The SseC translocon component in Salmonella enterica serovar Typhimurium is chaperoned by SscA
© Cooper et al.; licensee BioMed Central Ltd. 2013
Received: 4 June 2013
Accepted: 1 October 2013
Published: 4 October 2013
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© Cooper et al.; licensee BioMed Central Ltd. 2013
Received: 4 June 2013
Accepted: 1 October 2013
Published: 4 October 2013
Salmonella enterica is a causative agent of foodborne gastroenteritis and the systemic disease known as typhoid fever. This bacterium uses two type three secretion systems (T3SSs) to translocate protein effectors into host cells to manipulate cellular function. Salmonella pathogenicity island (SPI)-2 encodes a T3SS required for intracellular survival of the pathogen. Genes in SPI-2 include apparatus components, secreted effectors and chaperones that bind to secreted cargo to coordinate their release from the bacterial cell. Although the effector repertoire secreted by the SPI-2 T3SS is large, only three virulence-associated chaperones have been characterized.
Here we report that SscA is the chaperone for the SseC translocon component. We show that SscA and SseC interact in bacterial cells and that deletion of sscA results in a loss of SseC secretion, which compromises intracellular replication and leads to a loss of competitive fitness in mice.
This work completes the characterization of the chaperone complement within SPI-2 and identifies SscA as the chaperone for the SseC translocon.
Bacterial pathogens exploit host niches using strategies that block or modify host defense pathways. One such strategy employed by the Gram-negative bacterium Salmonella enterica, is the translocation of effector proteins into the host cell through a type three secretion system (T3SS). S. enterica serovar Typhimurium (S. Typhimurium) has two T3SSs encoded within Salmonella pathogenicity island-1 (SPI-1) and SPI-2 that facilitate invasion and intracellular survival within host cells [1–3]. The assembly of the T3SS is complex, involving the formation of membrane channels in the bacterial inner and outer membrane, and a terminal translocon that forms a pore in host membranes.
Both SPI-1 and SPI-2 encode a distinct group of chaperones that bind to their cognate cargo proteins to coordinate T3SS assembly and secretion of effectors. Virulence chaperones belong to one of three defined classes : class I chaperones bind to single (IA) or multiple (IB) effectors, class II chaperones interact with translocon components, and class III chaperones partner with apparatus components. Among each of the different classes, chaperones share structural similarity yet their amino acid sequence can be poorly conserved. As such, many chaperones have been first identified based on low sequence identity with previously characterized proteins, and by shared physical properties such as isoelectric point (pI). Class I chaperones tend to be small proteins (~9-15 kDa) with acidic pI, and function as dimers adopting a horseshoe-like shape [5–7]. Class II chaperones also form dimers but do not have an acidic pI, which reflects a different interaction surface required for substrate binding [8, 9].
In addition to directing secretion events, chaperone-cargo pairs can function as regulatory proteins for T3SS gene expression . The FlgN chaperone interacts with FlgK-FlgL to form a repressive complex that inhibits expression of late flagellar genes . The Yersinia virulence plasmid encodes the chaperone SycD (also known as LcrH) that aids in the secretion of the translocon components YopB and YopD, the latter of which is responsible for establishing a negative feedback loop to prevent effector gene expression at early stages of infection . As SycD is required for YopD stability in the cytosol, both chaperone and cargo are necessary for proper coordination of Yop expression.
In S. enterica, over twenty effectors secreted by the SPI-2 T3SS have been identified yet the full complement of virulence chaperones involved in their secretion remains to be identified or functionally analyzed. To date, three virulence chaperones have been characterized; we showed that SrcA chaperones the effectors SseL and PipB2 and binds to the T3SS ATPase SsaN . The SscB chaperone directs the secretion of SseF , and the class II chaperone, SseA, is responsible for the secretion of the putative translocon platform protein SseB and one of the two translocon proteins, SseD, but not SseC [14–16]. Comparative sequence analysis of SPI-2 identified a putative chaperone gene called sscA but its function had yet to be demonstrated. In light of these findings, we set out to identify and characterize the chaperone involved in secretion of the SseC translocon protein, with an a priori focus on the sscA gene in SPI-2. In this study we demonstrate that SscA interacts with SseC and is required for its secretion but is dispensable for secretion of the other translocon components SseD and SseB. Both SscA and SseC were required for fitness in infected mice and in vitro macrophage infection assays.
