Analysis of growth phase-dependent proteome profiles reveals differential regulation of mRNA and protein in Helicobacter pylori
Young Wook Choi1, Shin Ae Park1, Hyang Woo Lee1*, Dong Su Kim2 and Na Gyong Lee1
1Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul, Korea
2Research Division, Genomine Inc., Pohang, Kyungbuk, Korea
Helicobacter pylori is a slow growing, microaerophilic bacterium that causes various gastric dis- eases. To understand the growth phase-dependent global regulation of protein in H. pylori, we analyzed the proteome profiles of H. pylori 26695 harvested during the course of in vitro culture. Temporal changes in protein profiles were assessed using three independent cultures harvested at 6, 12, 24, 36, 48, and 60 h. Compared with the protein spots obtained at 6 h, 151 protein spots obtained at other time points exhibited significantly altered intensity, with 57 of these protein spots identified by MALDI-TOF MS analysis. Clustering analysis showed that overall protein profile was coordinated in accordance with the growth phases of the culture. When we compared mRNA transcript levels of the identified proteins, obtained from RT-PCR analysis, with their protein levels, we observed substantial discrepancies in their patterns, suggesting that the tran- scriptome and proteome of H. pylori were differentially regulated during in vitro culture. Prote- omic analysis also suggested that several H. pylori proteins underwent PTMs, some of which were modulated as a function of the growth phase of the culture. These findings indicate that H. pylori utilizes modulation of protein regulation and PTM as mechanisms to cope with changing growth environments. These observations should provide insight into the adaptive mechanisms employed by H. pylori within the context of growth environments.
Received: July 30, 2007 Revised: February 1, 2008 Accepted: March 10, 2008
Keywords:
Growth phase / Helicobacter pylori
1Introduction
Helicobacter pylori is a microaerophilic human pathogen that resides in the mucus layer of the stomach and is often asso- ciated with various gastric diseases [1, 2]. Several factors are involved in the successful adaptation of H. pylori to the harsh environment of the stomach, and the regulation of expres-
sion of these factors has been extensively investigated [3–12]. The expression of genes encoding factors responsible for survival and virulence of pathogenic bacteria is influenced by the growth state of the bacteria [13–15]. H. pylori is a spiral- shaped bacillus during infection of the gastric mucosae [16]
but, when exposed to unfavorable conditions, it transforms from a bacillary to a full coccoid form via an intermediate U- shaped form [17]. Like many other pathogenic bacteria, H. pylori has a stringent response [18, 19], which is required for
Correspondence: Professor Na Gyong Lee, Department of Bio- science and Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Korea
E-mail: [email protected] Fax: 182-2-3408-4334
activation of virulence genes, interaction with host cells or persistence within the host [13–15, 20]. These reports sug- gest that the virulence of H. pylori may also be influenced by bacterial growth phases.
Abbreviations: PBST, PBS containing 0.05% Tween-20; RT, reverse transcription
* Current address: Hyang Woo Lee, M.S. Tegoscience, 448 Gasan- dong, Gumcheon-gu, Seoul 153-803, Korea
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Studies using high-density DNA microarrays to eluci- date the global regulation of gene expression in H. pylori during in vitro culture have shown that there is a tran- scriptional switch in virulence gene expression between the late log and stationary phases [21, 22]. Genes related to virulence were induced at this stage of culture, suggesting that the late log phase may correspond to the most virulent phase of growth of H. pylori [21]. In addition, expression of genes involved in iron homeostasis changed dramatically at the switch, and bacteria in log and stationary phases responded differently to iron starvation [21, 22], suggesting that H. pylori cells differ physiologically during various growth phases and respond differently to environmental changes.
DNA microarray technology has proven powerful in characterizing the global changes in mRNA expression of cells or organisms during responses to environmental stim- uli. These microarrays, however, can measure only changes in mRNA level, whereas most biological phenomena result from protein functions. In contrast, proteomic analysis can provide direct information on global changes in gene reg- ulation at the protein level. Comparisons of mRNA and pro- tein profiles in mammalian cells, yeast, and bacteria have often disclosed poor correlation, likely due to the post-tran- scriptional and post-translational regulation of genes [23– 27]. In recent years, the proteome of H. pylori has been extensively investigated by several groups [28–33], but direct comparison of mRNA and protein expression has not been reported. Here, to elucidate the global regulation of H. pylori protein in response to changing growth conditions, we assessed the proteome profiles of H. pylori cultured in vitro and observed a substantial discordance in mRNA and pro- tein patterns.
2Materials and methods
2.1Bacteria and culture conditions
H. pylori strain 26695 was purchased from the American Type Culture Collection (Manassas, VA, USA). After revival from frozen stocks, the bacteria were precultured for 24–48 h on Brucella broth (Difco, Sparks, MD, USA) agar plates con- taining 10% horse serum (Gibco BRL, Life Technologies, Rockville, MD, USA) at 377C in an incubator under 10% CO2 atmosphere. Cultured H. pylori were collected from the agar plates, and the suspensions were adjusted with Brucella broth to an OD at 600 nm of 1.0 and diluted to an OD600 of 0.05 with Brucella broth supplemented with 10% new born bovine serum (Gibco BRL). Twenty-milliliter aliquots were distributed into six 100 mL flasks, which were filled with mixed gas (N2/CO2/O2 = 85:10:5 v/v/v). Bacterial cells were cultured at 377C with agitation at 200 rpm, and one flask each was taken for cell harvest at 6, 12, 24, 36, 48, and 60 h, respectively.
