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Quantitative Proteomic Analysis of Salmonella enterica Serovar Typhimurium under PhoP/PhoQ Activation Conditions Jian-Lan Yu† and Lin Guo*,†,‡ † ‡
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, P. R. China Key Laboratory of Analytical Chemistry for Biology, Medicine (Ministry of Education), Wuhan University, Wuhan, P. R. China
bS Supporting Information ABSTRACT:
The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg2þ concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg2þ concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg2þ concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg2þ concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg2þconcentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella’s adaptation to the host environment. KEYWORDS: Salmonella enterica serovar Typhimurium, PhoP/PhoQ system, SILAC, quantitative proteomics, low Mg2þ concentrations, cationic antimicrobial peptides
’ INTRODUCTION To survive and replicate within host tissues, bacterial pathogens modulate their gene expression by sensing host environmental signals. This adaptation process often involves twocomponent regulatory systems. In the facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), a two-component regulatory system (PhoP/PhoQ) serves as a master regulator of S. Typhimurium virulence and is often used as a study model for this type of effective bacterial defense mechanism. In the PhoP/PhoQ system, PhoQ serves as a membrane-bound sensor kinase that senses extracellular signals and then modifies the phosphorylation state of the response regulator PhoP. Phosphorylated PhoP r 2011 American Chemical Society
in turn functions as a transcriptional regulator and modulates the expression of a variety of genes directly or indirectly.13 Since the discovery of the PhoP/PhoQ system more than 20 years ago,1,2 various aspects of this regulatory system have been extensively studied. One of the focal points is to identify environmental signals sensed by PhoQ.48 In the early studies, starvation stress and acidic pH were found to increase the level of expression of several PhoP-activated genes (pag genes).911 A low concentration of Mg2þ (micromolar level) was later discovered Received: November 26, 2010 Published: May 12, 2011 2992
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Journal of Proteome Research to activate more pag genes, while a high concentration of Mg2þ (millimolar level) represses them.4 Initial clues linking low Mg2þ concentrations with PhoP/PhoQ activation came from the finding that PhoP is a direct transcriptional activator of MgtA and MgtB, two of the three Mg2þ transporters in S. Typhimurium.4,12 In recent years, reconstitution studies using purified PhoQ in membrane vesicles and NMR analysis showed that sublethal concentrations of cationic antimicrobial peptides (CAMP) can activate the PhoQ sensor kinase,5,13 and acidic pH can be additive with CAMP.6 Although the studies mentioned above demonstrate that PhoP/PhoQ activation can be triggered by several signals in vitro, the relevance of each signal to this system in vivo is widely debated.7,8 The current consensus is that the PhoP/PhoQ system is activated after S. Typhimurium enters phagosomes of host macrophages,3,11,14 so the ideal experiment for answering these questions is to measure all related factors in vivo. However, until this challenging task becomes experimentally feasible, in vitro experiments under conditions that mimic host environments remain the most widely accepted methods. We hypothesized that through comparative analysis of proteomic changes of S. Typhimurium in response to low Mg2þ concentrations or CAMP, we could identify the key differences between low-Mg2þ concentration- and CAMP-induced protein expression. These key differences and their functional implications in turn would provide some clues related to sensing and adaptation events in vivo. Several proteomic studies using S. Typhimurium isolated from infected RAW 264.7 macrophage15 or Salmonella strains grown in cultures with different phagosome-mimicking conditions1618 have provided abundant information about how S. Typhimurium responds to host microenvironmental conditions. However, very limited information regarding PhoP/PhoQ sensing and its signaling network can be extracted from these studies. Therefore, in this work, we conducted quantitative proteomic study focusing on proteome-wide alterations of S. Typhimurium under two PhoP/PhoQ activation conditions, low Mg2þ concentrations or CAMP. We used a SILAC (stable isotope labeling by amino acids in cell culture)-based protein labeling technique combined with liquid chromatographymass spectrometry (LCMS) analysis to generate large-scale quantitative data. This quantitative proteomic strategy was used to monitor the proteomic changes of the wild-type strain (WT) of S. Typhimurium separately exposed to low Mg2þ concentrations or a sublethal concentration of antimicrobial peptide (C18G5,19), with the wild-type strain or a PhoP-constitutive strain (PhoPc)10,20 at physiological Mg2þ concentrations as controls. By examining the known PhoPregulated proteins, we found that although both CAMP and low Mg2þ-concentration signals can activate the PhoP/PhoQ regulatory network, the scopes of their activation were different. Systematic analysis of metabolic pathways revealed that the low Mg2þ-concentration condition selectively altered expression levels. Our data also showed the influence of low Mg2þ concentrations on proteins regulated by RpoS and IHF and on proteins with undefined functions. Our quantitative proteomic analysis may provide new clues about Salmonella’s survival mechanisms within the host environment.
