INFECTION AND IMMUNITY, Oct. 2008, p. 4405–4413 0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00575-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 10
Mycoplasma pneumoniae Infection Induces Reactive Oxygen Species and DNA Damage in A549 Human Lung Carcinoma Cells䌤 Gongping Sun,1 Xuefeng Xu,2 Yingshuo Wang,2 Xiaoyun Shen,1 Zhimin Chen,2* and Jun Yang1,3,4* Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang, 310058, China1; The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310008, China2; National Key Laboratory for Infectious Disease Diagnosis and Therapy, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310008, China3; and Zhejiang California International NanoSystems Institute, Hangzhou, Zhejiang, 310030, China4 Received 11 May 2008/Returned for modification 13 June 2008/Accepted 16 July 2008
Mycoplasma pneumoniae is a frequent cause of community-acquired bacterial respiratory infections in children and adults. In the present study, using a proteomic approach, we studied the effects of M. pneumoniae infection on the protein expression profile of A549 human lung carcinoma cells. M. pneumoniae infection induced changes in the expression of cellular proteins, in particular a group of proteins involved in the oxidative stress response, such as glucose-6-phosphate 1-dehydrogenase, NADH dehydrogenase (ubiquinone) Fe-S protein 2, and ubiquinol-cytochrome c reductase complex core protein I mitochondrial precursor. The oxidative status of M. pneumoniae-infected cells was evaluated, and the results revealed that M. pneumoniae infection indeed caused generation of reactive oxygen species (ROS). It was further shown that M. pneumoniae infection also induced DNA double-strand breaks, as demonstrated by the formation of ␥H2AX foci. On the other hand, an ROS scavenger, N-acetylcysteine, could inhibit the ROS generation, as well as decrease ␥H2AX focus formation. This is the first report showing that M. pneumoniae infection can directly induce DNA damage, at least partially, through the generation of ROS, and thus this report strengthens the powerful application of proteomics in the study of the pathogenesis of M. pneumoniae. Mycoplasma pneumoniae is one of the smallest self-replicating organisms capable of cell-free existence. As a human pathogen, M. pneumoniae is a major cause of community-acquired respiratory infections in children and adults, which can lead to tracheobronchitis and primary atypical pneumonia (17). Besides causing diseases in the respiratory system, M. pneumoniae has been implicated in several extrapulmonary complications arising from infection; for example, it is a factor in the development of arthritis, cardiovascular diseases, and neurotropic infections (32, 34). Considerable progress in our understanding of M. pneumoniae pathogenesis has been made over the years. The activation of the host immune response and direct invasion of cells are believed to contribute to this pathogenesis (32, 37). It has been shown that attachment of M. pneumoniae to the respiratory epithelium is a prerequisite for disease (22). The close interaction between M. pneumoniae and host cells protects the bacterium from removal by the host’s mucociliary clearance mechanism and allows it to proliferate and produce metabolites, which in turn can cause cytotoxic effects (32, 34). Simultaneously, M. pneumoniae attachment induces the cells’ inflammatory reaction and the host’s immune response. For example, upregulation of interlukin-2, -4, -5, -6, -10, -12, and -18 and
interferon has been detected in bronchoalveolar lavages, blood, and lungs of M. pneumoniae-infected patients (34). Recently, it was found that the M. pneumoniae protein MPN372, which contains key amino acids similar to the pertussis toxin S1 subunit, might be responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in humans, thus leading to the hypothesis that MPN372 could be the pathogenic determinant of M. pneumoniae (11). Despite these advances in our understanding, the mechanisms underlying M. pneumoniae pathogenesis are still not completely clear. The rapid development of proteomic techniques has revolutionized our ability to study protein interactions and cellular changes on a global scale, revealing previously unknown and unanticipated associations (6). Therefore, in the current study we investigated the effects of M. pneumoniae infection on A549 host cell protein profiles to elucidate the pathogenic mechanism(s) by using two-dimensional gel electrophoresis (2-DE) and matrixassisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Interestingly, a group of proteins that are involved in the regulation of cellular redox status were found to be upregulated, indicating that M. pneumoniae infection may induce oxidative stress. Therefore, we further evaluated the generation of reactive oxygen species (ROS) in M. pneumoniae-infected cells. In addition, as ROS are known to induce DNA damage, we also used ␥H2AX focus formation, a new indicator of DNA double-strand breaks (DSBs) (38, 39), to determine whether M. pneumoniae can cause DNA damage through the action of ROS.
