Transcriptional Analysis of 10 Selected Genes in a Model of Penicillin G Induced Persistence of Chlamydophila psittaci in HeLa Cells

Abstract

Chlamydophila psittaci is an important intracellular pathogen. Persistent infection is an important state of the host-parasite interaction in this chlamydial infection, which plays a significant role in spreading the organism within animal populations and in causing chronic chlamydiosis and serious sequelae. In this study, a C. psittaci persistent infection cell model was induced by penicillin G, and real-time quantitative PCR was used to study the transcriptional levels of 10 C. psittaci genes (dnaA, dnaK, ftsW, ftsY, grpE, rpsD, incC, omcB, CPSIT_0846, and CPSIT_0042) in acute and penicillin-G-induced persistent infection cultures. Compared with the acute cultures, the penicillin-G-treated cultures showed a reduced chlamydial inclusion size and a significantly decreased number of elementary body particles. Additionally, some enlarged aberrant reticulate body particles were present in the penicillin-G-treated cultures but not the acute ones. The expression levels of genes encoding products for cell division (FtsW, FtsY) and outer membrane protein E encoding gene (CPSIT_0042) were downregulated (p < 0.05) from 6 h post-infection onward in the persistent infection cultures. Also from 6 h post-infection, the expression levels of DnaA, DnaK, IncC, RpsD, GrpE, and CPSIT_0846 were upregulated (p < 0.05); however, the expression level of OmcB in the persistent infection was< almost the same as that in the acute infection (p > 0.05). These results provide new insight regarding molecular activities that accompany persistence of C. psittaci, which may play important roles in the pathogenesis of C. psittaci infection.

Introduction

Chlamydophila psittaci is an intracellular pathogen that is most frequently associated with Psittaciformes but can also infect many other avian species as well as a wide range of mammalian hosts [27]. It can cause abortion, respiratory disorders, enteritis, and arthritis in infected cattle, sheep, horses, and other domestic animals [27]. This zoonotic infection can also occur in humans, usually resulting from close contact with infected mammals and birds, and human-to-human transmission has also been suggested [6, 23, 25, 31, 42]. The clinical presentations and outcomes of C. psittaci infections range from asymptomatic disease to severe systemic disease and even death. Unlike Chlamydia trachomatis, which mainly causes sexually transmitted infections, it can cause psittacosis, community-acquired pneumonia, chronic obstructive pulmonary disease, arthritic diseases, endocarditis, and encephalitis and is believed to be a possible etiologic agent of lymphomas of the ocular adnexa [9, 12, 16, 28, 31, 43]. However, its exact pathogenic mechanism remains unclear.

It is well known that Chlamydia is characterized by a unique biphasic developmental cycle during its conventional growth course, including the infectious metabolically inert extracellular elementary bodies (EBs) and the non-infectious metabolically active and dividing intracellular reticular bodies (RBs) [1]. However, when Chlamydia is cultured in conditions deleterious to its growth, including antibiotic treatment [33], amino acid depletion [11], iron depletion [39], gamma interferon (IFN-γ) exposure [2], co-infection with herpes simplex virus [40], and ATP, and heat-shock protein and adenosine induction [26, 38], this pathogen usually forms a persistent infection that is reversible upon removal of the stressor [40]. Persistence is an important state of the host-parasite interaction in this chlamydial infection, which plays a significant role in spreading the organism within animal populations and in causing chronic chlamydiosis and serious sequelae.

Chlamydial persistence is defined as a viable but noninfectious growth stage resulting in a long-term relationship with the infected host cell [3]. It is characterized by a third non-infectious, non-replicating, non-cultivable, metabolically altered but viable, abnormal, enlarged RB form, and these RBs are generally termed aberrant bodies (ABs).

Chlamydiae undergoing such infections are morphologically aberrant and display an unusual transcription profile [24]. It has been reported that the expression levels of many chlamydial genes, including omp1, ftsK, and others, are severely attenuated during persistence [17, 34, 36]. This alteration in transcriptional activity is thought to be a highly flexible means of adjusting the metabolic characteristics of the bacterium to support long-term infection of the host under varying circumstances, as observed in Mycobacterium tuberculosis and other organisms [5, 8,18].