2.2Preparation of bacterial cell lysates
Bacterial cells were harvested by centrifugation at 50006g for 10 min at 47C, washed in ice-cold PBS (pH 7.0) and kept frozen at 2807C until used. Bacterial cells were lysed by a motor-driven homogenizer in a solution of 7 M urea, 2 M thiourea containing 4% CHAPS, 1% DTT, and 2% v/v phar- malyte (pH range 4–10; Amersham Biosciences, Buck- inghamshire, UK). After centrifugation at 15 0006g for 60 min at 157C, insoluble material was discarded and the soluble fractions were saved for 2-DE analyses. The protein concentration of bacteria cell lysates was measured using Bradford protein assay reagent (Sigma, St. Louis, MO, USA) with BSA as a standard [34], and the cell lysates were stored at 2707C until 2-DE analysis.
2.32-DE analysis
IEF and second dimension SDS-PAGE were performed as described previously, with some modifications [35]. Briefly, 200 mg protein was applied to each 23 cm IPG strip (non- linear, pH gradient 4–10; Genomine, Pohang, Korea) that had been equilibrated overnight with a solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, and 1% v/v pharmalyte (pH range 4–10, Amersham Biosciences). For IEF, voltage was linearly increased from 150 to 3500 V dur- ing the first 3 h for sample entry and maintained at 3500 V for a total of 98 kV ? h. Following IEF, each strip was sub- jected to second dimension electrophoresis on a 10–16% gradient SDS-polyacrylamide gel (20624 cm2) using the ISO-DALT 2D gel system (Amersham Biosciences). Each gel was silver-stained as described previously [36], except that the fixing and sensitization steps with glutaraldehyde were omitted.
2.4Image acquisition and analysis of 2-DE gels
Image analysis of 2-DE gels was performed using PDQuest software, as described by the manufacturer (BioRad, Her- cules, CA, USA). A gel run with a culture harvested at 12 h was used as a reference pattern for spot analysis, and all spot files were matched to the reference pattern. Each matched spot in each pattern was numbered, and the quantity of each spot was normalized by total valid spot intensity. Two-DE analysis was repeated three times using independently grown cultures, and results were expressed as mean 6 SD. Statistical differences between the same spots at different time points were determined using Stu- dent’s t-test, and p values less than 0.05 were considered significant. Self-organizing map (SOM) analysis using the Avadis program (Strand Genomics, Bangalore, India) was used to group and display protein spots with similar pro- files.
2.5Enzymatic digestion of protein in-gel
Protein spots showing altered intensity on 2-DE gels were excised from the gels and enzymatically digested in-gel with sequence grade porcine trypsin (Promega, Madison, WI, USA) as described previously [37]. Gel pieces were washed with 50% ACN to remove SDS, salt and stain, and vacuum- dried to remove solvent, rehydrated with trypsin (8–10 ng/
mL) and incubated for 8–10 h at 377C. The proteolytic reac- tion was terminated by addition of 5 mL of 0.5% TFA. Tryptic peptides were recovered by combining the aqueous phases from several extractions of gel pieces with 50% aqueous ACN. After concentration, the peptide mixture was desalted using ZipTip (Millipore, Bedford, MA, USA), and the pep- tides were eluted in 1–5 mL ACN. An aliquot of this solution was mixed with an equal volume of a saturated solution of CHCA in 50% aqueous ACN, and 1 mL of mixture was spot- ted onto a target plate.
2.6MALDI-TOF MS analysis and database search
Protein analysis was performed using an Ettan MALDI-TOF mass spectrometer (Amersham Biosciences). Peptides were evaporated with a N2 laser at 337 nm using a delayed extrac- tion approach. The peptides were accelerated with a 20 kV injection pulse for TOF analysis. Each spectrum was the cumulative average of 300 laser shots. The search program ProFound, developed by Rockefeller University (http://
prowl.rockefeller.edu/prowl-cgi/profound.exe), was used to search the Swiss-Prot and NCBI databases. Spectra were calibrated using trypsin autodigestion ion peaks m/z (842.510, 2211.1046) as internal standards. The general pa- rameters for PMF searching were considered to allow max- imum one missed cleavage, a peptide charge state of 11, peptide mass tolerance of 60.2 Da (monoisotopic mass), partial oxidation of methionine, and carbamidomethylation of cysteine. Searches were restricted to the taxonomy cate- gory of Proteobacteria (purple nonsulfur bacteria). The number of matching/used peptides and the percent coverage of the identified protein were considered.
2.7Semiquantitative RT-PCR analysis
Total cellular RNA was isolated from harvested bacteria using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Grand Island, NY, USA). Each preparation was incubated with 2 U of RNase-free DNase (Promega) at 377C for 30 min, extracted with phenol/chloro- form (1:1 v/v) and precipitated with ethanol. Purified RNA was quantified spectrophotometrically using GeneQuant Pro (Amersham Biosciences) and resolved on an ethidium bro- mide-containing agarose gel to check integrity and correct quantification.
For reverse transcription (RT), 3 mg of total RNA was incubated with 4 mg of random hexamers (Amersham Bio- sciences) at 657C for 10 min and cooled on ice for 2 min. To
each aliquot was added 2 U of M-MLV RT (Invitrogen), the volume was adjusted to 50 mL, and each was incubated at 427C for 1 h, followed by heating at 957C for 5 min to termi- nate the reaction. Each RT reaction product was diluted to 50 mL with water, and 5 mL was used as the template cDNA for PCR. PCR was carried out in 50 mL reaction mixture containing a DNA template, 25 pmol of each gene-specific primer (Table 1), 16Taq buffer, 2.5 mM MgCl2, and 1 U of Taq DNA polymerase (Takara Korea, Seoul, Korea). The amplification protocol consisted of different numbers of cycles of denaturation at 957C for 30 s, annealing at 557C for 30 s, and extension at 727C for 60 s, followed by a final extension at 727C for 5 min. The resultant PCR products were resolved on ethidium bromide-containing 2.5% agarose gels, which were scanned on a UV-illuminator. The DNA bands were quantitated using a Gel Logic 100 Imaging sys- tem (Eastman Kodak, Rochester, NY, USA).
