Research Article pubs.acs.org/synthbio
A Secretion-Amplification Role for Salmonella enterica Translocon Protein SipD Anum Azam Glasgow,† Han Teng Wong,‡ and Danielle Tullman-Ercek*,§,∥ †
UC Berkeley-UCSF Graduate Program in Bioengineering, University of California Berkeley, Berkeley, California 94720, United States ‡ Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California 94720, United States § Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States ∥ Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States S Supporting Information *
ABSTRACT: The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is posttranscriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation. KEYWORDS: protein engineering, type III secretion, translocon, gene networks, Salmonella, virulence mechanisms
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facilitate infection by hijacking cellular processes and allowing bacteria to colonize the cell while evading an immune response.8−10 Known signal sequences selectively target cargo proteins to the T3SS. These signal sequences can additionally be repurposed to target heterologous proteins for export via the T3SS.5,7 Type III secretion has two distinct benefits as an engineering target. First, although the T3SS is essential to pathogenicity, this secretion apparatus is not essential for growth under lab conditions in organisms such as Salmonella enterica. Second, the T3SS can translocate proteins from the cytosol to outside the cell in a single step, as it spans both the inner and outer bacterial membrane. These advantages allow the T3SS to be repurposed for protein secretion without negative consequences for bacterial survival, growth, and production yield. Moreover, advances in manipulating the
rotein production at industrial scales relies heavily on bacterial systems.1−3 Bacteria grow quickly to high cell density, are genetically tractable, and give high yields of heterologous protein. However, downstream processing to recover and purify the protein of interest can be expensive and time-consuming, and yields from many bacterial systems remain too low for commercialization. Secreting proteins of interest from bacteria could ameliorate these problems, and several microbial secretion pathways are promising routes to exporting heterologous proteins from production hosts. A secretion strategy would eliminate the cell lysis step and many purification steps in industrial protein production. However, these systems suffer from incomplete characterization resulting in nonhomogenous product, low-yield secretion, and unpredictable specificity. The type III secretion system (T3SS) is a promising mechanism for industrial-level secreted protein production that overcomes these issues.3−7 Many pathogenic Gram-negative bacteria use T3SS to secrete toxic effector proteins into host cells, where they © XXXX American Chemical Society
Received: November 6, 2016
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DOI: 10.1021/acssynbio.6b00335 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Research Article
ACS Synthetic Biology system may also apply to in vivo applications of bacteriamediated targeted protein delivery.11,12 The T3SS includes a membrane-anchored apparatus composed of hundreds of copies of 25−30 different proteins,13 including structural proteins, chaperones, transcription factors, effector proteins and regulators. A hallmark of the T3SS is the “injectisome”, a needle-like structure that extends from the membrane into the extracellular space, through which proteins are secreted in an unfolded state. In S. enterica, a genomic island known as Salmonella Pathogenicity Island-1 (SPI-1) encodes a T3SS that is required for invasion into epithelial cells.14 The SPI-1 T3SS is a tightly controlled, spatiotemporally coordinated genetic circuit in which transcription factors control the expression and secretion of downstream effector proteins.5,6 This complex regulation ensures that the injectisome is constructed only under proper conditions, and that effector expression follows its assembly.4,6 Effector proteins are then secreted when the T3SS needle comes into contact with a host cell. The mechanism underlying this signal transduction event to activate effector secretion continues to be an area of active research.15−18 On the basis of evidence from several T3SS-expressing organisms, several groups have hypothesized that that the translocon proteinsstructural proteins that cap the T3SS needle and interact with host cellsare involved in regulating effector secretion.15,19−22 The translocon genes in S. enterica, sipB, sipC, and sipD, are encoded on SPI-1 in the sicA operon immediately preceding several effector genes and their chaperones. SipD is secreted by a nascent T3SS filament, binds to the tip of the structure, and makes contact with an endothelial cell membrane in the invasion phase of S. enterica infection.19,21 Immediately following the invasion event, SipB and SipC are secreted and bind to SipD to form a pore in the host cell membrane. These steps are followed by a dramatic increase in expression and secretion of other effectors. Interestingly, ΔsipD strains lose their ability to invade endothelial cells but retain secretion-competence, constitutively secreting effector proteins.23,24 Constitutive secretion by the T3SS is desirable for increased heterologous protein production because the complex regulation of the native T3SS limits protein secretion. Previously, we used the native T3SS to secrete degradation-prone biopolymer proteins that form intracellular inclusion bodies, establishing secretion as a viable strategy to improve the purity and yield of full-length proteins.3 However, secretion titers for these heterologous proteins were modest at 10−30 mg/L. In this study, we discovered that complementation of SipD in trans via exogenous addition of the protein to T3SS-expressing cultures increases heterologous secretion titers rather than restoring the native phenotype. We capitalized on these observations to engineer a hyper-secreting strain with 100fold improved secretion titers over the wild-type S. enterica SL1344-derived strain (SL1344*). Moreover, we determined that exogenous addition of purified SipD to T3SS-induced cultures enables access to the intracellular pool of heterologous proteins that have already been expressed for higher secretion efficacy. We characterized this effect by (1) correlating the increased secretion phenotype to the activities of several T3SS promoters in the regulon under the control of HilA as well as the pHilA promoter, (2) mapping this new function of SipD to a specific structural domain, and (3) determining that the effect is post-transcriptional and post-translational. To our knowledge, this study is the first to show that the presence of
extracellular SipD also promotes secretion independent of any cytoplasmic SipD depletion effect, and in addition to its established structural role in promoting the interaction with the host cell. This dual structural and regulatory role is uncommon in proteins and suggests that the T3SS presents a valuable engineering target for achieving controllable, high-titer secretion.