’ MATERIALS AND METHODS Bacterial Strains and Growth Conditions
S. serovar Typhimurium strains were kindly provided by S. I. Miller (University of Washington, Seattle, WA). Unless specified
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otherwise in the text, bacteria were grown in N-minimal medium, consisting of Buffer N21 (pH 7.4), 38 mM glycerol, 0.1% casamino acids, and MgCl2 3 6H2O for various Mg2þ concentrations. The low, physiological, and high concentrations of Mg2þ used in this study were 8 μM, 1 mM, and 10 mM, respectively.4,6 For SILAC, N-minimal medium was modified via replacement of casamino acids with 40 μg/mL lysine and 40 μg/mL arginine (adapted from ref 22 with minor modifications). “Heavy” and “light” SILAC media contained [13C6,15N2]-L-lysine and [13C6,15N4]-L-arginine (Sigma) and natural abundance lysine and arginine (Sigma), respectively. Bacterial cells from a single colony were grown in LB medium overnight, washed twice with the corresponding inoculation media before 1:100 dilutions, and grown for the indicated time. Protein Sample Preparation and Two-Dimensional Gel Electrophoresis
At each time point (3, 9, or 24 h), bacterial cells were harvested by centrifugation at 7000g for 20 min and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.3), 5 mM EDTA, 80 μg/mL lysozyme, 1 mM PMSF, and protease inhibitor cocktail (Roche Applied Science). Cell lysis was performed by sonication in an icewater bath, and unbroken cells were removed by sedimentation at 6000g for 10 min at 4 °C. The bacterial cell extract was then subjected to centrifugation at 200000g for 1 h at 4 °C. The supernatant containing soluble proteins was stored at 80 °C. A chloroform/methanol extraction-based desalting step23 was performed to remove salts from 450 μg of proteins before twodimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were resuspended in 250 μL of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.002% bromophenol blue, 2.8 mg/mL DTT, and 0.5% IPG buffer) for 30 min at room temperature. Insoluble material was removed by centrifugation at 14000g for 5 min. Isoelectric focusing (IEF) was conducted with 13 cm IPG strips in the pH range of 47 (GE Healthcare) on a Pharmacia Multiphor II electrophoresis unit. After IEF, the strips were soaked in equilibration buffer A [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 1% DTT] with rotation for 10 min and then in equilibration buffer B (the same as buffer A but with 2.5% iodoacetamide replacing DTT) for an additional 10 min. Each strip was then transferred on top of a 12% polyacrylamide gel. For the second dimension, the current was set as 20 mA/gel for the first 30 min and then at 40 mA/gel for the rest of the electrophoresis. The gels were stained with Coomassie Brilliant Blue G-250 and scanned. Gel comparison was performed using ImageMaster 2D Platinum version 6.0 (Amersham Biosciences). For spot detection, three key parameters (smooth, minimal area, and saliency) were set to 7, 30, and 3, respectively. Spots with more than 2-fold changes in abundance were considered as differentially expressed. They were then manually checked to exclude wrongly matched spots. SILAC, Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDSPAGE), and Mass Spectrometry
To prepare protein samples for SILAC analysis, bacterial cells were harvested and broken and cell debris was removed. Each SILAC sample was a 1:1 mixture of a light isotope- and heavy isotope-labeled sample. In the first set of experiments, we analyzed four groups of SILAC samples: low, Phy, C18G, and PhoPc. The heavy components of the four SILAC samples were 2993
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Figure 1. SILAC-based quantitative proteomic strategy. (A) Flowchart of our SILAC-based strategy. (B) List of the 9 h samples in the four experimental groups. (C) Number of proteins quantified in each group. (D) Examples of mass spectra used for peptide quantitation. Three MS/MS spectra identified as those of peptides from PhoP in the Low group are given to show the feasibility and accuracy of our quantitative proteomic strategy, with peptide sequences, charges, and peptide ratios indicated.
