Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium
© Uchiya et al; licensee BioMed Central Ltd. 2009
Received: 27 February 2009
Accepted: 25 August 2009
Published: 25 August 2009
SpiC encoded within Salmonella pathogenicity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.
Quantitative RT-PCR shows that the expression levels of the class 3 fliD and motA genes that encode for the flagella cap and motor torque proteins, respectively, were lower for a spiC mutant strain than for the wild-type Salmonella. Further, this mutant had lower expression levels of the class 2 genes including the fliA gene encoding the flagellar-specific alternative sigma factor. We also found differences in flagella assembly between the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain, whereas the spiC mutant had only few flagella. The absence of spiC led to reduced expression of the FlhD protein, which functions as the master regulator in flagella gene expression, although no significant difference at the transcription level of the flhDC operon was observed between the wild-type strain and the spiC mutant.
The data show that SpiC is involved in flagella assembly by affecting the post-transcription expression of flhDC.
Salmonellae are gram-negative bacteria causing a variety of disease syndromes in humans and animals. For example, Salmonella enterica serovar Typhi causes a systemic disease in human known as typhoid fever, whereas S. enterica serovar Typhimurium is responsible for gastroenteritis in humans and a systemic disease in mice similar to human typhoid fever. The ability of Salmonellae to survive within macrophages is required for systemic disease . Important virulence factors are introduced into the host environment including the host cell cytosol using two different type III secretion systems (TTSSs) encoded on the Salmonella pathogenicity islands, SPI-1 and SPI-2 . SPI-1 TTSS mediates bacterial entry into non-phagocytic cells  and SPI-2 TTSS is required for survival and replication in the intracellular environment of host cells and contributes to systemic infection in animals [4–6].
The spiC gene is adjacent to spiR (ssrA)/ssrB, a two-component regulatory gene, and is the initial gene for the operons encoding the structural and secretory components of SPI-2 . Previous studies show that a strain carrying a mutation in the spiC gene is unable to survive within macrophages and has greatly reduced virulence in mice. The SpiC protein is necessary to inhibit the fusion of Salmonella-containing phagosomes with endosomal and lysosomal compartments . SpiC is translocated by SPI-2 TTSS to the cytosol of the macrophages where it interacts with host proteins, i.e. TassC  or Hook3 , to alter intracellular trafficking. Further, several investigators report that SpiC is required for the translocation of SPI-2 effector proteins into the target cells by interacting with SsaM, a SPI-2 encoded protein [10–12]. In addition to these reports, we have shown that SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages, leading to the production of mediators such as interleukin-10, prostaglandin E2, and the expression of the suppressor in cytokine signaling 3 (SOCS-3) that are thought to have important roles in Salmonella virulence [13–15]. Additionally, our recent study shows that SpiC is involved in the expression of FliC, a component of the flagella filaments, where FliC plays a significant role in SpiC-dependent activation of the signal transduction pathways in macrophages following Salmonella infection . However, the mechanism of how SpiC affects the expression of FliC remains unknown.
The flagellum is essential for bacterial motility. Its structure consists of a basal body, a hook, and a filament. In Salmonella, synthesis of the flagellum involves over 50 genes. The expression of these genes is organized into three hierarchies. At the top hierarchy is the class 1 flhDC operon and it is essential for transcription of all of the genes for the flagellar cascade. flhDC expression is influenced at the transcription or post-transcription level by a number of global regulatory factors. The class 2 operons contain genes encoding the hook-basal body-associated proteins, a few regulatory proteins, and a component of the flagellum-specific type III export pathway. The class 3 operons contain genes involved in filament formation, flagella rotation and chemotaxis [17, 18]. Flagellin, a component of the filament, is transported from the cytoplasm using the flagellum-specific type III export system in the basal body where it is polymerized with the help of the cap protein FliD [19, 20]. This results in the assembly of the long helical flagella filaments. S. enterica serovar Typhimurium expresses two antigenically distinct flagellins encoded by the fliC and fljB genes and are coordinately expressed using a phase-variation mechanism .