* Corresponding author. Mailing address for Jun Yang: National Key Laboratory for Infectious Disease Diagnosis and Therapy, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310008, China. Phone and fax: 86 571 88208140. E-mail:
[email protected]. Mailing address for Zhimin Chen: Department of Pediatric Pulmonology, The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310008, China. Phone: 86 571 87061007, ext. 70411. Fax: 86 571 87033296. E-mail:
[email protected]. 䌤 Published ahead of print on 28 July 2008.
MATERIALS AND METHODS Mycoplasma culture. M. pneumoniae strain 29342 (American Type Culture Collection, Rockville, MD) was grown in mycoplasma broth containing myco-
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FIG. 1. Effects of M. pneumoniae infection on cell viability. A549 cells were infected with different concentrations (1, 5, 10, 20, 40, 60, 80, and 100 CFU/cell) of M. pneumoniae for different times (2, 12, and 24 h). Cell viability was measured by the MTT test. The data are the means ⫾ standard deviations for six biological replicates.
plasma broth base CM403 (Oxoid, Hampshire, United Kingdom), mycoplasma selective supplement G SR59 (Oxoid), 0.5% glucose, and 0.002% phenol red at 37°C in the presence of 5% CO2. Agar plates used for colony counting were prepared similarly and contained mycoplasma agar base CM401 (Oxoid) and mycoplasma selective supplement G SR59. Viable M. pneumoniae was quantified by counting the CFU in 10-fold serial dilutions of a mycoplasma broth solution spread on mycoplasma agar plates. Cell culture and infection. A549 human lung epithelial carcinoma cells (CCL185; American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (PAA, Pasching, Austria) at 37°C in the presence of humidified 5% CO2. For M. pneumoniae infection, 1 ml of M. pneumoniae was added to 10 ml of cell medium (1:10, vol/vol; approximately 1 ⫻ 107 CFU/106 cells) unless otherwise specified. Cells were then incubated for 12 h. As controls, 1 ml of mycoplasma broth without bacteria was added to cell cultures. Cell viability measurement. Cell viability after infection was examined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test as described previously (7). Briefly, cells were seeded into 96-well culture plates at a density of 1 ⫻ 104 cells/well. At the end of each infection time, 20 l of MTT (5 mg/ml in PBS) was added to each well. Three hours later, the solution was discarded, and 100 l of dimethyl sulfoxide was added to each well. After 10 min of vigorous vibration, the solution was transferred to a new plate, and the absorbance at 570 nm was determined with a microplate reader (Infinite M200; Tecan, Switzerland). Relative survival was determined by dividing the absorbance of the infected group by the absorbance of the control group. Protein extraction. Control and M. pneumoniae-treated A549 cells were washed with phosphate-buffered saline (PBS) at least three times and then detached with 0.25% trypsin (Sigma, St. Louis, MO) and centrifuged at 2,000 rpm for 5 min. The cell pellets were washed twice with ice-cold PBS and lysed for 30 min at 4°C in a lysis buffer consisting of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), and 0.2% Bio-Lyte (pH 3 to 10) (Bio-Rad, Hercules, CA). The cell lysates were frozen and thawed with liquid nitrogen three times. Insoluble cellular debris was removed by centrifugation at 20,000 rpm for 1 h. All steps were performed on ice to prevent protein degradation. The protein concentration was measured using the Bradford assay (Bio-Rad). The resulting supernatants were stored in aliquots at ⫺70°C until they were used. 2-DE and image analysis. 2-DE and image analysis were conducted by using the protocol established previously in our laboratory (9, 10). Briefly, approximately 250 g of extracted protein was placed in 350 l (final volume) of a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte (pH 3 to 10), and a trace of bromophenol blue and applied to linear IPG Readystrips (17 cm; pH 4 to 7; Bio-Rad) by in-gel rehydration for 12 h at 20°C. Isoelectric focusing (IEF) was performed with a protein IEF cell (Bio-Rad) under the following conditions: 20°C, 250 V for 30 min, 1,000 V for 2 h, 10,000 V for 5 h, and 10,000 V until 60,000 V 䡠 h was achieved. After IEF during the first-dimension electrophoresis, the strips were equilibrated at room