Various stress conditions, such as iron deprivation, IFN-γ exposure, and penicillin treatment, have been thoroughly studied in the promotion of persistence in Chlamydia trachomatis [10, 30, 46] and Chlamydophila pneumoniae [37, 45] but not in C. psittaci. Other reports showed that penicillin can block chlamydial binary fission, prevent RB to EB transition, and then induce a persistance state [30, 46]. In the current study, we used penicillin G to induce the persistence of C. psittaci and to demonstrate its morphologically abnormal, persistent forms and the altered expression of genes involved in DNA replication, cell division, energy metabolism, and cell membrane structures and the type III secreted protein.

 

Materials and Methods

C. psittaci and Cell Culture

Human cervical epithelial HeLa 229 cells (ATCC CCL-2.1) were used for culturing Chlamydia. HeLa 229 cells were maintained at 35℃ in 5% CO2 in DMEM (Hyclone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA) and 2 mmol/l L-glutamine (Sigma-Aldrich, Munich, Germany). C. psittaci 6BC (ATCC VR-125) was propagated in confluent HeLa 229 cell monolayers in complete growth medium with 10% FBS and 2 μg/ml cycloheximide as described previously. Chlamydial EBs were harvested at 48 h post-infection (p.i.). Cells containing mature EBs were centrifuged at 1,000 rpm for 10 min and then washed twice with phosphate-buffered saline (PBS), pH 7.4. Cell pellets were resuspended in sucrose-phosphate-glutamate (SPG) acid buffer (0.2 mol/l sucrose, 3.8 mmol/l KH2PO4, 6.7 mmol/l Na2HPO4, and 5 mmol/l L-glutamic acid, pH 7.4), disrupted by sonication, and stored at -80℃ until use. C. psittaci was used in all experiments at a multiplicity of infection (MOI) of 2. The MOI was determined by standard serial dilution methods to count the number of chlamydial inclusion-forming units (IFU) in the HeLa 229 cells using rabbit anti-C. psittaci antiserum.

Infection and Induction of Persistence

Penicillin G (Sigma-Aldrich) was used to establish a state of C. psittaci persistence in HeLa 229 cells using protocols described below. HeLa 229 cells were trypsinized from stock cultures, plated onto 24- or 6-well cell culture plates (0.5 × 106 and 1.5 × 106 per well, respectively) and incubated overnight at 35℃ in 5% CO2. Sterile coverslips were placed into the 24-well cell culture plates before culture. After 16 h of incubation, confluent HeLa 229 cell monolayers were infected with C. psittaci at a MOI of 2 for 2 h, and then the medium was replaced with complete DMEM containing 10% FBS, 2 μg/ml cycloheximide, and different concentrations of penicillin G (0, 50, 100, 200, and 400 U/ml). The infected cells were inoculated at 35℃ in 5% CO2 for the indicated time periods.

Transmission Electron Microscopy

The culture supernatant was replaced with 2.5% glutaraldehyde in phosphate buffer (0.1 mol/l, pH 7.4) at the indicated time points after infection. After 2 h of fixation at 4℃, the cells were removed from the cell culture plates using a cell scraper, collected in an Eppendorf tube, and centrifuged at 1,000 rpm for 5 min to remove dead and apoptotic cells at 4℃. Then, the cell pellets were postfixed in 1% osmic acid. After being washed twice with PBS, samples were dehydrated in increasing grades of ethanol (50%, 70%, and 100%) and acetone (90% and 100%) prior to infiltration and embedding in a mixture of Spurr epoxide resin (EPON812, DDSA, MNA, and DMP30) and pure acetone. Ultrathin sections (approximately 70 nm) were stained with uranyl acetate and lead citrate prior to examination and photography with a Hitchi HT-7100 transmission electron microscope.

Indirect Immunofluorescence Staining

During penicillin-G-induced persistence, infected cells were fixed with 4% paraformaldehyde for 30 min at 48 h p.i., permeabilized with 0.1% (v/v) Triton X-100 for 10 min, and blocked with DMEM containing 10% FBS at 37℃ for 1 h. Then, the samples were inoculated with rabbit anti-C. psittaci antiserum at 37℃ for 1 h. After being washed twice with PBS, the infected monolayer cells were inoculated with Cy2-conjugated goat-anti-rabbit IgG (green) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and DAPI (Thermo Scientific, Rockford, IL, USA) for 1 h. Images of Chlamydia inclusion-containing cells were obtained using a fluorescent microscope at 400-fold magnification.

