Protein Expression Profiles of Chlamydia pneumoniae in Models of Persistence versus Those of Heat Shock Stress Response
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《感染与免疫杂志》
Division of Infectious Diseases, Department of Medicine
Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, Kentucky
Infectious Diseases Program, School of Life Sciences, Queensland University of Technology, Brisbane, Australia
ABSTRACT
Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42°C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.
INTRODUCTION
Chlamydia pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes both respiratory and systemic diseases of humans. In addition to acute infections, such as pneumonia or other respiratory diseases (18, 19), C. pneumoniae has been associated with a wide range of chronic diseases that are characterized by a local and/or systemic inflammatory response (33, 38). More importantly, there is growing evidence of an association of this pathogen with atherosclerosis, although the precise role of C. pneumoniae in this disease remains unclear. The chlamydial developmental cycle begins when the extracellular elementary body (EB) attaches and enters the host cell by endocytosis and is contained within an inclusion, which continues to enlarge, while the EB differentiates into the reticulate body (RB). The RB undergoes logarithmic division by binary fission and subsequently redifferentiates into an EB. These infectious EB are released by host cell lysis at 60 to 84 h postinfection (hpi) and initiate a new cycle of replication (47).
The ability of chlamydiae to "persist" in host tissue for extended periods of time is attributed to the activation of host-cell-mediated immune response, which plays an important role in pathogenesis of C. pneumoniae infections (20, 21). Chlamydial persistence has been described by Beatty et al. (4) as a viable, but nonculturable state, resulting in a long-term relationship with the infected host cell. In vitro, persistence is characterized by a loss of infectivity due to the development of smaller inclusions, which, ultrastructurally, contain large pleomorphic noninfectious RBs, referred to as aberrant bodies (ABs), inhibited in both binary fission and their ability to redifferentiate into infectious EB. These characteristics are generally reversible upon the removal of the persistence-inducing factor(s) (for reviews, see references 4 and 23).
Earlier studies (5, 9, 32, 35) have proposed that in order to cause chronic (persistent) infections, Chlamydia would likely have differences in gene and protein expression patterns compared to Chlamydia causing acute infections. Interest in genetic studies, as well as expression profiles of genes and proteins during chronic infections, has led to the establishment of several in vitro models of persistence that attempt to mimic in vivo conditions (4, 23). Among the in vitro models of persistence, gamma interferon (IFN-)-induced persistence is the best-studied model thus far (7, 8, 30, 36, 37, 42). Using RNA analysis, Byrne et al. (9) showed that the expression of genes related to cell division (ftsK and ftsW) was downregulated, whereas DNA replication genes (dnaA, polA, mutS, and minD) were expressed at normal levels during IFN--mediated persistence. Previous studies in our laboratory have shown differentially expressed C. pneumoniae proteins due to IFN--induced persistence (29, 32, 35). Mathews et al. (29) described the upregulation of genes involved in the cell envelope (ompA and ompB), glycolysis (pyk), and peptidoglycan synthesis (nlpD) under persistent conditions. Molestina et al. (32) described a marked upregulation of proteins such as MOMP (ompA), GroEL, and proteins involved in DNA replication (GyrA), transcription (RpoA and PnP), translation (Rrf), glycolysis (Pgk and GlgP), and type III secretion (SctN) at 24 hpi. Furthermore, Mukhopadhyay et al. (35) reported altered expression profiles of proteins involved in amino acid biosynthesis (Adk), the cellular process (AhpC), the cell envelope (CrpA), translation (Map), and a hypothetical protein CPn0710, at 24 and 48 h.
Another in vitro model of chlamydial persistence is induced by iron-limiting (IL) growth conditions. The protein expression pattern under conditions of iron limitation has been examined in both C. trachomatis (39) and C. pneumoniae (1, 45). It is well established that iron is an important component in numerous cellular metabolic processes, as well as a mediator of virulence in several bacterial pathogens (31). Interestingly, as a proposed risk factor for heart disease in humans, excess iron in the atherosclerotic lesions could play a key role in promoting the growth of C. pneumoniae (44).
A wide range of evidence supports the role of heat shock proteins (hsp's) in rescuing the bacterial cell from stress-related damage by facilitating protein refolding, preventing the aggregation of partially folded polypeptides, and ensuring proper folding (22, 24). Chlamydial hsp-70 (DnaK), hsp-60 (GroEL), and hsp-10 (GroES) have been implicated as primary antigens for eliciting host immune response (12, 28). Furthermore, hsp gene regulation (43, 46), as well as its response to heat shock treatment in the developmental cycle (26), was determined in C. trachomatis.
Given the description of the IFN and IL models of C. pneumoniae persistence, it was of interest to further characterize their similarities and differences in order to better understand their potential role in chronic diseases. As a means of distinguishing the C. pneumoniae persistence response from a classic bacterial stress response, we examined the protein expression profiles of C. pneumoniae under IFN- treatment (primarily tryptophan limitation) and iron limitation and compared those with the response due to heat shock stress. Since the potential mechanism(s), in vivo, to restrict chlamydial growth could be due to induction of tryptophan catabolism (which are primarily induced by IFN-), iron deprivation, and/or other undefined stress-inducing conditions, we analyzed and compared the protein expression profiles of nine subcategories of C. pneumoniae proteins under these two models of induced persistence, along with heat shock stress, with that from the normal growth condition, during the mid-to-late (48 hpi) stage of its developmental cycle.
MATERIALS AND METHODS
Cell line. HEp-2 cells (ATCC CCL-23) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Iscove's maintenance medium (IMM; Cellgro, Washington, D.C.) as described previously (34). Cells were grown in 75-cm2 flasks (Costar, Cambridge, MA) at 37°C in 5% CO2 for 24 h to achieve confluence of the monolayer and harvested with trypsin-EDTA. HEp-2 cells used in our experiments are free of Mycoplasma contamination as determined by PCR testing.
Bacterial isolate. C. pneumoniae A-03 (ATCC VR-1452) was previously isolated in our laboratory from an atheroma of a patient with coronary artery disease during heart transplantation at the Jewish Hospital Heart and Lung Institute, Louisville, KY (38). C. pneumoniae organisms were propagated in HEp-2 cell monolayers in Iscove's growth medium (IGM) as described previously (34). Briefly, EBs were harvested and purified by disruption of HEp-2 cell monolayers with a cell scraper, sonication, and centrifugation over a renografin density gradient (34). EB suspensions were stored in sucrose-phosphate-glutamic acid buffer at –80°C, and the viability of frozen stocks was determined by titrating EB suspensions onto HEp2 cell monolayers by standard methods.
Infection protocols. In all three infection models used, HEp-2 cells were seeded in six-well tissue culture plates at 106 cells per well in IMM and incubated in 5% CO2 at 37°C overnight. Subsequently, each model was treated as described below.
IFN model. HEp-2 monolayers were inoculated with 1.0 x 108 inclusion forming units (IFU) per well of C. pneumoniae in 2 ml of IGM with or without human recombinant gamma interferon (IFN-) at 50 and 100 U per ml (Promega, Madison, Wis.), centrifuged at 675 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 for 48 h. For proteomic analysis, infected monolayers were pulse-labeled for 2 h (from 46 to 48 hpi) in methionine-cysteine-free RPMI 1640 medium (Cellgro, Herndon, Va.) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix; Amersham Pharmacia, Piscataway, N.J.) and 500 μg of cycloheximide per ml in the presence or absence of IFN- as described previously (35).
Iron limitation (IL) model. Uninfected Hep-2 monolayers were cultured in 2 ml of IGM with or without exposure to deferoxamine mesylate (DAM) at 50 μM (Sigma, St. Louis, Mo.) for 24 h. The monolayers were subsequently inoculated the following day with 1.0 x 108 IFU per well of C. pneumoniae in 2 ml of IGM, with or without DAM at 50 μM, centrifuged at 676 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 for 48 h. For proteomic analysis, infected HEp-2 cells were pulse-labeled for 2 h (from 46 to 48 hpi) in methionine-cysteine-free RPMI 1640 medium (Cellgro) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix) and 500 μg of cycloheximide per ml in the presence or absence of DAM.
