Analysis of Altered Protein Expression Patterns of Chlamydia p

Mar 18, 2004 - Chlamydia pneumoniae by an Integrated Proteome-Works System ... C. pneumoniae has a unique biphasic replicative cycle involving two ...
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Analysis of Altered Protein Expression Patterns of Chlamydia pneumoniae by an Integrated Proteome-Works System Sanghamitra Mukhopadhyay,† Richard D. Miller,‡ and James T. Summersgill*,†,‡ Division of Infectious Diseases, Department of Medicine, and Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky 40292 Received March 18, 2004

Abstract: We have identified, analyzed, and quantified differential protein expression profile of five C. pneumoniae proteins, Adk (adenylate kinase), AhpC (thiol-specific antioxidant), CrpA (15 KD cysteine rich protein), Map (methionine aminopeptidae), and Cpn0710 (hypothetical protein) under normal versus persistent growth conditions induced by interferon-γ, at different time intervals of their replicative cycle by successfully employing the latest proteomic analysis tool, PDQuest 2-D analysis software. We have also determined that this software represents a reliable analytical tool for mapping protein expression patterns in C. pneumoniae. Keywords: proteomics • protein expression • PDQuest software • Chlamydia pneumoniae • 2-D gel analysis

Introduction Achievements in the genomic era have significantly reshaped the understanding of host-pathogen interactions and therapeutics. Now, the challenge is to establish the functional interpretation of the proteins expressed by a genome, referred to as proteomics. We studied the protein expression patterns of the bacterium Chlamydia pneumoniae. This obligate intracellular pathogen causes both acute and chronic respiratory diseases and has been associated with cardiovascular-related clinical manifestations, like atherosclerosis.1,2 C. pneumoniae has been detected in atherosclerotic plaques and has been isolated in culture from atheromas,1,3,4 providing evidence for an association between C. pneumoniae and atherogenesis, although the mechanisms by which C. pneumoniae contributes to atherogenesis is still unknown. Several in vitro studies suggest that C. pneumoniae has the potential to “persist” in infected vascular cells and promote atherogenic responses.2,5-7 Vascular cells are susceptible to persistent infection and C. pneumoniae produces productive infection in human macrophages, endothelial cells, and arterial smooth muscle cells,8-10 which are the key cellular components of atherosclerosis. However, several questions need to be answered regarding the association of C. pneumonaie and atherosclerosis. * To whom correspondence should be addressed. Dr. James T. Summersgill, Infectious Diseases Laboratory, Instructional Building, Room 311, 500 South Preston Street, University of Louisville, Louisville, KY 40292. E-mail: [email protected]. Phone: (502) 852-5132. Fax: (502) 852-1512. † Division of Infectious Diseases. ‡ Department of Medicine, and Department of Microbiology and Immunology.

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Journal of Proteome Research 2004, 3, 878-883

Published on Web 05/25/2004

Figure 1. Transmission electron micrograph of a typical C. pneumoniae inclusion containing EBs and RBs. (Magnification: ×11 900).

C. pneumoniae has a unique biphasic replicative cycle involving two developmental forms: a spore-like infectious form known as the elementary body (EB) and an intracellular replicative form called the reticulate body (RB). The chlamydial replicative cycle requires the intracellular differentiation of EBs to RBs (within approximately 2 h following internalization), logarithmic division of RBs (between 8 and 12 h post infection [hpi]), followed by reorganization of RBs back to EBs (after about 18-22 hpi), which are released upon subsequent host cell lysis. Depending upon the species or strain, lysis or release from the infected host cell occur approximately 36 to 72 hpi. Figure 1 shows transmission electron micrograph (TEM) of a typical C. pneumoniae inclusion containing EBs and RBs. Most of the essential tasks such as adhesion, host cell colonization, and the ability to cope with host defense mechanisms when outside the host cell rely on the EBs. In addition to this lytic cycle, a nonlytic (persistent) phase may develop if induced with IFN (interferon)-γ, certain antibiotics or nutrient deprivation.11-13 Persistence may lead to frequent re-infection or develop into 10.1021/pr0400031 CCC: $27.50

