Global Analysis of a Plasmid-Cured Shigella flexneri Strain: New Insights into the Interaction between the Chromosome and a Virulence Plasmid Li Zhu,‡,¶ Xiankai Liu,‡,¶ Xuexue Zheng,‡ Xin Bu,‡ Ge Zhao,‡ Chaohua Xie,‡ Jingfei Zhang,‡ Na Li,‡ Erling Feng,‡ Jie Wang,§ Yongqiang Jiang,† Peitang Huang,*,‡ and Hengliang Wang*,‡ State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, 100071 Beijing, China, National Center of Biomedical Analysis, 100850 Beijing, China, and State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071 Beijing, China Received August 24, 2009
Shigella flexneri is an important human pathogen that causes dysentery, and remains a significant threat to public health, particularly in developing countries. The virulence of this pathogen is dependent on an acquired virulence plasmid. To investigate the crosstalk between the bacterial chromosome and the exogenous virulence plasmid, a virulence plasmid-cured strain was constructed using plasmid incompatibility. The global patterns of gene expression of this strain compared with the wild-type strain were analyzed using 2-DE combined with MALDI-TOF MS. Most known virulence factors of S. flexneri were identified in the 2-DE gels. Interestingly, the expression of the glycerol 3-phosphate (glp) regulonencoded proteins was increased when the virulence plasmid was absent. Microarray analysis confirmed that regulation occurred at the transcriptional level. Purification and identification of DNA binding proteins with affinity for the regulatory region of the glp genes revealed that regulation mediated by the virulence plasmid to control the expression of the glp regulon might in turn be mediated by protein GlpR. To our knowledge, this is the first study analyzing the interaction between a pathogen chromosome and a virulence plasmid at the proteomic level. Keywords: Shigella flexneri • comparative proteomics • 2-DE • plasmid cured • plasmid incompatibility • glycerol 3-phosphate regulon
Introduction Shigella spp. is a group of Gram-negative, nonspore forming, facultative pathogenic bacteria closely related to Escherichia coli. There are four species of Shigella classified based on differences in the O antigen and some biochemical reactions.1 Among these, Shigella flexneri is responsible for the majority of cases of endemic dysentery prevalent in developing countries where sanitation is poor. Because of the low infectious dose (10-100 bacteria) and the emergence of multiple resistance strains, S. flexneri is still a great threat to human health today. The virulence of S. flexneri is largely dependent on the products of a 230 kb virulence plasmid (VP). The complete DNA sequences of the VP pWR1002 and its derivative pWR5013 from a serotype 5 strain and the VP pCP3014 from a serotype 2a * Correspondence: Prof. Hengliang Wang, State Key Laboratory of Pathogen and Biosecurity, 20 Dongdajie Street, Fengtai District, Beijing, 100071; Prof. Peitang Huang, State Key Laboratory of Pathogen and Biosecurity, 20 Dongdajie Street, Fengtai District, Beijing, 100071. Tel: +86-10-63802181. Fax: +86-10-63802181. E-mail: (H.W.)
[email protected]; (P.H.) zhouxiaow@ nic.bmi.ac.cn. ¶ These authors contributed equally to this work. ‡ State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology. § National Center of Biomedical Analysis. † State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology. 10.1021/pr9007514
2010 American Chemical Society
strain have been determined. These DNA sequences showed that genes required for entry of S. flexneri into host cells are clustered within a 30 kb region (called the entry region) of the VP. Further sequence analysis of these plasmids revealed that most virulence-related genes exhibited a low G + C content (approximately 34%), suggesting that these genes might be acquired by horizontal gene transfer. The acquisition of plasmid-encoded virulence-related gene clusters is likely to have been a crucial step in the evolution of this pathogenic organism, enabling S. flexneri to evade the host immune system and invade epithelial cells. This might have given S. flexneri a survival advantage over its nonpathogenic ancestor. In contrast, some of the indigenous chromosome-encoded genes were selectively inactivated or lost. These genes, called ‘antivirulence genes’, were no longer compatible with the organisms pathogenic lifestyle. The best example is the case of the cadA gene in S. flexneri. A study had shown that the virulence phenotype of a S. flexneri serotype 2a strain transformed with the cadA gene from E. coli strain K-12 was significantly attenuated due to the expression of lysine decarboxylase, encoded by the cadA gene.5 Recently, the nadA and nadB genes were also identified as antivirulence genes in S. flexneri.6 S. flexneri has also evolved to control the expression of plasmid-encoded genes, such as virulence genes, which is another important step in the evolution of this pathogenic Journal of Proteome Research 2010, 9, 843–854 843 Published on Web 12/15/2009
research articles organism. VPs can be seen as extrachromosomal selfish genetic elements that inevitably place some burden on their host bacteria. In many cases, mechanisms have evolved to shut down the expression of plasmid-encoded virulence genes in nonhost environments. This evolutionary mechanism has been identified and extensively studied in S. flexneri.7 Expression of the genes that reside in the entry region of the VP has been found to be regulated by temperature; these genes are expressed at 37 °C but not at 30 °C.8 This regulation is dependent upon the chromosomally encoded protein H-NS (histone-like protein H1), which can bind to the virF and virB promoters to inhibit a series of cascade reactions. At 37 °C, changes in DNA conformation at the virF and virB promoters lead to the release of H-NS and the activation of downstream virulence genes.9 The fact that bacterial chromosomes can regulate the expressions of genes located in the VP might mean that the VP can similarly regulate the expression of genes located in the chromosome. In other words, there might be some crosstalk between the VP and the chromosome of S. flexneri. However, such regulatory hierarchy has scarcely been reported to date.10 In this study, we carry out a proteomic survey to investigate crosstalk between the VP and the chromosome of S. flexneri. We first constructed a VP-cured avirulent S. flexneri strain using plasmid incompatibility techniques. Then, the protein expression profile of this derivative strain, compared with the wildtype strain, in stationary phase at 30 and 37 °C was analyzed by means of two-dimensional gel electrophoresis. Differentially expressed proteins were identified by MALDI-TOF MS. Our findings showed that the expression of some glycerol 3-phosphate (glp) regulon-encoded proteins were increased in the absence of the VP. These findings will further our understanding of the crosstalk between the bacterial chromosome and the VP in S. flexneri. This study also indicated that comparative proteomic analysis is a powerful tool for the study of pathogen evolution.