HS response. HEp-2 monolayers were inoculated with 1.0 x 108 IFU per well of C. pneumoniae in 2 ml of IGM, centrifuged at 676 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 until heat shock (HS) treatment. The schedule for HS was as follows; monolayers for 8 h HS were incubated at 42°C in 5% CO2 from hpi 40 to 48, for 6 h HS from 42nd to 48th hpi and for 4 h HS from 44th to 48th hpi. Control monolayers were incubated at 37°C in 5% CO2 for 48 h. For protein expresssion analysis, all infected HEp-2 monolayers were washed once in phosphate-buffered saline (prewarmed to 42°C), and then pulse-labeled for 2 h (from hpi 46 to 48) in methionine/cysteine-free RPMI 1640 medium (Cellgro) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix) and 500 μg of cycloheximide per ml in the presence or absence of HS conditions.
Infectivity studies. Parallel monolayers were set up to conduct infectivity studies for each model. After the respective treatments described above, C. pneumoniae-infected HEp-2 cells were harvested at 48 h and titrated for viable IFU/ml. Recovery of infectivity from the inducing conditions was determined by the addition of 500 μg of tryptophan (Sigma-Aldrich)/ml or 8.0 mg of holotransferin (Calbiochem)/ml to the IFN and IL models, respectively. The results of four to six experiments (in triplicate) were expressed as a normalization to the untreated controls.
TEM analysis. A second set of parallel infected HEp2 monolayers was used for transmission electron microscopy (TEM) analysis to validate the level of persistence each model by analyzing the infected monolayers as follows. Samples from each model (each sample being the entire contents of one well of a six-well plate) were fixed with 3% glutaraldehyde (Sigma-Aldrich, St. Louis, Mo.), fixative in 0.1 M phosphate buffer at pH 7.4. After fixation for 1 h at room temperature, cells were scraped off, washed in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, and embedded in Spurr epoxy resin according to standard procedures (40). Ultrathin sections (50 to 100 nm) were cut and stained with uranyl acetate and lead citrate stains prior to examination and photography with a JEOL 1200EX transmission electron microscope.
Protein expression analysis. (i) Protein extraction. At the end of the 2-h labeling period (46 to 48 h), C. pneumoniae-infected HEp-2 cells were washed in cold phosphate-buffered saline (PBS), scraped with a cell scraper, and pelleted by centrifugation at 16,000 x g. Pellets were resuspended in 30 μl of buffer containing 2% (wt/vol) Sarkosyl, 1% (wt/vol) OBG (octyl--D-1-thioglucopyranoside), and 2 mM tributyl phosphine (TBP) and 5 μl of protease inhibitors (levpeptin and aprotinin). Samples were sonicated (Cell Dismembrator, Model 100; Fisher Scientific) for 5 s, boiled for 5 min, and then cooled to room temperature. A total of 100 μl of thiourea lysis buffer (7 M urea, 2 M thiourea, 10 mM Tris, 2 mM TBP, 2% [vol/vol] ampholine) was added, followed by the addition of 3.0 μl of a mixture of 50 mM MgCl2, 476 mM Tris-HCl, 24 mM Tris base, 1.0 mg of DNase I per ml, and 0.250 mg of RNase A per ml (pH 8.0); the mixture was then incubated on ice for 10 min and stored at –80°C.
Two-dimensional gel electrophoresis. Radiolabeled protein extracts were mixed with immobilized pH gradient (IPG) rehydration buffer containing 8 M urea, 4% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 0.04 M Tris base, 0.065 M dithiothreitol, and 0.01% (wt/vol) bromophenol blue in a final volume of 400 μl as follows. (i) To generate an electrophoretic map of purified C. pneumoniae EB, a 750-μg portion of protein extracts was mixed with IPG rehydration buffer. (ii) To study intracellular C. pneumoniae protein expression, 7.6 x 105 DNA copies/μl of 35S-labeled chlamydial proteins was mixed in IPG rehydration buffer. Protein samples were loaded onto Immobiline DryStrip (IPG strips 4-7, 6-11, or 3-10) [Amersham Biosciences, Uppsala, Sweden]), allowed to rehydrate overnight, and isoelectrofocused to 98,000 Vhours in a PROTEAN IEF cell (Bio-Rad, Inc.). The following focusing parameters were applied: a rapid advance voltage ramping method was used, in which 250 V for 15 min was used for ramping in the first step, 5,000 V for 5 h was used in the second step, and finally 10,000 V for 12 h was used for the final focusing step. A hold step was programmed to maintain the voltage at 500 V till the run was stopped. The maximum current limit per gel was set at 50 μA. After focusing was completed, IPG strips were equilibrated in buffer containing 6 M urea, 2% (wt/vol) dithiothreitol, 30% (vol/vol) glycerol, and 1x Tris-acetate.
Two-dimensional electrophoresis was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) on 20-by-20-cm slab gels in PROTEAN II xi multicell system (Bio-Rad) at 4°C for 4.5 h under a 500-V maximum voltage and 20 W per gel, with 200 mM Tricine used as a cathode buffer and 0.4% (wt/vol) sodium dodecyl sulfate plus 625 mM Tris-acetate (pH 8.3) used as an anode buffer. Gels were fixed in 40% ethanol-7.5% acetic acid for 30 min. Gels containing purified EB proteins were silver stained, whereas gels containing radiolabeled chlamydial proteins were treated for 30 min with Amplify fluorographic reagent (Amersham Pharmacia, Buckinghamshire, England), vacuum dried, and exposed to high-density phosphorimaging screens (Bio-Rad) for two and a half days. Images were scanned in a Molecular Imager FX Pro Plus system (Bio-Rad). This imaging equipment is fully integrated to the ProteomeWorks system (Bio-Rad) with a high-resolution image acquisition interface within the PDQuest software.
Image analysis. Protein spots were analyzed for differential expression patterns by using PDQuest software version 7.1 (Bio-Rad). For quantification, the spots were normalized to the total quantity of the valid spots in each of the gel images for the respective models of induced persistence. After normalization, gels or a portion of the gel were analyzed as mentioned previously (35). For statistical analysis of differential expression pattern between the protein spots in the gels under study, both manual and PDQuest automated analytical tools were used to represent the spot's quantity in each individual gel and grouped as the fold (or percent) increase or decrease.
MALDI-TOF-MS. Protein spots from C. pneumoniae EBs were excised from silver-stained gels and treated with 20 μg of trypsin per ml in 50 mM ammonium bicarbonate at 37°C overnight. A 2-μl aliquot of supernatant was mixed in an equal volume of saturated -cyano-4-hydroxycinnamic acid, and 50% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid, and 0.8 μl of the resulting solution was applied to the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) template. Masses of peptide fragments were determined by MALDI-time-of-flight (TOF) analysis with a Micromass mass spectrometer. Patterns of measured masses were matched against theoretical masses of proteins found in the annotated databases Swiss-Prot and TrEMBL, accessible in the ExPASy Molecular Biology server (http://expasy.cbr.nrc.ca/). Searches were performed by Profound-peptide mapping (The Rockefeller University Edition, version 4.10.5) with restrictions to proteins from 5 to 100 kDa and mass tolerances of 100 ppm. Partial enzymatic cleavages leaving one cleavage site, oxidation of methionine, and modification of cysteine with iodoacetamide were considered in these searches. Sequence coverage of >45% (P < 0.05) was considered as a positive match.
Alternative approach to protein identification using bioinformatics. Distinctly upregulated or unaltered protein spots that could not be identified by MALDI-TOF-MS (either resulting from insufficient amount of protein in the spot and/or a clearly resolved spot was not obtained in the silver-stained EB map) were identified by bioinformatics approach using the TagIdent tool (http://ca.expasy.org/tools/tagident.html). The molecular weight (Mw) and isoelectric point (pI) of such spots were estimated from the analytical gels and applied to TagIdent with very stringent parameters and specified error margins (pI range = 0.25 and Mw range = 10%) in order to match the proteins from the database (Swiss-Prot/TrEMBL/NCBI) near to the defined range of Mw and pI with a P value of <0.05.
Statistical analysis. The statistical significance between experimental groups was determined by using the Student t test. P values of <0.05 were considered statistically significant.