temperature for 15 min in a buffer containing 6 M urea, 2% sodium dodecyl sulfate (SDS), 0.375 M Tris-HCl (pH 8.8), 20% glycerol, and 2% DTT and then for another 15 min in the same buffer except that the DTT was replaced by 2.5% iodoacetamide. The equilibrated IPG strips were attached to the top of a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel with low-melting-point agarose to ensure firm contact. SDS-PAGE was performed for second-dimension separation with a constant voltage of 200 V for 8 h at 16°C. A total of six gels, three containing extracts of mock-infected cells and three containing extracts of M. pneumoniae-infected cells, were symmetrically assembled into a Protean Plus Dodeca cell (Bio-Rad) and run simultaneously in an effort to ensure reproducibility. Finally, the gels were stained by using an improved silver-staining method as described previously (9, 10). The silver-stained 2-DE gels were scanned with a GS-800 calibrated imaging densitometer (Bio-Rad) at a resolution of 150 dots per inch. An analysis of protein spot distribution and intensity was performed with PDQuest software (Bio-Rad). Briefly, the background was filtered, and spots were located and matched. The gel with the most spots and least background staining was selected as the reference gel, and the spot patterns of other gels were compared with the reference gel. Unmatched spots that were goodquality spots were manually added to the reference gel, and matched spots were considered the same spot. Protein spots separated on 2-DE gels were quantified by determining their relative volumes (i.e., the sum of the intensities of all the pixels contributing to a spot). The resulting data were exported to Microsoft Excel, and a statistical analysis was performed with Student’s t test. A P value of ⬍0.05 was considered significant (9, 10). In-gel digestion. Protein spots were manually excised from the silver-stained gels and transferred into 1.5-ml Eppendorf tubes. Each spot was washed twice in deionized water and destained by washing it with a solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. The gels were then washed twice in deionized water, dehydrated by addition of acrylonitrile, and dried in a SpeedVac (Thermo Savant, Holbrook, NY) for 30 min. The spots were subsequently rehydrated in 10 to 20 l of a proteomics-grade trypsin (Sigma) solution (20 l/ml in 40 mM NH4HCO3 plus 9% acrylonitrile) and incubated at 37°C for 6 to 8 h. Peptides were extracted twice by adding 30 l of a solution containing 50% acrylonitrile and 5% Trifluoroace. The extracted solutions were lyophilized in a vacuum centrifuge (Heto Drywinner, Germany). MALDI-TOF MS analysis and database search. Peptide extracts were dissolved in 5 to 10 l of 0.1% Trifluoroace, and 1 l of the solution was mixed with an equal volume of a 10-mg/ml ␣-cyano-4-hydroxycinnamic acid (CHCA) (Sigma) solution saturated with 50% acrylonitrile in 0.05% Trifluoroace and analyzed with a Voyager-DE STR MALDI-TOF MS (Applied Biosystems, Foster City, CA). The instrument was operated in reflector mode with a 160-ns delay extraction time, positive polarity, 60 to 65% grid voltage, and an accelerating voltage of 20,000 V. Laser shots (200 per spectrum) were used to acquire the spectra in the molecular mass range from 1,000 to 4,000 Da. External calibration was carried out using P14R and oxidized insulin chain B (Sigma), and internal calibration was performed using the autolytic peaks of trypsin. This procedure typically resulted in mass accuracies of 50 ppm. For each spectrum, all peaks with
FIG. 2. 2-DE analysis of protein level changes induced by M. pneumoniae infection. (A) Comparison of the 2-DE protein patterns of the control (Con) and M. pneumoniae-infected A549 cells (Mp). Extracted proteins were separated on a pH 4 to 7 IPG strip, followed by 12% SDS-PAGE and silver staining. The arrows indicate proteins upregulated or downregulated in M. pneumoniae-infected A549 cells. The number adjacent to a spot is its index number. (B) Enlarged 2-DE images of spots 8509 and 2501, which are upregulated in M. pneumoniae-infected A549 cells. (C) Western blot results showing that the level of vimentin was increased in M. pneumoniae-infected cells. (D) Annotated mass spectrum for vimentin. 4407