 2004 American Chemical Society

technical notes

Figure 2. Transmission electron micrograph of an atypical C. pneumoniae (IFN-γ-treated) inclusion containing large reticulatelike ABs (magnification: ×16 000).

long-term sequelae of chlamydial disease.13 Stimulation of host cells with IFN-γ inhibits growth of C. pneumoniae, primarily by induction of indoleamine 2,3-dioxygenase activity, which deprives the organisms of tryptophan. However, a subinhibitory concentration of IFN-γ can lead to an altered (persistent) replicative state of C. pneumoniae, in vitro, which is clearly manifested after about 36 hpi. Figure 2 depicts TEM of IFN-γ treated C. pneumoniae, showing small atypical inclusions containing large reticulate-like aberrant bodies (ABs) at 48 hpi. Studies indicate that under IFN-γ-induced persistence, C. pneumoniae expresses an altered gene transcription profile.14-16 The availability of proteomic analysis tools has augmented the investigation of potential functions of expressed proteins at different growth and infectious stages of C. pneumoniae. In this article, we have elucidated the plausible roles of the five identified proteins with relation to persistent infections caused by C. pneumoniae by using some of the salient features of the latest proteomic analysis tool, PDQuest 2-D analysis software.

Experimental Procedures Cell Line. HEp2 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.] supplemented with 10% (v/v) fetal bovine serum, 2mM L-glutamine, 1% (v/v) nonessential amino acids, 10 mM HEPES buffer, 10 µg/mL gentamycin, 25 µg/mL of vancomycin. Cells were grown in 75-cm2 flasks (Costar, Cambridge, MA) at 37 °C in 5% CO2 for 24 h to achieve confluency of the monolayer, and harvested with Trypsin-EDTA. Bacterial Isolate. C. pnuemoniae 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.1 C. pnuemoniae A-03 were propagated in HEp2 cell monolayer in Iscove’s Growth Medium (IGM) [IMM with 8.8% (v/v) glucose and 1.0 µg/mL cyclohexamide] at a multiplicity of infection of 100:1 and centrifuging the flask at 3500 × g (Sorvall RT 6000D) for 30 min at 10 °C and incubating at 37 °C in 5% CO2.

