Bacterial Nanobioreactors–Directing Enzyme ... - ACS Publications

Oct 19, 2015 - Pictured above is a schematic representation of N-terminal OmpA-SpyTag and PTE-SpyCatcher forming an isopeptide bond at the outer membr...
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Bacterial Nanobioreactors−Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles Nathan J. Alves,† Kendrick B. Turner,‡ Michael A. Daniele,‡ Eunkeu Oh,§,∥ Igor L. Medintz,‡ and Scott A. Walper*,‡ †

National Research Council, 500 Fifth Street NW (Keck 576), Washington, DC 20001, United States Center for Bio/Molecular Science & Engineering and §Optical Science Division, U.S. Naval Research Laboratory, Washington, DC 20375, United States ∥ Sotera Defense Solution, Inc. 7230 Lee DeForest Drive, Columbia, Maryland 21046, United States ‡

S Supporting Information *

ABSTRACT: All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing. KEYWORDS: outer membrane vesicle (OMV), phosphotriesterase (PTE), directed packaging, enzyme, E. coli, SpyCatcher, SpyTag



INTRODUCTION All bacteria studied to date, both Gram-negative and Grampositive, produce outer membrane vesicles (OMVs) from their surface.1−3 These small (30−200 nm) unilamellar proteoliposomes serve various functions from cell−cell signaling to packaging of virulence factors in pathogenic bacterial strains to infect host cells.4−7 Various studies have been performed demonstrating use of bacterial OMVs for packaging material and facilitating delivery of proteins of interest.8−11 Due to the diverse circumstances for which OMVs are formed and the complex composition of both their packaged contents and their lipid−protein shell a discrete pathway for the packaging of cellular components has not yet been elucidated.12,13 Despite the fact that the exact mechanisms of OMV biogenesis are not well characterized and differ between bacteria, we wanted to develop a method for engineering bacteria to simultaneously produce, package, and secrete an active enzyme of interest.14,15 To help drive packaging of the enzyme into the vesicles we sought to create a synthetic linkage between the enzyme and a known protein present in the outer membrane at high abundance. There are various synthetic © XXXX American Chemical Society

strategies for pairing two different proteins within a biological system that include the following: split proteins,16 coiled coils,17 and split inteins,18 just to list a few. For the purposes of this application the SpyCatcher/SpyTag bioconjugation system was selected which employs a fibronectin-binding protein (FbaB) from Streptococcus pyogenes which is a split protein that employs two subunit domains referred to as the SpyCatcher (SC) and SpyTag (ST) domains.19 Unlike many split protein systems, the SC/ST system provides for the formation of an isopeptide bond between proximal aspartic acid and lysine amino acid residues.20 This interaction and bond formation happens spontaneously as it does not require the addition of chaperone proteins, catalytic enzymes, or cofactors. The reaction occurs at room temperature and over a wide range of physiologically relevant conditions. We selected OmpA as our membrane tethering protein since it is a highly expressed porin protein present in the bacterial Received: September 17, 2015 Accepted: October 19, 2015

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DOI: 10.1021/acsami.5b08811 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Crystal structures for the proteins utilized in the biorthogonal membrane conjugation of PTE for packaging into outer membrane vesicles: OmpA, PTE, SpyTag, and SpyCatcher (PDB: 2GE4, 1PTA, 4MLI, 4MLI, respectively). Three separate OmpA-SpyTag fusion constructs were synthesized: C-terminal (C), N-terminal (N), and an internal (I) OmpA loop fusion. Pictured above is a schematic representation of N-terminal OmpA-SpyTag and PTE-SpyCatcher forming an isopeptide bond at the outer membrane surface of the bacteria. This membrane fusion facilitates incorporation of the PTE within the OMVs that are released from the bacteria surface due to the directional insertion of OmpA into the bacterial membrane.

outer membrane and subsequent OMVs.21 Native OmpA is a 37.2 kDa transmembrane porin protein implicated in the transport of small molecules 0.99 (Figure 6). KM, Vmax, and kcat values were calculated for the CA free PTE-SC of 103.7 μM, 0.119 μM/s, and 2320 s−1, respectively. These values were consistent with the literature values of PTE isolated from B. diminuta with a Zn/Zn binuclear metal ion active site under similar assay conditions.35 While we were able to quantify the amount of free PTE-SC in solution accurate quantification of the total amount of active PTE-SC encapsulated within the OMV for each construct, necessary for the kcat determination, was not feasible via absorbance or densitometry due to the expression levels observed and sample complexity. The kcat value of 2320 s−1, as F

