Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages
© Haneda et al; licensee BioMed Central Ltd. 2010
Received: 1 September 2010
Accepted: 21 December 2010
Published: 21 December 2010
The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established.
Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2.
A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.
The facultative intracellular bacterium Salmonella enterica causes a broad spectrum of diseases, such as gastroenteritis and bacteremia, which are typically acquired by oral ingestion of contaminated food or water. S. enterica serovar Typhimurium (S. Typhimurium) causes enterocolitis in humans and a typhoid-like systemic infection in mice.
Several virulence genes associated with Salmonella pathogenicity islands (SPIs) and the virulence plasmid have been characterized in S. Typhimurium. Two type III secretion systems (T3SS) encoded by SPI-1 and SPI-2 play central roles in Salmonella pathogenesis. SPI-1 is essential for the invasion of host cells and the induction of apoptosis in infected macrophages [1, 2]. SPI-2 T3SS primarily confers survival and replication on macrophages and is required for systemic infection in the mouse infection model [3, 4]. Expression of SPI-2 genes is induced within a modified phagosome, called the Salmonella-containing vacuole (SCV), in infected macrophages . Induction of SPI-2 genes depends on a two-component regulatory system, SsrA/SsrB, encoded within the SPI-2 region . Expression of SsrAB is also mediated by two-component regulatory systems, OmpR/EnvZ and PhoP/PhoQ, which sense osmotic stress and cation limitation, respectively [7, 8]. In addition, a global transcriptional regulator, SlyA, which interacts directly with the ssrA promoter region, is involved in the expression of SPI-2 T3SS [9–11].
During infection of mammalian hosts, S. Typhimurium has to rapidly adapt to different environmental conditions encountered in its passage through the gastrointestinal tract and its subsequent uptake into epithelial cells and macrophages. Thus, establishment of infection within a host requires coordinated expression of a large number of virulence genes necessary for the adaptation between extracellular and intracellular phases of infection. It has been demonstrated that the stringent response plays an important role in the expression of Salmonella virulence genes during infection [12–14].
The stringent response is mediated by the signal molecules, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (both are referred to as ppGpp in this manuscript), which accumulate in bacterial cells and exert both positive and negative effects on the transcription of many genes. ppGpp plays an important role in the virulence of pathogenic bacteria . In Gram-negative bacteria, ppGpp is synthesized by two tynthases, the synthase I and the synthase II, which are encoded by the relA and spoT genes, respectively . These enzymes respond differently to environmental conditions. RelA is activated by the binding of uncharged tRNA to ribosomes upon amino acid starvation. SpoT is induced during the exponential growth phase and responds to other changes in environmental conditions, specifically a lack of carbon sources or energy deprivation . ppGpp binds directly to the β and β' subunits of RNA polymerase (RNAP), leading to destabilization of the RNAP-rRNA promoter open complex . Moreover, the stringent response is increased by the availability of free RNAP, which gives rise to σ competition . ppGpp indirectly activates the expression of many stress-induced genes by its release from RNAP σ70-dependent promoters and by facilitating the use of alternativeσ factors. It has been shown that ppGpp is not only essential for surviving periods of stress but also for the interaction of bacteria with their host .
In case of S. Typhimurium, a mutant strain deficient in both relA and spoT (ΔrelA ΔspoT) shows marked reductions in both bacterial invasion into host cells and proliferation in macrophages [12, 13]. Furthermore, the virulence of the ΔrelA ΔspoT mutant is severely attenuated in mice [12, 13]. ppGpp controls the expression of SPI-1 to -5 and Spv through their transcriptional regulators HilA, InvF, RtsA, SsrA, SlyA, and SpvR [12–14, 21]. These observations indicate that ppGpp may play a major role in Salmonella virulence via the altered expression of regulatory genes. Because ppGpp has been shown to affect the expression of many virulence genes in S. Typhimurium, it is likely that there are additional virulence genes among the ppGpp-regulated genes.