Infectivity and Reactivation Analysis

Infectivity in the course of penicillin-G-induced persistence was determined by harvesting chlamydial organisms from infected monolayers by sonication at 48 h p.i. and re-inoculating them onto fresh HeLa 229 monolayers. Forty-eight hours after re-infection, infected monolayers were fixed with 4% paraformaldehyde and stained as described above. The numbers of inclusion bodies were counted in 30 random high-power fields and calculated as IFUs per milliliter.

For the reactivation analysis experiments, HeLa 229 monolayers grown on coverslips were infected at a MOI of 2, and persistence was induced as described above. Twenty-four hours p.i., the medium was replaced with complete growth medium without penicillin. After 24 h of incubation, chlamydial organisms were harvested by sonication and inoculated onto fresh HeLa 229 cell monolayers using complete standard medium. Forty-eight hours later, the monolayers were fixed with methanol, samples were stained as described above, and the infectious titer was estimated by counting 30 random fields per coverslip.

Preparation of RNA Samples

The HeLa 229 monolayers were grown in 6-well cell culture plates at a density of 1.5 × 106 per well. Two independent rounds of infection experiments were performed, including acute and persistent Chlamydia infections. At 6, 12, 24, 48, 72, and 96 h p.i., infected cells were removed using Trizol reagent (Tiangen, China), and total RNA was isolated through delamination in chloroform, precipitation in isopropyl alcohol, and washing with 75% ethanol. After being centrifuged at 12,000 rpm at 4℃ for 10 min, the RNA sediments were dissolved into 50 μl of sterile DEPC-treated water, aliquoted, titrated, and then stored at -80℃ until use.

Design of Gene-Specific Primers

Primers were designed using Primer Premier 5.0 software and were tested by conventional PCR using chromosomal DNA of C. psittaci 6BC as the template. The primers for the target genes are shown in Table 1.

Table 1.Sequences of primers used for quantitative real-time PCR.

Quantitative Real-Time PCR Analysis

Total RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Measurement of mRNA expression levels using quantitative real-time PCR was performed using a fluorescent quantitation PCR system (Thermo Scientific), with the following temperature-time profile: initial denaturation at 95℃ for 10 min and 40 cycles of 95℃ for 15 sec and 60℃ for 1 min. The melting curves of the amplification products were analyzed subsequently. Each reaction tube contained a reaction mixture of 1 μl of cDNA template, 0.5 μl of each primer (final concentration, 500 nmol/l), 15 μl of FS Universal SYBR Green Master Mix (Roche, USA), and 13 μl of deionized water. All quantitative real-time PCR assays from two independent series of infections were performed, at least, in triplicate for each target gene with samples collected from the persistence model at 6, 12, 24, 48, and 72 h p.i.. The relative gene expression ratios of the target genes were calculated based on the real-time amplification efficiency and cycle threshold deviation of a given sample versus the control (acute infection) in comparison with a reference gene, the 16S rRNA gene.

Statistical Analysis

To compare the relative transcriptional profiles of the candidate genes in acute and persistent C. psittaci infection, statistical comparisons were made using the one-way analysis of variance and Student’s t test. Differences at p values < 0.05 were considered to be statistically significant at a confidence level of 95%. Data were expressed as the means of at least three independent experiments ± standard deviation (SD).

 

Results

Infectivity of C. psittaci Cultures in Penicillin-G-Induced Persistent Infection

An in vitro cell culture model of persistent infection was induced by penicillin G, and the infectivity of C. psittaci was studied by immunofluorescence assay. When compared with the untreated cultures, the infectivity of the C. psittaci strain was drastically reduced at 48 h post infection in a significantly dose-dependent manner (Fig. 1). At penicillin G concentrations of 50, 100, 200, and 400 U/ml, the infectivity of C. psittaci was reduced to 90.0%, 32.2%, 9.6%, and 1.6%, respectively. Thus, it could be concluded that 50 U/ml penicillin G did not show obvious inhibition to the infectivity of C. psittaci; conversely 200 or 400 U/ml showed excessive inhibiting effects, and 100 U/ml penicillin G could effectively inhibit C. psittaci growth and induce a typical persistent infection state. Thus, the optimal concentration of penicillin G was 100 U/ml, and this concentration was used in all subsequent trials.