Experimental Section Bacterial Strains and Growth Conditions. E. coli strain DH5R, used for plasmid constructions, was grown in LB (Luria-Bertani) agar or LB broth (Difco). Wild-type S. flexneri serotype 2a strain 2457T was grown in TSA (tryptic soy agar) (Difco) containing 0.01% Congo red or LB broth. When necessary, antibiotics were added at the following concentrations: 100 µg of ampicillin/mL, 50 µg of kanamycin/mL, and 30 µg of chloramphenicol/mL. Construction of Recombinant Plasmids and Mutations in S. flexneri. A VP-cured strain ∆pSF was constructed using plasmid incompatibility methods (see schematic in Supplemental Figure 1). Briefly, a fragment (about 500 bp) of the ori region of the VP was amplified using primers incp1/incp2. Then, a derivative plasmid from pKD4611 (pKDinc) containing this fragment was transformed into the wild-type S. flexneri strain. Transformants were selected on LB/ampicillin agar plates, and then cultured for three generations at 37 °C to eliminate the vector. The loss of VP was confirmed by PCR using four different primer pairs (designed within genes ipaA, ipgB, mxiD and virG). Strain ∆pSF is, therefore, a useful derivative of S. flexneri serotype 2a strain 2457T with no exogenous genes. According to this procedure, the VP was removed from the wild-type strain in two independent experiments, and independently obtained plasmid-cured strains were phenotypically identical. 844
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Zhu et al. The glpR::kan mutant of S. flexneri (2457T∆glpR) was constructed by a modified method of the lambda red recombination protocol originally described by Datsenko and Wanner.11 Briefly, 500 bp upstream and 500 bp downstream of the glpR gene were amplified by PCR using the primers glpR5p1/ glpR5p2 and glpR3p1/glpR3p2, respectively. The resulting products and the kan cassette amplified from the plasmid pKD411 were cloned into pET22b (Novagen, Apr) to generate the targeting box (upstream arm-kan-downstream arm) for recombination. S. flexneri strain 2457T carrying pKD46 containing the red recombinase genes was grown at 30 °C in the presence of 10 mM arabinose (to induce the recombinase genes) and transformed with the gel-purified PCR product of the targeting box amplified using primers glpR5p1 and glpR3p2. Recombinants were selected on LB plates containing kanamycin (50 µg/mL). Mutant strains deoR::kan (2457T∆deoR), hns::kan (2457T∆hns) and mxiE::kan (2457T∆mxiE) were constructed in a similar way. All of the primers used to amplify the region (about 500 bp) upstream and downstream of the target genes are listed in Supplemental Table 1. For genetic complementation, the glpR gene of S. flexneri with its own promoters was amplified by PCR from the genome of strain 2457T using primers glpRRp1/glpRRp2. The PCR products were cloned into the BamHI and EcoRI sites of the low copy number vector pAK constructed in our lab [kanamycin resistance, containing only ori from pACYC184, and kan from pKD4], generating plasmid pAK-glpR. This plasmid was then introduced into strain 2457T∆glpR to generate the complemented strain 2457T∆glpR/pAK-glpR. To validate the differential expression of the glp regulon, a reporter vector was constructed. The 5′ upstream region of gene glpQT was amplified from the S. flexneri genome using primers glpREGp1/glpREGp2 and the chloramphenicol acetyltransferase reporter gene cat was amplified from plasmid pKD311 using primers kanp1/kanp2. These two DNA fragments were then fused into the low copy number vector pAK to generate the reporter vector pAK-cg. The wild-type and ∆pSF strains harboring this vector, 2457T/pAK-cg and ∆pSF/pAK-cg, respectively, were cultured to midlog phase (OD600nm ) 1.0), diluted 104-fold and then plated on LB agar plates containing different concentrations of chloramphenicol (2, 5, 10, and 20 µg/mL) to test the expression of the cat reporter gene in these two strains. Preparation of Whole-Cell Protein Extracts and OMP Samples. S. flexneri serotype 2a wild-type and mutant strains were grown aerobically in 100 mL LB medium at 37 °C. Cells were harvested in stationary phase (about 9 h) and whole cell protein extracts and outer membrane proteins (OMPs) were prepared as described previously.12,13 The protein concentration of samples was measured using the PlusOne 2-D Quant Kit (GE Healthcare), and 0.8 mg aliquots were stored at -80 °C. Two-Dimensional Polyacrylamide Gel Electrophoresis (2-DE) and In-Gel Protein Digestion. Isoelectric focusing (IEF) was performed with commercially available IPG-strips (18 cm, pH 4-7, pH 6-11; GE Healthcare) using an Ettan IPGphor IEF System (GE Healthcare). The second dimension was performed using 12.5% polyacrylamide gels in the Protean II XL vertical electrophoresis system (BioRad). Each 2-DE gel was repeated at least three times for each sample. For each analysis, the samples were treated with the 2-D Clean-Up Kit (GE Healthcare) according to the manufacturer’s instructions and resuspended in 350 µL of rehydration buffer [7 M urea, 2 M thiourea,
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Global Analysis of a Plasmid-Cured S. flexneri Strain 4% (w/v) CHAPS, 50 mM DTT, 0.5% IPG Buffer (same pH range as the IPG strips)]. The following procedure and the in-gel protein digestion were carried out as described previously.14 Peptides from digested proteins were resolubilized in 2 µL of 0.5% trifluoroacetic acid. The complete experiment including bacterial culture and sample preparation was repeated three times. For each sample, 2-DE analysis was repeated at least three times to get one high resolution image. Image analysis was processed by ImageMaster 2D Platinum software (Amersham Biosciences). The best three images obtained from one sample were used for quantitative analysis. The relative volume of each spot was determined from the spot intensities in pixel units and normalized to the sum of the intensities of all the spots on the gel. MALDI-TOF-MS/MS. The MALDI-TOF MS measurement was performed on a Bruker Ultraflex III MALDI-TOF-MS (Bruker