grown at low Mg2þ concentrations (Low) or physiological Mg2þ concentrations (Phy, C18G, and PhoPc) for 9 h at 37 °C, while the light components were derived from the wild-type strain grown for 3 h at physiological Mg2þ concentrations (1 mM) to serve as controls. In the second study, a minor modification was made. For direct measurement of proteomic changes during bacterial growth, the heavy isotope components and light isotope controls were prepared at the same Mg2þ concentrations. The four groups of samples were wild-type S. Typhimurium grown at a low Mg2þ concentration (8 μM, designated as Low-2nd), a
physiological Mg2þ concentration (1 mM, designated as Phy2nd), or a high Mg2þ concentration (10 mM, designated as High) or grown in the presence of a sublethal concentration (5 μg/mL) of antimicrobial peptide C18G5,19 (with a physiological Mg2þ concentration, designated as C18G-2nd). For quantitative mass spectrometry analysis, 200 μg of proteins of each group was separated by 5 to 15% SDSPAGE and each lane was cut into 20 gel bands. Protein bands were diced into small pieces and destained by being washed in a 200 mM NH4HCO3/acetonitrile mixture (1:1). Proteins were reduced with DTT, alkylated with iodoacetamide, and digested in gel 2994
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with trypsin (Promega, Madison, WI) as described previously.24 Tryptic peptides were concentrated under vacuum prior to mass spectrometric analysis. Peptide mixtures were analyzed using a QSTAR-ELITE mass spectrometer (Applied Biosystems) coupled with an Eksigent Tempo nano MDLC system. Peptides were first collected on a CapTrap column (0.5 mm 2 mm, MICHROM Bioresources, Inc.) and then eluted into an integrated nanoscale analytical column (MAGIC C18AQ, 100 μm 150 mm, 3 μm particle size, 200 Å pore size, MICHROM Bioresources, Inc.). Mobile phase A (2% ACN and 0.1% formic acid) and mobile phase B (98% ACN and 0.1% formic acid) were used to establish a 130 min gradient with a flow rate of 300 nL/min. The mass spectrometer was operated in a data-dependent MS/MS mode. MS scans were conducted from 400 to 1800 amu, at a rate of 1.0 s/ scan. For MS/MS analysis, each scan cycle consisted of one fullscan MS1 mass spectrum (m/z 4001800, charge states from þ2 to þ5) followed by five product ion scans, MS2. The threshold count was set at 30; the exclusion window was set at 90 s, and the mass tolerance was set at 50 mDa. Automatic Collision Energy and Automatic MS/MS Accumulation were selected. Data Processing
Raw MS data files were directly submitted by Mascot Daemon (version 2.2.2) (Matrix Science, London, U.K.) to an in-house MASCOT server (version 2.2) (Matrix Science) and Distiller (version 2.2.1.2) (Matrix Science). In brief, peak lists were generated by Distiller and searched against a target/decoy S. Typhimurium database downloaded from the Uniprot Web site (http://www.uniprot.org) (October 11, 2009; 6914 proteins) by MASCOT server. Mascot search parameters were set as follows: parent ion tolerance of 100 ppm and fragment ion mass tolerance of 0.40 Da. We selected carbamidomethyl Cys as a fixed modification and oxidation of methionine as a variable modification. Additionally, Arg10 and Lys8 were set as exclusive modifications. The peptide charges were þ2, þ3, þ4, or þ5, allowing up to two missed cleavages. The significance threshold was set at p < 0.05. The FDRs (false discovery rates) of Mascot search results were from 2.30% (PhoPc group) to 3.68% (Phy group) at the peptide level. For quantitation analysis, the rov files obtained from database searches were opened by Mascot Distiller. The quantitation processes in brief were as follows. (1) For the 20 LCMS/MS analyses in each sample group, 20 individual rov files were processed. (2) After these 20 quantitation reports had been combined (as Microsoft Excel files), only peptides passing stringent tests (threshold values for fraction, correlation, and standard error set to >0.5, >0.9, and 95% confidence), and it is a rough parameter reflecting protein abundance. (B) Quantitation data for PhoP-regulated proteins in all four groups.