FliC also has a role as a potent stimulator of the immune and pro-inflammatory responses [21, 22]. Several reports show that FliC activates the signal transduction pathways via Toll-like receptor 5 (TLR5) in cultured cells (e.g. epithelial cells) leading to the induction of immune and pro-inflammatory genes [23–26]. In addition to TLR5, flagellin was recently shown to be recognized in the host cell cytosol by two different Nod (nucleotide-binding oligomerization domain)-like receptors, Ipaf and Naip5 (also known as Birc1e) [27, 28].
Here, we investigate the mechanism of how SpiC regulates flagellum synthesis in S. enterica serovar Typhimurium. We found that SpiC is involved in flagella assembly by affecting the post-transcription expression of the flhDC operon.
Results and discussion
Transcription of the spiC gene is induced during the post-exponential phase of bacterial growth in LB medium
At the same time, we determined the transcription start site for spiC using a primer extension analysis (Fig. 1C). The size of the extension product showed that the transcription start site of spiC is an adenine that lies 18 nucleotides upstream of the spiC initiation codon (ATG) in agreement with the result of Walthers et al . This indicates that the SpiC protein consists of 127 amino acids with a predicted molecular mass of 14.7 kDa.
Effect of the spiC mutation on the expression of class 3 flagellar genes
In a previous study, proteomic analysis using matrix-assisted desorption/ionization-time of flight mass spectroscopy showed that the level of the FliC protein, a component of the flagella filaments, was lower in the culture supernatant of a spiC mutant, which carries a non-polar mutation in the spiC gene, than in the supernatant from wild-type Salmonella. Further, SpiC is involved in the expression of the fliC gene at the transcription level . These results suggest the possibility that SpiC participates in flagellar phase variation or the fliC gene expression directly. However, in addition to the FliC protein, we newly identified a FliD flagella protein that was decreased in the spiC mutant using proteomic analysis with liquid chromatography-tandem mass spectrometry (K. Uchiya, unpublished result). Taken together, these results suggest that SpiC contributes to the flagellar system by mechanisms other than phase variation or direct expression of the fliC gene in S. enterica serovar Typhimurium.
Flagella expression in S. enterica serovar Typhimurium is controlled in a hierarchical manner. At the top of the hierarchy is the class 1 flhDC operon that is essential for transcription of all of the genes in the flagellar cascade. The class 2 operons contain the genes encoding the hook-basal body-associated proteins, a few regulatory proteins, and a component of the type III export pathway. The class 3 operons contain genes involved in filament formation, flagella rotation and chemotaxis [17, 18].
The spiC mutant is defective in flagella filament formation
Because the flagella filament is made from the flagellin proteins FliC and FljB, we examined flagella of the respective Salmonella strains using electron microscopy. We found differences between the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain (Fig. 3A), whereas the spiC mutant had few flagella (Fig. 3B). Additionally, the defective flagella filament formation in the spiC mutant was rescued by introducing pEG9127 (Fig. 3C). The data suggest that SpiC affects the formation of flagella filaments by controlling the expression of flagellar genes. We next examined the involvement of other SPI-2-encoded virulence factors in flagella assembly. As expected, a mutation in the spiR gene , a two-component regulatory gene involved in the expression of SPI-2-encoded genes, resulted in the defective formation of flagella filaments, similar to the spiC mutant (Fig. 3D); however, the defective phenotype was not seen in the ssaV mutant that lacks a putative component of the SPI-2 TTSS (Fig. 3E) . This suggests the specific involvement of SpiC in the assembly of flagella filaments. Further, we examined the effect of SpiC on formation of flagella filaments using N-minimal medium containing low Mg2+ (pH 5.8) that is effective in inducing SPI-2 gene expression . However, we did not observe flagella even in the wild-type strain (data not shown).