Protein-chaperone interactions are essential for T3SS function because they coordinate the delivery and secretion of substrate cargo. Class II virulence chaperones are particularly important since they direct translocon secretion as a prerequisite step for the proper delivery of all subsequent effectors into the host cell. Given the modest sequence similarity between the Yersinia class II virulence chaperone SycD and SscA, we analyzed SscA as the potential chaperone for the SseC translocon in the Salmonella SPI-2 T3SS. The structure of SycD shows a crescent shape molecule with the concave face possessing protein interaction sites that are common between SycD and SscA (i.e. Y40, Y52, Y93) . The Shigella class II chaperone IpgC possesses a similar structure with the concave face binding an amino acid region of its cognate cargo IpaD , suggesting that a common cargo-binding region may exist among class II virulence chaperones.
Using protein-protein interaction studies and secretion assays we demonstrated that SscA is the class II virulence chaperone for SseC and showed that this interaction is important for Salmonella pathogenicity as deletion of either sscA or sseC lead to similar attenuated phenotypes in mouse infections. As documented previously, effectors can be secreted to the cell surface of the bacteria in the absence of a functional translocon, however delivery of effector proteins into host cells requires an assembled translocon apparatus [23, 24]. Interestingly, the sseC mutant had a more pronounced negative effect on replication in RAW264.7 cells suggesting an additional role for SseC that does not depend on its secretion, or that a very small number of bacteria assemble a functional translocon in the absence of the SscA chaperone, allowing for some measure of phenotype recovery in vitro. This latter possibility was suggested for Yersinia LcrH point mutants that had reduced secretion of translocon proteins but retained some ability to intoxicate host cells from a minimal number of T3SS . In our system, we find this possibility unlikely because we found no evidence for SseC secretion in the absence of SscA chaperone even for highly concentrated samples, and the attenuation level of the sscA and sseC mutants was similar in animal infections.
All experiments with animals were conducted according to guidelines set by the Canadian Council on Animal Care. The local animal ethics committee, the Animal Review Ethics Board at McMaster University, approved all protocols developed for this work.
Strains, plasmids and oligonucleotides used in this study
Strains, plasmids and primers
Reference or source
Salmonella enterica serovar Typhimurium wild type
SL1344, deletion of sscA
SL1344, deletion of sseC
SL1344, ushA::Cm; CmR (10 μg/ml)
oriRγ, KanR cassette flanked by FRT sites
RepA1019(Ts), λ, γ, β and exo expressed from ParaBAD, AmpR
Cytoplasmic expression of C-terminal FLAG fusion protein under control of the Ptac, AmpR
pFLAG-CTC, expresses in-frame fusion of SscA-FLAG
pFLAG-CTC, expresses in-frame fusion of SseC-FLAG
General cloning, TetR, CmR
pACYC; expresses in-frame fusion of SscA-6His
Arabinose-inducible gene expression, pBR322ori, PBAD, rrnBT1,2, araC, AmpR
sseA-sscA with endogenous sseA promoter in pWSK29, pSC101ori, AmpR
Co-immunoprecipitations (co-IP) were performed with S. Typhimurium expressing SscA-FLAG or SseC-FLAG from the IPTG-inducible pFLAG-CTC plasmid. A strain carrying empty plasmid was used as a control. Strains were grown overnight in Luria-Bertani broth (LB) and sub-cultured 1:50 into 50 ml of LPM medium and grown to an optical density (OD600) of 0.6 at 37°C. Cultures were then centrifuged at 3000 × g for 10 min, and re-suspended in phosphate buffered saline (PBS) containing mini-EDTA protease inhibitor cocktail (PBS-PI; Roche). Cells were lysed by 6 pulses of sonication for 30 sec each, with 60 sec intervals between sonication (Misonix Sonicator 3000, Misonix). Lysates were centrifuged at 3000 × g for 15 min at 4°C and the supernatant removed to obtain the cytosolic protein fraction. M2-agarose beads conjugated with anti-FLAG antibodies (F-gel; Sigma) was equilibrated with PBS-PI containing 10 μg/ml bovine serum albumin (BSA) for 60 min at 4°C with rocking and washed with PBS-PI three times. The beads were mixed with the cytosolic protein fractions and incubated for 16 h at 4°C with end-on-end mixing. Unbound proteins were removed by centrifuging the F-gel at 1000 × g for 5 min and removing the supernatant. The F-gel was washed ten times with PBS-PI containing 0.1% Triton-X100 before eluting bound proteins into sodium dodecyl sulfate (SDS)-sample buffer (1M Tris pH 8.0, 20% SDS, 0.5 M EDTA pH 8, 10% glycerol, 200 mM dithiothreitol). Bound proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Western blots were probed with antibodies to SseC (a gift from Dr. Michael Hensel), the FLAG epitope (Sigma), or the His6 tag (Qiagen). For reciprocal co-immunoprecipitations, a strain containing a plasmid encoding sscA-HIS 6 and a second compatible plasmid encoding sseC-FLAG was used. SscA-His6 was induced with arabinose and SseC-FLAG was induced with IPTG as above. In this experiment, the anti-FLAG gel was used for immunoprecipitations and anti-His antibody used in immunoblotting as described above.