Fig. 1.Infectivity of C. psittaci cultures with the addition of penicillin G. The number of inclusion-forming units was determined from methanol-fixed immunostained cell cultures at 48 h p.i. through fluorescence microscopy. The data represent the mean ± SD for three independent experiments. Statistical significance denoted by ** represents p < 0.01.

Morphological Features of C. psittaci Cultures in Penicillin-G-Induced Persistent Infection

In the persistence model, chlamydial bodies exhibiting abnormal morphology were observed from 24 h p.i. onward. In the absence of penicillin G, inclusion bodies were normal, had more irregular outlines, were pleomorphic in shape, and were relatively larger and much less homogenous than in the presence of penicillin G, which showed more regular and smaller inclusion bodies (Fig. 2).

Fig. 2.Immunofluorescence staining of chlamydial cells in acute and penicillin-G-induced persistent infection. (A) C. psittaci-infected cells at 24 h p.i.. (B) C. psittaci-infected cells at 48 h p.i.. The concentration of penicillin G is 100 U/ml; Magnification, 400×.

Under electron microscopy, as reported previously [19, 32], at 48 h p.i., untreated cultures of C. psittaci formed large inclusion bodies containing numerous EBs, numerous intermediate condensing bodies, and low numbers of RBs. After being treated with penicillin G, the inclusion bodies were smaller with enlarged RBs, and few or no EBs, indicating a general failure of the chlamydial bodies to complete rounded development into EBs (Fig. 3). In acute infection cultures, the inclusion bodies contained normal EBs and RBs; the EBs were electron opaque, spherical-oval shaped particles with little periplasmic space and were surrounded by an undulating cell membrane, whereas the RBs were round to oval in shape, with a typical electron translucent center and a cytoplasm condensed towards the periphery. In the penicillin-G-treated cultures, the inclusion bodies were smaller than normal and contained considerably lower numbers of chlamydial particles. The RBs (named aberrant bodies, AB) were larger than those in normal inclusion bodies, were highly variable in shape, and had an electron-lucent cytoplasm with a loose network of filaments and multiple electron-dense foci.

Fig. 3.Transmission electron micrographs of the chlamydial inclusion bodies in acute and persistent infection. (A) C. psittaci-infected cells at 24 h p.i.. (B) C. psittaci-infected cells at 48 h p.i.. Bar = 2 μm. EB, elementary body; RB, reticulate body; AB, aberrant body.

Activity of C. psittaci Released from Penicillin G-Induced Persistent Culture

To check the reversibility of the persistent state in the penicillin-G-induced model, the capability of persisting chlamydial bodies to rescue was examined using freshly infected host cells, with the persistent inducer removed. The morphological features of C. psittaci were found to be rescued in this model. Briefly, immunofluorescent staining showed that the inclusion bodies were larger and more pleomorphic in shape, indicating acute infection (Fig. 4A). Additionally, the inclusion bodies were larger, and more EBs and less ABs and RBs could be observed in the inclusion bodies by electron microscopy (Fig. 4B).

Fig. 4.Reversibility of chlamydial morphological characteristics from persistent infection. The morphological characteristics of chlamydial inclusion bodies from acute and penicillin-G (100 U/ml)-induced persistent infection were compared with those from the reactivation assay, which included removal of persistence inducer at 24 h p.i. and subsequent culture in complete growth medium for 24 h. (A) Immunofluorescence staining of chlamydial inclusion bodies. Magnification, 400×. (B) Transmission electron microscopy of chlamydial inclusion bodies. Bar = 5 μm.

In addition to the morphological data, IFUs were also compared to evaluate the infectivity of the organisms after persistent infection. After the removal of penicillin G (100 U/ml), C. psittaci recovered and returned to its infectious state. At 24 h post infection, compared with after acute infection, the recovery rate after persistent infection was 31.7%. However, when the concentration was too high, the Chlamydia cells were excessively inhibited and could not recover effectively after removing penicillin G (Fig. 5).

