mean. The control group in (A) represents the cells without any treatment and that in (B) represents the cells only treated with penicillin.
The degradations of Golgin-84 and GM130 are different in the lysates of chlamydial infected cells Golgin-84 and GM130 are markers for the Golgi apparatus and are often used to examine fragmentation. Therefore, we examined the expression/degradation of Golgin-84 and GM130 in the lysates of both acutely and persistently infected cells. Immunoblot analysis of Golgin-84 confirmed that the level of degradation of Golgin-84 in the lysates of the persistently infected cells was much less than that in acutely infected cells (Fig. 5). Golgin-84 was processed into two distinct fragments (∼78 kDa and ∼65 kDa) in the lysates of the acutely infected cells. The 78 kDa fragment was predominant in the lysates of the cells at 20 h p.i., whereas the smaller fragment was predominant in the lysates of the cells at 30 h p.i. and later (Fig. 5A and B). Golgin-84 in the lysates of the persistently infected cells was degraded into two fragments too (Fig. 5A); however, the ratio between the two almost remained constant in the lysates of the cells at 30 h p.i. and later (Fig. 5B). The protein appeared as a band of 84 kDa in the lysates of both control and penicillin-treated cells (Fig. 5A and B). We also found that GM130 was not degraded in the lysates of the control cells treated with or without penicillin (Fig. 5A and C). However, there was a marked reduction of GM130 in the lysates of acutely infected cells. By contrast, there was no obvious difference in the expression/degradation of GM130 in the lysates of the persistently infected cells between 20 h and 30 h p.i. Both of them were similar to those in control cells, suggesting GM130 degradation hardly existed in the lysates of persistently infected cells in the early phase. Although the amount of GM130 had a gradual and slight decline from 40 h p.i., it was clearly higher than that in acutely-infected cells (Fig. 5A and C). This indicated that persistent infection had little effect on GM130 expression/degradation. The Golgin-84 transcript levels are different in the acutely and persistently infected cells HeLa cells either acutely or persistently infected with Chlamydia trachomatis, and the control cells (with/without penicillin treatment) were collected at 16 h, 30 h and 48 h p.i. Total RNA was obtained and reverse transcribed into cDNA using random hexamers as primers. To rule out the possibility that the changes in target gene transcript levels in acutely and persistently infected cells may be caused by penicillin, we also monitored the expression of gapdh as an internal reference gene, gapdh transcription is not affected by Chlamydia infection (Johnson, 2004). We examined the expression of golga5, which encodes Golgin-84. The levels of golga5 relative to gapdh were calculated using the 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001).
The results showed that the expression of golga5 in control cells with/without penicillin was almost the same. However, the transcript levels in acutely infected cells increased by 52.6% at 16 h, then decreased by 64.3% at 30 h and by 92.6% at 48 h (Fig. 6A) compared to those in the control cells. The transcript levels in persistently infected cells remained almost constant at 16 h p.i., showing a slight increase of 3.1%, but then decreased by 50.5% at 30 h and by 10.7% at 48 h (Fig. 6B) compares to those in the control cells. Discussion Fragmentation of the Golgi is a common feature of a number of physiological processes, including mitosis and apoptosis. Several studies have showed that acute chlamydial infection can cause the Golgi to fragment (Christian et al., 2011; Elwell and Engel, 2012; Heuer et al., 2009). However, the immunofluorescence and TEM results presented herein showed that persistent chlamydial infection had little effect on the structure of the Golgi. Chlamydia tachomatis cannot synthesize sphingolipids or cholesterol and other Lipids (Hackstadt et al., 1996; Wylie et al., 1997). It completely relies on host-derived lipids for survival and multiplication and has evolved efficient methods to acquire glycerophospholipids, sphingolipids and cholesterol from the host cell (Elwell and Engel, 2012). One important way is to induce the Golgi to fragment. These fragments then relocate and surround the inclusions (Heuer et al., 2009). The results of the present and previous (Heuer et al., 2009) studies indicated that the Golgi in acutely infected host cells underwent fragmentation, whereas in persistently infected cells, no fragmentation was observed (or showed only minor signs). Previous biochemical and molecular biology studies have shown that chlamydial metabolism slows during persistent infections (Hogan et al., 2004). Thus, it can be inferred that the lipid requirements during this state will be less than those in the acute state, resulting in less fragmentation of the Golgi. Several proteins were reported to be involved in Golgi fragmentation (Heuer et al., 2009; Rejman Lipinski et al., 2009; Shitara et al., 2013). Among them, Golgin-84 was reported that its cleavage could cause Golgi fragmentation and increases the transport of sphingolipids to the inclusions to ensure the chlamydial reproduction (Heuer et al., 2009). But recent researches demonstrated that the cleavage only occurred when the Chlamydiae infected cells were harvested under standard lysis conditions in RIPA buffer and is due to CPAF activity in the lysates of the infected cells. And there is CPAF activity in the Chlamydia acutely infected cells (Chen et al., 2012). So concerns have been raised about CPAF mediated proteolysis of host proteins occurs during cell harvest and lysis. Harsh extraction conditions such as 8 M urea can block the CPAF activity, while in a much less stringent RIPA buffer there is continued