In this study, we constructed an agarose 2-dimensional electrophoresis (2-DE) reference map of S. Typhimurium grown under amino acid starvation to identify ppGpp-regulated proteins from whole-cell preparations. By comparative proteomic analysis of ppGpp-regulated and Salmonella- specific proteins, we identified a novel virulence factor, STM3169, required for intracellular survival within macrophages.
Results and Discussion
Agarose 2-DE reference map of S. Typhimurium with induced stringent responses
Next, we classified proteins identified on the map using the KEGG pathway database. While 156 proteins (45.3%) were classified into several metabolic categories (carbohydrate, energy, lipid, nucleotide, amino acid, and other amino acids), 70 proteins (22.8%) were grouped in the no entry category, which means that these proteins do not belong to the other categories. This category contained 20 known virulence-associated proteins, including flagella and flagella biosynthesis proteins (FliC, FljB, FliY, FliG, FliM, and FliD), SPI-1 effectors (SipD, SopB, and SopE2), an SPI-1 translocase (SipC), an iron transporter (SitA), superoxide dismutases (SodA, SodB, SodC1, and SodC2), a quorum-sensing protein (LuxS), a two-component response regulator (PhoP), peptidyl-prolyl cis-trans isomerases (FkpA and SurA), and a periplasmic disulfide isomerase (DsbA).
Identification of ppGpp-regulated proteins using comparative proteomics
S. Typhimurium proteins regulated by ppGpp
Average fold change determined by qRT-PCR
Proteins expressed lower in Δ relA Δ spoT strain
0.67 ± 0.22
0.18 ± 0.01c
0.25 ± 0.06c
0.25 ± 0.03c
0.60 ± 0.35
0.88 ± 0.35
0.17 ± 0.06c
0.15 ± 0.05c
0.15 ± 0.05c
0.54 ± 0.22
0.12 ± 0.05c
0.28 ± 0.12c
0.30 ± 0.02c
14.22 ± 2.22c
Proteins expressed higher in Δ relA Δ spoT strain
Of these proteins, six genes (treA, ugpB, ynhG, yliB, ugpB, degQ) had previously been identified as ppGpp-regulated genes in E. coli at the transcriptional level . In S. Typhimurium, it has been shown that ppGpp controls the expression of known virulence-associated genes, including sipC, fliY, sopB, and sodC1, in response to growth conditions relevant to host infection . Thus, to confirm the results from the comparative proteomic analysis, mRNA levels of the remaining 13 genes were assessed by qRT-PCR. As a result, mRNA expression levels of eight genes (stm3169, cpdB, tolB, ydgH, oppA, yajQ, yhbN, ytfJ) were significantly higher in SH100 than in TM157 under stringent conditions (Table 1).
Identification of novel virulence-associated factors regulated by ppGpp
Because it is believed that intracellular Salmonella is likely to be restricted to the acquisition of nutrient substrates from infected host cells, the stringent response could occur in SCV. Thus, we next analyzed the contribution of STM3169 to intracellular survival of S. Typhimurium in macrophages. In accordance with previous data that a ppGpp0 mutant strain deficient in both spoT and relA genes resulted in a severe reduction of intracellular proliferation and suvival . In contrast to the wild-type level of invasion, intracellular survival of TH973 in RAW264.7 cells was reduced, compared with that of the wild-type strain. The reduced CFU of TH937 in IFN-γ treated-RAW264.7 cells was not more severe than that in the ΔrelA ΔspoT double mutant, ΔssaV (SH113, SPI-2 T3SS component-defected mutant), and ΔssrB (YY1, SPI-2 regulator mutant) strain, but was equal to that in the ΔsseF (TM548, SPI-2 effector mutant) strain (Figure 5B and 5C). These results suggest that the expression of additional virulence factors, like STM3169, in macrophages might be affected in a highly avirulent phenotype of a ppGpp-deficient strain in mice.
stm3169 is regulated by the SPI-2 transcriptional regulator ssrB
It has been reported that ppGpp regulates SPI-2-encoded genes under aerobic condition . To further characterize the transcriptional regulation of stm3169 by ppGpp and SsrB, we constructed a ΔrelA ΔspoT ΔssrB triple mutant strain (YY2), and examined the affect of the transcriptional activity on stm3169::lacZ fusion gene. While the transcriptional activity of stm3169::lacZ fusion in the triple mutant strain was significantly reduced at the same level of ΔrelA ΔspoT double mutant strain, it could be restored by introduction of plasmid pSsrB expressing SsrB-FLAG but not pRelA expressing His6-tagged RelA (Figure 6C). These results indicate that ppGpp is controlled the expression of stm3169 through SsrB.