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RESULTS AND DISCUSSION Knocking Out sipD and Exogenously Adding SipD Increase Secretion Titer. Previous work indicates that the absence of SipD results in an ungated constitutive secretion of native effectors.23,24 We set out to assess the secretion competency of an SL1344* ΔsipD strain for heterologous proteins targeted for secretion. We employed strains carrying an export plasmid encoding a fusion of a FLAG-tagged target gene with the N-terminal domain of the native effector gene sptP under the control of the native SPI-1 sicA promoter.3,5 The N-terminus of SptP forms a flexible loop structure that binds its cognate chaperone, SicP, and targets heterologous proteins to the T3SS.26 SicP is also encoded on the export plasmid to ensure that there are sufficient chaperones to keep the expressed heterologous proteins in a secretion-competent state. The export plasmids harbored genes encoding the following target proteins: magainin-1, an antimicrobial peptide from frog skin; ADF-3 and pro-resilin, which are silk and resilin monomer proteins, respectively; and 2XTE, a protein derived from two repeats of the elastomeric domain of tropoelastin3,27 (see Tables S1 and S2 for plasmids and primers). All proteins are secreted from wild-type SL1344* at 90% sequence similarity at the C-terminus but very little sequence similarity with LcrV and PcrV, so it is possible that the absence of a small IpaD chaperone in Shigella is also compensated by the activity of the IpaD N-terminal region. Indeed, through random mutagenesis, IpaD has been shown to act as both a regulator and as part of a signal transduction pathway in type III secretion, though the mechanism for these activities remains F
DOI: 10.1021/acssynbio.6b00335 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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ACS Synthetic Biology
unless noted. Protein samples were prepared for sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) by boiling for 10 min in 1× Laemmli buffer with 2% SDS (Fisher Scientific). The samples were then loaded onto 15% polyacrylamide gels and subjected to 130 V for 70 min. For samples analyzed by Coomassie staining, the gels were stained according to the method of Studier.50 For samples analyzed by Western blotting, the samples were then blotted from the gels to polyvinylidene fluoride membranes (Millipore) following standard procedures, at 4 °C using 100 V for 1 h in a wet transfer apparatus (Mini Trans-Blot Cell, Bio-Rad) in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8). Western blots were probed with either anti-FLAG or anti-His primary monoclonal antibodies produced in mice (Sigma, product numbers F3165 and H1029) against the incorporated C-terminal FLAG tags and 6×-N-terminal His tags on all proteins of interest. After washing, the blots were then probed using antimouse secondary antibodies conjugated to horseradish peroxidase (HRP) (Sigma). To control for lysis, the blots were incubated with anti-GroEL primary antibodies produced in rabbit (Sigma) and then antirabbit secondary antibodies conjugated with HRP (Sigma). Detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for whole culture lysate samples over a total exposure time of 10 min on a Bio-Rad Chemidoc Imager. Either the last image, or the last image before the Imager detected pixel saturation, was used. A standard curve using 0−5 μg of a FLAG-BAP standard (Sigma) was generated for exact quantification. Relative quantification was performed using SL1344*/pLac-hilA pSicA-DH without addition of SipD as the standard. For secretion fraction samples, detection was performed with SuperSignal West Femto Chemiluminescent Substrate (ThermoFisher Scientific). Blots were autocorrected for contrast. Liquid Chromatography−Mass Spectrometry. Trypsindigested protein samples were analyzed using a Thermo-Dionex UltiMate3000 RSLCnano liquid chromatograph that was connected in-line with an LTQ-Orbitrap-XL mass spectrometer equipped with a nanoelectrospray ionization (nanoESI) source (Thermo Fisher Scientific, Waltham, MA). This instrumentation is located in the QB3/Chemistry Mass Spectrometry Facility at the University of California, Berkeley. The LC was equipped with a C18 analytical column (Acclaim PepMap 100, 150 mm length ×0.075 mm inner diameter, 3 μm particle size, 100 Å pore size, Thermo) and a 1 μL sample loop. Acetonitrile, formic acid (1 mL ampules, Fisher Optima grade, 99.9%), and water purified to a resistivity of 18.2 MΩ·cm (at 25 °C) using a Milli-Q Gradient ultrapure water purification system (Millipore, Billerica, MA) were used to prepare mobile phase solvents. Solvent A