Daltonics, Germany) operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. A saturated solution of R-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. One microliter of the matrix solution and the sample solution at a ratio of 1:1 was applied to the Score384 target well. Mass accuracy for peptide mass fingerprint (PMF) analysis was calibrated with a 0.1-0.2 Da external standard, and internal calibration was carried out with enzyme autolysis peaks, at a resolution of 12 000. The SNAP algorithm (S/N threshold, 5; Quality Factor Threshold, 30) in FlexAnalysis 2.4 was used to select the 150 most prominent peaks in the mass range m/z 700-4000. The subsequent MS/MS analysis was performed in a data-dependent manner, and the five most abundant ions fulfilling certain preset criteria (S/N higher than 25 and Quality Factor higher than 50) were subjected to high energy collisioninduced dissociation (CID) analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas. Data Interpretation and Database Searching. Search for PMFs was done using the program Mascot 2.1 (Matrix Science Ltd.) licensed in-house against the database of S. flexneri serotype 2a strain 2457T (4068 sequences) to eliminate redundancy resulting from multiple members of the same protein family, and the results were checked using Mascot 2.2 (http:// www.matrixscience.com) against the NCBInr database (version 20080221, 6 122 577 sequences). As for those identified in the NCBInr database, the proteins of S. flexneri spp. were selected as the best hits from the homologue protein lists. Monoisotopic masses were used to search the databases, allowing a peptide mass accuracy of 0.2 Da and one partial cleavage. Oxidation of methionine and carbamidomethyl modification of cysteine was considered. Scores greater than 49 and 78 were considered significant (p < 0.05) for local and Internet PMF searches, respectively. For unambiguous identification of proteins, more than five peptides had to be matched by MALDI-TOF data. RNA Extraction and Microarray Analysis. Total RNA was extracted from S. flexneri strain 2457T and mutant strain ∆pSF cultivated at 30 °C using Trizol reagent (Invitrogen). The RNA was purified using the Rneasy Mini kit (Qiagen) and treated with the RNase-free DNase (Qiagen). RNA concentration and purity were evaluated by spectrophotometric analysis at 260 and 280 nm. The experimental procedures for microarray analysis were carried out according to the instructions provided by the manufacturer (Catalog Number: A4319-00-01, Roche Nimblegen), with no modifications. We followed the protocol to scan one-color (Cy3) NimbleGen arrays with a GenePix 4000B Scanner and associated software. NimbleScan v2.5
software was used to analyze the raw data obtained from the first step. The RMA (Robust Multi-Array Analysis) algorithm15 was applied to perform array data normalization. The default settings of the software were used to perform RMA normalization. The raw data set has been submitted into the GEO database (http://www.ncbi.nlm.nih.gov/geo), accession number GSE12535. Purification and Identification of Sequence-Specific DNA Binding Proteins. The 5′ upstream regulatory region of the glpQ gene was amplified using the biotinylated primers glpbiotp1/glpbiotp2. About 3 µg of PCR product was then coupled to Dynabeads M-280 streptavidin (Dynal) using the conditions described by the manufacturer. About 15 mL of overnight bacterial culture was harvested, resuspended in 1 mL of TGED buffer [20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, 0.01% Triton X-100]16 containing 100 mM NaCl, and then lysed by sonication. The DNA-coupled magnetic beads were incubated with the cell lysate for 30 min at room temperature in TGED buffer containing 100 mM NaCl and washed three times with this buffer. Proteins were eluted on ice in 50 µL of TGED buffer containing 1 M NaCl. After purification using the 2-D Clean-Up Kit (GE Healthcare), the pellet was resuspended in 125 µL of rehydration buffer, loaded onto a 7 cm pH 3-10 IPG strip and analyzed as described above.
Results Phenotypic Characteristics of the S. flexneri ∆pSF (VPCured) Strain. Incompatible plasmids share elements of the same replication machinery and, therefore, compete with each other during both replication and partitioning into daughter cells, often resulting in the exclusion of one of the plasmids.17 A VP-cured avirulent S. flexneri strain, ∆pSF strain, was constructed using plasmid incompatibility in our study (see Experimental Section and the schematic in Supplemental Figure 1). Unlike virulent S. flexneri,8 strain ∆pSF did not cause keratoconjunctivitis in guinea pigs and exerted no cytotoxicity to cultured epithelial cells (Supplemental Figure 2A). The removal of VP was confirmed by PCR. The PCR results showed that all of the virulence genes tested (ipaA, ipgB, mxiD, and virG) and a fragment of the ori region (see Experimental Section) were missing in strain ∆pSF. However, another plasmid18 homologous to Salmonella typhi R27 plasmid (Genbank Accession number: AF250878) and a small plasmid (2Md plasmid, Genbank Accession number: AY028316) were still present (Supplemental Figure 2B), suggesting that other genetic elements were unchanged during our genetic manipulations. Strain ∆pSF formed bigger colonies on TSA and LB medium and had a shorter doubling time than the wild-type strain at 37 °C. The maximum growth density for strain ∆pSF (OD600nm ) 4.0) was much higher than that of the wild-type strain (OD600nm ) 3.3) at 37 °C in LB broth (Supplemental Figure 2C). These observations suggested that strain ∆pSF had a growth advantage over the wild-type strain, potentially indicating that the VP places some burden on its host. The ∆pSF and wild-type strains shared the same biochemical profile in the API 20E and API 50CH systems. In addition, there were no significant differences between these two strains in their antibiotic resistance patterns (Supplemental Figure 2D), Journal of Proteome Research • Vol. 9, No. 2, 2010 845
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Figure 1.
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Zhu et al.