RESULTS
Validation of models. In order to validate the models used in the present study, infectivity studies were conducted with C. pneumoniae-infected Hep-2 cells. Figure 1A shows the reduction in viable C. pneumoniae IFU by approximately 3 and 5 logs due to treatment with IFN- at 50 U/ml (P < 0.05) and 100 U/ml (P < 0.05), respectively. Replenishing the tryptophan (500 μg/ml) led to the almost complete recovery of infectivity, indicating that the decrease in infectivity was due primarily to the limitation of intracellular tryptophan. Similarly, Fig. 1B shows a reduction in C. pneumoniae infectivity of approximately 2 logs in the presence of DAM (50 μM) (P < 0.05). The addition of iron-saturated holotransferin (8.0 mg/ml) to the monolayers led to a return to equivalent IFU/ml as seen in the untreated monolayers, thus supporting the limitation of host cell iron as the cause of reduction in viable IFU. In comparison to these models of persistence, heat shock at 42°C for 4, 6 (P < 0.05), and 8 h (P < 0.05) showed a sequential loss of viable IFU/ml (Fig. 1C). Prolonged heat shock treatment for up to 12 or 24 h resulted in the complete loss of infectivity (data not shown).
FIG. 1. Infectivity studies showing loss of viable C. pneumoniae due to treatment with IFN- (A), DAM (B), and HS at 42°C (C). , P < 0.05 (compared to the untreated control).
For additional validation of all experimental conditions used in the present study (i.e., untreated, IFN- treated, IL treated, and HS treated) and also to assess the morphological features of each, C. pneumoniae-infected cultures were examined by TEM. Ultrastructural analysis of untreated C. pneumoniae-infected HEp-2 cells revealed typical inclusion and bacterial morphologies for cultures grown for 48 h (Fig. 2A). On average, inclusions were approximately 5 μm in diameter (range, 2.5 to 7 μm in diameter), with an average of 50 to 70 chlamydial particles (80% RBs, 15% IBs, and 5% EBs) per inclusion. Inclusions identified in IFN-treated (50 U/ml), C. pneumoniae-infected HEp-2 cells (Fig. 2B) appeared much smaller in diameter (ranging from 2 to 4 μm) than those observed in untreated samples. Fewer chlamydial particles were observed within these smaller inclusions. Commonly, three to five chlamydial particles filled the entire space of the smaller inclusion, since these chlamydial particles often were larger and more irregular in morphology (defined as aberrant bodies [ABs]) than those seen in untreated samples. Similarly, C. pneumoniae-infected HEp-2 cells treated with the iron chelator DAM (50 U/ml) exhibit inclusions much smaller than those in the untreated cultures, where the most common inclusion size observed was 3 μm in diameter (Fig. 2C). Also, the chlamydial particles were few (average of three chlamydial particles) within these inclusions and maintained the aberrant morphology (AB), as seen in the IFN-treated cultures. When exposed to 42°C for 4 h (HS model), C. pneumoniae inclusions were, on average, 4 μm in diameter and contained varied morphologies of C. pneumoniae (Fig. 2D), some of which resembled the ABs seen in the IFN and IL models. After prolonged exposure to heat (6 and 8 h), the cytoplasm of the bacteria became more electron lucent, and there appeared to be some disruption of the bacterial cell membrane, indicating the bacteria were undergoing cell death (Fig. 2E and F). Clearly, the C. pneumoniae morphologies seen in the HS model, especially at the 6- and 8-h time points, were distinct from the classical ABs observed in the IFN and IL models.
FIG. 2. TEM images showing morphological analysis of C. pneumoniae under normal growth conditions at 48 hpi (A), after IFN- treatment (50 U/ml) (B), after DAM treatment (50 μM) (C), and after HS at 42°C for 4 h (D), 6 h (E), and 8 h (F). The arrows in panels D, E, and F denote altered morphological forms of C. pneumoniae. EB, elementary body; IB, intermediate body; RB, reticulate body; AB, aberrant body.
Differential protein expression analysis. Intracellular expression of C. pneumoniae proteins at 48 hpi under untreated, as well as inducer-specific conditions under IFN, IL, and HS conditions, were determined by pulse-labeling each C. pneumoniae-infected HEp-2 cell monolayer. Of the 168 differentially expressed protein targets identified by the image analysis software, 52 C. pneumoniae proteins, constituting nine functional categories, were identified by either MALDI-TOF (41 proteins) or bioinformatics (11 proteins). Only these 11, of the 127 spots analyzed by bioinformatics, could be matched to C. pneumoniae proteins, using the strict exclusion criteria (see Materials and Methods). For quantitative analysis of differential regulation, the expression profiles of each protein in each of the three models examined was compared to their respective untreated (control) condition (Fig. 3).
FIG.3. Representative two-dimensional electrophoretic maps of C. pneumoniae proteins expressed at 48 hpi in untreated HEp2 cells (A), after IFN- treatment at 50 U/ml (i) or 100 U/ml (ii) (B), after treatment with 50 μM DAM (C), and after treatment at 42°C for 4 h (i), 6 h (ii), and 8 h (iii) (D). Representative gels out of six experiments are shown.
Common expression patterns under IFN and IL induced persistence and HS stress response. Proteins which showed common trends in expression pattern in all three treatment conditions are listed in Table 1. These include one amino acid/nucleotide biosynthesis protein (Ndk), and the majority of the stress response proteins, which belong to the functional categories of protein folding, assembly, and modification and seven hypothetical proteins (Table 1). The stress response proteins (DnaK, HtrA, ClpP_1, and ClpP_2) were found to be 1.5-fold upregulated, depending on the condition, in all three treatment conditions (Fig. 3B to D). There were a few exceptions to the expression trends in the stress response category. Although the expression of GroES and DksA showed a general inclining trend in the IFN model, GroES and DksA showed unaltered expression profiles in the IL model (Table 1 and Fig. 3C). Endopeptidases ClpC and ClpX, as well as GrpE, were found to be upregulated under IFN and IL conditions; however, they were found to be downregulated under HS treatment. Similarly, ClpB was upregulated under IL and HS treatments but remained unchanged under IFN. Yet another common feature of the HS response and both persistence models was the downregulation of Gcp_1 and Ndk (Table 1). Extending the HS period for the C. pneumoniae-infected monolayers to 8 h resulted in the downregulation of 8 of the 12 stress response proteins (Table 1 and Fig. 3D), perhaps suggesting the beginning of a cell death phase. Finally, most of the hypothetical proteins identified and analyzed—CPn0011, CPn0710, CPn0518, CPn0742, CPn1032, CPn0216, and CPn0917—showed decreased or unchanged protein levels under all experimental conditions.
Unique protein expression patterns. Considerable variation in profiles was exhibited by the proteins involved in biosynthesis, cell envelope, cellular processes, energy metabolism, transcription, and translation when subjected to IFN, IL, and HS treatments (Table 2). For the HS response, most of the proteins involved in the biosynthesis of amino acids and cofactors, cell envelope, cellular processes, energy metabolism, transcription, and translation were prominently downregulated, especially when HS was applied for up to 6 and 8 h (Table 2 and Fig. 3D). All four cell envelope proteins (CrpA, OmpA, OmcB, and PorB) showed a marked decrease in expression pattern compared to the IFN and IL persistent models (Table 2). Furthermore, the expression profiles of energy metabolism proteins involved in the pentose phosphate pathway (Tal), the glycolysis pathway (GapA, Pgk, Eno, and Pyk), and the tricarboxylic acid cycle (SucC) were mostly reduced in the HS model (Table 2). Only five proteins, those involved in cellular and transport-related processes (AhpC, GspD, LcrE, ExbD, and GreA), showed an increase in expression in response to 4-h HS. Apart from ExbD, two GspD and GreA levels were increased under HS at 4 and 6 h, and RplL levels were increased after 8 h of HS. ExbD was the only protein found to be upregulated throughout the HS treatment.
The most notable response of C. pneumoniae to IFN was the upregulation of the amino acid biosynthesis proteins (TrxA, TrxB, Adk, and PyrH) (Fig. 3B). These proteins were downregulated in the other two models, except for Adk, which was found to be upregulated under IL as well and remained unchanged in response to HS for 4 h (Table 2). Proteins involved in cellular and transport related processes (AhpC, GspD, LcrE, YscL, and ExbD) were either unchanged or downregulated under IFN conditions. However, they showed an inclining trend under IL and HS (4-h) treatments.
In the IL model, most proteins examined showed an inclining trend. Specifically, proteins involved in the biosynthesis of cofactors (BirA, BioA, and BioD) were markedly upregulated only under IL conditions compared to the untreated controls. The other protein in this category, RibC, which remained unchanged under IL treatment, also showed a similar expression pattern under IFN treatment. The three proteins involved in transcription (GreA, RpoA, and TctD) also showed consistent upregulation in expression profile under IL condition compared to the varied pattern observed for the other two conditions (Table 2 and Fig. 3C). Furthermore, of the six proteins attributed to translation, three proteins involved in peptide chain elongation (Tsf, TufA, and Efp1) and other proteins including Map, PfrA, and ribosomal proteins RplL and RpsA showed a general increasing trend under IL, whereas the reverse trend was observed in the expression pattern in the IFN and HS response (Table 2).