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Elementary bodies (EBs) were harvested and purified by disruption of HEp2 cell monolayers with a cell scraper, sonication, and centrifugation over a renograffin density gradient.17 EB suspensions were stored in sucrose-phosphate-glutamic acid buffer at -80 °C. Infection Protocol. HEp-2 cells were seeded in six-well plates at 0.5 × 106 cells per well in IMM and incubated in 5% CO2, at 37 °C overnight. Cells were subsequently inoculated with 0.5 × 108 IFU per well (MOI 100:1) of C. pneumoniae A-03 in 2 mL of Iscove’s growth media with or without human recombinant IFN-γ 50 U and 100 U per mL (Promega, Madison, Wisconsin), centrifuged at 3500 × g for 1 h at 10 °C, and incubated at 37 °C in 5% CO2 for 24 and 48 h. To detect chlamydial protein synthesis, infected HEp-2 cells were pulse-labeled for 2 h 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, NJ) and 500 µg of cycloheximide per mL in the presence or absence of IFN-γ. At the end of the 2-h labeling period, HEp-2 cells were washed in cold phosphate buffered saline, scraped with a cell scrapper, and pelleted by centrifugation at 16 000 × g. Protein Extraction. To extract proteins from C. pneumoniaeinfected HEp-2 cells, pellets were re-suspended in 30 µL of buffer containing 2% (w/v) sarkosyl, 1% (w/v) OBG (Octyl β-D1-thioglucopyranoside), and 2mM tri-butyl phosphine (TBP) and 5 µL protease inhibitors (levpeptin and aprotinin). Samples were sonicated (Cell dismembrator, Model 100 [Fisher Scientific]) for 5 s, boiled for 5 min, then cooled to room temperature. A 100-µL portion of thiourea lysis buffer (7M urea, 2M thiourea, 10 mM Tris and 2mM TBP and 2% (v/v) 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-1 per mL, and 0.250 mg of RNase A per mL (pH 8.0) on ice for 10 min and stored at -80 °C. 2D-Gel Electrophoresis. Radio-labeled protein extracts were mixed with IPG (Immobilized pH gradient) rehydration buffer containing 8 M urea, 4% (w/v) CHAPS, 0.04 M Tris Base, 0.065 M DTT (Dithiothreitol), 0.01% (w/v) bromophenol blue in a final volume of 400 µL as follows. (i) To generate an electrophoretic map of purified C. pneumoniae EBs, 750 µg of protein extracts was mixed with IPG rehydration buffer. (ii) To study intracellular C. pneumoniae protein expression, 75 µL of [35S]labeled chlamydial proteins was mixed in IPG rehydration buffer. Diluted samples were loaded onto IPG strips [pI 4-7, 6-11 or 3-10] (Bio-Rad ReadyStrip IPG strip, strip length 17 cms), allowed to swell overnight and isoelectrofocused to 95 000 Vhours in PROTEAN IEF cell (Bio-Rad, Inc.). The following focusing parameters were applied: 5000 V of maximum voltage, 500 V of holding voltage, 50 µA of maximum current per gel, 99 000 V‚h, and for a duration of 18 h. After focusing was completed, IPG strips were equilibrated in buffer containing 6 M urea, 2% (w/v) DTT, 30% (v/v) glycerol, and 1× Tris-acetate. 2D electrophoresis was carried out with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide) on 20 × 20 cm slab gels in PROTEAN II xi multicell system (Bio-Rad, Inc.) at 4 °C for 4.5 h under a 500-V maximum voltage and 20,000 mW per gel, with 200 mM Tricine used as a cathode buffer, and 0.4% (w/v) SDS 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 stained with silver, whereas gels containing radiolabeled chlamydial proteins were treated for 30 min with Amplify Journal of Proteome Research • Vol. 3, No. 4, 2004 879

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technical notes

fluorographic reagent (Amersham Pharmacia, Buckinghamshire, England), vacuum-dried, and exposed to high-density phosphor-imaging screens (Bio-Rad, Inc.) for 2.5 days. Images were scanned in Molecular Imager FX Pro Plus system (BioRad, Inc.). This imaging equipment is fully integrated to the ProteomeWorks system with a high-resolution image acquisition interface within the PDQuest software. Image Analysis. Protein spots were analyzed and quantified for differential expression patterns between gels treated with and without IFN-γ using PDQuest software version 7.1. This software enables automated analysis of the differences in protein expression profiles in complex samples. After selecting the gels or portion of the gels to be analyzed, spots of interest are detected and a Matchset is created. In a Matchset, protein spots from different gels are matched to each other and are represented in a synthetic image called a Master Image. The Master Image includes all information about all the spots in all the gels in the Matchset. The spot review tool displays histograms of all spots in a Matchset. Bars in each histogram represent the spot’s quantity in each individual gel of the Matchset. For statistical analysis of differential expression pattern, analytical tools are available in PDQuest software which allows spots to be grouped as fold increase or decrease. Identification of Protein Spots. 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. Two microliters of supernatant was mixed in an equal volume of saturated R-cyano-4-hydroxycinnamic acid, 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (TFA), and 0.8 µL of the resulting solution was applied to the 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 with the Profound-peptide mapping (The Rockefeller University Edition version 4.10.5) with restrictions to proteins from 1 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.