STM3169 is homologous to DctP in Rhodobacter capsulatus with a 31% identity and a 73% similarity. DctP, along with DctQ and DctM, constitutes a tripartite ATP-independent periplasmic transporter (TRAP-T) system involved in succinate utilization, and DctP plays a role as an extracytoplasmic solute receptor in this transporter . STM3170 and STM3171, which are located immediately downstream from STM3169, have a 66% and an 80% similarity with DctQ and DctM, respectively. These suggest that the TRAP-T in S. Typhimurium is composed of stm3169, stm3170, and stm3171 genes. In addition, two hypothetical operons, yiaOMN and stm4052-4054, are annotated as TRAP-T in the S. Typhimurium strain LT2 . Recently, it has been reported that the TRAP-T (SiaPQM) in Haemophilus influenzae is essential for LPS sialylation and virulence . Further research is necessary to determine the role of these transporters in S. Typhimurium virulence.
We constructed an agarose 2-DE reference map of amino-acid starved S. Typhimurium and identified a novel virulence-associated factor, STM3169, regulated by ppGpp by applying the map to comparative proteomics. stm3169 is also regulated by an SPI-2 two-component regulator, SsrB. Recently, it has been reported that the lack of ppGpp synthesis in Salmonella strains attenuates virulence and induces immune responses in mice . Thus, further analysis of proteins regulated by ppGpp may lead to the development of new vaccines.
Bacterial strains, primers, and culture conditions
Bacterial strains and plasmids used.
Spontaneous nalidixic acid resistant derivative of wild-type 14028
SH100 ΔrelA::cat ΔspoT::kan
SH100 ΔrelA::cat ΔspoT::kan ΔssrB::tet
K-12 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 (lacXYA-argR)U169 deoR (80 dlac (lacZ)M15)
thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpir
TA cloning vector
pSC101-based low copy number plasmid
p15A-based low copy number plasmid, tet template
New England Biolabs
FLAG tag expression vector
Integrational plasmid with promoterless lacZ gene
Expression vector for His6 fusion protein
stm3169 gene in pMW118
stm3169::lacZ operon fusion in pLD-lacZ Ω
ssaG::lacZ operon fusion in pLD-lacZ Ω
pBAD-HisA expressing relA gene
pFLAG-CTC expressing ssrB gene
Red recombinase expression plasmid
kan cassette used for gene deletion
Nucleotide sequence (5' to 3')a
Construction of the deletion mutants
Construction of plasmids for the complementations
Construcion of the lacZ fusions
Comfirmation of gene expression by qRT-PCR
Construction of mutants
Nonpolar mutants of relA and spoT were constructed by allele exchange using the temperature- and sucrose-sensitive suicide vector pCACTUS . The relA and spoT genes were amplified by PCR with the following primers: (1) relA-FW and relA-RV for relA and (2) spoT-FW and spoT-RV for spoT. S. Typhimurium strain SH100 genomic DNA was used as the template. The PCR products were cloned into TA cloning vector pGEM-T Easy (Promega) generating plasmid pGEM-relA and pGEM-spoT, respectively. A disruption mutation of relA was created by the insertion of the HincII-digested promoterless cat gene into a unique NruI site in the coding sequence of relA on pGEM-relA. The resulting plasmid pGEM-relA::cat was digested with BglII and then self-ligated, yielding plasmid pGEM-ΔrelA::cat. In contrast, the spoT gene was disrupted by the insertion of a SmaI-digested Kmr-encoding gene (kan) cassette from pUC18K  into NruI sites in the coding sequence of spoT on pGEM-spoT, thus generating pGEM-ΔspoT::kan. The disrupted gene was then subcloned using SalI and SphI into similarly digested pCACTUS, and the resulting plasmid was introduced into strain SH100 by electroporation for allele exchange mutagenesis, which was carried out as described previously . ΔrelA ΔspoT mutant strain was created by phage P22-mediated transduction .