Wild type S. Typhimurium and ΔsscA strains were grown overnight in LB and sub-cultured 1:50 into LPM and grown to OD600 of 0.6. Cultures were then centrifuged for 2 min at 10,000 × g and the supernatant was filtered through a 0.2 μm filter (Pall Scientific) and precipitated with 10% trichloroacetic acid (TCA). Precipitated secreted proteins were centrifuged at 16,000 × g at 4°C for 30 min and the pellets were washed with acetone and dissolved in SDS-sample buffer. The whole cell lysate fraction was made by dissolving the bacterial pellet in SDS-sample buffer. To ensure equal protein loading in SDS-PAGE, the volume of SDS sample buffer was adjusted according to the optical density of the original culture. Protein samples were analyzed by Western blot using antibodies to DnaK (Convance), SseC (a gift from Dr. Michael Hensel), SseB, SseD, and SseG (a gift from Dr. John Brumell).
RAW264.7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C with 5% CO2. Cells were seeded 16 h prior to infection into 24-well plates at a density of 2 × 105 cells per well. Overnight cultures of bacteria were washed with PBS, diluted in DMEM/10% FBS, and used to infect macrophages at a multiplicity of infection (MOI) of 50 for 30 min at 37°C, 5% CO2. Infected cells were washed three times with PBS and the media was replaced with DMEM/10% FBS/100 μg/mL gentamicin for 1.5 h to kill extracellular bacteria. Cells were then washed twice with PBS and incubated for 20 h in DMEM/10% FBS with10 μg/mL gentamicin. At 2 h and 20 h after infection, the cells were washed twice with PBS then lysed with 1% Triton X-100, 0.1% SDS in PBS to release intracellular bacteria. Colony forming units (cfu) were determined by plating serially-diluted lysates onto LB agar plates containing appropriate antibiotics. Experiments were performed twice independently using 3 technical replicates per assay.
Competitive infections were performed in female C57BL/6 mice (Charles River) by oral inoculation of a 0.1 ml mixture containing equal numbers (1×108 cfu) of a chloramphenicol resistant wild type strain (ushA::Cm) and mutant strains as described previously . The marked wild type strain was previously shown to be phenotypically neutral . Three days after infection, the spleen, liver and cecum was removed, homogenized in ice-cold PBS (Mixer Mill, Retsch) and serially diluted in PBS. The competitive index (CI) was determined by plating dilutions of the homogenized tissue lysates on agar plates containing streptomycin and incubating overnight at 37°C to recover both wild type and mutant bacteria. Colonies were then replica-stamped onto separate plates containing streptomycin and chloramphenicol to enumerate wild type and mutant bacteria. The CI was calculated as (cfu mutant/cfu wild type)output/(cfu mutant/cfu wild type)input. Mouse experiments were performed twice using groups of 5 mice for each experiment. Statistical analysis was performed using a Student t test.
In summary, we have verified that SscA is the chaperone for the SseC translocon component in the T3SS encoded by SPI-2. This work completes the characterization of the known chaperone complement within SPI-2. In future work, it will be useful to investigate whether this particular chaperone-cargo pair has any additional regulatory function on gene expression within SPI-2.
Conducted experiments and analyzed data: CAC, DTM, SEA. Wrote manuscript: CAC, DTM, BKC. Edited manuscript and provided essential discussion: CAC, DTM, SEA, AVCP, BKC. All authors read and approved the final manuscript.
Salmonella pathogenicity Island
Type 3 secretion system
Salmonella enterica serovar Typhimurium
This work was supported by the Canadian Institutes of Health Research (CIHR; MOP-82704), the Natural Sciences and Engineering Research Council (NSERC) and the Canada Research Chairs program. CAC is the recipient of an NSERC postgraduate scholarship; DTM and SEA are each supported by a Canada Graduate Scholarship from the CIHR. BKC is the Canada Research Chair in Infectious Disease Pathogenesis.
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