Results and Discussion Upon treatment with IFN-γ, a significant differential expression pattern of the Chlamydial proteins were observed. Figure 3 shows IFN-γ-mediated alterations in five identified proteins, Adk (Figure 3A), AhpC (Figure 3B), CrpA (Figure 3C), Cpn0710 (Figure 3D) and Map (Figure 3E). Our results show (Table 1, Figure 3A) that Adk is significantly down-regulated (absent) at early time points (24 hpi), but shows remarkable up-regulation (7-fold) later in the replicative cycle at 48 hpi in HEp2 cells at both the concentrations of IFNγ. Adenylate kinase is essential for maintenance and cell growth. It catalyzes the reversible high-energy transfer of terminal phosphate from ATP/GTP to AMP to form ADP.18 This reaction plays a central role in balancing the pool of mono-, di-, and tri-phosphate forms of adenine nucleotides and thus contributes to the homoeostasis of cellular adenine nucleotide composition.19 Recently, Munier-Lehmann et al.,20 observed that Adk-driven maintenance of adenine nucleotide homoeostasis previously known to be essential for bacterial growth, in vitro, is also crucial for growth and for virulence of Yersinia 880

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Figure 3. Statistical analysis of the differential expression pattern of C. pneumoniae proteins at different time intervals under IFNγ-induced persistence. Red bars in panel A and D indicate the protein spots which are absent (down-regulated).

technical notes

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Table 1. Differential Expression Pattern of C. pneumoniae Proteins at Different Time Intervals under IFN-γ-Induced Persistence 24 h C. pneumoniae proteina

Adk 7.2/23.5 AhpC 4.9/23.5 CrpA 5.1/18.0 CPn0710 4.5/9.5 map 4.9.33.5

a

function

adenylate kinase: essential for maintenance and cell growth alkyl hydroperoxide reductase 15 kD cysteine rich protein: cell envelope/ membrane protein hypothetical protein methionine aminopeptidase: removes the amino-terminal methionine from nascent proteins

48 h

IFN-γ (50 U/mL)

IFN-γ (100 U/mL)

IFN-γ (50 U/mL)

IFN-γ (100 U/mL)

absent (down) b

absent (down)b

7 (up)

7 (up)

2 (up) 7 (down)

6 (up) 3 (down)

unchanged unchanged

5 (down) 2 (up)

unchanged 6 (down)

unchanged 8 (down)

6 (up) unchanged

absent (down)c 3 (up)

Observed pI/Mw of the protein. b See Figure 3A. c See Figure 3E.

pestis, in vivo. Furthermore, it has also been shown that Pseudomonas aeroginosa is capable of secreting Adk, previously known as cytosolic enzyme, and that it played a role in macrophage cell death.21 Thus, the possibility that, in addition to adenine nucleotide homoeostasis, Adk exerts certain pathogenic effects inside a human host cannot be excluded. By comparing the role of Adk in other virulent bacteria 20,21 it is evident that the up-regulation of Adk in the persistent state may play some role in chronic pathogenesis of C. pneumoniae. Peroxiredoxins are ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels which mediate signal transduction in mammalian cells. This family contains proteins related to Alkyl hydroperoxide reductase (AhpC) and thiol specific antioxidant (TSA). AhpC is responsible for directly reducing organic hyperoxide in its reduced dithiol form. We have observed that earlier in the replication cycle at 24 hpi, AhpC is 2-fold and 6-fold up-regulated with 50 and 100 U/mL INF-γ, respectively, but is unchanged and 5-fold downregulated with 50 and 100 U/mL INF-γ at the later time point of 48 hpi (Table 1, Figure 3B). In recent studies of a human pathogen, Burkholderia pseudomallei,22 Loprasert et. al.,23 showed compensatory increase in ahpC gene expression in a B. pseudomallei catalase (katG) mutant and the role AhpC plays in protecting B. pseudomallei against reactive nitrogen intermediates (RNI). Similar observations have been reported in the studies with Mycobacterium tuberculosis.24 In cultured epithelial cells, cytokine-induced nitric oxide synthase (iNOS) activity leads to a reduced infectivity of both C. pneumoniae and C. trachomatis.25 In vivo studies also support the role of iNOS as a mediator of defense against Chlamydial26,27 and Salmonella typhimurium28 infections. Up-regulation of AhpC would protect the bacteria against killing by iNOS-mediated RNI, and thereby rendering ability to the organism to cause long-term latent infection. In inflammatory environments, nitric oxide can react with superoxide to form peroxynitrite, a highly reactive oxidant which, in turn, leads to the oxidation of LDL and the foam cell formation.29 The protective effect of AhpC against RNI may be due to an intrinsic peroxynitritase activity. While AhpC may play an important role in C. pneumoniae persistent infections, an explanation for our observed differences in AhpC expression in persistent cultures at 24 h, compared to 48 h, will require additional investigation. The function of 15 kD cysteine rich protein, CrpA still remains unclear, though it has been shown to be associated with the Chlamydial inclusion membrane. At the molecular level, the inclusion membrane defines the critical interface between Chlamydia and the host cell.30 Several recent reports have indicated CrpA as an antigenic determinant.31,32 Our