Global Analysis of a Plasmid-Cured S. flexneri Strain
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Figure 1. Comparison of 2-DE profiles of strain ∆pSF (without virulence plasmid) with the wild-type strain at 30 °C (A) and 37 °C (B). Most virulence factors encoded by the virulence plasmid (including IpaABCD, IpgCD and SepA) were identified by this comparison. Some proteins of the sn-glycerol 3-phosphate regulon (glp regulon) were also noted to be upregulated in strain ∆pSF. Journal of Proteome Research • Vol. 9, No. 2, 2010 847
research articles suggesting that the structural integrity of the bacterial cells was not affected by the removal of VP. 2-DE Profiles of the ∆pSF and Wild-Type Strains and Identification of Differentially Expressed Proteins. Whole cell protein preparations and OMP preparations of S. flexneri wildtype and ∆pSF strains grown at 30 and 37 °C were analyzed and their 2-DE profiles were highly comparable. Whole cell protein extracts were separated using IPG strips with a linear gradient of pH 4-7 and pH 6-11 in the first dimension. About 700 and 350 Coomassie-stained protein spots were located in acidic gels (pH 4-7) and alkaline gels (pH 6-11), respectively (Figure 1). In previous experiments, all of the OMPs were scattered in the pI ranges of pH 4-7 when pH 3-10 immobilized pH gradient (IPG) strips were used (data not shown). To obtain better separation, we only used pH 4-7 IPG strips in the IEF analysis for OMP samples. About 130 Coomassiestained protein spots were detected in this region. A protein was regarded to be differentially expressed if the spot density (volume ‰) in one strain was significantly different [>2-fold for common, or >1.5-fold for abundant proteins (vol‰ > 6‰)] from that in the other strain. These protein spots are indicated in Figure 1. After destaining and in-gel trypsin digestion, a total of 45 spots representing 34 proteins were identified by MALDITOF MS (Table 1). The database search results obtained from Mascot analysis showed that most downregulated proteins (which were actually spots that disappeared) in strain ∆pSF were virulence related proteins, which were missing in previous proteomic studies of S. flexneri probably due to the instability of VP.19 In our 2-DE gels, many virulence effectors of the S. flexneri type III secretion system (TTSS), such as IpaA/C/D and their chaperons, were separated and identified. However, the effector IpaB and the membrane apparatus proteins of TTSS were not identified. This is possibly because these proteins were not soluble in the 2-DE lysis buffer used for sample preparation. It certainly indicated that the physical chemistry of IpaB is quite different from IpaD, although they both located at the tip of the TTSS needle complex.20 The identification of known virulence factors in 2-DE will inevitably benefit future proteomic research on this organism and studies on the pathogenesis mechanisms of S. flexneri. The expression of heat shock proteins DnaK and IbpA was noted to be maximal at 37 °C in the wild-type strain (see Table 1), suggesting that the function of these proteins might be related to the virulence of S. flexneri. It had been reported that IbpA/IbpB, ClpB, and the DnaK system form a functional triade of chaperones.21 We, therefore, propose that this complex might play an important role in the correct folding of virulence factors. Four of the upregulated proteins (GlpA, GlpB, GlpK and GlpQ) in strain ∆pSF are associated with the glycerol degradation pathway. These proteins enable the bacteria to utilize a wide variety of glycerophosphodiesters to produce energy. The genes encoding these proteins are located in different operons in the genome of S. flexneri. The expression of these genes is, therefore, possibly regulated at the level of transcription. In addition, the expression of a putative OMP YciD was upregulated in strain ∆pSF, as shown in the 2-DE profiles of OMPs (Figure 1). The expression of this protein was also found to be upregulated in bacteria at stationary phase compared with exponentially growing bacteria in a previous study.22 Since there are no TTSS complexes assembled on the outer membrane of strain ∆pSF, upregulation of YciD might be a strategy 848
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Zhu et al. to complement the reduction of the protein content of the bacterial outer membrane to maintain structure integrity. Microarray Analysis of Wild-Type and ∆pSF Strains of S. flexneri. In this study, microarray analysis was carried out to examine expression at the transcriptional level of all chromosomal genes in the ∆pSF strain compared with the wild-type strain. Since the VP is missing in strain ∆pSF, the expression of VP genes was of no relevance in this case. The Nimblegen oligonucleotide microarrays designed for the S. flexneri serotype 2a strain 2457T chromosome (without VP probes) were used to analyze the total RNA from the wild-type and ∆pSF strains. Because the expression of VP genes at 37 °C would interfere with the quantitative analysis of mRNA, these experiments were performed at 30 °C but not at 37 °C. A gene was regarded to be differentially expressed only if its normalized intensity in one strain was significantly different (>3-fold) from that in the other strain. Since the products of genes with a low abundance of RNA transcript were difficult to detect in our proteomic studies, these genes (‰ intensity was less than 0.1 in both strains) were not investigated further. The normalized intensity values whose variation coefficients in three replicates were greater than 20% were also discarded. The differently expressed genes are listed in Table 2. Eight out of the first 20 upregulated genes were members of the glp gene family, confirming the results of our proteomic studies, while most downregulated genes were hypothetical genes and insertion sequence (IS) elements. Since the expression of glp regulon genes were increased at both the transcriptional and translational levels in strain ∆pSF, we investigated these genes further. According to the expression intensity of these genes, we selected glpQ as a representative gene to use in further studies. Validation of