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intensities over 3% (normalized to the highest peak) were selected by Data Explorer 4.0 (Applied Biosystems) and searched in the NCBI database (release date, 19 September 2006) with MASCOT software (version 2.1; Matrix Science, London, United Kingdom). The database searches were performed using the following parameters: Homo sapiens (human), trypsin digestion, cysteine as carbamidomethylated, methionine as oxidized, and mass tolerance of 100 ppm using internal calibration. A total of 359 proteins were actually searched. Scores greater than 53 were considered significant (P ⬍ 0.05). To eliminate redundancy of proteins with different names and accession numbers or isoforms or individual members of a protein family, the protein with the highest scores was selected for the protein family. Western blotting. Vimentin was detected by Western blotting as described previously, with some modifications (35). Briefly, equal amounts of protein were loaded into the wells of a 12% Tris-HCl Ready gel (Bio-Rad). After electrophoresis, proteins were transferred to an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad) and incubated with an antivimentin antibody (Sigma), which was followed by addition of an IRDye 680-labeled goat anti-mouse secondary antibody (LI-COR Biosciences). -Actin was used as control, and the membrane was incubated with an anti--actin antibody (Upstate, Lake Placid, NY), followed by an IRDye 800CW goat anti-rabbit secondary antibody (LICOR Biosciences). The membrane was then scanned using the Odyssey infrared imaging system (LI-COR Biosciences). Measurement of intracellular ROS. The production of intracellular ROS was measured using 2⬘,7⬘-dichlorofluorescein diacetate (DCFH-DA) (33). DCFH-DA reacts with ROS to form the highly fluorescent compound dichlorofluorescein. Briefly, a 10 mM DCFH-DA stock solution (in methanol) was diluted 500-fold in PBS to obtain a 20 M working solution. After M. pneumoniae treatment, the cells in a 96-well plate were washed twice with PBS and then incubated in a 100-l working solution of DCFH-DA at 37°C for 30 min. Fluorescence was then determined with an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a microplate reader (Infinite M200; Tecan, Switzerland). To determine the role of ROS in the induction of DNA damage, cells were first incubated with a ROS scavenger, N-acetylcysteine (NAC) (Sigma), for 2 h, which was followed by M. pneumoniae infection. The ROS level was determined by dividing the absorbance of the infected group by the absorbance of the control group. Immunofluorescence microscopy. Immunofluorescence microscopy to observe the formation of ␥H2AX foci was conducted essentially as described previously (39). In short, 1 ⫻ 105 cells were seeded into a six-well culture plate containing a glass coverslip in each well. After treatment, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized in 0.2% Triton X-100. After blockage with blocking serum for 1 h, samples were incubated with a mouse monoclonal anti-␥H2AX antibody (1:3,000; Upstate Technology) overnight at 4°C, followed by fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:300; Beijing Zhongshan Biotechnology Co., China) for 1 h. To stain the nuclei, DCFH-DA and 4⬘,6⬘-diamidino-2-phenylindole (DAPI) (Sigma) were added to the cells, which were then incubated for another 15 min. Each coverslip was then removed from the plate, mounted on a glass slide, and observed with an Olympus DP70 fluorescence microscope (Olympus, Tokyo, Japan). Statistical analysis. Each experiment was conducted at least three times. Statistical analysis was performed by using the 2 test and Student’s t test. A P value of ⬍0.05 was considered significant. Data are presented below as means ⫾ standard deviations.
RESULTS Effects of M. pneumoniae infection on cell viability. First, we wanted to determine the effects of M. pneumoniae infection on A549 cell viability. As shown in Fig. 1, it was found that at concentrations below 10 CFU/cell, M. pneumoniae infection had little effect on cell viability, which is consistent with our previous finding that M. pneumoniae did not affect cell viability or cell proliferation at a concentration of 10 CFU/cell (36). However, higher concentrations of M. pneumoniae did affect cell viability, particularly after longer incubation times. For example, with 100 CFU of M. pneumoniae per cell, the cell viability decreased to 70 to 80% at 12 or 24 h after infection. Therefore, in the experiments described below, unless other-
TABLE 1. Differentially expressed proteins in M. pneumoniaeinfected A549 cells Relative vol
Index no.