The PCR-based λ Red recombinase system using pKD46 and pKD4 was performed to disrupt stm3169 or sseF. The growth rate of these mutant strains in LB and MgM (pH5.8) broth showed the same levels to wild-type strain.
To construct ΔrelA ΔspoT ΔssrB mutant strain, the cloned ssrB gene was disrupted by the insertion of a Tetr-encoding gene (tet) cassette, which was amplified with pAC-tet-FW and pAC-tet-RV primers using pACYC184 (New England Biolabs) as template. The ΔssrB::tet fragment was amplified by PCR using ssrB-FW and ssrB-RV primers, and the resulting PCR product was introduced into S. Typhimurium SH100 carrying pKD46. The disrupted genes were transferred by phage P22 transduction into ΔrelA ΔspoT mutant strain TM157.
To construct ssaG::lacZ and stm3169::lacZ transcriptional fusions, pLD-ssaGZ and pLD-stm3169Z were transferred from Escherichia coli SM10λpir to S. Typhimurium SH100 by conjugation. The fusions were introduced into SH100, ΔrelA ΔspoT (TM157), ΔssrB::tet (YY3), and ΔssaV (SH113) mutant strains by phage P22-mediated transduction. All constructs were verified by PCR or DNA sequencing.
Construction of plasmids
For construction of the complementing plasmid, pMW-Stm3169, stm3169 gene was amplified by PCR with stm3169-FW and stm3169-RV primers. S. Typhimurium SH100 genomic DNA was used as the template. The PCR products were digested with BglII and XhoI, and cloned into the Bglll-XhoI site on pMW118 (Nippon Gene), generating plasmid pMW-Stm3169.
To construct pRelA and pSsrB, the target genes were amplified by PCR with the following primers: relA-FW2 and relA-RV2 for relA and ssrB-FW and ssrB-RV for ssrB. The PCR product containing relA was digested with XhoI-HindIII and cloned into the same sites on pBAD-HisA (Invitrogen). The PCR product containing ssrB was digested with XhoI-BamHI and cloned into the same sites on pFLAG-CTC (Sigma). pRelA and pSsrB expressed His6-tagged RelA and SsrB-FLAG fusion protein, respectively.
To construct lacZ transcriptional fusions, the DNA fragments containing (predicted) promoter regions of ssaG were amplified by PCR using the primers ssaG-Pro-FW and ssaG-Pro-RV, and those containing promoter regions of stm3169 were amplified using stm3169-Pro-FW and stm3169-Pro-RV. The PCR products digested with SalI and BamHI were ligated into the same sites of pLD-lacZ Ω .
Sample preparation for agarose 2-DE
Agarose 2-DE samples were prepared from amino-acid starved S. Typhimurium strain SH100, as well as relA and spoT double knockout strain TM157 (ΔrelA ΔspoT). The cell pellets were washed twice with cold phosphate-buffered saline (PBS) and dissolved in lysis buffer containing 5 M urea, 1 M thiourea, 0.05% w/v β-mercaptoethanol, and one tablet of protein inhibitor (Complete Mini EDTA-free; Roche Diagnostics, Mannheim, Germany), which was dissolved in 10 mL of the solution. The lysates were centrifuged (104,000 × g, 20 min, 4°C) and the clear supernatant was used.
We performed proteome analysis according to the procedures of Oh-Ishi et al. and Kuruma et al.. An aliquot of 200-300 μL (containing 500 μg of protein) of sample solution was subjected to first-dimension IEF at 667 V for 18 h at 4°C, followed by second-dimension SDS-PAGE. The slab gel was stained with CBB R-350 (PhastGel Blue R; GE Healthcare).