the Differential Expression of the glp Regulon Using the Chloramphenicol Acetyltransferase Reporter Gene. To validate the differential expression of the glp regulon in the wild-type and ∆pSF strains, we constructed a vector pAK-cg carrying the 5′ upstream region of gene glpQT fused with the chloramphenicol acetyltransferase reporter gene cat and introduced it into the wild-type and ∆pSF strains. If the VP is able to affect the expression of the glpQ gene, the expression of the cat gene should also be affected and the host bacteria should have a different chloramphenicol resistance profile. As shown in Supplemental Figure 3, after 15 h of culture in LB broth containing 10 µg/mL chloramphenicol, ∆pSF/pAK-cg grew to a higher cell density than 2457T/pAK-cg. After 36 h of culture at 37 °C, ∆pSF/pAK-cg grew on the LB plate with 5 µg/ mL chloramphenicol, while 2457T/pAK-cg did not grow. These results demonstrated that the presence of VP could affect the expression of the glp regulon at the transcriptional level. Next, we investigated whether the VP encodes a regulatory protein that directly controls expression of the glp regulon. Identification of Regulatory Proteins Binding to the 5′ Upstream Region of the glpQ Gene. PCR products amplified using biotinylated primers and linked to streptavidin coated magnetic beads were used as baits to capture DNA-binding proteins. As shown in Figure 2 and Table 3, proteins that could directly bind to the 5′ upstream region of the glpQ gene were GlpR, DeoR and H-NS. Protein H-NS has been reported to be a major temperature-sensitive global regulator, controlling the expression of most virulence genes.23 Identifying H-NS as one of the regulatory proteins of the glpQ gene explains our finding from proteomic analysis that the expression of GlpQ protein was higher at 37 °C than at 30 °C (see the relative abundance of GlpQ and GlpA/B/K in Table 1). Unfortunately, we did not
99
151 112
107
W05
W06 W07
W08
91 108
137
W04
W21 W22
167
W03
251
160
W02
W19
103 169 66 252
D05 D06 D07 W01
145 73 405 176 141 137 159
248
D04
W11 W12 W13 W14 W15 W16 W17
190 146
D02 D03
231 72
194
D01
W09 W10
scorea
spot ID
gi|10957203 gi|18462547
gi|56383093
gi|18462541 gi|30043258 gi|30039826 gi|18462529 gi|18462543 gi|18462540 gi|56383093
gi|18462530 gi|18462539
gi|18462537
gi|56383102 gi|18462578
gi|18462586
gi|18462568
gi|18462559
gi|18462578
gi|30040787 gi|30043380 gi|30041027 gi|18462581
gi|30041930
gi|30043041 gi|30041931
gi|30041928
GI
0.91 ( 0.07
0.08 ( 0.01
ND ND ND ND ND
0.82 ( 0.01 6.42 ( 0.63 1.54 ( 0.33 2.13 ( 0.08 1.14 ( 0.05
3.07 ( 0.40 0.86 ( 0.18
ospC3
ND ND
ND
36.71 ( 2.46
ipaC
parA ibpA dnaK stbA mkaD parB ipaC
ND ND 8.00 ( 0.83 ND ND ND ND
ND ND
ND
0.77 ( 0.14
0.34 ( 0.13 6.36 ( 1.53
ND
10.59 ( 1.63
0.65 ( 0.03 16.76 ( 1.49 298.25 ( 14.17 ND
5.72 ( 0.64 1.24 ( 0.05
3.69 ( 0.17 0.36 ( 0.08
0.17 ( 0.03 4.44 ( 0.54 131.26 ( 23.76 9.53 ( 1.08
21.65 ( 1.89
7.17 ( 0.81
∆pSF
37 °C (mean ( SD) wild-type
0.98 ( 0.16 0.46 ( 0.04 21.24 ( 3.16 1.78 ( 0.14 5.95 ( 0.71 0.71 ( 0.05 3.23 ( 0.11
spa47 phoN1
sepA
virA ipaD
phoN2/apy
ipgC
mxiD
ipaD
wrbA lldD yciD ipaA
glpA
glpK glpB
glpQ
gene
-
-
0.38 -
-
-
-
-
-
-
-
3.91 3.78 2.27 -
11.06
1.55 3.50
3.02
ratiod
0.54 ( 0.05 ND
8.93 ( 0.85
0.57 ( 0.05 ND 9.91 ( 0.85 1.23 ( 0.03 ND 1.33 ( 0.33 ND
ND 2.62 ( 0.03
0.47 ( 0.03
0.35 ( 0.03 ND
0.48 ( 0.05
0.46 ( 0.09
ND
1.28 ( 0.05
0.22 ( 0.03 5.46 ( 2.34 46.33 ( 4.85 0.72 ( 0.03
0.26 ( 0.03
3.32 ( 0.13 0.39 ( 0.03
5.75 ( 0.14
wild-type
ND ND
ND
ND ND 8.90 ( 0.69 ND ND ND ND
ND ND
ND
ND ND
ND
ND
ND
ND
0.64 ( 0.07 8.05 ( 0.49 242.10 ( 26.17 ND
0.79 ( 0.01
6.17 ( 0.27 1.26 ( 0.10
12.67 ( 1.19
∆pSF
30 °C (mean ( SD)
relative abundance (volume‰)b
Table 1. Identification of Differentially Expressed Proteins by MALDI-TOF MS
-
-
0.90 -
-
-
-
-
-
-
-
2.97 1.47 5.23 -
3.08
1.86 3.23
2.20
ratiod
58% 46%
68%
54% 41% 61% 68% 46% 49% 57%
70% 56%
15%
67% 52%
44%
80%
40%
61%
62% 74% 48% 62%
78%
63% 65%
66%
sequence coverage
10/57 16/65
22/40
21/68 9/63 40/63 23/89 14/42 11/25 15/35
25/60 10/82
15/36
25/99 16/45
12/43
14/62
17/39
21/60
13/42 41/176 9/49 29/61
33/114
29/98 21/83
23/45
matched/ searched
-
-
-
D O O K -
NU I
MU
I
-
NU
-
R C M -
C
C E
C
COGc product
glycerophosphodiester phosphodiesterase glycerol kinase anaerobic glycerol-3-phosphate dehydrogenase subunit B sn-glycerol-3-phosphate dehydrogenase (anaerobic), large subunit TrpR binding protein WrbA L-lactate dehydrogenase putative outer membrane protein IpaA, secreted by the Mxi-Spa machinery, modulates entry of bacteria into epithelial cells IpaD, secreted by the Mxi-Spa machinery, required for entry of bacteria into epithelial cells MxiD, outermembrane protein of the secretin family, component of the Mxi-Spa secretion machinery IpgC, cytoplasmic chaperone for IpaB and IpaC PhoN2 (Apy), periplasmic phosphatase, apyrase, ATP diphosphohydrolase secreted VirG-processing protein IpaD, secreted by the Mxi-Spa machinery, required for entry of bacteria into epithelial cells SepA, extracellular serine protease of the IgA1 protease family, secreted by a C-terminal autotransporter domain type III secretion system ATPase PhoN1, periplasmic nonspecific acid phosphatase plasmid segregation protein heat shock protein molecular chaperone DnaK plasmid stable inheritance protein mouse killing factor plasmid segregation protein IpaC, secreted by the Mxi-Spa secretion machinery, required for entry into epithelial cells IpaC, secreted by the Mxi-Spa secretion machinery, required for entry into epithelial cells hypothetical protein R27_p014 OspC3, probably secreted by the Mxi-Spa secretion machinery, function unknown
Global Analysis of a Plasmid-Cured S. flexneri Strain
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Journal of Proteome Research • Vol. 9, No. 2, 2010 849
850
Journal of Proteome Research • Vol. 9, No. 2, 2010
168
105
197 209
136
92
225
109 144 193
84
105 114
167
W23
W24
W25 W26
W27
W28
W29
W30 W31 W32
W33
W34 W35
W36
gi|30040710
gi|56383102 gi|18462537
gi|18462559
gi|18462664 gi|18462530 gi|18462559
gi|18462548
gi|18462546
gi|18462570
gi|18462582 gi|18462567
gi|56383078
gi|56383114
GI
ND ND ND ND ND ND
ND
ND ND
7.32 ( 1.38 2.14 ( 0.07 5.91 ( 0.44 5.77 ( 0.44 0.39 ( 0.01 7.80 ( 0.97