Expression of class 2 flagellar genes in the spiC mutant
To examine the mechanism by which SpiC is involved in the expression of the class 3 genes, we focused on the class 2 fliA gene encoding the flagellar-specific alternative sigma factor σ28, which is required for transcription of the class 3 promoters [33, 34]. The activity of the transcription factor σ28 is negatively regulated by direct interaction with an anti-σ28 factor, the FlgM in the cell [35, 36]. FlgM is excreted out of the cell through the flagellum-specific type III export apparatus, leading to the induction of fliA gene transcription [37–39]. SpiC is reported to be required for secretion of some virulence factors from the cytoplasm using the SPI-2 TTSS [10, 11], although the molecular mechanism is not known. Several genes encoding the SPI-2 TTSS and the flagellum-specific type III export system show sequence similarities [18, 40]. Therefore, in addition to its role in SPI-2 TTSS, SpiC might participate in the export of FlgM proteins from the cytoplasm via the type III flagellar protein export system. To examine this possibility, cell lysates were prepared and the level of intracellular FlgM was assessed using Western blot with anti-FlgM antibody. Western blot analysis showed that the level of FlgM in the wild-type cell was higher than that in the spiC mutant (data not shown), indicating that a decrease in class 3 genes expression in the spiC mutant is due to an FlgM-independent mechanism.
SpiC is required for the post-transcriptional expression of the master regulator, FlhDC
The class 1 genes products FlhD and FlhC form a heterotetramer that activates the σ70 promoter in the class 2 genes by interacting with the RNA polymerase α subunit [41, 42]. flhDC expression is influenced at the transcription or post-transcription level by a number of global regulatory factors. For example, cyclic AMP-CRP [43–46], H-NS [46, 47], QseBC , CsrA , and the heat shock-induced chaperones, DnaK, DnaJ, and GrpE , function as positive regulators, while negative regulation is mediated by OmpR , RcsCDB , LrhA , and ClpXP .
Although the molecular mechanism by which SpiC contributes to the post-transcription regulation of the flhD expression remains unknown, it is thought that SpiC directly or indirectly participates in either flhD translation or in the stability of the FlhD protein. Almost all of the positive regulators that involved in flhDC expression regulate their expression at the transcription level [45–47, 50], while CsrA, a RNA-binding protein, stimulates flhDC expression using a post-transcription mechanism . CsrA is thought to allow flhDC translation by binding to the 5' segment of the flhDC mRNA and stabilizing its mRNA. The Csr system consists of CsrA and the two small regulatory RNAs, csrB and csrC. The activity of CsrA is reported to be antagonized by csrB and csrC RNAs  where gene expression is controlled by the BarA/SirA two-component regulatory system that is involved in the expression of SPI-1-encoded genes [56–58]. One hypothesis is that SpiC affects FlhDC expression via a Csr post-transcription regulatory system. Therefore, we investigated the effect of SpiC on csrB and csrC expression using quantitative RT-PCR. However, no differences in the expression levels of these genes were observed between the wild-type strain and the spiC mutant (data not shown). More research is required to clarify the molecular mechanism in how SpiC regulates the post-transcriptional expression of the flhDC.
We next examined the expression of FlhD at bacterial growth phase of OD600 of 0.7 in LB, because the spiC expression is induced at over an OD600 of 1.5 when the bacteria are grown in LB. However, the expression level of FlhD in the spiC mutant was reduced compared to the wild-type strain even in the exponential growth phase (data not shown), indicating that the FlhD expression is not strictly growth phase-dependent. We cannot explain this phenomenon until the mechanism by which SpiC regulates the post-transcriptional expression of the flhDC or the molecular mechanism of SpiC become clear.