Table 2. Differential Expression Pattern of C. Pneumoniae Proteins (unidentified) at Different Time Intervals under IFN-γ-Induced Persistence 24 h

48 h

C. pneumoniae protein spot numbers

IFN-γ (50 U/mL)

IFN-γ (100 U/mL)

IFN-γ (50 U/mL)

IFN-γ (100 U/mL)

2201 3201 3203 4201 9201 9202

2 (up) 3 (down) 4 (up) 5 (down) 5 (down) 6 (down)

4 (up) 3 (up) 1.5 (up) 4 (down) 1.5 (down) 5 (down)

3 (down) 1.5 (up) 2 (down) 3 (up) unchanged 8 (down)

5 (down) 3 (down) 8 (down) 2 (down) 5 (up) 8 (down)

studies show significant down-regulation of CrpA (Table 1, Figure 3C) after 24 hpi, but shows up-regulation after 48 hpi as delayed expression under induced persistence. Methionine aminopeptidase (MAP) catalyzes removal of the initiator methionine from nascent polypeptides Bacterial protein synthesis starts with a formylated methionine residue, and this residue is sequentially cleaved away by a unique peptide deformylase (PDF) and a methionine aminopeptidase to generate mature proteins. The formylation-deformylation of proteins is a unique hallmark of bacterial metabolism. We have observed a 6-8-fold down-regulation of Map after 24 hpi, but about a 3-fold up-regulation after 48 hpi at persistent conditions, compared to the normal growth of C. pneumoniae (Table 1, Figure 3E). The expression pattern of MAP is similar to CrpA under persistent conditions. This provides evidence to the fact that C. pneumoniae have ability to persist in the host cell with a basal level of protein synthesis, even though they remain in a culture negative stage. We have also identified a hypothetical protein (CPn0710) which does not show any alteration at 24 hpi, but interestingly, after 48 hpi, shows 6-fold up-regulation at IFN-γ 50 U/mL, followed by a remarkable down-regulation at IFN-γ 100 U/mL (Table 1, Figure 3D). The characterization of differential protein expression patterns of C. pneumoniae is ongoing. To demonstrate the effectiveness of the PDQuest software in this process, we have shown the quantitation of six additional representative unidentified protein spot clusters (Table 2, Figure 4A-C) which represents differential expression profiles of the spots at 24 and 48 hpi under IFN-γ-induced persistence. The standard spot (SSP) numbers for un-identified spots are generated by the software as 2201, 3201, 3203 (Figure 4A), 4201 (Figure 4B), and 9201, 9202 (Figure 4C). Future identification of these spots will aid in a more complete understanding of the pathogenesis of C. pneumoniae. Journal of Proteome Research • Vol. 3, No. 4, 2004 881

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Protein Expression Patterns of Chlamydia pneumoniae

Figure 4. Statistical analysis of the differential expression pattern of C. pneumoniae proteins (un-identified) at different time intervals under IFN-γ-induced persistence.