Control
M. pneumoniae
3211 1306 1411 1412 1509 2501 3810 4712 5706 6810 7807 8101 8201 8504 8506 8515 8609 8623 5214 5304 5413 6206 6312 2008 6010 9016 2705 6316 7809
11.47 ⫾ 6.04 6.00 ⫾ 13.53 25.20 ⫾ 12.12 24.23 ⫾ 16.45 150.27 ⫾ 25.36 41.73 ⫾ 27.45 21.83 ⫾ 9.15 28.93 ⫾ 14.3 27.00 ⫾ 12.93 19.33 ⫾ 7.92 7.47 ⫾ 1.36 24.43 ⫾ 3.38 27.67 ⫾ 10.86 21.67 ⫾ 1.50 35.03 ⫾ 2.42 80.47 ⫾ 26.8 34.07 ⫾ 11.61 83.90 ⫾ 8.16 22.90 ⫾ 4.96 36.90 ⫾ 23.97 35.77 ⫾ 17.18 20.63 ⫾ 7.93 36.93 ⫾ 13.57 2.20 ⫾ 2.16 13.27 ⫾ 5.76 53.03 ⫾ 17.12 27.73 ⫾ 7.96 27.43 ⫾ 12.29 8.50 ⫾ 3.65
41 ⫾ 5.55 28.00 ⫾ 3.32 52.97 ⫾ 8.88 103.07 ⫾ 21.90 503.10 ⫾ 68.40 101.53 ⫾ 15.46 45.97 ⫾ 7.03 92.10 ⫾ 23.19 70.47 ⫾ 18.69 51.70 ⫾ 9.48 15.27 ⫾ 0.71 58.50 ⫾ 1.30 82.23 ⫾ 31.37 51.97 ⫾ 10.39 76.00 ⫾ 12.86 325.70 ⫾ 79.68 98.37 ⫾ 26.62 285.73 ⫾ 54.50 52.13 ⫾ 11.67 90.40 ⫾ 20.95 159.97 ⫾ 70.95 49.90 ⫾ 8.26 95.83 ⫾ 31.23 37.17 ⫾ 8.57 43.23 ⫾ 13.64 121.70 ⫾ 22.79 8.20 ⫾ 1.93 3.60 ⫾ 2.91 1.07 ⫾ 0.90
Ratioa
P value
3.571 4.671 2.101 4.251 3.351 2.431 2.111 3.181 2.611 2.671 2.041 2.391 2.971 2.391 2.171 4.051 2.891 3.411 2.281 2.451 4.471 2.421 2.591 16.71 3.261 2.291 3.382 7.622 7.942
0.0034 0.029 0.0329 0.0076 0.0011 0.0303 0.0223 0.0159 0.0296 0.0105 0.0009 ⬍0.0001 0.0465 0.0411 0.0056 0.0072 0.0185 0.0215 0.0162 0.0437 0.0421 0.0115 0.0401 0.0024 0.0248 0.014 0.0145 0.0308 0.0266
a 1, protein upregulated in M. pneumoniae-infected A549 cells; 2, protein downregulated in M. pneumoniae-infected A549 cells.
wise specified, A549 cells were infected with M. pneumoniae using a concentration of 10 CFU/cell. M. pneumoniae infection induces changes in the protein expression profile of A549 cells. High-resolution 2-DE was performed with the cellular proteins from A549 cells with or without exposure to M. pneumoniae for 12 h. Three pairs of gels were analyzed for quantitative spot comparison with the image analysis software. Representative silver-stained gels are shown in Fig. 2A. Digitized images were analyzed by using PDQuest software. After spot detection, background subtraction, and volume normalization, approximately 967 ⫾ 101 protein spots were detected for control A549 cells and 978 ⫾ 83 spots were detected for M. pneumoniae-infected A549 cells. Compared to the control, no protein spot appeared or disappeared in M. pneumoniae-infected A549 cells, but 26 protein spots were upregulated and three protein spots were downregulated. These protein spots are indicated in Fig. 2A, and the relative changes are shown in Table 1. Enlarged images of spots 8509 and 2501, which were upregulated by M. pneumoniae infection, are shown in Fig. 2B. The 29 protein spots that were either up- or downregulated after M. pneumoniae infection were excised from the corresponding 2-DE gels, digested in the gel with trypsin, and identified by using MALDI-TOF MS analysis and database searching. Using the MASCOT software to search the nrNCBI database, 15 protein spots were tentatively identified. The identified proteins are summarized in Table 2. The functions of
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TABLE 2. Fifteen proteins upregulated in M. pneumoniae-infected A549 cells and GeneCards database search results for them Spot no.
Score
8609
116
2501
Gene
Description
gi no.