was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1% formic acid (v/v). The elution program consisted of isocratic flow at 2% B for 4 min, a linear gradient to 30% B over 38 min, isocratic flow at 95% B for 6 min, and isocratic flow at 2% B for 12 min, at a flow rate of 300 nL/min. Full-scan mass spectra were acquired in the positive ion mode over the range m/z = 350 to 1800 using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 60 000 (at m/z = 400, measured at full width at halfmaximum peak height). In the data-dependent mode, the eight most intense ions exceeding an intensity threshold of 50 000 counts were selected from each full-scan mass spectrum for tandem mass spectrometry (MS/MS) analysis using collisioninduced dissociation (CID). Data acquisition was controlled
cultures were grown aerobically in lysogeny broth (LB)-Lennox (VWR) supplemented with the required antibiotics, kanamycin (50 μg/mL) or chloramphenicol (34 μg/mL), with shaking at 225 rpm and 37 °C (MaxQ 8000, 443, Thermo Scientific). T3SS-inducing growth conditions used were as previously described.3 The conditions include low aeration at 120 rpm and high-salt inducing media, LB-Lennox media with 17 g/L NaCl (Fisher Scientific). The cells were cultured overnight from 12 to 16 h and subcultured from the overnight culture at approximately 1:100 dilution to give a starting OD of 0.02 in 5 mL inducing media. The cultures were supplemented with antibiotics as needed, then grown for 8 h at 37 °C, and shaking at 120 rpm. For strains harboring the pLac-hilA plasmid, gene expression was induced at the time of subculture using 1 mM IPTG (Fisher Scientific) for hilA overexpression. Cells were pelleted by spinning at 4000g for 10 min in a tabletop plate centrifuge (Beckman Coulter). The supernatant was collected for analysis and filtered using 0.22 μM syringe filters (Thermo Scientific) and stored at 4 °C. Whole culture lysate and secretion fraction samples for Western blotting and protein gel electrophoresis were prepared immediately and stored at 4 °C until used. Experiments were performed in inducing conditions unless otherwise stated. For noninducing conditions, cells were subcultured in 5 mL of LB-Lennox media (VWR), supplemented with antibiotics as needed, and grown for 8 h at 37 °C with shaking at 225 rpm. The ΔsipD strain was generated using the FLP recombinase system described by Datsenko and Wanner48 using the plasmid pKD46,48 and primers KJM156F and KJM157R (Table S2). The strain retains the KanR cassette and the FRT scar sites. Recombinant SipD Purification. E. coli BL21 DE(3) pLysS cells harboring pET28b(+) protein expression vectors (Novagen) encoding SipD or the various SipD mutants were cultured overnight from frozen stocks for 12−16 h. Saturated overnight cultures were added into 1 L Terrific Broth at 37 °C at a 1:50 dilution, and induced with 1 mM IPTG after 2 h of growth at 225 rpm. After 6 h of growth at 225 rpm at 37 °C, the cells were harvested by centrifugation at 6000g for 20 min and resuspended in binding buffer (20 mM sodium phosphate (Fisher Scientific), 500 mM NaCl (Fisher Scientific), 25 mM imidazole (Acros Organics), pH 7.3). The cell pellets were then lysed by sonication. The insoluble fractions were removed by centrifugation at 12 000g for 10 min twice. The soluble fraction of the cell lysate was passed through a Ni2+-NTA affinity column (GE Healthcare) at room temperature under native conditions. The column was washed with 60 mL of binding buffer. Proteins were eluted with binding buffer supplemented with 250 mM imidazole, pH 7.4. Samples were desalted using PD-10 columns (GE Healthcare) and then buffer exchanged into phosphate buffered saline (PBS). SipD purity was assessed by confirming a single band at 35 kDa with SDS-PAGE, and purified SipD concentrations were determined by absorbance at 280 nm using the extinction coefficient for SipD (as calculated using the ExPASy ProtParam tool).49 The identity of the protein was confirmed multiple times with tandem mass spectrometry using the UC Berkeley QB3 Mass Spectometry Core Facility with 98% protein coverage (Table S4). SDS-PAGE and Western Blotting. Whole culture lysate and secretion fractions were analyzed for protein content using protein gel electrophoresis followed by Western blotting. For whole culture lysates, 10 μL of culture was collected before pelleting cells. For secretion fractions, 10 μL of sample was collected after cells were pelleted without further concentration G
DOI: 10.1021/acssynbio.6b00335 ACS Synth. Biol. XXXX, XXX, XXX−XXX
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All experiments were conducted in biological triplicate on different days.