0.96 ( 0.07
1.19 ( 0.17 22.65 ( 2.93
ospC2
spa47 mxiD
ompF
virA sepA
mxiD
ospD1
ipgB1
0.53 ( 0.01
ND ND
8.26 ( 0.71 3.93 ( 0.24
icsB ipgD
7.91 ( 1.92
ND
0.98 ( 0.15
ipgB2
icsP/sopA
ND
∆pSF
37 °C (mean ( SD) wild-type 0.86 ( 0.11
gene
0.07
-
-
-
-
-
-
-
-
-
ratiod
6.99 ( 1.33
ND ND
ND
1.73 ( 0.61 ND 0.51 ( 0.06
ND
1.05 ( 0.18
ND
ND ND
ND
ND
wild-type
1.25 ( 0.10
ND ND
ND
ND ND ND
ND
ND
ND
ND ND
ND
ND
∆pSF
30 °C (mean ( SD)
relative abundance (volume‰)b
0.18
-
-
-
-
-
-
-
-
-
ratiod
75%
27% 18%
19%
72% 33% 33%
49%
62%
62%
45% 50%
50%
73%
sequence coverage
20/87
9/15 22/55
9/22
15/53 14/32 21/39
29/59
9/44
14/25
20/36 23/52
11/21
23/142
matched/ searched
M
MU
NU
NU NU
-
-
-
-
-
M
COGc
product IcsP (SopA), outermembrane protease of the OmpP family, involved in cleavage of surface exposed IcsA IpgB2, probably secreted by the Mxi-Spa secretion machinery IcsB, invasion protein IpgD, secreted by the Mxi-Spa machinery, modulates entry of bacteria into epithelial cells IpgB1, secreted by the Mxi-Spa machinery, function unknown OspD1, secreted by the Mxi-Spa secretion machinery, function unknown OspC2, probably secreted by the Mxi-Spa secretion machinery, function unknown orf, hypothetical type III secretion system ATPase MxiD, outermembrane protein of the secretin family, component of the Mxi-Spa secretion machinery MxiD, outermembrane protein of the secretin family, component of the Mxi-Spa secretion machinery secreted VirG-processing protein SepA, extracellular serine protease of the IgA1 protease family, secreted by a C-terminal autotransporter domain outer membrane protein 1a (Ia;b;F)
a PMF scores greater than 49 are significant (p < 0.05). b The relative abundance of protein spot is represented by the relative volume calculated in the correspondent 2-DE gel. ND indicates that the protein spot was not detected. c Each letter represents a particular functional category. These single letter codes can be decoded using the COG service (http://www.ncbi.nlm.nih.gov/COG/). d The ratio is calculated by dividing the relative intensity in the ∆pSF strain by that in the wild-type strain.
scorea
spot ID
Table 1. Continued
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Global Analysis of a Plasmid-Cured S. flexneri Strain Table 2. Differentially Expressed Genes Detected by Microarray Analysis of Cells Grown at 30°C normalized intensity (mean ( SD)
GI
gene synonym
wild-type
∆pSF
gi|30041500 gi|30041501 gi|30043322 gi|30040123 gi|30042089 gi|30041542 gi|30041705 gi|30040675 gi|30040825 gi|30041243 gi|30042505 gi|30043788 gi|30043359 gi|30041978 gi|30043813 gi|30043223
osmE tktA efp waaY pta purA atpG
S1909 S1910 S4063 S0327 S2645 S1960 S2181 S0950 S1123 S1604 S3127 S4570 S4103 S2508 S4600 S3955
8752.41 ( 788.39 14822.97 ( 1267.06 18414.05 ( 251.03 18920.20 ( 2735.53 2120.70 ( 418.82 17826.03 ( 1017.29 15015.85 ( 1231.75 31865.73 ( 911.36 18190.47 ( 968.07 7315.93 ( 486.58 3641.23 ( 539.52 6179.35 ( 1011.09 2154.62 ( 308.93 2295.77 ( 228.31 1757.17 ( 261.38 4634.95 ( 194.19
1518.23 ( 70.88 3802.51 ( 195.32 5500.53 ( 394.17 5834.84 ( 188.77 674.24 ( 96.28 5677.85 ( 335.08 4855.61 ( 308.80 10391.91 ( 1001.37 6009.14 ( 366.46 21958.67 ( 756.40 10932.17 ( 1500.33 18814.67 ( 1494.28 6631.23 ( 511.10 7077.94 ( 776.45 5426.80 ( 1051.19 14375.80 ( 1539.11
gi|30040915 gi|30040979 gi|30041928 gi|30040432 gi|30041298 gi|30041170 gi|30041871 gi|30041732 gi|30041734 gi|30042608 gi|30043553 gi|30041266 gi|30043041 gi|30040114 gi|30041735 gi|30041929 gi|30040205 gi|30043755 gi|30041932
prsA glpQ cspE relF cspC spr hybA glpD rplT glpK gtrII glpT ybaJ ytfK glpC
S1219 S1294 S2454 S0680 S1668 S1518 S2391 S2213 S2215 S3244 S4316 S1633 S3743 S4803 S2216 S2455 S0413 S4534 S2458
660.45 ( 95.75 3419.75 ( 498.47 17764.75 ( 1197.29 9762.45 ( 734.68 14189.83 ( 1609.80 14776.99 ( 1569.45 7621.99 ( 1269.74 2799.40 ( 359.24 4427.15 ( 462.98 1725.71 ( 303.79 1692.39 ( 259.51 4448.34 ( 594.88 5859.16 ( 616.05 2246.26 ( 125.58 5377.91 ( 856.48 8011.78 ( 370.22 2181.09 ( 256.72 3149.16 ( 494.47 1900.82 ( 174.28
2108.05 ( 115.21 11327.78 ( 1043.86 60196.87 ( 1033.85 34336.06 ( 6427.27 50403.59 ( 4786.61 52996.92 ( 1810.75 28721.36 ( 689.14 10648.80 ( 1465.90 17424.17 ( 1169.66 6892.19 ( 552.07 6877.37 ( 1031.19 18098.74 ( 1412.31 24008.33 ( 913.92 9499.53 ( 1637.60 23030.23 ( 3953.70 35579.00 ( 2766.69 10081.34 ( 523.48 19706.50 ( 2317.87 14832.36 ( 1808.32
gi|30041930 glpA
S2456
941.42 ( 44.41
7372.18 ( 1283.23
gi|30043040 glpF gi|30041931 glpB
S3742 S2457
2465.67 ( 284.27 1081.14 ( 94.93
20329.35 ( 2262.21 13664.19 ( 591.95
ratioa COGb
0.17 0.26 0.30 0.31 0.32 0.32 0.32 0.33 0.33 3.00 3.00 3.04 3.08 3.08 3.09 3.10
product
S
IS1294 orfB IS629 orfB IS629 orfB IS629 orfB hypothetical protein L IS629 orfB IS629 orfA L hypothetical bacteriophage protein P IS629 orfB R activator of ntrL gene PR transketolase 1 isozyme L elongation factor P (EF-P) putative LPS biosynthesis protein IQ phosphotransacetylase V adenylosuccinate synthetase L membrane-bound ATP synthase, F1 sector, gamma-subunit 3.19 hypothetical bacteriophage protein 3.31 phosphoribosylpyrophosphate synthetase 3.39 glycerophosphodiester phosphodiesterase 3.52 L cold shock protein 3.55 UNTP prophage maintenance protein 3.59 P cold shock protein 3.77 C putative lipoprotein 3.80 M hypothetical protein 3.94 hypothetical protein 3.99 hydrogenase-2 small subunit 4.06 sn-glycerol-3-phosphate dehydrogenase (aerobic) 4.07 C 50S ribosomal subunit protein L20/regulator 4.10 glycerol kinase 4.23 C putative glucosyl tranferase II 4.28 hypothetical protein 4.44 L sn-glycerol-3-phosphate permease 4.62 G hypothetical protein 6.26 L hypothetical protein 7.80 L sn-glycerol-3-phosphate dehydrogenase (anaerobic), K-small subunit 7.83 E sn-glycerol-3-phosphate dehydrogenase (anaerobic), large subunit 8.24 glycerol diffusion facilitator protein 12.64 C sn-glycerol-3-phosphate dehydrogenase (anaerobic), membrane anchor subunit
a The ratio is calculated by dividing the relative intensity in the ∆pSF strain by that in the wild-type strain. b Each letter represents a particular functional category. These single letter codes can be decoded at the COG service (http://www.ncbi.nlm.nih.gov/COG/).