’ RESULTS Establishment of an Experimental Strategy and Overview of the Data
To study the proteome-wide changes along with S. Typhimurium adaptation, initially we needed to define the growth time points for the pre- and postadaptation proteomes. 2D-PAGE analysis of soluble proteins from S. Typhimurium grown in various media (N-minimal medium with different Mg2þ concentrations) revealed that after the samples had grown for 3 h (early log phase), few differences (95% probability) were identified in each group, of which around 500 proteins were quantified (with at least one quantitation event) (Figure 1C). See Figure 1D for examples of mass spectra used. Low Mg2þ Concentrations and C18G Had Different Activation Effects on the PhoP/PhoQ Regulatory Network
We selected previously known PhoP-related proteins from four groups of data sets, compared their abundance by mass spectra counts (Figure 2A), and analyzed changes of their expression levels based on quantitative data (Figure 2B). It was clear that proteins in the low-Mg2þ concentration group showed the highest relative abundance in general (Figure 2A). By analyzing the protein ratios, we found that while the PhoPc and C18G groups increased the levels of expression of several PhoP-regulated proteins, many more proteins were upregulated at the low Mg2þ concentration (Figure 2B). To ensure that the different expression profiles of PhoP-regulated proteins in each group were not caused by differences in sample preparation and MS analysis, we conducted a replicate of the entire experiment and obtained similar results (Figure S4 of the Supporting Information). The PhoPc strain is a widely used mutant that can increase the net level of phosphorylation of PhoP and lead to an increased level of expression of PhoP-activated genes (pag genes).10,20,26 In this study, we found levels of only PhoP and SlyB increased 2996
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Figure 4. Metabolic pathway analysis revealed low-Mg2þ-concentration-specific regulation in certain metabolic activities. The log2 value of each protein ratio (listed in Table S2 of the Supporting Information) was used in the heat maps. Each box represents a protein, and gray boxes indicate missing data. (A and B) Heat maps of pathways differentially regulated in the low-Mg2þ concentration group. (C) Expression levels of three proteins for phosphate uptake. Asterisks refer to proteins not quantified. (D) Heat map showing expression levels of ribosomal subunits. (E) Growth curves of S. Typhimurium under four conditions.
(>2-fold change) in the PhoPc group, while levels of some other well-recognized PhoP-regulated proteins, such as PhoN, PagC, and IraP, were unchanged (Figure 2B). A possible reason for the discrepancy is that the upregulation of PhoN, Udg (also named PagA or PmrE), EptA (PagB or PmrC), and PagC was found in a previous study in which PhoPc was grown in LB medium,20 while N-minimal medium were used in our study. Slightly different culture conditions can differentially affect the S. Typhimurium proteome.18 Therefore, differences between rich LB medium and nutrient-limited N-minimal medium may affect the regulation output of different PhoP-regulated proteins. Another explanation is that as a mutant strain that intrinsically activates the PhoP/ PhoQ system, PhoPc may express PhoP-regulated proteins early, without the steps of signal sensing and transduction.