DISCUSSION
To our knowledge, this is the first study to examine the HS stress response in C. pneumoniae. Of the 12 classical HS stress response proteins analyzed, nine (GroEL_1, DnaK, DksA, GrpE, HtrA, ClpP_1, ClpP_2, ClpB, and AhpC) exhibited elevated expression levels after HS for 4 h, and several of these remained upregulated after 6 h of HS. This indicates that C. pneumoniae, like C. trachomatis (26), exhibits a classic bacterial HS stress response. Of interest was the fact that the expression of GroEL_2 or GroEL_3 was not detected, in keeping with the reported lack of transcription of these genes in C. trachomatis after HS (27). It should be noted that the lack of detection of these, or perhaps other proteins, may be due to their presence below the lower limits of the assay or to differences in turnover rates for a given protein.
Our qualitative protein profiling analysis of C. pneumoniae under two models of persistence, compared to HS, has enabled us to suggest several important observations of chlamydial persistence. First, there is a significant translational level stress response seen in both IFN and IL persistence. Of the 12 stress response proteins analyzed, 8 (groEL, groES, dnaK, dksA, htrA, gcp1, clp1, and clp2) showed the same trends in both persistence models that they showed under HS. This suggests that a stress response is one part of the persistence response. Our data support the findings of Gerard et al. (17), who reported the increased expression of C. trachomatis groEL genes under persistent conditions in both in vitro and in synovial tissue.
Several other functional groupings also showed quite different trends between HS and persistence. In some cases this persistence response was common to both models, while in other cases it was different between the two models of persistence. In both the IFN and the IL models, the cell envelope proteins CrpA and OmpA were significantly increased in expression compared to untreated controls, whereas OmcB was downregulated. OmpA functions as an integral membrane protein, providing structural rigidity to the EB outer membrane and permitting diffusion of solutes through the intracellular RB membrane, while the cysteine-rich 60-kDa OmcB functions as an outer membrane cross-linking protein vital for EB formation. The data in the literature has been discordant with respect to these cell envelope proteins. DNA microarray analysis, immunofluorescence, and immunoblotting studies with C. trachomatis persistence induced with IFN- showed significant downregulation of both OmpA and OmcB (3, 5). In addition, downregulation of omp1 (ompA) was observed in persistent C. trachomatis in fallopian tubes and synovial tissues, as well as in the monocyte model of persistence (13-15). By comparison, quantitative reverse transcription-PCR analysis on the gene transcripts of ompA and omcB showed upregulation in C. pneumoniae under IFN--induced persistence (29). However, it appears that the structural appearance of the inclusion, particularly the RB morphology, is aberrant, since they are incapable of normal binary fission and redifferentiation into infectious EB. Perhaps, CrpA and OmpA are an important part of AB formation during C. pneumoniae persistence.
Upregulation of several key amino acid/nucleotide biosynthesis enzymes (TrxA, TrxB, Adk, and PyrH) was the major observation in the IFN model, suggesting a realignment of the amino acid biosynthetic machinery as the bacteria attempt to adapt to tryptophan limitation. Of these four proteins, Adk was also upregulated in the IL model of persistence, suggesting a broader role for this protein in the cellular adaptation to nutrient limitation. In contrast, Ndk, a nucleoside-2-phosphate kinase, was downregulated (Table 1), a common observation in all three treatment conditions investigated in the present study. Ndk aids in the survival of some pathogens through cellular cytotoxicity (10).
Overall, in the IL model of persistence, most of the identified proteins involved in cofactor biosynthesis, cellular processes, energy metabolism, transcription, and translation were increased after treatment with DAM. This response was not surprising, since major alterations in cellular transport and metabolism in response to low iron (particularly the upregulation of high-affinity iron transport systems) is a common feature of most bacterial pathogens during growth in iron-limited conditions (11, 31), which includes in the human host. Interestingly, three of the upregulated proteins were part of the biotin biosynthetic pathway. Biotin (vitamin H) is a necessary cofactor for various metabolic enzymes and several biotin-dependent carboxylases and decarboxylases (2, 6). By genome comparison of Escherichia coli and two Bacillus species, it is apparent that C. pneumoniae possesses all of the genes required for complete biotin synthesis. In IL conditions, all three biotin synthesis proteins—BirA, BioA, and BioD—were found to be upregulated. An increase in BioA expression under IL conditions in C. pneumoniae has also reported by Wehrl et al. (45). Although biotin plays a key role in many metabolic processes, the specific reason for the increased demand for biotin by C. pneumoniae under IL conditions (but not during tryptophan limitation) is unknown.
Further evidence of metabolic realignment under low-iron conditions was seen in the upregulation of five proteins (AhpC, GspD, LcrE, YscL, and ExbD) involved in cellular process, transport, and secretion. Although this number of proteins is small, some possible mechanisms are emerging. For example, three of these proteins (GspD, LcrE, and YscL) are part of the type III secretion system (TTSS) that is thought to be required for invasion of host cells. Although some of these TTSS proteins are downregulated in IFN-treated cells (Table 2) (41), the current results suggest that the TTSS might also be used for intracellular maintenance under iron-limiting conditions. Furthermore, ExbD is a transport protein shown to be involved in TonB-linked iron transport in other bacteria (11). Although a TonB homolog has not been identified in C. pneumoniae, it is tempting to speculate that ExbD is part of a high-affinity iron transport system that is induced under low-iron conditions.
Four of the six proteins in the energy-intermediary metabolism category (Pgk, Eno, Pyk, and SucC) were upregulated in the IL model. Two of these (Eno and SucC) were also upregulated in the IFN model. GapA, Pgk, Eno, and Pyk, belong to the glycolytic pathway, whereas Tal and SucC belong to the pentose phosphate pathway and the tricarboxylic acid pathway, respectively. In addition to the catabolism of sugars, these pathways provide the cell with intermediates for the synthesis of amino acids, nucleotides, vitamins, and cell wall constituents. Both Pyk and Tal have previously been shown to increase by C. pneumoniae and C. trachomatis in vitro, providing ATP for energy metabolism (16, 25). This observation is not surprising, since the tricarboxylic acid cycle is important for generating NADH2, FADH, and GTP, as well as precursor molecules for intermediary metabolism.
Taken together, these data indicate that rather than simply slowing into a state of hibernation, the persistent state is one of active metabolism with the bacteria adapting to the new conditions (particularly in IL conditions). The evidence for active metabolism is also seen in the upregulation of proteins involved in transcription and translation. It would seem that a similar alteration process is at work during the differentiation from RBs to ABs in the persistence models. The transcription proteins GreA, RpoA, and TctD were all upregulated in the IL model, and the latter two were upregulated in the IFN model. Seven proteins involved in translation (Map, Tsf, TufA, Efp1, PfrA, RplL, and RpsA) showed an increasing trend in the IL model, and several were also upregulated in the IFN model. These data support the finding of Belland et al. (5) that transcription of elongation factor TS is upregulated 2.5-fold in C. trachomatis after IFN-induced persistence.
Although our studies were limited to the proteins translated under persistence during the period from 46 to 48 hpi and represent only ca. 4% of the genome, this approach has shown that C. pneumoniae expresses a unique set of proteins in response to various environmental stimuli. The determination of the protein expression patterns of these two models of persistence provides an insight into the coordination of some specific proteins, such as generating a "stress response" under induced persistence conditions, as observed in the proteins required for protein folding, assembly, and modification. On the other hand, proteins required for the cell envelope, cellular processes, energy metabolism, transcription, translation, and biosynthesis showed unique responses to IFN treatment (tryptophan depletion), IL, and HS. Within host tissue chronically infected with C. pneumoniae, we might expect to find aspects of both IFN and IL persistence, as well as an HS aspect; therefore, the identification of inducer-specific proteins may be useful in the development of treatment or interventional strategies.
ACKNOWLEDGMENTS
This study was supported by NIH grants AI51255 and HL68874 (J.T.S.).
We thank Ned Smith, Jian Cai, and William Pierce for excellent technical support with the MALDI-TOF-MS.
FOOTNOTES
Present address: Biological Defense Research Directorate, Naval Medical Research Center, 12300 Washington Ave., Rockville, MD 20852.