2.8Antibody preparation
Antibodies specific for H. pylori proteins were prepared by immunizing mice with protein spots excised from 2-DE gels (Pfr and UreB) or recombinant proteins expres- sed in E. coli (NapA, HydB, FtsA, and TsaA). For expres- sion of recombinant proteins, H. pylori genes were PCR amplified from genomic DNA using gene-specific prim- ers and cloned into the expression vector pET-28a(1) (Novagen, Madison, WI, USA). Recombinant proteins were expressed in E. coli BL21(DE3)pLysS (Novagen) and purified by affinity chromatography using His-bind col- umns (Novagen).
Six-wk-old male ICR mice (SLC, Hamamatsu, Japan) were immunized intraperitoneally with H. pylori proteins emulsified in complete Freund’s adjuvant (Sigma) and boost- ed twice at 2-wk intervals with the same proteins emulsified in incomplete Freund’s adjuvant. Blood was collected 2 wk after the final immunization, allowed to clot at room tem- perature for 4 h and centrifuged. The sera were divided into aliquots and kept frozen at 2707C.
2.9Immunoblot analysis
Aliquots containing 20 mg protein from whole cell lysates were fractionated on 12.5% SDS-PAGE gels and transferred to NC membranes (Hybond ECL; Amersham Biosciences). The membranes were incubated for 2 h at room temperature in PBS containing 0.05% Tween-20 (PBST) and 5% nonfat dry milk, followed by incubation with specific antibodies (1:500 in PBST containing 5% nonfat dry milk) overnight at 47C. After extensive washing in PBST, the membranes were incubated with HRP-conjugated donkey anti-mouse IgG antibody (Serotec, Kidlington, Oxford, UK; 1:2000) for 1 h at room temperature, and immunoreactive proteins were detected using the ECL Western blot substrate (Amersham Biosciences) and exposure to X-ray film.
Table 1. Forward and reverse primers used for RT-PCR
TIGR ORF no.
Gene
name
Forward primer
Reverse primer
Product size (bp)
23S RNA 50 -aacgagattccctaagtagt-30 50 -tcaggttctatttcactccg-30 320
HP0294 amiE 50 -cacactaagaatgaggtgtt-30 50 -cttatcattgacaagaatca-30 297
HP1038 aroQ 50 -gacccaaggctttatggtatg-30 50 -cacgcctccacaagccgctcc-30 324
HP1134 atpA 50 -acttgattgcgatatggctg-30 50 -tcacccaaagcgtttagcac-30 279
HP0526 cag6 50 -gatagcaacgatccgcaaga-30 50 -tctatgaatggcggtaaatact-30 302
HP0544 cag23 50 -gcattttgcagacaataattttg-30 50 -ttcactcaaatcgtcatttaaga-30 313
HP0545 cag24 50 -cgaatttagattttgatagtttcaa-30 50 -gcagcgttgtttgttggtcg-30 290
HP0109 dnaK 50 -acaaccaactccgcaatggc-30 50 -gaaatctcaatcgcgcaagc-30 284
HP0687 feoB 50 -gcacaagcgttatgggattt-30 50 -ctcttcccatgtaagccaaa-30 300
HP0601 flaA 50 -ggataaggctatggatgagcag-30 50 -taacgcacctgtagcgatacg-30 306
HP0978 ftsA 50 -atagcggcaattccatacgc-30 50 -caaatgctcttctaagccgc-30 314
HP1325 fumC 50 -ggcattatcaaggcgtgcga-30 50 -ttaacagattctccaaactagg-30 292
HP1027 fur 50 -tagagcgcttgagaatgtcta-30 50 -atgtcatggctaatcagcttg-30 376
HP0960 glyQ 50 -gagtattggaagaatcaagg-30 50 -atgctcataaggtttatccc-30 284
HP0010 groEL 50 -aagaagtagcgagcaaaacc-30 50 -catagcgtcagcgatgagtt-30 275
HP0011 groES 50 -tttcagccattaggagaaag-30 50 -tgtatgacaacaagagcctg-30 285
HP0697 HP0697 50 -gatgacaaaatcgtcttaccc-30 50 -tcatggatcacaggataccaa-30 290
HP0632 hydB 50 -gtagtcgatcctatcactagga-30 50 -agcagcgccatgttcatcaa-30 287
HP1379 lon 50 -cgcagcggccttgcatttaa-30 50 -aatctccttaaacgcttcttctt-30 313
HP1355 nadC 50 -acgctttagagttgttggaa-30 50 -caaagatccttaaaaggggt-30 261
HP0243 napA 50 -gcaagcggatgcgatcgtgt-30 50 -cagcggtgttagagagctct-30 311
HP0653 pfr 50 -agttgctaaacgaacaagtg-30 50 -gttcagacacataccattgc-30 357
HP0397 serA 50 -gatgcgctcatcactcgcag-30 50 -gtgccataccaatcttctct-30 281
HP1563 tsaA 50 -atgttagttacaaaacttgccccag-30 50 -gtaatatcagccaccataggga-30 307
HP0824 trxA 50 -atgagtcactatattgaattaac-30 50 -tgatggacgacttcgccatc-30 259
HP0072 ureB 50 -tgagccaatccaacaaccct-30 50 -catggttgttacaccgcttg-30
3Results and discussion
3.1Analysis of proteome profiles of H. pylori at various growth stages
To obtain protein profiles of H. pylori at various phases of growth, H. pylori 26695 was cultured in liquid media and harvested at 6, 12, 24, 36, 48, and 60 h. Consistent with our previous results [38], the growth curve obtained from three independent cultures showed a continuous increase in cell density until 48 h, after which it reached a plateau (Fig. 1).