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CPAF activity during cell lysis. The immunoblotting results presented herein showed that Golgin-84 was degraded in the lysates of acutely infected cells which is consistent with the results previously reported (Chen et al., 2012; Heuer et al., 2009), but much less so upon the lysate of the persistently infected cells, though both acutely and persistently infected cells were harvested under the “standard lysis conditions”. This result might imply that the amount of activated CPAF and/or the activity of the CPAF in the lysate of persistently infected cells was lower than that in the acutely infected cells. GM130-mediated tethering is required for cisternal stacking during Golgi assembly (Diao et al., 2008) and the phosphorylation of the GM130 is required for Golgi fragmentation in mitosis (Lowe et al., 1998). No report directly indicates that the amounts of GM130 would decrease after chlamydial infection or the Golgi fragmentation. The immunoblotting results presented herein show that GM130 was also degraded in the lysates of Chlamydiae acutely infected cells, while there was very little GM130 degradation in the lysates of persistent chlamydial infected cells. Since GM130 is not in the list of the substrates of CPAF (Chen et al., 2012), further evidences are needed to make sure whether the degradation of GM130 occurs in the intact chlamydial acutely infected cells. To our knowledge, no previous research exists addressing the changes in the transcription of the gene encoding Golgin-84 during acute or persistent chlamydial infections. Here, we showed that the transcript levels of golga5 varied in acutely infected cells, but were much more stable in persistently infected cells. Since inducing Golgi fragmentation is the main way in which Chlamydiae obtain lipids, acute infection may expand the Golgi network to achieve this goal and lead to an associated increase in golga5 transcription in the early infection stage. As the infection proceeded, the level of fragmentation increased possibly in line with changes in golga5 transcription. There might be a mechanism for the host cell to respond to the chlamydial infection and to regulate the level of the golga5 transcription. However, because Chlamydiae show reduced metabolism during persistent infections, they require fewer lipids. This results in a smaller increase in golga5 transcription during the early stages of a persistent infection. Once the Chlamydiae settle into the persistent state, they require a small but steady lipid supply, which may explain why the transcript levels of Golgin-84 in persistently-infected cells were less changed than those in control cells. Taken together, our results indicate that the suppression of golga5 transcription may play a role in the Golgi fragmentation in acutely infected cells. Rather than using data obtained from conventional TEM examination of ultrathin sections, which is not accurate enough, we performed ET examination of semi-ultrathin sections. This technique enables much more detailed examination of intracellular structures, including their spatial arrangement and relationships (Gan and Jensen, 2012; Lidke and Lidke, 2012). Using ET, we were able to observe Golgi fragmentation unambiguously, along with the process of Golgi segmentation. Furthermore, we also could identify the different types of the transport vesicles. Of the different types of sorting vesicle presented in the Golgi, clathrin-coated vesicles meditate selective transport events (Pearse and Bretscher, 1981). While non-clathrin-coated vesicles act as the non-selective carriers in constitutive biosynthetic pathways (Griffiths et al., 1985) and are involved in recycling between the Golgi apparatus and the ER (Serafini et al., 1991). We found that the numbers of both non-clathrin-coated and clathrin-coated vesicles increased in acutely infected cells though the numbers were small, and the results may suggest that the Chlamydiae could affect the vesicles to supply their the nutrients during the acute infection. But the numbers of these two types vesicles were too small for us to propose a conclusive mechanism of the Chlamydiae affecting the vesicles. We didn’t find significant differences
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Persistent and acute chlamydial infections induce different structural changes in the Golgi apparatus Huiling Zhu a,1 , Hongmei Li a,1 , Pu Wang a,1 , Mukai Chen a,1 , Zengwei Huang a,b , Kunpeng Li a , Yinyin Li a , Jian He a , Jiande Han a,∗ , Qinfen Zhang a,∗∗ a State Key Laboratory of Biocontrol, School of Life Sciences, Department of Dermatology in the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China b Guangzhou Sugarcane Industry Research Institute, Guangzhou, China
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Article history: Received 17 October 2013 Received in revised form 20 March 2014 Accepted 24 March 2014 Keywords: Chlamydia trachomatis Acute infection Persistent infection Golgi fragmentation
a b s t r a c t Chlamydia trachomatis causes a wide range of diseases that have a significant impact on public health. Acute chlamydial infections can cause fragmentation of the Golgi compartment ensuring the lipid transportation from the host cell. However, the changes that occur in the host cell Golgi apparatus after persistent infections are unclear. Here, we examined Golgi-associated gene (golga5) transcription and expression along with the structure of the Golgi apparatus in cells persistently infected with Chlamydia trachomatis. The results showed that persistent infections caused little fragmentation of the Golgi. The results also revealed that Golgi fragmentation might be associated with the suppression of transcription of the gene golga5. © 2014 Elsevier GmbH. All rights reserved.
Introduction Chlamydia trachomatis is an obligate intracellular bacterium that causes many diseases in human beings. It is the leading pathogen of preventable blindness worldwide, and the most frequently reported sexually transmitted disease (Schachter, 1999). Indeed, about 40% of sexually transmitted diseases are caused by Chlamydia trachomatis (Stephens et al., 2011). The bacterium has a biphasic developmental cycle that alternates between infectious extracellular elementary bodies (EBs) and metabolically active intracellular reticulate bodies (RBs); the latter multiply within vacuoles called inclusions (Abdelrahman and Belland, 2005; Stephens et al., 2011). The normal life cycle of the bacterium can be disturbed by antibiotics, nutrient depletion, immunological factors, phage infection, and monocytic infection, resulting in a persistent infection (Beatty et al., 1994). Persistent Chlamydiae are characterized by morphologically enlarged RBs, with a slow metabolism and weakened
∗ Corresponding author at: Build 415#, Room 101, School of Life Sciences, Sun Yat-sen University, Xingang West Road 135, Guangzhou, China, 510275. Tel.: +86 20 84112286; fax: +86 20 84110108. ∗∗ Corresponding author. E-mail addresses: hanjd [email protected] (J. Han), [email protected] (Q. Zhang). 1 These authors contributed equally.