As described above, the BarA/SirA system is involved in not only the flagella gene expression but also the SPI-1 gene expression. Phosphorylated SirA directly interacts with promoters of the hilA and hilC genes that are the SPI-1-encoded transcription regulator genes . HilA, a member of the OmpR/ToxR family, directly activates transcription of the inv/spa and prg/org promoters on SPI-1 . In addition to the BarA/SirA system, the AraC-like regulator RitA directly controls the hilA expression leading to SPI-1 gene expression, while RitB, a helix-turn-helix DNA binding protein, negatively regulates the expression of the flhDC . Reports also show that the ATP-dependent ClpXP protease negatively regulates the expression of flagella and SPI-1 gene [54, 61]. Interestingly, mutation in the SPI-2 genes also affects the expression of the SPI-1 gene . And thus many reports show the relationship of flagella synthesis and SPI-1 gene expression.
Our recent studies show that the SpiC-dependent expression of FliC plays a significant role in activation of the signaling pathways leading to the induction of SOCS-3, which is involved in the inhibition of cytokine signaling, in Salmonella-infected macrophages . Lyons et al.  also reported that infection of polarized epithelial cells by Salmonella leads to IL-8 expression by causing the SPI-2-dependent translocation of flagellin to a basolateral membrane domain expressing TLR5. Together with our previous results, these findings suggest the involvement of FliC in SPI-2-dependent events in the pathogenesis of Salmonella infection.
In conclusion, here we show that SpiC encoded within SPI-2 is required for flagella assembly in S. enterica serovar Typhimurium. We concluded that the mechanism is due to the involvement of SpiC in the post-transcriptional expression of FlhDC. The data indicate the possibility that SPI-2 plays a role in Salmonella virulence by making use of the flagellar system.
Bacterial strains, plasmids, and growth conditions
The bacterial strains used in this study were derived from the wild-type S. enterica serovar Typhimurium strain 14028s. The spiC::kan derivative EG10128 was described by Uchiya et al. . The deletion mutant in the flhD gene was constructed using the Red recombination system . To delete the flhD or spiC gene, a kanamycin resistance gene flanked by FLP recognition target sites from plasmid pKD4 was amplified using PCR with primer regions homologous to the flhD gene (5'-TGCGGCTACGTCGCACAAAAATAAAGTTGGTTATTCTGGATGGGAGTGTAGGCTGGAGCTGCTTC-3' and 5'-CGCGAGCTTCCTGAACAATGCTTTTTTCACTCATTATCATGCCCTCATATGAATATCCTCCTTAGT-3') or the spiC gene (5'-TTGTGAGCGAATTTGATAGAAACTCCCATTTATGTCTGAGGAGGGGTGTAGGCTGGAGCTGCTTC-3' and 5'-AGATTAAACGTTTATTTACTACCATTTTATACCCCACCCGAATAACATATGAATATCCTCCTTAGT-3'). Kanamycin-resistant strains were obtained by transforming the PCR products into strain 14028s harboring λ Red recombinase on the plasmid pKD46. Disruption of the flhD or spiC gene was confirmed using PCR with flhD or spiC gene-specific primers. The kanamycin resistance gene was then removed by transforming the strain with plasmid pCP20 that expresses FLP recombinase, resulting in an in-frame deletion of the flhD or spiC gene. Plasmid pEG9127 is a derivative of pBAC108L containing the cloned spiC gene . The bacteria were grown at 37°C in Luria broth (LB). Kanamycin was used at 50 μg/ml.
RNA preparation and primer extension analysis
Bacteria were grown in LB. When the OD600 reached 0.3, 0.7, 1.1, and 1.5, the total RNA was isolated using an RNeasy kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's protocol. The RNA (50 μg) was mixed with 32P-end-labeled synthetic oligonucleotide (5'-GCAGGATGCCCATCAATAGTCATT-3'), and 50 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) was added to 30-μl reaction mixtures containing 1 mM of deoxynucleoide triphosphates, 5 mM dithiothreitol, and 1 unit of RNasin/μl. The reaction was performed at 42°C for 1 h. The extension products were analyzed using electrophoresis on a 6% polyacrylamide-7 M urea gel and compared to sequence ladders initiated with the same primer.