Gene product
No. of peptide matches
Coverage (%)
Mr (103)
pI
13/28
26
59
6.42
Xq28
NM000402
Chromosome location
Reference sequence
Subcellular location
Function
G6PD
G6PD
120731
72
VIM
Vimentin
57471646
9/24
29
49
5.19
10p13
NM003380
1411
69
RCN1
Reticulocalbin 1 precursor
4506455
6/16
20
38
4.86
11p13
AF318337
Endoplasmic reticulum
8504
82
NDUFS2
4758786
8/21
20
52
7.21
1q23
NM004550
Mitochondrion
8201
131
NADH dehydrogenase (ubiquinone) Fe-S protein 2 Proteasome subunit alpha type 1
130848
13/42
54
30
6.15
11p15.1
NM002786
Cytoplasm, nucleus
8623
64
90109864
19/67
15
149
6.05
4q28.1
NM020337
4712
128
14165435
21/77
45
50
5.39
9q21.32-33
NM002140
Cytoplasm, nucleus
Nucleic acid binding, DNA binding, RNA binding
5413
64
UQCRC1
92090651
8/23
16
53
5.94
3p21.3
NM003365
Mitochondrion
Ubiquinol-cytochrome c activity, oxidoreductase activity
6810
86
GARS
21264523
17/55
25
83
6.61
7p15
NM002047
6312
79
PSMD13
26S proteasome subunit p40.5
3618343
8/30
26
42.9
5.53
11p15.5
AK092845
5304
77
EIF3S2
4503513
9/43
30
36
5.38
1p34.1
NM003757
6010
94
KRT1
Eukaryotic translation initiation factor 3, subunit 2 beta Keratin 1
11935024
15/65
30
66
8.16
12q12-13
NM006121
8515
60
CENPF
Centromere protein F
1345731
38/67
10
36.7
5.03
1q32-41
NM016343
Nucleus
9016
53
ZNF605
Zinc finger protein 605
74762445
17/68
25
74.3
9.32
12q24.33
NM183238
Nucleus (probably)
2008
56
TAP2
Antigen peptide transporter 2 (APT2)
549044
8/28
16
75.6
8.24
6p21.3
NM000544
Endoplasmic reticulum
PSMA1 ANKRD50 HNRPK
Ankyrin repeat domain-containing protein 50 Heterogeneous nuclear ribonucleoprotein K isoform Ubiquinol-cytochrome c reductase complex core protein I, mitochondrial precursor Glycyl-tRNA synthetase
these identified proteins are also shown in Table 2 and were determined by searching the GeneCards database (http://www .genecards.org/index.shtml). These proteins are involved in cellular metabolism, mobility, and stress reaction. The rest of
Produces pentose sugars for nucleic acid synthesis and main producer of NADPH reducing power Structural molecule activity, structural constituent of cytoskeleton, protein binding May regulate calciumdependent activities in the endoplasmic reticulum lumen or post- endoplasmic reticulum compartment Transfer of electrons from NADH to the respiratory chain RNA binding, threonine endopeptidase activity
Nucleotide binding, glycine-tRNA ligase activity, protein binding, ATP binding, ligase activity Acts as a regulatory subunit of the 26S proteasome involved in ATP-dependent degradation of ubiquitinated proteins Translation initiation factor activity, protein binding Receptor activity, structural constituent of cytoskeleton protein binding, sugar binding Probably required for kinetochore function, involved in chromosome segregation during mitosis, interacts with retinoblastoma protein, CENP-E, and BUBR1 May be involved in transcriptional regulation Nucleotide binding, homoaconitate hydratase activity, transporter activity, protein binding, ATP binding
protein spots either were not identified by peptide mass fingerprinting or produced no spectrum at all. To support the accuracy of protein identifications based on 2-DE analyses, one identified protein, vimentin, was chosen
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FIG. 3. M. pneumoniae infection increases intracellular ROS levels. A549 cells were infected with different concentrations (1, 10, and 100 CFU/cell) of M. pneumoniae for different times (2, 12, and 24 h). Intracellular ROS levels were measured as described in Materials and Methods. The data are the means ⫾ standard deviations for six biological replicates.
from the cell lysate of M. pneumoniae-infected cells and subjected to Western blot analysis. As shown in Fig. 2C, M. pneumoniae treatment significantly increased the amount of vimentin, thus corroborating the proteomics results. M. pneumoniae infection induces ROS in A549 cells. Among the 15 proteins identified, the following 3 proteins are involved in regulating cellular oxidative status: glucose-6-phosphate 1-dehydrogenase (G6PD), NADH dehydrogenase (ubiquinone) Fe-S protein 2, and ubiquinol-cytochrome c reductase complex core protein I mitochondrial precursor (Table 2). This suggests that M. pneumoniae infection might induce oxidative stress in cells. Therefore, the generation of ROS in M. pneumoniae-infected A549 cells was examined. As shown in Fig. 3, there was clear dose- and time-dependent induction of ROS following M. pneumoniae infection. It was found that M. pneumoniae infection-induced ROS generation peaked at 12 h and then subsided by 24 h, although it was still significantly higher than the basal level. Thus, the increased expression of G6PD and other redox-related proteins should reflect a cellular defense mechanism against oxidative stress induced by M. pneumoniae infection. M. pneumoniae infection induces ␥H2AX focus formation in A549 cells. It is well established that ROS mediate the DNA damage elicited by many genotoxic agents (20, 29). Since the experiments described above linked M. pneumoniae infection with elevated ROS levels, we pursued a logical question: does M. pneumoniae also induce DNA damage? Among the various types of DNA damage, DSBs are the most severe. ␥H2AX (the phosphorylated form of H2AX) focus formation has been gradually accepted as a sensitive indicator of DSBs (38, 39). Hence, A549 cells were infected with 10 CFU/cell of M. pneumoniae and then subjected to immunofluorescence microscopy to evaluate the formation of ␥H2AX foci. As shown in Fig. 4A, ␥H2AX foci were readily observed 2 h after M. pneumoniae infection and persisted until at least 24 h postinfection. Again, more ␥H2AX foci were observed at 12 h postinfection than at other time points (Fig. 4B), which correlated well with the time