infectivity, which are called aberrant bodies (ABs). The persistent chlamydial infection is thought to be related to many chronic diseases, including arthritis and trachoma (Gerard et al., 2010; Mpiga and Ravaoarinoro, 2006). In subfertile women with tubal pathology, serological markers of persistent Chlamydia trachomatis infections are significantly more common compared to women without tubal pathology (den Hartog et al., 2005). Therefore persistent infection of the urogenital tract in women could be an important cause of infertility. More seriously, persistent infection of Chlamydia trachomatis is difficult to diagnose and treat because it is often culture-negative and resistant to antibiotics (Patton et al., 1994). During the persistent state, Chlamydiae display a transcriptional profile that is significantly different from cells in the acute infection (Belland et al., 2003; Nicholson et al., 2003). Previous studies have undertaken genomic transcriptional profiling using microarray and differential gene expression analyses of Chlamydiae cells in the persistent and acute infections (Belland et al., 2003; Nicholson et al., 2003). Alterations in host gene expression after chlamydial infection has also been studied; however, most of these genes are involved in inflammatory response (Ren et al., 2003). Not much is known about how the expressions of the genes associated with host Golgi apparatus respond to the induction of persistent chlamydial infections. Chlamydiae interact with host cells to ensure their own survival and multiplication. Such methods include subverting the host cytoskeleton (Carabeo et al., 2002), hijacking membrane trafficking
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pathways (Grieshaber et al., 2003), inducing Golgi fragmentation (Heuer et al., 2009), and inhibiting apoptosis (Fan et al., 1998). The Golgi apparatus processes and packages proteins, lipids, and other macromolecules. It receives proteins and other molecules from the endoplasmic reticulum and modifies, sorts, and packages them for storage within the cell or secretion to the cell exterior. Golgi fragmentation is a common feature of a number of physiological processes such as mitosis and apoptosis (Heuer et al., 2009; Rabouille and Jokitalo, 2003). During mitosis, fragmentation is related to Golgin-84 (Diao et al., 2008) and GM130 (Lowe et al., 1998), whereas during apoptosis, fragmentation is caused by caspase-dependent cleavage of giantin and golgin-160 (Mancini et al., 2000). Like many other intracellular pathogens, Chlamydia trachomatis requires host cell lipids, such as sphingolipids and cholesterol, for multiplication (Hatch and McClarty, 1998; van Ooij et al., 2000). Acute infection with Chlamydia trachomatis induces Golgi fragmentation. The breakdown of the Golgi structure into smaller elements around the inclusions increases lipid transport into the inclusions. However, no study has yet examined changes in Golgi structure that occur in cases of persistent infection. Golgi fragmentation is reported to be triggered by the successive cleavage of the Golgi matrix protein Golgin-84 (Heuer et al., 2009). While another research demonstrated that 11 previously reported protein cleavages in Chlamydia-infected cell are due to proteolysis during cell lysis and Chlamydia-induced Golgi reorganization occurs in the absence of detectable golgin-84 cleavage, which highlights the need to re-evaluate the Chlamydia literature on CPAF (chlamydial protease or proteasome-like activity factor) (Chen et al., 2012). However, there is currently no evidence on whether Golgin-84 is degraded in the persistent Chlamydia infection. Here, we used immunofluorescence analysis, transmission electron microscopy (TEM), electron tomography (ET), immunoblot analysis, and RT-PCR to examine the process of Golgi fragmentation during both persistent and acute chlamydial infections. The results indicated that chlamydial persistent infection of host cells caused much less Golgi fragmentation than acute infection.
organisms were then scraped from culture dishes 48 h later. The supernatant was analyzed to determine the number of infectious forming units (IFU) per ml. Data presented were the means with standard errors of three representative experiments (Fig. S1). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.03.002.
Immunofluorescence analysis Cells grown on coverslips as previously described (Huston et al., 2008) were fixed with cold 4% paraformaldehyde, permeabilized in 0.5% (v/v) Triton X-100 (Sigma–Aldrich, USA), and then blocked with 1% (w/v) BSA (Thermo Scientific, USA). The cells were then incubated for 1.5 h at room temperature with a mouse anti-CT MOMP antibody (Abcam, UK) diluted 1 in 400 of 1% BSA followed by washed three times with PBS and blocked with 1% (w/v) BSA before incubation for 1 h with goat anti-mouse IgG-FITC:sc-2010 (Santa Cruz Biotechnology, USA) diluted 1 in 500 of 1% BSA (w/v). The cells were then washed three times with PBS, and followed by incubated for a further 1.5 h with a rabbit anti-GM130, anti-Golgin-84 and anti-Giantin antibody (Abcam, UK) diluted 1 in 200 of 1% (w/v) BSA. Because of the similar results from using these three markers (Figs. S2 and S3), we finally used GM130 as the markers in all the experiments. After washing three times with PBS, the samples were incubated with goat ant-rabbit IgG-PE:sc-3739 (Santa Cruz Biotechnology, USA) diluted 1 in 250 of 1% (w/v) BSA. Finally, the samples were labeled with the nucleic acid stain, ProLong Anti-Fade DAPI (Invitrogen, USA) and observed under a ZEISS Axio Imager Z1 immunofluorescence microscope (Zeiss, Germany) fitted with a 100× oil objective lens. Images were captured using a Carl Zeiss Axio Cam, and fluorescence was quantified using the AxioVision 4.7 (Zeiss). Supplementary Figs. S2 and S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm. 2014.03.002.