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Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, Kentucky
Infectious Diseases Program, School of Life Sciences, Queensland University of Technology, Brisbane, Australia
ABSTRACT
Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42°C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.
INTRODUCTION
Chlamydia pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes both respiratory and systemic diseases of humans. In addition to acute infections, such as pneumonia or other respiratory diseases (18, 19), C. pneumoniae has been associated with a wide range of chronic diseases that are characterized by a local and/or systemic inflammatory response (33, 38). More importantly, there is growing evidence of an association of this pathogen with atherosclerosis, although the precise role of C. pneumoniae in this disease remains unclear. The chlamydial developmental cycle begins when the extracellular elementary body (EB) attaches and enters the host cell by endocytosis and is contained within an inclusion, which continues to enlarge, while the EB differentiates into the reticulate body (RB). The RB undergoes logarithmic division by binary fission and subsequently redifferentiates into an EB. These infectious EB are released by host cell lysis at 60 to 84 h postinfection (hpi) and initiate a new cycle of replication (47).
The ability of chlamydiae to "persist" in host tissue for extended periods of time is attributed to the activation of host-cell-mediated immune response, which plays an important role in pathogenesis of C. pneumoniae infections (20, 21). Chlamydial persistence has been described by Beatty et al. (4) as a viable, but nonculturable state, resulting in a long-term relationship with the infected host cell. In vitro, persistence is characterized by a loss of infectivity due to the development of smaller inclusions, which, ultrastructurally, contain large pleomorphic noninfectious RBs, referred to as aberrant bodies (ABs), inhibited in both binary fission and their ability to redifferentiate into infectious EB. These characteristics are generally reversible upon the removal of the persistence-inducing factor(s) (for reviews, see references 4 and 23).
Earlier studies (5, 9, 32, 35) have proposed that in order to cause chronic (persistent) infections, Chlamydia would likely have differences in gene and protein expression patterns compared to Chlamydia causing acute infections. Interest in genetic studies, as well as expression profiles of genes and proteins during chronic infections, has led to the establishment of several in vitro models of persistence that attempt to mimic in vivo conditions (4, 23). Among the in vitro models of persistence, gamma interferon (IFN-)-induced persistence is the best-studied model thus far (7, 8, 30, 36, 37, 42). Using RNA analysis, Byrne et al. (9) showed that the expression of genes related to cell division (ftsK and ftsW) was downregulated, whereas DNA replication genes (dnaA, polA, mutS, and minD) were expressed at normal levels during IFN--mediated persistence. Previous studies in our laboratory have shown differentially expressed C. pneumoniae proteins due to IFN--induced persistence (29, 32, 35). Mathews et al. (29) described the upregulation of genes involved in the cell envelope (ompA and ompB), glycolysis (pyk), and peptidoglycan synthesis (nlpD) under persistent conditions. Molestina et al. (32) described a marked upregulation of proteins such as MOMP (ompA), GroEL, and proteins involved in DNA replication (GyrA), transcription (RpoA and PnP), translation (Rrf), glycolysis (Pgk and GlgP), and type III secretion (SctN) at 24 hpi. Furthermore, Mukhopadhyay et al. (35) reported altered expression profiles of proteins involved in amino acid biosynthesis (Adk), the cellular process (AhpC), the cell envelope (CrpA), translation (Map), and a hypothetical protein CPn0710, at 24 and 48 h.
Another in vitro model of chlamydial persistence is induced by iron-limiting (IL) growth conditions. The protein expression pattern under conditions of iron limitation has been examined in both C. trachomatis (39) and C. pneumoniae (1, 45). It is well established that iron is an important component in numerous cellular metabolic processes, as well as a mediator of virulence in several bacterial pathogens (31). Interestingly, as a proposed risk factor for heart disease in humans, excess iron in the atherosclerotic lesions could play a key role in promoting the growth of C. pneumoniae (44).
A wide range of evidence supports the role of heat shock proteins (hsp's) in rescuing the bacterial cell from stress-related damage by facilitating protein refolding, preventing the aggregation of partially folded polypeptides, and ensuring proper folding (22, 24). Chlamydial hsp-70 (DnaK), hsp-60 (GroEL), and hsp-10 (GroES) have been implicated as primary antigens for eliciting host immune response (12, 28). Furthermore, hsp gene regulation (43, 46), as well as its response to heat shock treatment in the developmental cycle (26), was determined in C. trachomatis.
Given the description of the IFN and IL models of C. pneumoniae persistence, it was of interest to further characterize their similarities and differences in order to better understand their potential role in chronic diseases. As a means of distinguishing the C. pneumoniae persistence response from a classic bacterial stress response, we examined the protein expression profiles of C. pneumoniae under IFN- treatment (primarily tryptophan limitation) and iron limitation and compared those with the response due to heat shock stress. Since the potential mechanism(s), in vivo, to restrict chlamydial growth could be due to induction of tryptophan catabolism (which are primarily induced by IFN-), iron deprivation, and/or other undefined stress-inducing conditions, we analyzed and compared the protein expression profiles of nine subcategories of C. pneumoniae proteins under these two models of induced persistence, along with heat shock stress, with that from the normal growth condition, during the mid-to-late (48 hpi) stage of its developmental cycle.
MATERIALS AND METHODS
Cell line. HEp-2 cells (ATCC CCL-23) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Iscove's maintenance medium (IMM; Cellgro, Washington, D.C.) as described previously (34). Cells were grown in 75-cm2 flasks (Costar, Cambridge, MA) at 37°C in 5% CO2 for 24 h to achieve confluence of the monolayer and harvested with trypsin-EDTA. HEp-2 cells used in our experiments are free of Mycoplasma contamination as determined by PCR testing.
Bacterial isolate. C. pneumoniae A-03 (ATCC VR-1452) was previously isolated in our laboratory from an atheroma of a patient with coronary artery disease during heart transplantation at the Jewish Hospital Heart and Lung Institute, Louisville, KY (38). C. pneumoniae organisms were propagated in HEp-2 cell monolayers in Iscove's growth medium (IGM) as described previously (34). Briefly, EBs were harvested and purified by disruption of HEp-2 cell monolayers with a cell scraper, sonication, and centrifugation over a renografin density gradient (34). EB suspensions were stored in sucrose-phosphate-glutamic acid buffer at –80°C, and the viability of frozen stocks was determined by titrating EB suspensions onto HEp2 cell monolayers by standard methods.
Infection protocols. In all three infection models used, HEp-2 cells were seeded in six-well tissue culture plates at 106 cells per well in IMM and incubated in 5% CO2 at 37°C overnight. Subsequently, each model was treated as described below.
IFN model. HEp-2 monolayers were inoculated with 1.0 x 108 inclusion forming units (IFU) per well of C. pneumoniae in 2 ml of IGM with or without human recombinant gamma interferon (IFN-) at 50 and 100 U per ml (Promega, Madison, Wis.), centrifuged at 675 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 for 48 h. For proteomic analysis, infected monolayers were pulse-labeled for 2 h (from 46 to 48 hpi) in methionine-cysteine-free RPMI 1640 medium (Cellgro, Herndon, Va.) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix; Amersham Pharmacia, Piscataway, N.J.) and 500 μg of cycloheximide per ml in the presence or absence of IFN- as described previously (35).
Iron limitation (IL) model. Uninfected Hep-2 monolayers were cultured in 2 ml of IGM with or without exposure to deferoxamine mesylate (DAM) at 50 μM (Sigma, St. Louis, Mo.) for 24 h. The monolayers were subsequently inoculated the following day with 1.0 x 108 IFU per well of C. pneumoniae in 2 ml of IGM, with or without DAM at 50 μM, centrifuged at 676 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 for 48 h. For proteomic analysis, infected HEp-2 cells were pulse-labeled for 2 h (from 46 to 48 hpi) in methionine-cysteine-free RPMI 1640 medium (Cellgro) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix) and 500 μg of cycloheximide per ml in the presence or absence of DAM.