Fig. 5.Reactivation of persistent C. psittaci after removal of penicillin G. The numbers of recoverable infectious chlamydial organisms (in IFU) from acute and penicillin-G-induced persistent infection were compared with those from the reactivation assay, which included removal of persistence inducer at 24 h p.i. and subsequent culture in complete growth medium for 24 h. The data represent the mean ± SD for three independent experiments. Statistical significance denoted by * represents p < 0.05.

Difference in C. psittaci Gene Expression in Penicillin-G-Treated vs Normal C. psittaci Infection

The relative mRNA expression levels of genes related to the course of development of persistence induced by penicillin G are shown in Fig. 6. From 6 h p.i. onward, six genes encoding proteins with functions in DNA replication (dnaA), inclusion membrane (incC), heat shock protein 60 (hsp60) (grpE), hsp70 (dnaK), δ factor (rpsD), and type III secretion protein (CPSIT_0846) were upregulated significantly in C. psittaci penicillin-G-induced persistence, with peak expression levels at 72, 48, 12, 48, 24, and 12 h p.i., respectively. In comparison with these upregulated genes, genes encoding products involved in cell division (ftsW and ftsY) and the gene encoding outer membrane protein E (CPSIT_0042) were downregulated in the penicillin-G-treated cultures from 6 h p.i. onward, but the expression levels of these genes increased over time. The gene encoding the 60 kDa cysteine-rich outer membrane complex protein B (omcB) showed approximately equal levels of transcription in the acute and penicillin-G-mediated persistent infections, except at 24 and 72 h p.i. when it was significantly downregulated in the penicillin-G-mediated persistent infections.

Fig. 6.Relative mRNA expression levels of C. psittaci genes in acute and persistent infection at different time points. HeLa229 cells were infected with C. psittaci 6BC (MOI = 2) and treated with 100 U/ml penicillin G to induce persistence in three independent experiments. Acute nontreated infections were used as controls. A total of 10 candidate chlamydial genes (dnaA, dnaK, ftsW, ftsY, incC, omcB, CPSIT-0042, CPSIT-0846, grpE, and rpsD) were determined by real-time quantitative PCR at 6, 12, 24, 48, and 72 h p.i.. The 16S rRNA gene was used as an internal standard for relative comparison of gene expression between treated and untreated cultures at each time point. The data that represent the mean ± SD for three independent experiments are calculated as relative expression by the 2-ΔCt method. Statistical significance denoted by * and ** represents p < 0.05 and p < 0.01, respectively.

 

Discussion

Active infection with C. psittaci has significant clinical symptoms. However, persistent infection with this organism often induces severe and difficult-to-treat sequelae. Considerable in vivo and in vitro evidence shows that a state of persistence can arise during chlamydial infections [24]. However, the mechanisms that evoke and maintain the persistence of Chlamydia remain unclear. To further investigate this mechanism, a state of C. psittaci persistence was established in HeLa cells using penicillin G treatment.

After treatment with penicillin G, the morphology of C. psittaci inclusion bodies showed significant abnormalities. We found greatly enlarged, irregular, and less dense RBs (i.e., ABs) in the culture treated with penicillin G, which suggests that penicillin exposure blocks binary fission and RB to EB differentiation, but does not prevent EB to RB differentiation; these results are consistent with those reported by Goellner et al. [19]. In general, agents that target bacterial proteins and RNA synthesis can inhibit chlamydial differentiation from EB to RB state or from RB to EB state, depending on when they are added. In contrast, agents that target DNA or peptidoglycan synthesis specifically inhibit RB to EB differentiation [24]. Furthermore, compared with the control culture, there were few or no EBs observed in the penicillin-G-treated cultures at the end of the developmental cycle over the 48 h cell culture (Fig. 3). This reduction of released EBs strongly suggests that penicillin G can prevent the normal developmental cycle of C. psittaci and, as part of that process, reduce the number of EBs released via cell lysis. This is similar to results reported in C. trachomatis and C. pneumoniae persistent infections induced by IFN-γ [29,35]. We also found that the capability of infectivity sharply decreased after exposure to penicillin and was recoverable upon the removal of penicillin. This suggests that removal of the persistence-inducing stimuli generally reverts C. psittaci from its persistent state back to its normal state.