For the C18G-treated group, we found that the levels of a number of PhoP network members (PhoP, SlyB, IraP, PagC, PhoN, and ArnC) were upregulated (Figure 2B and Figure S4 of the Supporting Information). In contrast, many more PhoPregulated proteins were upregulated in the low Mg2þ-concentration group (Figure 2B and Figure S4 of the Supporting Information). To distinguish between the profiles of PhoPregulated proteins induced by low Mg2þ concentrations and C18G, we divided these proteins into several functional groups (Figure 2B). All PhoP-regulated proteins in the C18G group and a portion in the low-Mg2þ concentration group belong to three functional groups: regulators and mediators, proteins for LPS modification and CAMP resistance, and virulence factors and other enzymes (Figure 2B). The group of regulators and 2997
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Salmonella Pathogenicity Island-2 (SPI-2). Levels of PhoPregulated proteins were decreased or not changed at high Mg2þ concentrations (Figure S4 of the Supporting Information), confirming a direct linkage between Mg2þ concentrations and PhoP activation. Full activation of the PhoP/PhoQ regulatory network by low Mg2þ concentrations and partial activation by C18G were consistent with previous gene-level studies.3,7,13 In theory, this CAMP-induced partial activation can also be used as a beneficial strategy for Salmonella infection, as CAMP and low-Mg2þ concentration signals may work under different situations to promote regulation of the PhoP/PhoQ system. Systematic Analysis of Metabolic Pathways Revealed a Unique Regulation Effect of Low Mg2þ Concentrations
Figure 5. RpoS, IHF, and low-Mg2þ concentration regulation. (A) Regulatory relationships among PhoP, RpoS, and IHF (adapted from ref 28). (B) Levels of RpoS-regulated proteins were decreased in all tested groups, while IHF and those IHF/RpoS coregulated proteins showed relatively high expression levels in the low-Mg2þ concentration group. Asterisks refer to proteins not quantified.
mediators contains PhoP, SlyB, SlyA, and IraP. phoP and slyB are the most conserved genes in PhoP regulons across the Enterobacteriaceae family, and SlyB can decrease PhoP protein activity.27 SlyA can enhance the overall transcription of PhoPactivated loci.22 IraP is transcriptionally activated by PhoP to stabilize another regulator RpoS.28 For the proteins in the group for LPS modification and CAMP resistance, we identified Udg, EptA, ArnA (also named PbgP3 or PmrI), ArnB (PbgP1 or PmrH), and ArnC (PbgP2 or PmrF). They are products of the PmrA regulon that can be activated by PhoP through an intermediate PmrD for LPS modification29 and resistance to antimicrobial peptides.30 The group of virulence factors and other enzymes consists of PhoP-regulated proteins with various functions. PagC is a virulence factor required for intramacrophage survival.1 VirK and SodC1 are enzymes for resistance to antimicrobial peptides and phagocytic respiratory burst.31,32 The functions of PhoN and PcgL are unknown.2,33 In addition to those found in the three functional groups mentioned above, we discovered more PhoP-regulated proteins at high relative abundance in the low-Mg2þ concentration group (Figure 2B and Figure S4 of the Supporting Information). These proteins include MgtA and MgtB, the major Mg2þ transporters;12,34 SsrB, the response regulator controlling Salmonella Pathogenicity Island-2 (SPI-2);35 and SseL, a secretion effector,36 which can be grouped under Mg2þ transporters and
In addition to PhoP-regulated proteins, our quantitative proteomic analysis also provided a large amount of data for metabolic enzymes. We sorted and analyzed all quantified proteins according to the KEGG PATHWAY (http://www. genome.jp/kegg/pathway.html) annotations.37 On the basis of the number of proteins with quantitative data, we were able to generate information regarding protein expression for 52 individual pathways following the KEGG pathway maps (see Table S1 of the Supporting Information for the list). In 11 of the 52 pathways, we noticed pathway-level regulation with similar trends in all four experimental groups. In 3 of the 52 pathways (glycolysis, purine biosynthesis, and ribosomal subunits), we observed specific regulation in the low-Mg2þ concentration group. For the remaining pathways, the trends were difficult to discern, because of branch circuits, reversible reactions, and (or) functional complexity. Several pathways or functional categories were upregulated in all four groups. These included proteins for the biosynthesis of siderophore group nonribosomal peptides, histidine and thiamine metabolism, and the ABC transporters for the uptake of Fe, Zn, thiamine, and methionine (Figure 3A). In contrast, the citrate cycle (TCA cycle) proteins and the ABC transporters for saccharides and glutamate/aspartate were downregulated (Figure 3B). The protein expression levels for several functional categories were found to be unchanged (