The significant steps in proteomic analysis process are, first, the separation of complex protein mixtures, followed by characterization and identification of the separated peptides. To fulfill these demanding requirements we have used the ProteomeWorks System from Bio-Rad Laboratories, Inc. (Hercules, CA) (www.bio-rad.com). This system integrates the tools and technology to meet the entire spectrum, from sample preparation to protein separation and mapping to protein identification by linking to the international protein databases. Rapid analysis of complex 2D gel patterns is very important for achieving high-throughput proteomics. Protein spots were analyzed and quantified for differential expression patterns between gels treated with and without IFN-γ at 24 h and 48 h using PDQuest software (Figures 3 and 4). This software enables analysis of most of the differences in protein expression profiles in complex samples. After selecting the gels or portion of the gels to be analyzed, spots of interest are detected and a Matchset is created establishing the experimental relationships between the samples. Figure 3 ( parts A-E) shows the statistical analysis of the differential expression pattern of the five identified proteins, and Figure 4 (parts A-C) shows some of the un-identified protein spots of C. pneumoniae at 24 and 48 h under induction of IFN-γ at 50 U/mL and 100 U/mL. The key to obtaining a quality Matchset is to select the most appropriate parameters during spot detection. The spot detection wizard function assists in the process of identifying and quantifying spots in the gel image. The spot review tool displays histograms of all spots in a Matchset. Bars in each histogram represent the spot’s 882

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quantity in each gel member of the Matchset. Each boxed area of both the Figures 3 and 4 show histograms depicting alteration in the profile between normal (IFN-untreated) with persistent (IFN-treated) protein spots. The pathogenic significance of C. pneumoniae in atherosclerosis remains unclear despite the detection of this bacterium atheromas by numerous studies,3,6,33 including a report from our laboratory documenting the isolation of C. pneumoniae from the coronary artery of a patient with severe atherosclerosis1 as the mechanisms involved in establishing persistent infection with this organism, is not well understood. This organism is capable of remaining viable in the host despite antibiotic treatment of chronic respiratory infections following acute illness.34 Vandahl et al.35 reported a 2D reference map of the C. pneumoniae proteome containing 263 identified proteins in EB. Our group had reported earlier, the differential expression pattern of 21 C. pneumoniae proteins under IFN-γ treatment with 50 U/mL at 24 h using Bioimage 2D analyzer software (Genomic Solutions).17 In the present study, we used an automated and advanced proteomic tool (PDQuest vs 7.1) to examine the potential regulatory effects of IFN-γ (50 U/mL and 100 U/mL) for a longer span up to 48 h to get a more complete picture of expression of chlamydial proteins under persistent conditions. The ultimate rewards in proteomics will not come from only displaying or quantifying as many spots as possible, nor from identifying as many protein spots as possible, even though these are the stepping stones to worthy and important technical goals. Rather, the rewards will come from applying proteomics tools to answer important biological questions which would aid in identifying potential bacterial virulence factors, diagnostic markers in patients with chronic infection or possible therapeutic targets. Our main goal in this article was to characterize the protein regulation in IFN-γ-induced persistence phase in C. pneumoniae. The advances in proteomics tools have been invaluable in approaching this goal. Further identification, analysis, and quantification are under way to understand the pathogenic mechanism of this intracellular pathogen that might lead us to comprehensive knowledge and contribute to the elucidation and validation of biological functions. List of Abbreviations: HEp2: human epithelial cells type 2; EB: elementary body; RB: reticulate body; IFN: interferon.

Acknowledgment. We thank George B. Harding for maintaining HEp2 cells and purifying elementary bodies of C. pneumoniae. We also thank Dr. Laura Pantoja Bollinger for her assistance with the TEM work. Our sincere gratitude is extended to Bruce Sadownick and Charles Martin for their excellent technical support with PDQuest 2-D analysis software. This project was supported by NIH grants HL68874 and AI51255. References (1) Ramirez, J. A.; Ahkee, S.; Summersgill, J. T.; Ganzel, B. L.; Ogden, L. L.; Quinn, T. C.; Gaydos, C. A.; Bobo, L. L.; Hammerschlag, M. R.; Robin, P. M.; LeBar, W.; Grayston, J. T.; Kuo, C.-C.; Campbell, L. A.; Patton, D. L.; Dean, D.; Schachter, J. Isolation of Chlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis. Ann. Intern. Med. 1996, 125, 979-982. (2) Campbell, L. A.; Kuo, C.-C.; Grayston, J. T. Chlamydia pneumoniae and cardiovascular disease. Emerg. Infect. Dis. 1998, 4, 571-579. (3) Kuo, C.-C.; Shor, A.; Campbell, L. A.; Fukushi, H.; Patton, D. L.; Gryaston, J. T. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J. Infect. Dis. 1993, 167, 841-849.