HS response. HEp-2 monolayers were inoculated with 1.0 x 108 IFU per well of C. pneumoniae in 2 ml of IGM, centrifuged at 676 x g for 1 h at 10°C, and incubated at 37°C in 5% CO2 until heat shock (HS) treatment. The schedule for HS was as follows; monolayers for 8 h HS were incubated at 42°C in 5% CO2 from hpi 40 to 48, for 6 h HS from 42nd to 48th hpi and for 4 h HS from 44th to 48th hpi. Control monolayers were incubated at 37°C in 5% CO2 for 48 h. For protein expresssion analysis, all infected HEp-2 monolayers were washed once in phosphate-buffered saline (prewarmed to 42°C), and then pulse-labeled for 2 h (from hpi 46 to 48) in methionine/cysteine-free RPMI 1640 medium (Cellgro) containing 100 μCi of [35S]methionine-cysteine per ml (Redivue Pro-Mix) and 500 μg of cycloheximide per ml in the presence or absence of HS conditions.
Infectivity studies. Parallel monolayers were set up to conduct infectivity studies for each model. After the respective treatments described above, C. pneumoniae-infected HEp-2 cells were harvested at 48 h and titrated for viable IFU/ml. Recovery of infectivity from the inducing conditions was determined by the addition of 500 μg of tryptophan (Sigma-Aldrich)/ml or 8.0 mg of holotransferin (Calbiochem)/ml to the IFN and IL models, respectively. The results of four to six experiments (in triplicate) were expressed as a normalization to the untreated controls.
TEM analysis. A second set of parallel infected HEp2 monolayers was used for transmission electron microscopy (TEM) analysis to validate the level of persistence each model by analyzing the infected monolayers as follows. Samples from each model (each sample being the entire contents of one well of a six-well plate) were fixed with 3% glutaraldehyde (Sigma-Aldrich, St. Louis, Mo.), fixative in 0.1 M phosphate buffer at pH 7.4. After fixation for 1 h at room temperature, cells were scraped off, washed in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, and embedded in Spurr epoxy resin according to standard procedures (40). Ultrathin sections (50 to 100 nm) were cut and stained with uranyl acetate and lead citrate stains prior to examination and photography with a JEOL 1200EX transmission electron microscope.
Protein expression analysis. (i) Protein extraction. At the end of the 2-h labeling period (46 to 48 h), C. pneumoniae-infected HEp-2 cells were washed in cold phosphate-buffered saline (PBS), scraped with a cell scraper, and pelleted by centrifugation at 16,000 x g. Pellets were resuspended in 30 μl of buffer containing 2% (wt/vol) Sarkosyl, 1% (wt/vol) OBG (octyl--D-1-thioglucopyranoside), and 2 mM tributyl phosphine (TBP) and 5 μl of protease inhibitors (levpeptin and aprotinin). Samples were sonicated (Cell Dismembrator, Model 100; Fisher Scientific) for 5 s, boiled for 5 min, and then cooled to room temperature. A total of 100 μl of thiourea lysis buffer (7 M urea, 2 M thiourea, 10 mM Tris, 2 mM TBP, 2% [vol/vol] ampholine) was added, followed by the addition of 3.0 μl of a mixture of 50 mM MgCl2, 476 mM Tris-HCl, 24 mM Tris base, 1.0 mg of DNase I per ml, and 0.250 mg of RNase A per ml (pH 8.0); the mixture was then incubated on ice for 10 min and stored at –80°C.
Two-dimensional gel electrophoresis. Radiolabeled protein extracts were mixed with immobilized pH gradient (IPG) rehydration buffer containing 8 M urea, 4% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 0.04 M Tris base, 0.065 M dithiothreitol, and 0.01% (wt/vol) bromophenol blue in a final volume of 400 μl as follows. (i) To generate an electrophoretic map of purified C. pneumoniae EB, a 750-μg portion of protein extracts was mixed with IPG rehydration buffer. (ii) To study intracellular C. pneumoniae protein expression, 7.6 x 105 DNA copies/μl of 35S-labeled chlamydial proteins was mixed in IPG rehydration buffer. Protein samples were loaded onto Immobiline DryStrip (IPG strips 4-7, 6-11, or 3-10) [Amersham Biosciences, Uppsala, Sweden]), allowed to rehydrate overnight, and isoelectrofocused to 98,000 Vhours in a PROTEAN IEF cell (Bio-Rad, Inc.). The following focusing parameters were applied: a rapid advance voltage ramping method was used, in which 250 V for 15 min was used for ramping in the first step, 5,000 V for 5 h was used in the second step, and finally 10,000 V for 12 h was used for the final focusing step. A hold step was programmed to maintain the voltage at 500 V till the run was stopped. The maximum current limit per gel was set at 50 μA. After focusing was completed, IPG strips were equilibrated in buffer containing 6 M urea, 2% (wt/vol) dithiothreitol, 30% (vol/vol) glycerol, and 1x Tris-acetate.
Two-dimensional electrophoresis was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) on 20-by-20-cm slab gels in PROTEAN II xi multicell system (Bio-Rad) at 4°C for 4.5 h under a 500-V maximum voltage and 20 W per gel, with 200 mM Tricine used as a cathode buffer and 0.4% (wt/vol) sodium dodecyl sulfate plus 625 mM Tris-acetate (pH 8.3) used as an anode buffer. Gels were fixed in 40% ethanol-7.5% acetic acid for 30 min. Gels containing purified EB proteins were silver stained, whereas gels containing radiolabeled chlamydial proteins were treated for 30 min with Amplify fluorographic reagent (Amersham Pharmacia, Buckinghamshire, England), vacuum dried, and exposed to high-density phosphorimaging screens (Bio-Rad) for two and a half days. Images were scanned in a Molecular Imager FX Pro Plus system (Bio-Rad). This imaging equipment is fully integrated to the ProteomeWorks system (Bio-Rad) with a high-resolution image acquisition interface within the PDQuest software.
Image analysis. Protein spots were analyzed for differential expression patterns by using PDQuest software version 7.1 (Bio-Rad). For quantification, the spots were normalized to the total quantity of the valid spots in each of the gel images for the respective models of induced persistence. After normalization, gels or a portion of the gel were analyzed as mentioned previously (35). For statistical analysis of differential expression pattern between the protein spots in the gels under study, both manual and PDQuest automated analytical tools were used to represent the spot's quantity in each individual gel and grouped as the fold (or percent) increase or decrease.
MALDI-TOF-MS. Protein spots from C. pneumoniae EBs were excised from silver-stained gels and treated with 20 μg of trypsin per ml in 50 mM ammonium bicarbonate at 37°C overnight. A 2-μl aliquot of supernatant was mixed in an equal volume of saturated -cyano-4-hydroxycinnamic acid, and 50% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid, and 0.8 μl of the resulting solution was applied to the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) template. Masses of peptide fragments were determined by MALDI-time-of-flight (TOF) analysis with a Micromass mass spectrometer. Patterns of measured masses were matched against theoretical masses of proteins found in the annotated databases Swiss-Prot and TrEMBL, accessible in the ExPASy Molecular Biology server (http://expasy.cbr.nrc.ca/). Searches were performed by Profound-peptide mapping (The Rockefeller University Edition, version 4.10.5) with restrictions to proteins from 5 to 100 kDa and mass tolerances of 100 ppm. Partial enzymatic cleavages leaving one cleavage site, oxidation of methionine, and modification of cysteine with iodoacetamide were considered in these searches. Sequence coverage of >45% (P < 0.05) was considered as a positive match.
Alternative approach to protein identification using bioinformatics. Distinctly upregulated or unaltered protein spots that could not be identified by MALDI-TOF-MS (either resulting from insufficient amount of protein in the spot and/or a clearly resolved spot was not obtained in the silver-stained EB map) were identified by bioinformatics approach using the TagIdent tool (http://ca.expasy.org/tools/tagident.html). The molecular weight (Mw) and isoelectric point (pI) of such spots were estimated from the analytical gels and applied to TagIdent with very stringent parameters and specified error margins (pI range = 0.25 and Mw range = 10%) in order to match the proteins from the database (Swiss-Prot/TrEMBL/NCBI) near to the defined range of Mw and pI with a P value of <0.05.
Statistical analysis. The statistical significance between experimental groups was determined by using the Student t test. P values of <0.05 were considered statistically significant.