Real-time PCR Materials and methods Acute and persistent chlamydial infection HeLa cells (HeLa S3 (ATCC® CCL-2.2TM ), USA) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Gibco, USA) using a six-well plate at 37 ◦ C in 5% CO2 . When the population reached 3–6 × 105 cells per well, Chlamydia trachomatis serovar D at an MOI of 8 was added into the wells and incubated with the cells by centrifugation at 3400 × g at 37 ◦ C for 1 hour (h). Samples were then incubated for another hour at 37 ◦ C in 5% CO2 . Extracellular bacteria were removed by aspirating the supernatant followed by the addition of fresh RPMI 1640 medium supplemented with 10% fetal calf serum, 0.5% glucose, and the cells were maintained under the same conditions until they were harvested at different time points respectively. For the persistent infection group, all the procedures were the same except for the addition of 100 U/ml penicillin G (Sigma Life Science, USA) in the medium after centrifugation (Skilton et al., 2009). Control HeLa cells (not infected by Chlamydia trachomatis) were also treated with the above medium with/without 100 U/ml penicillin G. The persistent infection was confirmed by the reactivation assay of treated samples (Goellner et al., 2006). The detailed steps are as follows. At 24 h post infection (p.i.) and 48 h p.i., the medium was replaced with fresh RPMI 1640 medium supplemented with 10% fetal calf serum, 0.5% glucose, with no penicillin. Chlamydial
Total RNA was extracted and purified using an RNeasy mini Kit (QIAGEN), and 31–62 g of RNA was eluted in 50 l of RNase-free H2 O. RNA (2 g) was added to RNase-free H2 O to a final volume of 15.5 l and then denatured at 70 ◦ C for 5 min. The RNA samples were then incubated with 2 l of random hexamers (Promega), 5 l of 5× reaction buffer, 1 l of 10 mM dNTP mix (Promega), 20 units of RNase inhibitor, and 200 units of Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Invitrogen) (final reaction volume, 25 l). Reverse transcription reactions were performed at 37 ◦ C for 60 min (with a final step at 72 ◦ C for 10 min). cDNA was analyzed by agarose gel electrophoresis. The primers for real-time PCR (see the supplementary table) were synthesized by Beijing Genomics Institute (BGI) and stored at a concentration of 5 M in ddH2 O. A Roche LightCycler 480 was used to measure the levels of golga5 and gapdh cDNA. Each reaction contained 1 l of gene-specific forward and reverse primers, 5 l of 2× SYBR Green Master Mix (Roche), 2 l of cDNA template, and 1 l of ddH2 O (final volume, 10 l). The conditions for real-time PCR were as follows: pre-denaturation at 95 ◦ C for 5 min, followed by 40 cycles at 95 ◦ C for 10 s, 54 ◦ C for 15 s, and 72 ◦ C for 6 s. All reverse transcription reactions and real-time PCR reactions were performed in triplicate. Roche LightCycler 480 software (version 1.5) was used for sample quantification. All experiments were repeated three times. Supplementary table related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.03.002.
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Immunoblot analysis HeLa cells (controls and infected cells) were lysed in RIPA buffer (Thermo Scientific, USA) at the indicated time points, and the protein concentrations were determined using the Pierce BCA kit (Thermo Scientific, USA). Total proteins (20 g/lane) were separated in a reducing SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was incubated with rabbit anti-GM130 (Abcam, UK) diluted 1 in 10,000 of 5% (w/v) BSA, mouse anti-Golgin-84 (BD Transduction Laboratories, USA) diluted 1 in 200 of 5% (w/v) BSA, or mouse anti-GAPDH (Bioworld, USA) diluted 1 in 15,000 of 5% (w/v) BSA to detect the respective proteins. The membranes were then washed and incubated with peroxidase conjugated goat anti-rabbit IgG (Thermo Scientific, USA) diluted 1 in 150,000 of 5% (w/v) BSA or peroxidase conjugated goat anti-mouse IgG (Thermo Scientific) diluted 1 in 20,000 of 5% (w/v) BSA and developed using the ECL reagent (Pierce, USA), as described previously (Huston et al., 2008). Sample preparation for transmission electron microscopy Cells were harvested by centrifugation at about 1000 g, fixed in 3% glutaraldehyde (SPI Supplies, USA) for 1 h, and then with 1% osmic acid (Electron Microscopy Sciences, EMS, USA) for 90 min. The cells were dehydrated in an acetone gradient and embedded in Epon812 resin (TED PELLA INC., USA), as described previously (Huang et al., 2010; Qinfen et al., 2004). Thin (∼100 nm) sections were cut with a LKB2188 microtome (LKB BROMMA, Sweden), transferred to 200 mesh carbon-coated copper grids, stained with 2% uranyl acetate for 30 min followed by Reynold’s lead citrate for 10 min, and observed under a JEM 100CX II transmission electron microscope (JEOL Ltd, Japan) at 100 kV to determine specimen quality and to select cells suitable for subsequent study. Blocks containing suitable cells were returned to the microtome and semi-ultrathin (∼250 nm) sections were cut and transferred to carbon-coated slot grids. The sections were post-stained with 2% uranyl acetate for 30 min followed by Reynold’s lead citrate for 10 min. Additional colloidal gold particles (15 nm) were deposited on both surfaces of the semi-ultrathin sections and used as fiducial markers during subsequent image alignment. Electron tomography data collection and reconstruction Semi-ultrathin sections (∼250 nm) were prepared for tomography as described previously (Ladinsky et al., 1999) and examined using a JEM 2010 TEM (JOEL, Japan). When a suitable Golgi region was selected, a single axis tilt series was digitally recorded using a 2k × 4k CCD (Gatan CA, USA); the samples were manually tilted at 2◦ increments over a range of 120◦ (±60◦ ). The nominal magnifica˚ tion was 10k and the final A/pixel was 6.55. Images were aligned using the IMOD software package (Kremer et al., 1996), using the colloidal gold particles as fiducial markers. The reconstructed 3D subcellular structures were then segmented and rendered using Chimera (Pettersen et al., 2004). Statistical analysis Statistical analyses were performed using the SAS program (version 8.1, 1999–2000, SAS Institute Inc., Cary, NC). Method was carried out by analysis of t-test. Data were expressed as mean ± standard error (SE). For the length of Golgi apparatus, the measuring tools in CCD (Megaview G2, OSIS, Germany) were used to determine the length of the Golgi stacks in all groups of HeLa cells at 48 h p.i. In total, we measured the lengths of 79, 77, 31 and 51 Golgi apparatus from
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the Hela cells without and with penicillin treatment, the acutely infected and persistently infected cells respectively. For the number of the different types of vesicles from the Golgi apparatus, we in all classified and calculated the vesicles from 15, 9 and 11 tomograms for the control (include the cells with and without penicillin treated), acutely infected and persistently infected cells respectively. In each tomogram, there are usually more than 10 vesicles. Results There is little Golgi fragmentation in cells harboring a persistent Chlamydia infection In order to confirm the persistent infection, reactivation assays of the samples were determined where the penicillin was removed from the culture medium at 24 h and 48 h p.i. The results (Fig. S1) showed that Chlamydiae in persistency hardly had any infecting ability, while those Chlamydiae rescued at two different time points p.i. from persistently infected cells still could regain their infectivity. These results confirmed the persistent chlamydial infection induced by penicillin. To determine whether the Golgi undergoes fragmentation in response to persistent infection, we examined its morphology by immunofluorescence microscopy. At all time points p.i., control cells treated either without (Fig. 1A, E, I, M, Q and U) or with penicillin (Fig. 1D, H, L, P, T and X) contained a normal condensed Golgi apparatus within the cytoplasm and close to the nucleus, indicating that penicillin did not affect the structure of the Golgi apparatus. At 20 h p.i., most of the cells acutely infected with Chlamydia showed a condensed Golgi apparatus that was closely associated with the chlamydial inclusions and the nucleus (Figs. 1B, S2 and S3). As the acute infection progressed, the Golgi apparatus showed significant morphological changes, resulting in the dispersed distribution of the Golgi stacks around the inclusions (Fig. 1F, S2 and S3). At 40 h p.i., not only the Golgi elements were further dispersed (Figs. 1J, S2 and S3), but also the amount of the Golgi apparatus decreased. In 18.9% of acutely infected cells, the Golgi apparatus had completely disappeared by 40 h p.i., while there was no disappeared in the persistently infected cells. This finding indicated that the lysis of the host cells occurred since 40 h p.i. in the acutely infected cells. The Golgi apparatus dispersing and lysis of the host cells became much more serious at 72 h and 96 h p.i., and it was difficult to find the intact cells with intact inclusion or Chlamydia at 120 h p.i. in acutely infected cells (Fig. 1V). These results indicate that acute chlamydial infection induces the Golgi apparatus to break down into small-dispersed fragments and finally can induce the lysis of the host cells. Surprisingly, at 30 h p.i. the Golgi apparatus in most persistently infected cells retained the intact condensed structure and remained closely associated with the inclusions and cell nucleus (Fig. 1G, S2 and S3). This was still the case at 40 h (Figs. 1K, S2 and S3), 72 h, 96 h, and 120 h p.i., although in some areas, there were a few dispersed fragments at 120 h p.i. in the persistently infected cells. And in all the time points, the chlamydial inclusions remained intact. These results suggest that persistent infection with Chlamydia trachomatis induces less fragmentation of the Golgi than acute infection. Electron microscopy analyses confirms that persistent chlamydial infection causes little Golgi fragmentation We next confirmed that the structure of the Golgi apparatus was still intact after persistent infection using TEM. We used a combination of conventional TEM and ET to examine the
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Fig. 1. Immunofluorescence analysis marked with antibody again GM130. Comparison of Golgi morphology in non-infected (control) (A, E, I, M, Q, and U), acutely infected (B, F, J, N, R and V), persistently infected (C, G, K, O, S and W), and penicillin-treated (D, H, L, P, T and X) cells at the indicated time points post-infection. Chlamydiae were stained with FITC-labeled anti-chlamydia MOMP (green channel). The Golgi was stained with rabbit monoclonal anti-GM130 antibody labeled with Cy5 (red) and then analyzed under an immunofluorescence microscope. DNA was stained with Hoechst (blue). Scale bar = 5 m.