Oligonucleotide primers and fluorescent probes used in RT-PCR
Nucleotide sequence (5'-3')
Preparation of whole-cell proteins
An overnight culture in LB was inoculated into 15 ml of fresh LB at a 1:100 dilution. The cultures were grown at 37°C with mild aeration to an OD600 of 1.6 (the spiC-inducing condition). After a 1-ml sample of the culture was centrifuged at 18,500 × g for 15 min, the bacterial pellet suspended in 1 ml of cold water was mixed with trichloroacetic acid (final concentration 6%), placed on ice for 30 min, and centrifuged at 14,000 × g for 20 min. After drying, the pellets were dissolved in 100 μl of sodium dodecyl sulfate (SDS)- sample buffer and boiled for 5 min.
Construction of the fliA or flhD-lacZ fusion on a plasmid
To construct the transcriptional fusion of the fliA or flhD promoter region to the promoterless lacZ gene using the promoter-probe vector pRL124 , a 0.51-kbp DNA fragment containing the fliA promoter region or a 0.73-kbp DNA fragment containing the flhD promoter region were amplified using PCR with the following primers: for fliA, 5'-ACGCGTCGACTATGCGCCTGTTAGGGCGCG-3' and 5'-CGGGGTACCCACCCAATCGCGGCTGCGTA-3'; and for flhD, 5'-ACGCGTCGACGCCACATTAATGTGAAGGAC-3' and 5'-CGGGGTACCCGGATGTATGCATTGTTCCC-3'. The PCR products digested with Sal1 and Kpn1 were ligated into the same site in pRL124, producing pRL-fliA and -flhD.
Bacteria were grown overnight in LB at 37°C and diluted to 1:100 in fresh LB and grown with aeration to an OD600 of 1.6. β-galactosidase activity was measured using the substrate o-nitrophenyl β-D-galactoside as described elsewhere . Each sample was assayed in triplicate.
Transmission electron microscopy
Bacterial cells grown in LB for 20 h at 37°C without shaking were deposited on carbon-film grids, partially dried, and stained with 2.0% uranyl acetate. The negatively stained samples were observed using a 2000EX electron microscope (JEOL) at an acceleration voltage of 100 kV.
Western Blot Analysis
Whole-cell proteins (150 μg) from bacteria were fractionated in 16% Tricine-SDS-polyacrylamide gel, electrophoresed, and then electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) as described previously . The bands were detected using the ECL plus Western blot detection system (GE Healthcare, Little Chalfont, UK) according to the manufacture's instructions. The peptide fragment, DHQTITRLTQDSRV, from the FlhD polypeptide was synthesized and an antiserum specific for the oligopeptide was obtained by immunization of rabbits with the peptide coupled to keyhole limpet hemocyanin using benzidine. The resulting anti-FlhD peptide antibody was used at a dilution of 1:300. DnaK was detected with a 1:1000 dilution of anti-DnaK antibody (Assay designs, Ann Arbor, MI). Bands were analyzed using a GS-800 calibrated densitometer (Bio-Rad).
Each experiment was performed at least three times. The results are expressed as means ± the standard deviations. The data were analyzed using analysis of variance with the Dunnett's test. A value of p < 0.05 was considered statistically significant.
We are grateful to Dr. Sunao Iyoda for helpful discussions. We wish to thank Hidetaka Iwamizu and Maya Sakakibara for technical assistance and the Hanaichi Ultrastructure Research Institute Co., Ltd. for assistance using electron microscopy. This work was supported by Grants-in-Aid for the Academic Frontier Project for Private Universities; matching fund subsidy from the MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2007–2011; and for Scientific Research (C) 20590460 from the Japan Society for the Promotion of Science; and for Specially Promoted Research of Meijo University Research Institute (to K.U.).
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