course of ROS generation. These results suggest that M. pneumoniae can induce DNA damage in A549 cells. Inhibition of ROS generation decreases M. pneumoniae-induced ␥H2AX focus formation. To determine whether ROS were indeed responsible for M. pneumoniae-induced DNA damage, A549 cells were first incubated with the ROS scavenger NAC for 2 h and then infected with M. pneumoniae. It was found that preincubation with NAC significantly decreased the ROS generation stimulated by M. pneumoniae infection (data not shown), and, apparently consequently, ␥H2AX focus formation was also significantly, although not completely, decreased (Fig. 4). Therefore, these data indicate that ROS have a significant role in M. pneumoniae-induced DNA damage. DISCUSSION Proteomics, which focuses on determination of the structure, expression, cellular location, biochemical activity, and interactions of as many proteins as possible, is an invaluable tool for the study of systems biology (6). This approach has been used in the study of bacterial pathogens, as well as in antibacterial drug design (14, 21). Specifically, in the study of human pathogens, proteomics has been widely used to determine the protein composition of pathogens, the structure and protein interactions of pathogens’ proteins, and the effects of infection and individual pathogenic proteins on the host cellular proteome. The resulting knowledge could lead to discovery of new molecular targets and biomarkers for medicine and biopharmacy (6, 15, 21). With these methods, a proteome map has been constructed for M. pneumoniae (23, 30). However, the effects of M. pneumoniae infection on host cell protein expression have not been investigated. This study used a proteomic approach to assess the effect of M. pneumoniae infection on protein expression in A549 cells. Fifteen proteins that were significantly upregulated in M. pneumoniae-infected A549 cells were identified, and all of them are involved in cellular metabolism, mobility, or the
FIG. 4. M. pneumoniae infection induces ␥H2AX focus formation partially through the ROS pathway. (A) Representative images obtained by immunofluorescence microscopy of A549 cells infected with 10 CFU/cell M. pneumoniae for different time intervals. (B) Quantification of ␥H2AX foci in A549 cells. Black bars, percentage of cells with no ␥H2AX foci; open bars, percentage of cells with 1 to 10 ␥H2AX foci/cell; light gray bars, percentage of cells with 11 to 20 ␥H2AX foci/cell; dark gray bars, percentage of cells with ⬎20 ␥H2AX foci/cell. Mp, M. pneumoniae-infected cells; NAC⫹Mp, cells infected by M. pneumoniae after 2 h of preincubation with NAC. *, P ⬍ 0.05 (compared with blank); **, P ⬍ 0.01 (compared with blank); #, P ⬍ 0.05 (compared with M. pneumoniae-infected cells); ##, P ⬍ 0.01 (compared with M. pneumoniae-infected cells). The data are the means and standard deviations for three biological replicates. 4411
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stress response. Among them, several proteins related to cell redox regulation were of particular interest to us. G6PD is the first and rate-limiting enzyme in the pentose phosphate pathway. G6PD deficiency is the most common enzyme deficiency worldwide and can cause diseases such as neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis (8). Its key function in metabolism is to catalyze the synthesis of riboses for nucleic acid production, and, more importantly, it is the principal intracellular source of NADPH, which participates in cellular antioxidation reactions against ROS, including superoxide anion, hydrogen peroxide, and hydroxyl radicals (28). Thus, G6PD is considered an essential factor in cellular resistance to oxidative stress. Indeed, it has been shown that G6PD expression could be induced in response to agents that cause oxidative stress (5, 31). However, induction of the G6PD protein as a consequence of bacterial or viral infection has not been widely documented, although increased G6PD activity has been detected in Rickettsia conoriiinfected mouse tissue and virus-infected tobacco leaves (24, 26). Our findings document a case of mycoplasmal infection that causes an increase in the G6PD level in a human cell line. Interestingly, two other upregulated proteins identified in this study, NADH dehydrogenase (ubiquinone) Fe-S protein 2 and ubiquinol-cytochrome c reductase complex core protein I mitochondrial precursor, are also involved in the cellular redox milieu. The finding that 3 of 15 upregulated proteins are involved in redox balance focused our attention on ROS, and we sought to examine ROS generation in M. pneumoniae-infected cells in an effort to substantiate the connection. As shown in Fig. 3, our results proved that infection with M. pneumoniae