RESULTS
Validation of models. In order to validate the models used in the present study, infectivity studies were conducted with C. pneumoniae-infected Hep-2 cells. Figure 1A shows the reduction in viable C. pneumoniae IFU by approximately 3 and 5 logs due to treatment with IFN- at 50 U/ml (P < 0.05) and 100 U/ml (P < 0.05), respectively. Replenishing the tryptophan (500 μg/ml) led to the almost complete recovery of infectivity, indicating that the decrease in infectivity was due primarily to the limitation of intracellular tryptophan. Similarly, Fig. 1B shows a reduction in C. pneumoniae infectivity of approximately 2 logs in the presence of DAM (50 μM) (P < 0.05). The addition of iron-saturated holotransferin (8.0 mg/ml) to the monolayers led to a return to equivalent IFU/ml as seen in the untreated monolayers, thus supporting the limitation of host cell iron as the cause of reduction in viable IFU. In comparison to these models of persistence, heat shock at 42°C for 4, 6 (P < 0.05), and 8 h (P < 0.05) showed a sequential loss of viable IFU/ml (Fig. 1C). Prolonged heat shock treatment for up to 12 or 24 h resulted in the complete loss of infectivity (data not shown).
FIG. 1. Infectivity studies showing loss of viable C. pneumoniae due to treatment with IFN- (A), DAM (B), and HS at 42°C (C). , P < 0.05 (compared to the untreated control).
For additional validation of all experimental conditions used in the present study (i.e., untreated, IFN- treated, IL treated, and HS treated) and also to assess the morphological features of each, C. pneumoniae-infected cultures were examined by TEM. Ultrastructural analysis of untreated C. pneumoniae-infected HEp-2 cells revealed typical inclusion and bacterial morphologies for cultures grown for 48 h (Fig. 2A). On average, inclusions were approximately 5 μm in diameter (range, 2.5 to 7 μm in diameter), with an average of 50 to 70 chlamydial particles (80% RBs, 15% IBs, and 5% EBs) per inclusion. Inclusions identified in IFN-treated (50 U/ml), C. pneumoniae-infected HEp-2 cells (Fig. 2B) appeared much smaller in diameter (ranging from 2 to 4 μm) than those observed in untreated samples. Fewer chlamydial particles were observed within these smaller inclusions. Commonly, three to five chlamydial particles filled the entire space of the smaller inclusion, since these chlamydial particles often were larger and more irregular in morphology (defined as aberrant bodies [ABs]) than those seen in untreated samples. Similarly, C. pneumoniae-infected HEp-2 cells treated with the iron chelator DAM (50 U/ml) exhibit inclusions much smaller than those in the untreated cultures, where the most common inclusion size observed was 3 μm in diameter (Fig. 2C). Also, the chlamydial particles were few (average of three chlamydial particles) within these inclusions and maintained the aberrant morphology (AB), as seen in the IFN-treated cultures. When exposed to 42°C for 4 h (HS model), C. pneumoniae inclusions were, on average, 4 μm in diameter and contained varied morphologies of C. pneumoniae (Fig. 2D), some of which resembled the ABs seen in the IFN and IL models. After prolonged exposure to heat (6 and 8 h), the cytoplasm of the bacteria became more electron lucent, and there appeared to be some disruption of the bacterial cell membrane, indicating the bacteria were undergoing cell death (Fig. 2E and F). Clearly, the C. pneumoniae morphologies seen in the HS model, especially at the 6- and 8-h time points, were distinct from the classical ABs observed in the IFN and IL models.
FIG. 2. TEM images showing morphological analysis of C. pneumoniae under normal growth conditions at 48 hpi (A), after IFN- treatment (50 U/ml) (B), after DAM treatment (50 μM) (C), and after HS at 42°C for 4 h (D), 6 h (E), and 8 h (F). The arrows in panels D, E, and F denote altered morphological forms of C. pneumoniae. EB, elementary body; IB, intermediate body; RB, reticulate body; AB, aberrant body.
Differential protein expression analysis. Intracellular expression of C. pneumoniae proteins at 48 hpi under untreated, as well as inducer-specific conditions under IFN, IL, and HS conditions, were determined by pulse-labeling each C. pneumoniae-infected HEp-2 cell monolayer. Of the 168 differentially expressed protein targets identified by the image analysis software, 52 C. pneumoniae proteins, constituting nine functional categories, were identified by either MALDI-TOF (41 proteins) or bioinformatics (11 proteins). Only these 11, of the 127 spots analyzed by bioinformatics, could be matched to C. pneumoniae proteins, using the strict exclusion criteria (see Materials and Methods). For quantitative analysis of differential regulation, the expression profiles of each protein in each of the three models examined was compared to their respective untreated (control) condition (Fig. 3).
FIG.3. Representative two-dimensional electrophoretic maps of C. pneumoniae proteins expressed at 48 hpi in untreated HEp2 cells (A), after IFN- treatment at 50 U/ml (i) or 100 U/ml (ii) (B), after treatment with 50 μM DAM (C), and after treatment at 42°C for 4 h (i), 6 h (ii), and 8 h (iii) (D). Representative gels out of six experiments are shown.
Common expression patterns under IFN and IL induced persistence and HS stress response. Proteins which showed common trends in expression pattern in all three treatment conditions are listed in Table 1. These include one amino acid/nucleotide biosynthesis protein (Ndk), and the majority of the stress response proteins, which belong to the functional categories of protein folding, assembly, and modification and seven hypothetical proteins (Table 1). The stress response proteins (DnaK, HtrA, ClpP_1, and ClpP_2) were found to be 1.5-fold upregulated, depending on the condition, in all three treatment conditions (Fig. 3B to D). There were a few exceptions to the expression trends in the stress response category. Although the expression of GroES and DksA showed a general inclining trend in the IFN model, GroES and DksA showed unaltered expression profiles in the IL model (Table 1 and Fig. 3C). Endopeptidases ClpC and ClpX, as well as GrpE, were found to be upregulated under IFN and IL conditions; however, they were found to be downregulated under HS treatment. Similarly, ClpB was upregulated under IL and HS treatments but remained unchanged under IFN. Yet another common feature of the HS response and both persistence models was the downregulation of Gcp_1 and Ndk (Table 1). Extending the HS period for the C. pneumoniae-infected monolayers to 8 h resulted in the downregulation of 8 of the 12 stress response proteins (Table 1 and Fig. 3D), perhaps suggesting the beginning of a cell death phase. Finally, most of the hypothetical proteins identified and analyzed—CPn0011, CPn0710, CPn0518, CPn0742, CPn1032, CPn0216, and CPn0917—showed decreased or unchanged protein levels under all experimental conditions.
Unique protein expression patterns. Considerable variation in profiles was exhibited by the proteins involved in biosynthesis, cell envelope, cellular processes, energy metabolism, transcription, and translation when subjected to IFN, IL, and HS treatments (Table 2). For the HS response, most of the proteins involved in the biosynthesis of amino acids and cofactors, cell envelope, cellular processes, energy metabolism, transcription, and translation were prominently downregulated, especially when HS was applied for up to 6 and 8 h (Table 2 and Fig. 3D). All four cell envelope proteins (CrpA, OmpA, OmcB, and PorB) showed a marked decrease in expression pattern compared to the IFN and IL persistent models (Table 2). Furthermore, the expression profiles of energy metabolism proteins involved in the pentose phosphate pathway (Tal), the glycolysis pathway (GapA, Pgk, Eno, and Pyk), and the tricarboxylic acid cycle (SucC) were mostly reduced in the HS model (Table 2). Only five proteins, those involved in cellular and transport-related processes (AhpC, GspD, LcrE, ExbD, and GreA), showed an increase in expression in response to 4-h HS. Apart from ExbD, two GspD and GreA levels were increased under HS at 4 and 6 h, and RplL levels were increased after 8 h of HS. ExbD was the only protein found to be upregulated throughout the HS treatment.
The most notable response of C. pneumoniae to IFN was the upregulation of the amino acid biosynthesis proteins (TrxA, TrxB, Adk, and PyrH) (Fig. 3B). These proteins were downregulated in the other two models, except for Adk, which was found to be upregulated under IL as well and remained unchanged in response to HS for 4 h (Table 2). Proteins involved in cellular and transport related processes (AhpC, GspD, LcrE, YscL, and ExbD) were either unchanged or downregulated under IFN conditions. However, they showed an inclining trend under IL and HS (4-h) treatments.
In the IL model, most proteins examined showed an inclining trend. Specifically, proteins involved in the biosynthesis of cofactors (BirA, BioA, and BioD) were markedly upregulated only under IL conditions compared to the untreated controls. The other protein in this category, RibC, which remained unchanged under IL treatment, also showed a similar expression pattern under IFN treatment. The three proteins involved in transcription (GreA, RpoA, and TctD) also showed consistent upregulation in expression profile under IL condition compared to the varied pattern observed for the other two conditions (Table 2 and Fig. 3C). Furthermore, of the six proteins attributed to translation, three proteins involved in peptide chain elongation (Tsf, TufA, and Efp1) and other proteins including Map, PfrA, and ribosomal proteins RplL and RpsA showed a general increasing trend under IL, whereas the reverse trend was observed in the expression pattern in the IFN and HS response (Table 2).