305
Whole cell lysates prepared from cultures harvested at each time point were subjected to 2-DE analysis, and their pro- teome profiles are obtained (see Fig. S1 in Supporting Infor- mation). A gel of H. pylori cultured for 12 h, which contained approximately 1200 spots, was used as the reference pattern for spot analysis. Protein spots on 2-DE gels were matched against the reference map, and matched spots were assigned numbers. To obtain reliable data, we repeated 2-DE analysis with three sets of independent cultures grown on different days. A total of 366 spots were detected on all 18 gel images and selected for clustering analysis. The mean spot intensity at each time point was compared with that at 6 h, and SOM clustering analysis was performed using the log10 ratio for each spot. The results showed that the overall protein profile
Figure 1. Growth curve of H. pylori 26695. Shown are the mean 6SD of values obtained from three independent cultures, which were also used for 2-DE and RT-PCR analyses. The arrow indicates the time point at which there were marked decreases in mRNA transcript levels for a number of genes, as determined by RT-PCR.
did not change dramatically during the culture period, but was in accordance with the growth phases of the culture (Fig. 2). Approximately 50% of the protein spots displayed increased intensity from the log to stationary phase while 30% of the spots were decreased in intensity over time. Sev- eral spots showed increased or decreased level only at the
Figure 2. Clustering analysis of H. pylori protein spots detected during in vitro culture. The cluster diagram displays the self- organizing map of the profiles of 366 protein spots detected on all 3 sets of 18 gels. Red bands indicate increased, and green bands indicate decreased, protein level over time. The values represent the mean fold-changes relative to the intensity at 6 h obtained from three different gels, with the color bar denoting log10 (fold- change in protein spot intensity).
early log phase but remained unchanged through the rest of the culture period. We performed a statistical analysis using Student’s paired t-test (95% confidence interval) to select spots showing significant changes in intensity at any time point relative to its intensity at 6 h. The intensity of 151 spots was significantly altered at any time point. The number of spots with altered intensity increased over time, from 37 spots at 12 h to 83 spots at 48 h, after which it remained constant. These results suggested that the expression of many proteins encoded by H. pylori genes is regulated in a growth phase-dependent fashion.
3.2Identification of H. pylori proteins showing differential regulation during in vitro culture
Among the 151 protein spots showing significant changes in intensity during in vitro culture, 118 H. pylori spots exhibited ti 2-fold increase (n = 84) or decrease (n = 34) rela- tive to the level of the culture at 6 h. To identify spots showing changes in intensity, each was excised, digested in-gel with
trypsin, and subjected to MALDI-TOF MS analysis. The pep- tide profiles were used for database searches (Fig. S2 in Sup- porting Information). In total, 57 protein spots were identi- fied; 46 spots with increased intensity during in vitro culture are listed in Table 2, and 11 spots with decreased intensity in Table 3. Some ofthe identified protein spots are also displayed in Fig. 3. Supporting Information Table S1 provides complete information supporting the identity of each protein spot including the peak lists of MALDI-TOF mass spectra, sequence coverage, the number of peptides matched/used, and theoretical/observed Mr and pI. The levels of protein spots at each time point are shown in Supporting Information Table S2. The identified proteins could be functionally classi- fied into various groups [39], including those involved in amino acid biosynthesis, energy metabolism, replication, transcription, translation, and production of envelope, indi- cating global changes in H. pylori protein regulation. Many of the proteins with increased levels are involved in energy me- tabolism, amino acid biosynthesis, and translation, in agree- ment with the fact that bacterial cells undergo high energy metabolism as well as vigorous protein synthesis during growth. The protein spot displaying the highest increase was single-strand DNA-binding protein (Ssb; 60.6-fold), followed by fumarate reductase iron–sulfur subunit (FrdB; 11.2-fold). Protein spots exhibiting the highest decrease were a hypo- thetical protein HP0697 (5.6-fold) and nicotinate-nucleotide pyrophosphorylase (NadC; 4.9-fold). HP0697 was detected as three spots on 2-DE gels, one of which was decreased while the other two were increased in intensity over time (see below). NadC was also present as two spots, one of which was increased while the other was reduced.
3.3Comparison of mRNA and protein levels of H. pylori genes
To determine whether the proteins showing altered levels according to growth phase are regulated at the transcriptional level, we selected 25 genes and measured their mRNA tran- script levels by semiquantitative RT-PCR. We found that these 25 genes could be classified into three groups based on their mRNA expression patterns (Fig. 4A). The seven genes in Group I, consisting of ferric uptake regulator (fur), neu- trophil activating protein (napA), fumarase (fumC), thio- redoxin (trxA), nonheme iron-containing ferritin (pfr), chap- erone and heat shock protein 70 (dnaK), and 3-dehy- droquinate dehydratase II (aroQ), exhibited a high mRNA transcript level from 6 to 24 h but showed marked reduction thereafter to a basal level. The 11 genes in Group II, including alkyl hydroperoxide reductase (tsaA), three cag pathogenicity island proteins (cag6, cag23, and cag24), nadC, ATP-depend- ent protease (lon), phosphoglycerate dehydrogenase (serA), iron(II) transport protein (feoB), and cell division protein (ftsA), were also highly expressed from 6 to 12 or 24 h and decreased thereafter. Unlike the genes of Group I, however, the mRNA expression of these genes was induced again at the stationary phase, being similar to that of 6 h, suggesting
Table 2. List of protein spots with increased intensity during in vitro culture
Spot TIGR ORF Protein name Gene Fold-change in protein levela) Reference
no. no.