Figure 2. Identification of the glp regulon-specific DNA binding proteins. Streptavidin coated Dynabeads were used to create a DNA affinity solid-phase to capture potential regulatory proteins. (A) Protein GlpR (spot: DB01), DeoR (spot: DB02) and H-NS (spot: DB03) were identified by MALDI-TOF/TOF using the glp regulatory DNA fragment. (B) No proteins were detected when blank beads were used as a control. Journal of Proteome Research • Vol. 9, No. 2, 2010 851
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Table 3. Identification of DNA-Binding Proteins by MALDI-TOF/TOF spot ID
scorea
GI
gene
sequence coverage
matched/ searched
DB01
391
gi|30043556
glpR
34%
5/12
repressor of the glp operon
DB02
504
gi|30040567
deoR
31%
9/30
Deoxyribose operon repressor
DB03
118
gi|30041005
hns
33%
4/15
DNA-binding protein H-NS
product
peptide sequences
16-QQGYVSTEELVEHFSVSPQTIR-37 39-DLNELAEQNLILR-51 148-DGGIIGEATLDFISQFR-164 165-LDFGILGISGIDSDGSLLEFDYHEVR-190 40-RDLNNHSAPVVLLGGYIVLEPR-61 41-DLNNHSAPVVLLGGYIVLEPR-61 225-RFDIVVSDCCPEDEYVK-241 226-RFDIVVSDCCPEDEYVK-241 20-ECTLETLEEMLEKLEVVVNER-40
a
PMF data and tandem MS data were merged into one file for MS/MS ion searches against the local database. Scores greater than 49 are significant (p < 0.05).
Figure 3. The expression of GlpQ in different mutant strains. Only the expression of GlpQ in glpR deletion mutant is similar to that of ∆pSF strain. And the expression of GlpQ can be reduced to a level similar to that of wide-type strain when gene glpR were introduced into ∆pSF strain and 2457T∆glpR strain.
identify any regulatory proteins encoded by the VP. In fact, the VP of S. flexneri serotype 2a strain 2457T encodes three common transcriptional regulators, VirF, VirB and MxiE (Orf81 was disrupted in S. flexneri serotype 2a strains).24 VirF controls transcription of the virB gene and is controlled by H-NS, which can directly regulate the expression of the glpQ gene. Therefore, bacteria do not need these two proteins to control the glp regulon. As for MxiE, deletion of its encoded gene did not affect expression of GlpQ (Figure 3). It, therefore, appears that the VP is more likely to repress the expression of the glp regulon via a more complex pathway. Our findings show that the VP could control the expression of the glp regulon at the transcriptional level. If this regulation is not directly related to any of the proteins encoded by VP, it might be mediated by one of the DNA-binding proteins that we identified. We, therefore, constructed hns, deoR and glpR deletion mutants to test the expression of GlpQ in these strains. The results showed that only expression of GlpQ in the glpR deletion mutant is similar to that of the ∆pSF strain (Figure 3). Although GlpR and DeoR belong to the same transcriptional regulator family and both bind to the upstream DNA sequence of glpQ in vitro, deletion of DeoR did not affect the expression of GlpQ in vivo. GlpR might, therefore, be associated with the regulatory mechanism used by the VP to control the expression of the glp regulon in S. flexneri. To validate the function of the glpR gene in the regulation of the VP, we constructed a low copy number vector pAK-glpR 852
Journal of Proteome Research • Vol. 9, No. 2, 2010
carrying the gene glpR with its own upstream regulatory region and transformed it into strains 2457T∆glpR and ∆pSF. The expression of GlpQ reduced to a level similar to that of the wildtype strain when the glpR gene was introduced (Figure 3). In fact, the transcriptional level of glpR was lower in strain ∆pSF than in wild-type strain according to the results of microarray analysis (with a ratio of 0.46). This also indicated that the VP might regulate the expression of GlpR, leading to expression changes of its downstream targets, that is, the glp regulon. We then tried to identify the regulatory proteins binding to the 5′ upstream region of the glpR gene using the same method described above. Unfortunately, we were only able to identify two possible regulators, deoR and glpR (Supplemental Figure 4). This indicated that glpR might regulate its own expression and that the mechanism used by the VP to control the expression of the glp regulon is more complex than expected. The pathway is still to be determined; however, we now know that GlpR is the last effector of this pathway controlling the glp target genes.