does increase ROS levels in A549 cells. In the 1980s there were reports suggesting that ROS contribute to the cytotoxic effects of M. pneumoniae (1–3), although these early reports have largely been ignored in descriptions of the pathogenesis of M. pneumoniae. The present study, in which we started from a proteomic perspective, resurrected the notion that ROS have an important role in the pathogenesis of M. pneumoniae, and the results fit well with the severe deleterious effects on cells known to be elicited by ROS. One of the deleterious consequences of ROS generation is DNA damage. In the literature, we found no reports that M. pneumoniae is able to induce DNA damage, but there have been a few reports suggesting that certain Mycoplasma species, such as Mycoplasma hyorhinis, may contribute to the DNA fragmentation process by providing a Mycoplasma nuclease in cells undergoing apoptosis induced by cycloheximide (18, 19). The results described here, which showed that M. pneumoniae infection induced ␥H2AX focus formation, a presumptive indicator of DSBs, thus came as a surprise to us. On the one hand, DNA damage from ROS stimulated by M. pneumoniae infection is quite plausible; on the other hand, since M. pneumoniae rarely affects cell viability, let alone inducing apoptosis (at least in our experimental setting with 10 CFU/cell), the profound DNA damage in infected cells was hard to imagine. Not that bacterium-induced DNA damage is uncommon. For example, a recent report showed that a pathogenic Escherichia coli strain (phylogenetic group B2) expresses a hybrid peptidepolyketide that causes DSBs and activates the DNA damage checkpoint pathway, ultimately leading to cell cycle arrest and cell death (16). The conundrum here is that the strong corre-
lation of M. pneumoniae-induced ROS with severe DNA damage (and the ability to break this correlation by treatment with NAC [Fig. 4]) occurred in a context in which a minimal decrease in cell viability was observed. Resolution of this paradox is challenging. On the one hand, it seems likely that the presence of ␥H2AX foci may not be directly related to a decrease in cell viability. This seems to be the case for the potent carcinogen benzo[a]pyrene, which, as we have observed previously, can induce strong ␥H2AX focus formation in human amnion FL cells, human osteosarcoma U2OS cells, and mouse embryonic fibroblasts without concomitant loss of cell viability (39; unpublished data). On the other hand, the disappearance of ␥H2AX foci is generally regarded as repair of DSBs (12, 25, 27). Since at 24 h the number of cells with over 20 foci/cell was less than the number at 12 h, indicating that there was repair of DNA damage, it is quite possible that the cellular DNA repair system could efficiently fix the problem, thus protecting the cells from apoptosis or other deleterious consequences. Similarly, many other issues remain to be resolved. For instance, early studies on oxidative stress in host cells during M. pneumoniae infection emphasized the production of hydrogen peroxide and superoxide by the mycoplasmas (2, 3). In this study we were unable to differentiate, qualitatively or quantitatively, M. pneumoniae-generated ROS from the general cellular pool of ROS. Nor could we exclude the possibility that the increase in the ROS level might be the consequence of a decrease in the rate of ROS destruction, for example, by inhibition of the host enzymes, such as catalase and superoxide dismutase, that normally remove these toxic oxygen metabolites. Furthermore, one has to recognize the complex response in different cell types (13) and in different environmental situations, notably the growth state of M. pneumoniae influenced by a permissive or nonpermissive growth medium (4). Finally, it should be borne in mind that the observed proteomic changes represent changes in steady-state levels, and even though these changes might represent changes in gene expression, other mechanisms, such as posttranslational modification, could also be responsible. All these possibilities are worthy of further study. In summary, by using 2-DE and MS techniques, we investigated the cellular response to M. pneumoniae infection in A549 cells and identified a group of proteins with different functions, which provided useful information for understanding the pathogenesis of M. pneumoniae better. In particular, the results of the proteomic study reintroduce the possibility that ROS are important factors in M. pneumoniae pathogenesis.
ACKNOWLEDGMENTS We thank C. Zhang for technical assistance. We thank S. B. Schwartz of the Centers for Disease Control and Prevention (Atlanta, GA) and Robert Wohlhueter, who recently retired from the Centers for Disease Control and Prevention, for their invaluable support during this study and for editing the manuscript. This work was supported in part by grants from the Department of Health, Zhejiang Province (grant 2006QN020); the Department of Science and Technology, Zhejiang Province (grant 2007C33023); the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents; the National Hi-Tech Development Program (grant 2004AA649120); and the National Natural Science Foundation, China (grant 30771826).
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