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Fig. 2. Electron micrographs of acutely and persistently infected cell. A–C show acutely infected cells at 36 h, 48 h and 68 h p.i., respectively, whereas D–F show persistently infected cells at the same time points. (A) In the acutely infected cells at 36 h p.i., about half of the Chlamydiae are RBs. (B) In the acutely infected cells at 48 h p.i., most of the Chlamydiae are EBs and the inclusion predominately occupied the cytoplasm of the host cell. (C) In the acutely infected cells at 68 h p.i., there are few Chlamydiae left in the inclusion which still predominately occupied the cytoplasm of the host cell. (D) Fewer Chlamydiae are present in the persistently infected cells at 36 h p.i., and three of them in the image are enlarged to be at least 2 times the size of a normal RB with extra empty vesicle attached, and usually they are named as ABs. In (E) and (F), the Chlamydiae are similar at 48 h and 68 h p.i. respectively, some of them contained multiple electron-dense granules (arrows). The host cells remain intact at all time points in persistent infection group. Bar = 500 nm. N: nuclear, EB: elementary body, RB: reticulate body, AB: aberrant body
Chlamydiae, the host HeLa cells, and the Golgi apparatus. At 36 h p.i., half of the Chlamydiae in acutely infected cells had developed into EBs (Fig. 2). At 48 h p.i., most had developed into EBs, and at 68 h p.i. the majority of HeLa cells were disrupted and the Chlamydia had been released. Most Chlamydiae in penicillin-treated infected cells enlarged to be 2–5 times the size of a normal RB, and some of them contained multiple electron-dense granules or with extra empty vesicles attached. Usually this type of Chlamydiae in EM was named as AB. They also showed a near-consistent morphology at all time points tested. Also, the number of Chlamydiae was much lower than that in acutely infected cells at all time points tested.
The majority of HeLa cells were intact at 68 h p.i. These EM results indicated that persistent chlamydial infection was successfully induced by penicillin (Fig. 2). There was no obvious difference in the size of the Golgi apparatus in persistently infected cells (∼908 nm in length), control cells (∼898 nm in length), or cells treated with penicillin (∼796 nm in length) (Fig. 3). The P-values between the control cells and the persistently infected cells or cells treated with pencillin were 0.470 and 0.194 respectively, which were both larger than 0.05. However, the Golgi apparatus in acutely infected cells was clearly shorter (∼510 nm in length) (Fig. 3). And the P-value was 0.0147, smaller
Fig. 3. Structure of the Golgi in infected cells. The Golgi is indicated by the orange contour. (A) Golgi in an acutely infected cell (length, ∼0.5 m). (B) Golgi in a persistently infected cell (length, ∼2 m). M: mitochondria, EB: elementary body, AB: aberrant body. Bar = 500 nm. (C) Lengths of the Golgi in control cells, acutely infected cells, and persistently infected cells. Columns show the average length of the Golgi in each group. The bars represent the standard error of the mean. Control: Normal HeLa cells. Control P: HeLa cells treated with Penicillin. Acute: Cells acutely infected by Chlamydia trachomatis. Persistent: Cells persistently infected by Chlamydia trachomatis.
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Fig. 4. Golgi secretory vesicles in infected cells. (A) Golgi vesicle gallery. Top row shows the non-coated vesicles with no projecting proteins on the membrane. Middle row demonstrates the non-clathrin-coated vesicles with short protruding proteins, which make the membrane appear thicker. Bottom row displays the clathrin-coated vesicles, with a regular arrangement of clathrins projecting from the vesicle membrane (arrow). All maps were slices in 26.2 nm thickness taken from the tomograms. Bar = 30 nm. (B) The average numbers of the non-coated vesicles and (C) The average numbers of the coated vesicles. The columns show the average numbers of different types of the vesicles in one Golgi tomogram of the normal HeLa cells (Control), acutely infected cells (Acute), and persistently infected cells (Persistent). The bars represented the standard error of the mean.
than 0.05. TEM analyses of persistently infected cells suggested the absence of any obvious Golgi fragmentation, whereas the length of the Golgi apparatus in acutely infected cells was about half that in persistently-infected and control cells. We next used ET to obtain 3D images of the Golgi structure. ET enables researchers to measure the length of the Golgi apparatus and makes the vesicles in the trans-Golgi network easier to be recognized (Ladinsky et al., 1999). We classified the Golgi secretory vesicles into three different types as previously (Ladinsky et al., 1999) described: vesicles without a coating, clathrin-coated vesicles, and vesicles with a non-clathrin coating (Fig. 4A). There were no obvious differences of the average numbers of the non-coated vesicles in the Golgi apparatus among the acutely infected, persistently-infected cells and the
control group (The P-values were 0.353 and 0.710 respectively, P > 0.05) (Fig. 4B). However, the numbers of the clathrin-coated and non-clathrin-coated vesicles in the Golgi apparatus of acutely infected cells obviously increased (The P-values were 0.026 and 0.023 respectively, P < 0.05) (Fig. 4C), whereas in the Golgi apparatus of persistently infected cells, they only showed a slight increase (The P-values were 0.375 and 0.345 respectively, P > 0.05) (Fig. 4C). Taken together, the immunofluorescence and TEM results showed that persistent chlamydial infection had little effect on the structure of the Golgi. Many studies showed that Golgin-84 and GM130 are associated with Golgi fragmentation. Therefore, we then used immunoblotting and real-time PCR to examine whether persistent infection induced changes of these proteins.