DISCUSSION
To our knowledge, this is the first study to examine the HS stress response in C. pneumoniae. Of the 12 classical HS stress response proteins analyzed, nine (GroEL_1, DnaK, DksA, GrpE, HtrA, ClpP_1, ClpP_2, ClpB, and AhpC) exhibited elevated expression levels after HS for 4 h, and several of these remained upregulated after 6 h of HS. This indicates that C. pneumoniae, like C. trachomatis (26), exhibits a classic bacterial HS stress response. Of interest was the fact that the expression of GroEL_2 or GroEL_3 was not detected, in keeping with the reported lack of transcription of these genes in C. trachomatis after HS (27). It should be noted that the lack of detection of these, or perhaps other proteins, may be due to their presence below the lower limits of the assay or to differences in turnover rates for a given protein.
Our qualitative protein profiling analysis of C. pneumoniae under two models of persistence, compared to HS, has enabled us to suggest several important observations of chlamydial persistence. First, there is a significant translational level stress response seen in both IFN and IL persistence. Of the 12 stress response proteins analyzed, 8 (groEL, groES, dnaK, dksA, htrA, gcp1, clp1, and clp2) showed the same trends in both persistence models that they showed under HS. This suggests that a stress response is one part of the persistence response. Our data support the findings of Gerard et al. (17), who reported the increased expression of C. trachomatis groEL genes under persistent conditions in both in vitro and in synovial tissue.
Several other functional groupings also showed quite different trends between HS and persistence. In some cases this persistence response was common to both models, while in other cases it was different between the two models of persistence. In both the IFN and the IL models, the cell envelope proteins CrpA and OmpA were significantly increased in expression compared to untreated controls, whereas OmcB was downregulated. OmpA functions as an integral membrane protein, providing structural rigidity to the EB outer membrane and permitting diffusion of solutes through the intracellular RB membrane, while the cysteine-rich 60-kDa OmcB functions as an outer membrane cross-linking protein vital for EB formation. The data in the literature has been discordant with respect to these cell envelope proteins. DNA microarray analysis, immunofluorescence, and immunoblotting studies with C. trachomatis persistence induced with IFN- showed significant downregulation of both OmpA and OmcB (3, 5). In addition, downregulation of omp1 (ompA) was observed in persistent C. trachomatis in fallopian tubes and synovial tissues, as well as in the monocyte model of persistence (13-15). By comparison, quantitative reverse transcription-PCR analysis on the gene transcripts of ompA and omcB showed upregulation in C. pneumoniae under IFN--induced persistence (29). However, it appears that the structural appearance of the inclusion, particularly the RB morphology, is aberrant, since they are incapable of normal binary fission and redifferentiation into infectious EB. Perhaps, CrpA and OmpA are an important part of AB formation during C. pneumoniae persistence.
Upregulation of several key amino acid/nucleotide biosynthesis enzymes (TrxA, TrxB, Adk, and PyrH) was the major observation in the IFN model, suggesting a realignment of the amino acid biosynthetic machinery as the bacteria attempt to adapt to tryptophan limitation. Of these four proteins, Adk was also upregulated in the IL model of persistence, suggesting a broader role for this protein in the cellular adaptation to nutrient limitation. In contrast, Ndk, a nucleoside-2-phosphate kinase, was downregulated (Table 1), a common observation in all three treatment conditions investigated in the present study. Ndk aids in the survival of some pathogens through cellular cytotoxicity (10).
Overall, in the IL model of persistence, most of the identified proteins involved in cofactor biosynthesis, cellular processes, energy metabolism, transcription, and translation were increased after treatment with DAM. This response was not surprising, since major alterations in cellular transport and metabolism in response to low iron (particularly the upregulation of high-affinity iron transport systems) is a common feature of most bacterial pathogens during growth in iron-limited conditions (11, 31), which includes in the human host. Interestingly, three of the upregulated proteins were part of the biotin biosynthetic pathway. Biotin (vitamin H) is a necessary cofactor for various metabolic enzymes and several biotin-dependent carboxylases and decarboxylases (2, 6). By genome comparison of Escherichia coli and two Bacillus species, it is apparent that C. pneumoniae possesses all of the genes required for complete biotin synthesis. In IL conditions, all three biotin synthesis proteins—BirA, BioA, and BioD—were found to be upregulated. An increase in BioA expression under IL conditions in C. pneumoniae has also reported by Wehrl et al. (45). Although biotin plays a key role in many metabolic processes, the specific reason for the increased demand for biotin by C. pneumoniae under IL conditions (but not during tryptophan limitation) is unknown.
Further evidence of metabolic realignment under low-iron conditions was seen in the upregulation of five proteins (AhpC, GspD, LcrE, YscL, and ExbD) involved in cellular process, transport, and secretion. Although this number of proteins is small, some possible mechanisms are emerging. For example, three of these proteins (GspD, LcrE, and YscL) are part of the type III secretion system (TTSS) that is thought to be required for invasion of host cells. Although some of these TTSS proteins are downregulated in IFN-treated cells (Table 2) (41), the current results suggest that the TTSS might also be used for intracellular maintenance under iron-limiting conditions. Furthermore, ExbD is a transport protein shown to be involved in TonB-linked iron transport in other bacteria (11). Although a TonB homolog has not been identified in C. pneumoniae, it is tempting to speculate that ExbD is part of a high-affinity iron transport system that is induced under low-iron conditions.
Four of the six proteins in the energy-intermediary metabolism category (Pgk, Eno, Pyk, and SucC) were upregulated in the IL model. Two of these (Eno and SucC) were also upregulated in the IFN model. GapA, Pgk, Eno, and Pyk, belong to the glycolytic pathway, whereas Tal and SucC belong to the pentose phosphate pathway and the tricarboxylic acid pathway, respectively. In addition to the catabolism of sugars, these pathways provide the cell with intermediates for the synthesis of amino acids, nucleotides, vitamins, and cell wall constituents. Both Pyk and Tal have previously been shown to increase by C. pneumoniae and C. trachomatis in vitro, providing ATP for energy metabolism (16, 25). This observation is not surprising, since the tricarboxylic acid cycle is important for generating NADH2, FADH, and GTP, as well as precursor molecules for intermediary metabolism.
Taken together, these data indicate that rather than simply slowing into a state of hibernation, the persistent state is one of active metabolism with the bacteria adapting to the new conditions (particularly in IL conditions). The evidence for active metabolism is also seen in the upregulation of proteins involved in transcription and translation. It would seem that a similar alteration process is at work during the differentiation from RBs to ABs in the persistence models. The transcription proteins GreA, RpoA, and TctD were all upregulated in the IL model, and the latter two were upregulated in the IFN model. Seven proteins involved in translation (Map, Tsf, TufA, Efp1, PfrA, RplL, and RpsA) showed an increasing trend in the IL model, and several were also upregulated in the IFN model. These data support the finding of Belland et al. (5) that transcription of elongation factor TS is upregulated 2.5-fold in C. trachomatis after IFN-induced persistence.
Although our studies were limited to the proteins translated under persistence during the period from 46 to 48 hpi and represent only ca. 4% of the genome, this approach has shown that C. pneumoniae expresses a unique set of proteins in response to various environmental stimuli. The determination of the protein expression patterns of these two models of persistence provides an insight into the coordination of some specific proteins, such as generating a "stress response" under induced persistence conditions, as observed in the proteins required for protein folding, assembly, and modification. On the other hand, proteins required for the cell envelope, cellular processes, energy metabolism, transcription, translation, and biosynthesis showed unique responses to IFN treatment (tryptophan depletion), IL, and HS. Within host tissue chronically infected with C. pneumoniae, we might expect to find aspects of both IFN and IL persistence, as well as an HS aspect; therefore, the identification of inducer-specific proteins may be useful in the development of treatment or interventional strategies.
ACKNOWLEDGMENTS
This study was supported by NIH grants AI51255 and HL68874 (J.T.S.).
We thank Ned Smith, Jian Cai, and William Pierce for excellent technical support with the MALDI-TOF-MS.
FOOTNOTES
Present address: Biological Defense Research Directorate, Naval Medical Research Center, 12300 Washington Ave., Rockville, MD 20852.
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