Discussion The proteins encoded by the glp regulon were required for the degradation of sn-glycerol 3-phosphate and its precursors. A detailed description of this system can be found in the Web site of Biocyc (http://biocyc.org/ECOLI/NEW-IMAGE?type) PATHWAY&object)PWY0-381). Briefly, the role of this system is believed to be utilization of glycerol and glycerol phosphates generated by the breakdown of phospholipids and triacylglycerol. The questions that we endeavored to answer in this study were why the VP represses the expression of the glp regulon and whether the function of this regulon leads to the instability of the VP or interferes with the virulence of S. flexneri. From the roles of the Glp proteins and our observations that these proteins are produced in different amounts in the two strains arose the hypothesis that the VP is involved in the metabolism of carbon source(s), which S. flexneri can utilize upon intracellular replication. If this is the case, the downregulation of these Glp proteins in the wild-type strain might be detrimental to the propagation of this pathogen. In fact, our preliminary results from an invasion assay showed that the ∆glpR strain, in which Glp proteins were overexpressed, appeared to be more virulent than the wild-type strain. Another hypothesis is that the downregulation of these Glp proteins in the wild-type strain might be beneficial to the stability of the VP. Our future work will focus on this issue. The regulation of these proteins has been investigated in E. coli,25 the ancestor of S. flexneri. It was reported that the repressor for this regulon was encoded by the glpR gene.26 This repressor specifically bound DNA fragments containing the
research articles
Global Analysis of a Plasmid-Cured S. flexneri Strain
Figure 4. Regulation was measured as a function of the glp regulon. Expression of the glp regulon was under the control of the virulence plasmid presence and temperature.
control regions for the glpD, glpK, glpTQ, and glpABC genes, whose expression was altered in this study. Binding of DNA by GlpR was diminished in the presence of sn-glycerol 3-phosphate.26 However, there were no other regulatory mechanisms regarding this regulon reported to date. Here, we present evidence that the VP of S. flexneri might regulate the expression of the glp regulon. When the VP is present, the expression of the glp regulon will be repressed. This regulation was mediated by protein GlpR. In addition, we also found that the expression of the glp regulon was controlled by temperature. These results are summarized graphically in Figure 4. The complete regulatory pathway from the VP to the glp regulon is more complex than expected. The first steps toward elucidation of this regulatory pathway have been made in this study. One hypothesis is that alteration of glp gene expression is the consequence of regulation by the quorum-sensing system, which might be affected by some unknown factors encoded by the VP. This is inferred from the finding that strain ∆pSF formed larger colonies and had a higher maximum growth density than the wild-type strain, indicating that the quorum-sensing systems might function differently in the two strains. Furthermore, the expression of Glp proteins was much more abundant at stationary phase than at exponential growth phase according to the results of 2-DE profiles of the wild-type grown at exponential phase,22 suggesting that the cell density affected the expression of these proteins whether or not the VP was present. Another possible hypothesis is that the outer membrane structure has significantly changed due to the absence of the TTSS apparatus in strain ∆pSF. Differences in the outer membrane structure lead to different fates for membrane phospholipids, which will be degraded by the Glp protein system. This is inferred from our finding that the expression of a putative OMP YciD was upregulated in strain ∆pSF (see the 2-DE profiles of OMPs in Figure 1). There have been many comparative proteomic studies focused on the differences between wild-type virulent pathogens and virulence plasmid cured strains.27,28 However, in these studies, the plasmid-cured strains were screened using traditional curing agents or by spontaneous loss of the plasmid by mutation. These processes probably caused some mutations to occur in the chromosome of the cured strains which could theoretically lead to phenotypic changes unrelated to the VP. Although these studies were useful in the identification of many virulence related proteins and might provide valuable clues regarding virulence regulation, they do not provide data suitable for the analysis of crosstalk between the pathogenic chromosome and the VP. The method used in this study to
remove the virulence plasmid is much more gentle minimizing the chances of unexpected mutations in the chromosome. In addition, there is no exogenous plasmid left in the cured strain, ensuring the confidence of the comparative proteomic results. This strategy is robust, and can easily be duplicated by other researchers interested in the field of pathogenic microbiology. We therefore believe that this strategy provides exciting advances in proteomic approaches to examine coevolution between bacterial chromosomes and endogenous plasmids. In conclusion, we found that the expression of some glycerol 3-phosphate (glp) regulon-encoded proteins was increased in the absence of the VP in this study. These findings will further our understanding of the crosstalk between the bacterial chromosome and the VP in S. flexneri. This study also indicated that comparative proteomic analysis is a powerful tool for the study of pathogen evolution. Abbreviations: 2-DE, two-dimensional gel electrophoresis; Apr, ampicillin resistance; bp, base pair(s); CHAPS, 3-[(3Cholamidopropyl) dimethylammonio] propanesulfonic acid; CID, collision-induced dissociation; DTT, dithiothreitol; glp, glycerol 3-phosphate regulon; H-NS, histone-like protein H1; IEF, isoelectric focusing; IPG, immobilized pH gradient; IS, insertion sequence; kan, kanamycin resistance gene; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight; MS, mass spectrometry; OMPs, outer membrane proteins; ori, origin of DNA replication; p, plasmid; PCR, polymerase chain reaction; PMF, peptide mass fingerprint; RMA, Robust Multi-Array Analysis; TSA, tryptic soy agar; TTSS, type III secretion system; VP, virulence plasmid; ∆, deletion; ::, novel junction (fusion or insertion); the protein/gene abbreviations used in text are listed in Table 1, except that glpR/GlpR, deoR/DeoR and hns/H-NS are listed in Table 3.
Acknowledgment. We are very grateful to Prof. Qinong Ye for critical, constructive, and helpful comments on the manuscript. This work was supported by the National Key Basic Research Program of China (973 Program, No. 2005CB522904), Mega-projects of Science Research for the 11th Five-year Plan (No. 2009ZX10004-103) and the National Natural Science Foundation of China (No. 30470101 and No. 30700035). Microarray analysis was carried out by the Chinese National Engineering Center for Biochip at Shanghai. Supporting Information Available: Primers used in this study for gene deletion; schematic of method used to cure the virulence plasmid in this study; the different phenotypic characteristics between ∆pSF strain and wild-type strain; validation of the differential expressions of glp regulon using chloramphenicol acetyltransferase reporter gene; identification of the glpR promoter-specific DNA binding proteins. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Hale, T. L. Genetic basis of virulence in Shigella species. Microbiol. Rev. 1991, 55 (2), 206–24. (2) Buchrieser, C.; Glaser, P.; Rusniok, C.; Nedjari, H.; D’Hauteville, H.; Kunst, F.; Sansonetti, P.; Parsot, C. The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol. Microbiol. 2000, 38 (4), 760–71. (3) Venkatesan, M. M.; Goldberg, M. B.; Rose, D. J.; Grotbeck, E. J.; Burland, V.; Blattner, F. R. Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect. Immun. 2001, 69 (5), 3271–85.
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PR9007514