Fig. 5. Immunoblot analysis of Golgin-84 and GM130 in the lysate of acutely and persistently infected cells. (A) Immunoblot analysis of cell lysates at 20 h, 30 h, 40 h, 72 h, 96 h and 120 h p.i. demonstrate the degradation of Golgin-84 and GM130 in the lysates of acutely (A) and persistently (P) infected cells. In acutely infected cell lysates, Golgin-84 was degraded into a 78 kDa fragment at 20 h post-infection and further degraded into a 65 kDa fragment by 30 h p.i. and later. In persistently infected cells lysates, two separate fragments were still visible even at 120 h p.i. and the ratio of the 78 kDa fragment and 68 kDa fragment almost remain constant after 72 h p.i. There was no degradation of Golgin-84 in the lysates of non-infected cells not treated (C) or treated (Cp) with penicillin. There was a clear reduction in GM130 in the lysates of the acutely infected cells (A), whereas levels remained relatively constant in the lysates of the persistently infected (P) and control (C and Cp) cells. GAPDH levels remained almost constant in all groups. (B) Quantification ratio of Golgin-84/GAPDH demonstrated in (A). Black: the intact Golgin-84 84 kDa/GAPDH ratio, Red: cleaved Golgin-84 78 kDa/GAPDH ratio and Blue: the cleaved Golgin-84 65 kDa/GAPDH ratio; (C) Quantification ratio of GM130/GAPDH demonstrated in (A).
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Fig. 6. Real-time PCR to examine the transcript levels of golga5. Samples of acutely infected (A) and persistently infected (B) cells were taken at 16 h, 30 h and 48 h p.i. and subjected to real-time PCR analysis. The mean cDNA copy number for golga5 was normalized to the level of host gapdh. Data are expressed as the mean from three independent experiments. The error bars represent the standard error of the mean. The control group in (A) represents the cells without any treatment and that in (B) represents the cells only treated with penicillin.
The degradations of Golgin-84 and GM130 are different in the lysates of chlamydial infected cells Golgin-84 and GM130 are markers for the Golgi apparatus and are often used to examine fragmentation. Therefore, we examined the expression/degradation of Golgin-84 and GM130 in the lysates of both acutely and persistently infected cells. Immunoblot analysis of Golgin-84 confirmed that the level of degradation of Golgin-84 in the lysates of the persistently infected cells was much less than that in acutely infected cells (Fig. 5). Golgin-84 was processed into two distinct fragments (∼78 kDa and ∼65 kDa) in the lysates of the acutely infected cells. The 78 kDa fragment was predominant in the lysates of the cells at 20 h p.i., whereas the smaller fragment was predominant in the lysates of the cells at 30 h p.i. and later (Fig. 5A and B). Golgin-84 in the lysates of the persistently infected cells was degraded into two fragments too (Fig. 5A); however, the ratio between the two almost remained constant in the lysates of the cells at 30 h p.i. and later (Fig. 5B). The protein appeared as a band of 84 kDa in the lysates of both control and penicillin-treated cells (Fig. 5A and B). We also found that GM130 was not degraded in the lysates of the control cells treated with or without penicillin (Fig. 5A and C). However, there was a marked reduction of GM130 in the lysates of acutely infected cells. By contrast, there was no obvious difference in the expression/degradation of GM130 in the lysates of the persistently infected cells between 20 h and 30 h p.i. Both of them were similar to those in control cells, suggesting GM130 degradation hardly existed in the lysates of persistently infected cells in the early phase. Although the amount of GM130 had a gradual and slight decline from 40 h p.i., it was clearly higher than that in acutely-infected cells (Fig. 5A and C). This indicated that persistent infection had little effect on GM130 expression/degradation. The Golgin-84 transcript levels are different in the acutely and persistently infected cells HeLa cells either acutely or persistently infected with Chlamydia trachomatis, and the control cells (with/without penicillin treatment) were collected at 16 h, 30 h and 48 h p.i. Total RNA was obtained and reverse transcribed into cDNA using random hexamers as primers. To rule out the possibility that the changes in target gene transcript levels in acutely and persistently infected cells may be caused by penicillin, we also monitored the expression of gapdh as an internal reference gene, gapdh transcription is not affected by Chlamydia infection (Johnson, 2004). We examined the expression of golga5, which encodes Golgin-84. The levels of golga5 relative to gapdh were calculated using the 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001).