12 h 24 h 36 h 48 h 60 h
2616R HP0010 Chaperone and heat shock protein groEL 1.5 2.1 2.2b) 1.6 2.0 [28,29,31,32]
3604R HP0010 Chaperone and heat shock protein groEL 1.7 1.9 2.2b) 1.8 2.1b) [28,29,31,32]
4320R HP0019 Chemotaxis protein cheV 21.3 1.3 1.8b) 2.0b) 2.0b)
7312R HP0096 Phosphoglycerate dehydrogenase 1.4 1.4 2.0b) 1.8 2.0b) [32]
3309R HP0107 Cystein synthetase cysK 1.1 2.7 2.5b) 3.3b) 2.9b)
3302R HP0153 Recombinase recA 21.1 2.0b) 2.5b) 2.9b) 3.4b)
3404R HP0154 Enolase eno 1.5 2.0b) 2.2b) 2.0b) 2.3b) [29]
3202R HP0191 Fumarate reductase, iron–sulfur subunit frdB 1.6 1.6 2.2b) 2.0 1.8 [33]
2214R HP0191 Fumarate reductase, iron–sulfur subunit frdB 7.7 6.6 11.2b) 10.9 7.5 [33]
5405R HP0197 S-adenosylmethionine synthetase 2 metX 1.1 1.1 1.7b) 1.7 2.2 [31, 32, 33]
8221 HP0240 Octaprenyl-diphosphate synthese ispB 1.0 1.6 1.6b) 1.7b) 2.1
2009R HP0243 Neutrophil activating protein napA 21.3 5.1 8.1b) 6.8b) 5.9b) [28, 29, 30,
31, 32]
4904R HP0243 Neutrophil activating protein napA 2.1b) 1.5 1.1 1.1 1.1 [28, 29, 30,
31, 32]
5914R HP0264 ATP-dependent protease binding subunit clpB 2.1b) 2.1b) 2.7b) 2.2b) 2.7b) [29]
8511R HP0380 Glutamate dehydrogenase gdhA 2.2 1.9 1.2 2.4 4.8b)
7605R HP0397 Phosphoglycerate dehydrogenase serA 1.8b) 2.4b) 2.6b) 2.9b) 3.1b) [29, 30]
7609R HP0397 Phosphoglycerate dehydrogenase serA 1.9 3.7b) 3.7 5.3b) 6.9b) [29, 30]
7603R HP0399 Ribosomal protein S1 rps1 3.2 3.4b) 3.6 3.9 5.2 [29]
3402R HP0500 DNA polymerase III beta-subunit dnaN 1.4 2.0b) 2.4b) 2.7b) 2.7b) [29]
2108R HP0591 Ferridoxin oxidoreductase gamma subunit oorC 1.6 2.7b) 3.4b) 4.4b) 4.1
7706R HP0617 Aspartyl-tRNA synthetase aspS 1.1 1.8 2.6b) 3.0b) 2.6b) [29]
8707R HP0632 Quinone-reactive Ni/Fe hydrogenase, large subunit hydB 2.6 1.6b) 1.8 2.7b) 1.2
8709R HP0632 Quinone-reactive Ni/Fe hydrogenase, large subunit hydB 1.2 1.8b) 2.4b) 2.5b) 3.4b)
8717R HP0632 Quinone-reactive Ni/Fe hydrogenase, large subunit hydB 1.7 2.0 2.1b) 2.7b) 2.0b)
2102R HP0692 3-Oxoadipate CoA-transferase subunit B yxjE 1.7 2.5 4.0b) 4.8b) 4.4b) [29, 31]
1116R HP0697 Hypothetical protein 2.5 6.5 4.8b) 6.1 3.3b) [32]
6511R HP0734 Conserved hypothetical protein 1.3 1.5 2.0 2.2b) 1.7
4418R HP0744 Tyrosyl-tRNA synthetase tyrS 21.4 1.2 1.5b) 2.2b) 1.9b)
4519R HP0858 ADP-heptose synthase rfaE 1.0 1.5b) 1.7b) 2.0b) 1.8b) [29]
7314R HP0859 ADP-L-glycero-D-mannoheptose-6 epimerase rfaD 2.2 3.1 5.1b) 3.4 6.7b) [29]
6601R HP0978 Cell division protein ftsA 1.1 1.8b) 1.2 1.7 1.4 [29]
1005R HP1038 3-Dehydroquinate dehydratase II aroQ 21.1 1.8 3.1 4.9b) 4.9 [32]
4315R HP1059 Holliday junction DNA helicase ruvB 21.1 1.6 2.2b) 2.2b) 2.3b)
6416R HP1112 Adenylosuccinate lyase purB 1.4 1.6b) 2.9 2.0 2.6b)
4419R HP1179 Phosphopentomutase deoB 1.4 2.9 4.2b) 5.2b) 4.2b)
5402R HP1179 Phosphopentomutase deoB 1.4 3.0b) 2.6b) 2.6b) 2.6b)
8301R HP1193 Aldo-keto reductase, putative 21.5 1.7 1.4 1.2 2.2b) [29]
8311R HP1193 Aldo-keto reductase, putative 1.6 1.9b) 1.8b) 2.2b) 2.5b) [29]
5111R HP1245 Single-strand DNA binding protein ssb 19.3 43.3b) 60.6b) 50.5b) 42.5b) [31]
0305R HP1293 DNA-directed RNA polymerase, alpha subunit rpoA 21.4 1.1b) 2.2b) 1.8 2.5 [29, 32, 33]
8502R HP1325 Fumarase fumC 1.0 1.6 3.6b) 2.2b) 2.5 [29]
1316R HP1373 Rod shape-determining protein mreB 1.2 5.7 2.5 2.7 3.3b) [33]
7201R HP1375 UDP-N-acetylglucosamne acyltransferase lpxA 1.3 2.1 3.1 3.2b) 3.4b) [29]
7918R HP1379 ATP-dependent protease lon 2.1b) 1.6 1.6 1.2 1.2
5530 HP1420 Flagella export protein ATP synthase flil 1.1 2.7b) 2.2 2.2 1.7
7107R HP1563 Alkyl hydroperoxide reductase
a)mean fold-increase when compared with 6 h cultures.