Protein spots were excised from a destained gel with 50% (v/v) ACN and dried under vacuum. The proteins were digested in the gel with trypsin. Digested fragments of 15 pmol were loaded on a Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), which consisted of Nanospace SI-2 (Shiseido Fine Chemicals), an HPLC (LCQ Deca), and an ion trap mass spectrometer (Thermo Finnigan). We identified a protein from measured masses of the tryptic peptides and their MS/MS fragments using the SEQUEST program. When the top-ranked candidates had SEQUEST scores lower than 90, we inspected the raw MS and MS/MS spectra of peptides to judge their qualities. We classified identified proteins according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database http://www.genome.ad.jp/kegg/pathway.html.
Gel-to-gel comparisons between SH100 and TM153 were performed for two separately prepared samples. Each scanned 2-DE gel image was analyzed with the gel image analysis software SameSpots (Progenesis).
RNA extraction and quantitative real-time PCR
S. Typhimurium strains were grown in LB and ppGpp expression was induced as described above. Total RNA was isolated from the bacterial culture using RNAprotect Bacteria Reagent and the RNeasy Protect Bacteria Mini Kit with the gDNA Eliminator spin column (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was performed with the primer pairs listed in Table 3 using QuantiTect SYBR Green and the 7900HT Sequence Detection System (Applied Biosystems). The data were analyzed using the comparative Ct method (Applied Biosystems). Transcription of the target gene was normalized to the levels of gyrA mRNA.
For the competitive index assay, female BALB/c mice (5-6 weeks old) were used for the mouse infection study and were housed at Kitasato University according to the standard Laboratory Animal Care Advisory Committee guidelines. Mice were inoculated by intraperitoneal infection with 100 μL of inoculum containing a total of 1 × 105 bacteria (each strain at 5 × 104), consisting of an equal number of wild-type and mutant strains. At 48 h after infection, the mice were sacrificed by carbon dioxide inhalation. The spleens were homogenized in cold PBS by mechanical disruption. The number of each strain in the spleen was determined by plating a dilution series of the lysate onto LB agar alone and LB agar with appropriate antibiotics. Each competitive index value was calculated as [mutant/wild-type] output/[mutant/wild-type] input and represented as the mean of at least three independent infections.
Macrophage survival assay
Cells of a mouse macrophage-like line, RAW264.7, were diluted in DMEM containing 10% FBS and seeded in 24-well plates at a density of 5 × 105 cells per well. S. Typhimurium strains were used to infect RAW264.7 cells at a multiplicity of infection of 1. The bacteria were centrifuged onto the cells (500 ×g, 5 min) and incubated for 25 min at 37°C in a 5% CO2 incubator. Cells were washed three times with PBS, and DMEM containing interferon-γ (IFN-γ) (100 units/well; Peprotech) and gentamicin (100 μg/mL; Sigma) was added. After 95 min of incubation, the medium was replaced with DMEM containing IFN-γ (100 units/well) and gentamicin (10 μg/mL). The number of intracellular bacteria was determined at 2 h and 24 h after infection. For the enumeration of intracellular bacteria, the cells were washed three times with PBS and lysed in 1% Triton X-100, and bacteria were quantified by spreading serial 10-fold dilutions of RAW264.7 cell lysates on LB agar plates to count the colony-forming units (CFU). Each experiment was repeated three times.
β-galactosidase activities of reporter gene fusions were determined according to a standard procedure .
The competitive index, mRNA expression, and bacterial proliferation in macrophage cells were compared using Student's t-test. For comparative proteomics, the intensity of the spot was compared by one-way ANOVA. Values of P < 0.05 were considered statistically significant.
We thank Toru Hattori (SCRUM inc, Japan) for 2-DE gel image analysis. We thank Kaori Dobashi, Nobue Nameki, Masato Hosono, Kohei Yamashita, and Ayako Mizuta for their technical assistance.
This work was supported in part by Grants-in-Aid for Young Scientists (B) (17790291 and 22790415 for TH) and for Scientific Research (C) (17590398 and 21590490 for NO) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Kitasato University Research Grant for Young Researchers (2010 for TH).
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