Although the morphologic and microbiologic features of C. psittaci were significantly altered in persistent infection, the abnormal organisms continued to replicate their genomes [4]. However, the transcript profile of C. psittaci in persistent infection was different from that in active infection and also varied in different persistent cultures. The transcript profile is widely used to determine if bacteria are viable and metabolically active. In this study, we selected 10 genes for analysis. 16S rRNA was used to standardize and accurately compare gene expression levels. In the penicillin-G-treated cultures, among the 10 genes analyzed, six were significantly upregulated, three were obviously downregulated, and one gene slowly decreased after a latent period.

One of the upregulated genes was dnaA, encoding a gene product required for DNA replication and repair, which was in accordance with the morphologic result that greatly enlarged RBs could be seen and EB to RB differentiation was not blocked by penicillin G. Lambden et al. [30] also reported that penicillin does not prevent C. trachomatis DNA replication. The proteins with the most highly conserved numbers in C. psittaci are hsp60-kDa (grpE) and hsp70-kDa (dnaK), which are expressed at steady-state levels throughout the chlamydial life cycle and are the major targets of host immune responses [44]. These two genes were upregulated in this penicillin-G-induced persistent infection model, which may have helped the organism escape the host’s immune response, thereby maintaining the persistent state of the infection.

The rpsD gene was also upregulated in this penicillin-G-induced persistent infection model, which supports its role in helping C. psittaci adapt to its environment and survive. The rpsD gene of Chlamydia encodes the alternative sigma factor (sigma28), which bears strong homology to many bacterial sigma factors, including Escherichia coli sigma8 and Bacillus subtilis sigmaB and sigmaD, and it may play a role in the regulatory network that allows Chlamydiae to survive changes in its environment, enabling it to complete its unique developmental cycle [41].

It is predicted that CPSIT_0846 encodes a type III secretion system (T3SS) effector protein located in the inclusion membrane during C. psittaci infection, and it was upregulated from 12 h post infection. T3SS effector proteins are injected into the host cell cytosol, chlamydial inclusion membranes, or inclusion lumens via T3SS, and they have critical roles in facilitating EB invasion, promoting the survival and replication of intracellular chlamydial organisms, and preparing chlamydial organisms for the dissemination of progeny EBs to new cells [47]. The upregulated CPSIT_0846 may be related to C. psittaci survival in this persistent infection state.

The gene encoding inclusion membrane protein C (IncC) was also upregulated in this persistent infection model. Reports previously showed that IncC may be involved in processes like inclusion formation, transportation to nuclear space, and evasion of early lysosomal fusion, and it also can upregulate the expression of cytokines, modulate host immune response, and play key roles in the hostpathogen interaction [20-22]. Therefore, the upregulation of incC in the model of penicillin-induced persistent infection may be a mechanism for C. psittaci to modulate its survial under sterssful conditions.

In contrast, ftsK and ftsW were expressed in untreated cells, but the expression was attenuated in cells treated with penicillin G. The same result was found in IFN-γ induced C. pneumoniae persistent infection [7]. The ftsW and ftsY encoded proteins were involved in cell division [13], which may explain the aberrant RB morphology and the decrease in number of EBs observed in persistent cultures.

Two outer membrane protein-encoding genes, omcB and CPSIT_0042, were also analyzed. CPSIT_0042 encoding outer E protein decreased dramatically, but omcB only reduced slightly after penicillin G treatment. As the second abundant outer membrane protein, the chlamydial outer membrane complex protein B (OmcB) is highly conserved among Chlamydia species and may function as an adhesion for chlamydial invasion into host cells [14, 15]. Mathews et al. [32] also reported that omcB transcripts did not significantly change in C. pneumoniae persistence induced by IFN-γ [32].

In the work described here, in vitro observations indicate that chlamydial persistence is not characterizable by a single transcript profile under all circumstances. This supports the idea that persistent infection in vivo is a complex, flexible strategy that promotes the long-term survival of these organisms, and provides new information regarding the basic biology of chlamydial persistence that may be helpful in the design of improved therapeutic interventions for chronic chlamydial infections.