b)p,0.05 when compared with the spot intensity of 6 h cultures.
tsaA 1.1 1.7b) 1.0 21.4 1.6 [28,29,31,32]
that their expression is inversely correlated with the growth pattern of the culture. The seven genes in Group III consist of those whose expression remained constant throughout the cul-
ture. These results suggest that the log-stationary switch at the transcriptional level occurs between 24 and 36 h, earlier than theswitchesobservedinproteinexpressionandgrowthprofiles.
Figure 3. Identification of H. pylori pro- tein spots showing changes in intensity during in vitro culture. The names of genes and spot numbers are shown.
Table 3. List of protein spots with decreased intensity during in vitro culture
Spot TIGR Protein name Gene Fold-change in protein levela) Reference
no. ORF no.
12 h 24 h 36 h 48 h 60 h
5006R HP0268 Hypothetical protein 3.4b) 3.7b) 3.6b) 3.1b) 4.5b)
8422R HP0360 UDP-glucose 4-epimerase galE 3.4b) 3.7 3.6 3.1 4.5
0112R HP0526 Cag pathogenicity island protein cag6 2.0 1.5 1.4b) 1.7 2.1b)
8716R HP0632 Quinone-reactive Ni/Fe
hydrogenase, large subunit
hydB 1.0
1.2 2.4 2.8b)
1.6
1109R HP0697 Hypothetical protein 2.2b) 1.8b) 3.8b) 3.9b) 5.6b) [32]
3301R HP0960 Glycyl-tRNA synthetase,
alpha subunit
glyQ
2.1
1.3
2.5b)
2.6b)
2.6b)
6305R HP1052 UDP-3-O-acyl N-acetylglucosamine
deacetylase
envA 2.4
2.3b)
3.1b)
3.1b)
2.7b)
5529 HP1190 Histidyl-tRNA synthetase hisS 2.5b) 2.6 2.0 1.7 1.7
7509R HP1325 Fumarase fumC 1.7 2.3b) 1.6 1.4 1.0 [29]
8201R HP1355 Nicotinate-nucleotide pyro-
phosphorylase
nadC 3.2b)
2.6b)
4.1b)
4.6b)
4.9b)
1205R HP1481 Hypothetical protein 2.5 1.9 2.4 3.4 4.2b)
a)Mean fold-decrease when compared with 6 h cultures.
b)p,0.05 when compared with the spot intensity of 6 h cultures.
Next, we compared the mRNA transcript and protein levels of each H. pylori gene, as determined from total spot intensity on 2-DE gels and scanning of RT-PCR products, respectively (Fig. 4B). We also performed immunoblot anal- ysis to confirm the total cellular levels of the proteins. When we compared the mRNA and protein levels of six selected genes, we found that while three proteins, urease beta sub- unit (UreB), Pfr, and NapA, showed similar patterns of reg- ulation, slightly increasing over time, their mRNA expres- sion profiles were quite different. Transcript encoding UreB was constant throughout, whereas those encoding Pfr and NapA remained high until 24 h, but markedly decreased thereafter. Two other proteins, TsaA and FtsA, showed reg- ulation patterns generally coinciding with the growth curve
of the culture. In contrast, their mRNA transcript levels exhibited the opposite response, indicating that the mRNA and protein levels of these two genes are inversely correlated. In addition, we found that for quinone-reactive Ni/Fe hydro- genase large subunit (HydB) the protein level as well as its mRNA expression was independent of the growth phase. Taken together, these findings demonstrate that the expres- sion of H. pylori genes is differentially regulated at the mRNA and protein levels during in vitro culture. Therefore, genes under the same type of transcriptional control may not be under the same type of control at the protein level, leading to diverse physiological responses to environmental stimuli.
Some of the H. pylori genes examined in this study exhibited mRNA expression profiles substantially different
Figure 4. Comparison of H. pylori mRNA and protein levels. (A) H. pylori gene mRNA transcript levels were assessed by semiquantitative RT-PCR. Gels shown are representative of three independent sets of six cultures. (B) Total protein spot intensities obtained from 2-DE analysis (bar) were compared with mRNA levels determined by RT-PCR (solid line). The growth curve of the culture is also shown (broken line). Immunoblot analysis was performed to confirm total protein levels (below graphs).
from those reported previously [21]. For example, we found that pfr mRNA expression was decreased between 24 and 36 h, the apparent time point for the switch from the log to the stationary phase, whereas it was previously found to be significantly induced at the log-stat switch [21]. In addition, we found that flaA mRNA expression was constant over time, whereas it was earlier shown to be induced at the sta- tionary phase [21]. Among the factors that may have influ- enced the expression of these genes are the growth condi- tions, which have been shown to substantially alter H. pylori protein expression profiles [32]. Another possibility is the difference between H. pylori strains, in that we used strain 26695 whereas the previous report used strain SS1. To assess this possibility, we assayed pfr mRNA expression in strain SS1 cultured under the same conditions as 26695. We found that pfr mRNA expression peaked at 48 h and decreased at 60 h, in good agreement with the previous report (data not shown). Therefore, it may not be appropri- ate to directly compare results obtained using different H. pylori strains.