technical notes (4) Maass, M.; Bartels, C.; Engel, P. M.; Mamat, U.; Sievers, H. H. Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J. Am. Coll. Cardiol. 1998, 31, 827-832. (5) Grayston, J. T.; Kuo, C.-C.; Coulson, A. S.; Campbell, L. A.; Lawrence, R. D.; Ming-Jong, L. Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery. Circulation. 1995, 93, 3397-3400. (6) Kuo, C.-C.; Gown, A. M.; Benditt, E. P.; Grayston, J. T. Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain. Artheroscler. Thromb. 1992, 13, 1501-1504. (7) Kutlin, A.; Roblin, P. M.; Hammerschlag, M. R. In vitro activities of azithromycin and ofloxacin against Chlamydia pneumoniae in a continuous-infection model. Antimicrob. Agents Chemother. 1999, 43, 2268-2272. (8) Gaydos, C. A.; Summersgill, J. T.; Sahney, J. A.; Ramirez, J. A.; Quinn, T. C. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect. Immun. 1996, 64, 1614-1620. (9) Godzik, K.; O’Brien, E. R.; Wang, S. W.; Kuo, C.-C. In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae. J. Clin. Microbiol. 1995, 168, 1231-1235. (10) Molestina, R. E.; Dean, D.; Ramirez, J. A.; Summersgill, J. T. Characterization of a strain of Chlamydia pneumoniae isolated from a coronary atheroma by analysis of the omp1 gene and biological activity in human endothelial cells. Infect. Immun. 1998, 66, 1360-1376. (11) Mehta, S. J.; Miller, R. D.; Ramirez, J. A.; Summersgill, J. T. Inhibition of Chlamydia pneumoniae replication in HEp-2 cells by interferon-γ role of tryptophan catabolism. J. Infect. Dis. 1998, 177, 1326-1331. (12) Pantoja, L. G.; Miller, R. D.; Ramirez, J. A.; Molestine, R. E.; Summersgill, J. T. Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon. Infect. Immun. 2001, 69, 7927-7932. (13) Hogan, R. J.; Mathews, S. A.; Mukhopadhyay, S.; Summersgill, J. T.; Timms, P. T. Chlamydial persistence: Beyond the biphasic paradigm. Infect. Immun. 2004, in press. (14) Byrne, G. I.; Ouellette, S. P.; Wang, Z.; Rao, J. P.; Lu, L.; Beatty, W. L.; Hudson, A. P. Chlamydia pneumoniae expresses genes required for DNA replication but not cytokines during persistent infection of HEp2 cells. Infect. Immun. 2001, 69, 5423-5429. (15) Mathews, S. A.; George, C.; Flegg, C.; Stenzel, D.; Timms, P. Differential expression of ompA, ompB, pyk, nlpD, and Cpn0585 genes between normal and interferon-γ treated cultures of Chlamydia pneumoniae. Microb. Pathog. 2001, 30, 337-345. (16) Hogan, R. J.; Mathews, S. A.; Kutlin, A.; Hammerschlag, M. R.; Timms, P. Differential expression of genes encoding membrane proteins between acute and continuous Chlamydia pneumoniae infections. Microbial Pathogenesis. 2003, 34, 11-16. (17) Molestina, R. E.; Klein, J. B.; Miller, R. D.; Pierce, W. H.; Ramirez, J. A.; Summersgill, J. T. Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells. Infect. Immun. 2002, 70, 2976-2981. (18) Noda, L. In The Enzyme; Boyer, P. D., Ed.; Academic Press: New York, Vol. 8; 1973 pp 279-305. (19) Atkinson, D. E. In Cellular Energy Metabolism and its Regulation; Atkinson, D. E. Ed.; Academic Press: Orlando, FL, pp 85-107. (20) Munier-Lehmann, H.; Chenal-Francisque, V.; Ionescu, M.; Christova, P.; Foulon, J.; Carnieal, E.; Barzu, O. Relationship between

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