using Xcalibur software (version 2.0.7, Thermo). Raw data were searched against the Salmonella typhimurium (strain SL1344) protein database (UniProtKB, www.uniprot.org, accessed March 14, 2014) using Proteome Discoverer software (version 1.3, SEQUEST algorithm, Thermo). Densitometry Analysis. Quantification was carried out on Western blots in which samples were compared with a standard curve. Dilutions of FLAG-BAP fusion protein (Sigma) or purified FLAG-tagged SptP-magainin-1 (0−5 μg) were used to generate a standard curve with which sample signal, normalized to final cell density (OD600), was compared. Band signals were determined using ImageJ densitometry analysis software and standard curves were calculated using a linear best-fit model. Each peak was manually bound and integrated to calculate the signal. To determine secretion titer, the amount of protein transferred to the blot was interpolated from the best fit line as described, corrected for sample loading dilution if applicable, and then multiplied to account for total protein mass in the secretion fraction volume. This value was then converted to a secretion titer in mg/L. For Western blots that were analyzed for relative secretion by different strains, the signal from each band was also calculated by integration by ImageJ and adjusted by multiplication by the gel loading factor. The percentage of secreted protein was calculated by dividing the signal from the secretion fraction by the signal from the whole culture lysate fraction. All samples from each time point were run together on Western blots. Transcriptional and Translational Arrest. Overnight cultures of S. enterica SL1344*/pLac-hilA pSicA-DH were grown aerobically as described above. These cultures were then subcultured at an OD600 of 0.4 for 1 h at 225 rpm and 37 °C. The cells were centrifuged at 4000g for 10 min and washed twice in LB-Lennox to remove previously secreted proteins. The washed cells were next resuspended in LB-Lennox to an OD600 of 0.6 and supplemented with 1.2 μM of SipD, 1 mM of IPTG (Fisher Scientific), and either 100 μg/mL rifampicin (Fisher Scientific) or 8 μg/mL tetracycline (Fisher Scientific) to inhibit transcription and translation, respectively. The resuspended cultures were grown for 4 h at 225 rpm and 37 °C. The cells were then pelleted at 4000g for 10 min. The supernatant was collected for analysis and concentrated 25-fold by spin concentration using a 30 kDa MWCO membrane (Sartorius). The concentrated supernatant was analyzed by SDS-PAGE and Western blotting as described above. Flow Cytometry. Wild-type SL1344*, ΔsipD, and ΔprgI cultures harboring native SPI-1 promoter-GFP fusion plasmids were grown in high-salt inducing media as described above. 1.4 μM exogenous SipD was also added at the point of subculture as needed. The transcriptional GFP fusions to four SPI-1 promoters that used in this study were previously used by Temme (2008).7 The promoters are pHilA, pInvF, pInvF-2, and pSicA. At hourly time points, 10 μL aliquots from each culture were added to 90 μL PBS with 20 mg/mL kanamycin (Fisher Scientific). Samples were stored at 4 °C overnight. The next day, 2−20 μL aliquots from the samples were added to 180− 198 μL PBS to a total volume of 200 μL, to a final cell density of 0.01, in transparent 96-well flat-bottom plates. Plates were loaded in a Millipore EasyCyte flow cytometer and viable cell populations were gated. The same gating parameters were used for all samples. For each sample, 10 000 events were collected based on the live cells gate. Data was analyzed using FlowJo software. GFP-expressing cells were counted and fluorescence was expressed in terms of percent of cells that were fluorescent.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00335.
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Additional methods and data (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: 847-491-7043. E-mail:
[email protected]. ORCID
Anum Azam Glasgow: 0000-0002-0938-881X Han Teng Wong: 0000-0001-9836-1338 Danielle Tullman-Ercek: 0000-0002-4555-4803 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Jeff Glasgow, Sergey Boyarskiy, Emily Hartman, Caroline Ajo-Franklin, Brittney Nguyen, Bill Burkholder and all the members of the Tullman-Ercek lab at UC Berkeley for valuable discussions, and Anthony T. Iavarone for help with mass spectrometry of SipD. The QB3/Chemistry Mass Spectrometry Facility at the University of California, Berkeley receives support from the National Institutes of Health (grant number 1S10OD020062-01). A.A.G. gratefully acknowledges the support of graduate fellowships from the NSF and Sandia National Laboratories. H.T.W. gratefully acknowledges the support of graduate fellowship from the A*STAR, Singapore. The authors would also like to thank the Hammond lab for the use of their equipment.
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