3.4Modification of H. pylori proteins as a function of growth stage
Many proteins were detected on 2-DE gels as more than one spot (Tables 2 and 3, and see also Supporting Information Table S2) as reported in previous studies on the proteome analysis of H. pylori [28–33]. Multiple forms of proteins on 2-DE gels may reflect multimerization, modification, or fragmentation of proteins, which may be due to PTM or artificial chemical modifications during sample preparation. We therefore compared the relative intensity of multiple spots for each protein at each time point (Fig. 5 and see also Supporting Information Table S2). Although the four Pfr spots did not show major changes in relative intensity over time in culture, other proteins represented by multiple spots, including NadC, AroQ, HP0697, FumC, and FtsA, showed different relative ratios of spots depending on growth phase. For example, of the three AroQ spots, Spot 1005R initially represented only 30% of the total AroQ protein but gradually increased over time, representing 60% of the total AroQ
Figure 5. Relative levels of spots of selected proteins detected during in vitro culture, as deter- mined by 2-DE analysis. Shown are the mean values of spots from three gels obtained using independent cultures.
protein at the end of the culture. In contrast, Spot 1020R showed a relative decrease over time, from 50 to 27% of the protein. The three HP0697 spots exhibited relative inten- sities similar to the AroQ spots. Two other proteins, FumC and FtsA, each of which was present as two spots on 2-DE gels, showed distinct differences in their relative ratios, in that one spot for each protein reached a maximum ratio at 24 h, when the transcriptional switch occurred, and decreased thereafter. Taken together, these findings suggest that PTM of H. pylori proteins is regulated as a function of growth phase.
4Concluding remarks
Although transcription is thought to be the major step at which gene expression is regulated, many genes are subject to post-transcriptional control, including mRNA stability, translation, PTM and protein stability. In this study, we ana- lyzed the global protein profiles of H. pylori cells harvested at various stages of growth in vitro and evaluated the correlation of mRNA and protein levels at each time point. We found that the proteome profiles of H. pylori were largely similar to
the bacterial growth pattern, while the mRNA expression profiles exhibited an earlier turn-off of transcription. These results suggest that the bacterial perception of environmen- tal changes during culture led to rapid alterations in mRNA level, but slower changes in protein level and bacterial cell physiology. We also observed considerable discrepancies be- tween mRNA and protein levels of several genes at different time points, indicating that mRNA and protein may not be coordinately regulated. The clustering of 2-DE protein pro- files did not show dramatic changes throughout the culture period. By contrast, mRNA expression profiles using DNA microarrays showed a dramatic shift of many genes during the log to stationary switch [21]. This may reflect a bacterial strategy to maintain the homeostasis of intracellular envi- ronments by regulating mRNA expression in response to changes outside the cells but maintaining protein levels relatively constant.
The approaches employed in this study have provided a comprehensive understanding of the systemic regulation of mRNA and protein of H. pylori genes during growth stages, a pattern of regulation which would not be evident from data obtained at any single time point. One advantage of prote- omics technology is that it can detect the modification of
expressed proteins. Our results suggest that several H. pylori proteins undergo PTMs, some of which are modulated as a function of the growth phase of the culture. Our findings clearly demonstrate that H. pylori utilizes modulation of protein regulation and modification as mechanisms to cope with changing growth environments and should provide insights into the adaptive mechanisms utilized by H. pylori within the context of its growth environments.
This work was supported by a grant from the Seoul R & BD Program (No. 10582), Republic of Korea.
The authors have declared no conflict of interest.
5References
[1]Dunn, B. E., Cohen, H., Blaser, M. J., Helicobacter pylori. Clin. Microbiol. Rev. 1997, 10, 720–741.
[2]Blaser, M. J., Atherton, J. C., Helicobacter pylori persistence: Biology and disease. J. Clin. Invest. 2004, 113, 321–333.
[3]Akada, J. K., Shirai, M., Takeuchi, H., Tsuda, M., Nakazawa, T., Identification of the urease operon in Helicobacter pylori and its control by mRNA decay in response to pH. Mol. Microbiol. 2000, 36, 1071–1084.
[4]Eaton, K. A., Krakowka, S., Effect of gastric pH on urease- dependent colonization of gnotobiotic piglets by Helico- bacter pylori. Infect. Immun.1994, 62, 3604–3607.
[5]Eaton, K. A., Suerbaum, S., Josenhans, C., Krakowka, S., Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect. Immun. 1996, 64, 2445–2448.
[6]Kavermann, H., Burns, B. P., Angermuller, K., Odenbreit, S. et al., Identification and characterization of Helicobacter pylori genes essential for gastric colonization. J. Exp. Med. 2003, 197, 813–822.
[7]Guillemin, K., Salama, N. R., Tompkins, L. S., Falkow, S., Cag pathogenicity island-specific responses of gastric epithelial cells to Helicobacter pylori infection. Proc. Natl. Acad. Sci. USA 2002, 99, 15136–15141.
[8]Segal, E. D., Cha, J., Lo, J., Falkow, S., Tompkins, L. S., Altered states: Involvement of phosphorylated CagA in the induction of host cellular growth changes by Helico- bacter pylori. Proc. Natl. Acad. Sci. USA 1999, 96, 14559– 14564.
[9]Higashi, H., Tsutsumi, R., Muto, S., Sugiyama, T. et al., SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 2002, 295, 683– 686.
[10]Cover, T. L., Blanke, S. R., Helicobacter pylori VacA, a para- digm for toxin multifunctionality. Nat. Rev. Microbiol. 2005, 3, 320–332.
[11]Satin, B., Del Giudice, G., Della Bianca, V., Dusi, S. et al., The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor. J. Exp. Med. 2000, 191, 1467–1476.