The results showed that the expression of golga5 in control cells with/without penicillin was almost the same. However, the transcript levels in acutely infected cells increased by 52.6% at 16 h, then decreased by 64.3% at 30 h and by 92.6% at 48 h (Fig. 6A) compared to those in the control cells. The transcript levels in persistently infected cells remained almost constant at 16 h p.i., showing a slight increase of 3.1%, but then decreased by 50.5% at 30 h and by 10.7% at 48 h (Fig. 6B) compares to those in the control cells. Discussion Fragmentation of the Golgi is a common feature of a number of physiological processes, including mitosis and apoptosis. Several studies have showed that acute chlamydial infection can cause the Golgi to fragment (Christian et al., 2011; Elwell and Engel, 2012; Heuer et al., 2009). However, the immunofluorescence and TEM results presented herein showed that persistent chlamydial infection had little effect on the structure of the Golgi. Chlamydia tachomatis cannot synthesize sphingolipids or cholesterol and other Lipids (Hackstadt et al., 1996; Wylie et al., 1997). It completely relies on host-derived lipids for survival and multiplication and has evolved efficient methods to acquire glycerophospholipids, sphingolipids and cholesterol from the host cell (Elwell and Engel, 2012). One important way is to induce the Golgi to fragment. These fragments then relocate and surround the inclusions (Heuer et al., 2009). The results of the present and previous (Heuer et al., 2009) studies indicated that the Golgi in acutely infected host cells underwent fragmentation, whereas in persistently infected cells, no fragmentation was observed (or showed only minor signs). Previous biochemical and molecular biology studies have shown that chlamydial metabolism slows during persistent infections (Hogan et al., 2004). Thus, it can be inferred that the lipid requirements during this state will be less than those in the acute state, resulting in less fragmentation of the Golgi. Several proteins were reported to be involved in Golgi fragmentation (Heuer et al., 2009; Rejman Lipinski et al., 2009; Shitara et al., 2013). Among them, Golgin-84 was reported that its cleavage could cause Golgi fragmentation and increases the transport of sphingolipids to the inclusions to ensure the chlamydial reproduction (Heuer et al., 2009). But recent researches demonstrated that the cleavage only occurred when the Chlamydiae infected cells were harvested under standard lysis conditions in RIPA buffer and is due to CPAF activity in the lysates of the infected cells. And there is CPAF activity in the Chlamydia acutely infected cells (Chen et al., 2012). So concerns have been raised about CPAF mediated proteolysis of host proteins occurs during cell harvest and lysis. Harsh extraction conditions such as 8 M urea can block the CPAF activity, while in a much less stringent RIPA buffer there is continued
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CPAF activity during cell lysis. The immunoblotting results presented herein showed that Golgin-84 was degraded in the lysates of acutely infected cells which is consistent with the results previously reported (Chen et al., 2012; Heuer et al., 2009), but much less so upon the lysate of the persistently infected cells, though both acutely and persistently infected cells were harvested under the “standard lysis conditions”. This result might imply that the amount of activated CPAF and/or the activity of the CPAF in the lysate of persistently infected cells was lower than that in the acutely infected cells. GM130-mediated tethering is required for cisternal stacking during Golgi assembly (Diao et al., 2008) and the phosphorylation of the GM130 is required for Golgi fragmentation in mitosis (Lowe et al., 1998). No report directly indicates that the amounts of GM130 would decrease after chlamydial infection or the Golgi fragmentation. The immunoblotting results presented herein show that GM130 was also degraded in the lysates of Chlamydiae acutely infected cells, while there was very little GM130 degradation in the lysates of persistent chlamydial infected cells. Since GM130 is not in the list of the substrates of CPAF (Chen et al., 2012), further evidences are needed to make sure whether the degradation of GM130 occurs in the intact chlamydial acutely infected cells. To our knowledge, no previous research exists addressing the changes in the transcription of the gene encoding Golgin-84 during acute or persistent chlamydial infections. Here, we showed that the transcript levels of golga5 varied in acutely infected cells, but were much more stable in persistently infected cells. Since inducing Golgi fragmentation is the main way in which Chlamydiae obtain lipids, acute infection may expand the Golgi network to achieve this goal and lead to an associated increase in golga5 transcription in the early infection stage. As the infection proceeded, the level of fragmentation increased possibly in line with changes in golga5 transcription. There might be a mechanism for the host cell to respond to the chlamydial infection and to regulate the level of the golga5 transcription. However, because Chlamydiae show reduced metabolism during persistent infections, they require fewer lipids. This results in a smaller increase in golga5 transcription during the early stages of a persistent infection. Once the Chlamydiae settle into the persistent state, they require a small but steady lipid supply, which may explain why the transcript levels of Golgin-84 in persistently-infected cells were less changed than those in control cells. Taken together, our results indicate that the suppression of golga5 transcription may play a role in the Golgi fragmentation in acutely infected cells. Rather than using data obtained from conventional TEM examination of ultrathin sections, which is not accurate enough, we performed ET examination of semi-ultrathin sections. This technique enables much more detailed examination of intracellular structures, including their spatial arrangement and relationships (Gan and Jensen, 2012; Lidke and Lidke, 2012). Using ET, we were able to observe Golgi fragmentation unambiguously, along with the process of Golgi segmentation. Furthermore, we also could identify the different types of the transport vesicles. Of the different types of sorting vesicle presented in the Golgi, clathrin-coated vesicles meditate selective transport events (Pearse and Bretscher, 1981). While non-clathrin-coated vesicles act as the non-selective carriers in constitutive biosynthetic pathways (Griffiths et al., 1985) and are involved in recycling between the Golgi apparatus and the ER (Serafini et al., 1991). We found that the numbers of both non-clathrin-coated and clathrin-coated vesicles increased in acutely infected cells though the numbers were small, and the results may suggest that the Chlamydiae could affect the vesicles to supply their the nutrients during the acute infection. But the numbers of these two types vesicles were too small for us to propose a conclusive mechanism of the Chlamydiae affecting the vesicles. We didn’t find significant differences
in the numbers of non-coated vesicles in the infected and control cells. Acknowledgements We thank Mr. Dong Chen for the helps of ultrathin sections and Prof. Qiang Zhou for the helps of data statistic analysis. The research was supported by the National Natural Science Foundation of China (31070654, 81071404), the Science Foundation of the State Key Laboratory of Biocontrol (SKLBC10B05), and the Natural Science Foundation of Guangdong Province (S2013010015360). References Abdelrahman, Y.M., Belland, R.J., 2005. The chlamydial developmental cycle. FEMS Microbiol. Rev. 29, 949–959. Beatty, W.L., Morrison, R.P., Byrne, G.I., 1994. Persistent Chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microbiol. Rev. 58, 686–699. Belland, R.J., Zhong, G., Crane, D.D., Hogan, D., Sturdevant, D., Sharma, J., Beatty, W.L., Caldwell, H.D., 2003. Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis. Proc. Natl. Acad. Sci. U. S. A. 100, 8478–8483. 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