[12]Merrell, D. S., Goodrich, M. L., Otto, G., Tompkins, L. S., Falkow, S., pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect. Immun. 2003, 71, 3529– 3539.
[13]Hammer, B. K., Swanson, M. S., Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol. Microbiol. 1999, 33, 721–731.
[14]Bachman, M. A., Swanson, M. S., RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase. Mol. Microbiol. 2001, 40, 1201– 1214.
[15]Primm, T. P., Andersen, S. J., Mizrahi, V., Avarbock, D. et al., The stringent response of Mycobacterium tuberculosis is required for long-term survival. J. Bacteriol. 2000, 182, 4889– 4898.
[16]Marshall, B. J., Warren, J. R., Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 1, 1311–1315.
[17]Benaissa, M., Babin, P., Quellard, N., Pezennec, L. et al., Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to the coccoid form. Infect. Immun. 1996, 64, 2331–2335.
[18]Wells, D. H., Gaynor, E. C., Helicobacter pylori initiates the stringent response upon nutrient and pH downshift. J. Bac- teriol. 2006, 188, 3726–3729.
[19]Mouery, K., Rader, B. A., Gaynor, E. C., Guillemin, K., The stringent response is required for Helicobacter pylori survi- val of stationary phase, exposure to acid, and aerobic shock. J. Bacteriol. 2006, 188, 5494–5500.
[20]Gaynor, E. C., Wells, D. H., MacKichan, J. K., Falkow, S., The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes. Mol. Microbiol. 2005, 56, 8–27.
[21]Thompson, L. J., Merrell, D. S., Neilan, B. A., Mitchell, H. et al., Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression. Infect. Immun. 2003, 71, 2643–2655.
[22]Merrell, D. S., Thompson, L. J., Kim, C. C., Mitchell, H. et al., Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect. Immun. 2003, 71, 6510– 6525.
[23]Lian, Z., Wang, L., Yamaga, S., Bonds, W. et al., Genomic and proteomic analysis of the myeloid differentiation program. Blood 2001, 98, 513–524.
[24]Chen, G., Gharib, T. G., Huang, C. C., Taylor, J. M. et al., Dis- cordant protein and mRNA expression in lung adenocarci- nomas. Mol. Cell. Proteomics 2002, 1, 304–313.
[25]Griffin, T. J., Gygi, S. P., Ideker, T., Rist, B. et al., Com- plementary profiling of gene expression at the tran- scriptome and proteome levels in Saccharomyces cerevi- siae. Mol. Cell. Proteomics 2002, 1, 323–333.
[26]Tian, Q., Stepaniants, S. B., Mao, M., Weng, L. et al., Inte- grated genomic and proteomic analyses of gene expres- sion in mammalian cells. Mol. Cell. Proteomics 2004, 3, 960–969.
[27]Basler, M., Linhartova, I., Halada, P., Novotna, J. et al., The iron-regulated transcriptome and proteome of Neisseria meningitidis serogroup C. Proteomics 2006, 6, 6194–6206.
[28]Jungblut, P. R., Bumann, D., Haas, G., Zimmyarndt, U. et al., Comparative proteome analysis of Helicobacter pylori. Mol. Microbiol. 2000, 36, 710–725.
[29]Cho, M. J., Jeon, B. S., Park, J. W., Jung, T. S. et al., Identifying the major proteome components of Helico- bacter pylori strain 26695. Electrophoresis 2002, 23, 1161–1173.
[30]Baik, S. C., Kim, K. M., Song, S. M., Kim, D. S. et al., Prote- omic analysis of the sarconsine-insoluble outer membrane fraction of Helicobacter pylori strain 26695. J. Bacteriol. 2004, 186, 949–955.
[31]Backert, S., Kwok, T., Schmid, M., Selbach, M. et al., Sub- proteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electro- phoresis and mass spectrometry. Proteomics 2005, 5, 1331– 1345.
[32]Uwins, C., Deitrich, C., Argo, E., Stewart, E. et al., Growth- induced changes in the proteome of Helicobacter pylori. Electrophoresis 2006, 27, 1136–1146.
[33]Lin, Y. F., Wu, M. S., Chang, C. C., Lin, S. W. et al., Com- parative immunoproteomics of identification and charac- terization of virulence factors from Helicobacter pylori related to gastric cancer. Mol. Cell. Proteomics 2006, 5, 1484–1496.
H-151
[34]Bradford, M. M., A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal. Biochem. 1976, 72, 248– 254.
[35]Bjellqvist, B., Pasquali, C., Ravier, F., Sanchez, J. C., Hoch- strasser, D., A nonlinear wide-range immobilized pH gra- dient for two-dimensional electrophoresis and its defini- tion in a relevant pH scale. Electrophoresis 1993, 14, 1357– 1365.
[36]Oakley, B. R., Kirsch, D. R., Morris, N. R., A simplified ultra- sensitive silver stain for detecting proteins in polyacryl- amide gels. Anal. Biochem. 1980, 105, 361–363.
[37]Mirgorodskaya, E. P., Mirgorodskaya, O. A., Dobretsov, S. V., Shevchenko, A. A. et al., Electrospray mass spectrometric study of melittin trypsinolysis by a kinetic approach. Anal. Chem. 1995, 67, 2864–2869.
[38]Lee, H. W., Choe, Y. H., Kim, D. K., Jung, S. Y., Lee, N. G., Proteomic analysis of a ferric uptake regulator mutant of Helicobacter pylori: Regulation of Helicobacter pylori gene expression by ferric uptake regulator and iron. Proteomics 2004, 4, 2014–2027.
[39]Tomb, J. F., White, O., Kerlavage, A. R., Clayton, R. A. et al., The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997, 388, 539–547.