DOI: 10.1021/acsami.5b08811 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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most instances with increased OMV production. We hypothesize that both OMV packaging and reduction in membrane integrity contribute to PTE export from the cell and therefore a reduction in the toxicity allowing for production of higher concentrations of the recombinant enzyme. While none of the SpyTag/SpyCatcher fusion constructs resulted in a complete conversion of free PTE-SC to covalently fused PTE-SC/OmpA-ST, it is evident that the presence of the ST and SC help drive packaging of the PTE into the OMVs. The location of the ST fusion was critical for the packaging efficiency of the PTE-SC, and we found that the C-terminal fusion of SpyTag to the mutant OmpA, in the presence or absence of IPTG activation, resulted in not only the highest total amount of PTE-SC produced but also the highest levels of PTE-SC packaged within the OMVs. Omitted in the OmpA-ST constructs described herein is a C-terminal domain that associates with the peptidoglycan layer. The C-terminal positioning of the ST peptide may have allowed for greater accessibility for the PTE-SC to interact with as this domain normally projects away from the outer membrane and toward the depths of the periplasmic space. This fusion was not without fault in that the C-terminal ST constructs also resulted in a significant amount of freely secreted PTE-SC into the culture media. We also showed that the packaged PTE-SC was much less susceptible to inactivation via exposure to multiple freeze−thaw cycles compared to free PTE-SC. This result is important as it demonstrates that the functional biological nanoparticles created by this method are far more robust allowing for implementation in more harsh environments providing for increased opportunities for use in applications that may not have been previously possible. The results of this study can be broadly applied to other outer membrane vesicle packaging systems across various applications to both increase vesiculation, drive enzyme packaging, and enhance enzymatic stability through OMV encapsulation. Though still in its infancy, careful design of bacterial synthesis pathways and the export of proteins, small molecules, and nucleic acids as OMV cargo will continue to be used to develop novel materials for environmental remediation, therapeutics, and methods of controlling the properties of microbial communities.

Figure 7. Freeze−thaw stability test of PTE-SC packaged within NAI and CAI OMVs compared to free PTE-SC purified from the UC supernatant of the CAI construct. Four cycles of freeze−thaw between −80 °C and room temperature were carried out, and the percent PTE activity was directly compared via initial velocity measurements utilizing paraoxon as a substrate. Packaged PTE-SC in both the NAI and CAI constructs exhibited heightened resistance to inactivation compared to free PTE-SC.

increased resistance to inactivation from freeze−thaw compared to the CAI packaged PTE-SC. This result was expected since the CAI construct exhibited heightened membrane destabilization compared with the NAI construct in the aforementioned experiments which likely would provide less protection to the encapsulated enzyme compared to a fully intact membrane. Through packaging the PTE within OMVs the enzyme is much less susceptible to inactivation making this functional material a powerful and robust reagent compared to free enzyme allowing for improved implementation under harsh conditions.



CONCLUSION The efforts described herein were undertaken to develop a reproducible packaging mechanism to utilize bacterial OMVs for the development of functional biological nanoparticles. The focus of our efforts was to develop a system of using bacterial cultures to produce enzyme-filled particles that could potentially serve as reagents for the remediation of biowarfare agents. Through careful manipulation we were able to demonstrate a method for increasing vesiculation and improving the packaging efficiency of a periplasmically produced active enzyme, PTE-SC. The OMV packaged PTESC was capable of breaking down paraoxon that passively entered the vesicle through transmembrane porin proteins and exhibited kinetic parameters comparable to native, free PTE. OmpA was successfully employed as a membrane anchor to facilitate packaging of the target enzyme. Later efforts will focus on replacing the chromosomal OmpA with the recombinant OmpA-ST to allow for expression levels that are more akin to wild-type. In addition, alternate known outer membrane associated proteins will be utilized to enhance isopeptide bond formation between the SpyTag and SpyCatcher as well as allow for the packaging of multiple discrete proteins within a single OMV. In addition to OMV packaging, it was observed that PTE-SC production was significantly increased when compared to traditional cytoplasmic and periplasmically targeted methods of protein production through the addition of the OmpA-ST mutant. While overall PTE-SC expression levels varied there was an increase in PTE-SC production across all ST fusion locations tested. The increased PTE production correlates in



MATERIALS AND METHODS

Materials. Ultra-Clear (25 × 89 mm) centrifuge tubes were purchased from Beckman Coulter (Brea, CA). Precast MiniPROTEAN TGX 4−15% gradient gels were purchased from BioRad (Hercules, CA). Paraoxon was purchased from Chem Service (West Chester, PA). A mouse anti6xHis antibody and goat antimouse alkaline phosphatase conjugated antibody were purchased from Life Technologies (Frederick, MD). GelCode Blue Protein Stain and 1Step NBT/BCIP precipitating substrate were purchased from Thermo Scientific (Rockford, IL). All other buffer salts and reagents were purchased from Sigma-Aldrich (St. Louis, MO). All measurements were carried out in triplicate, and data represents means ± standard deviations. Construction of E. coli Expression Plasmids. Genes encoding for a truncated OmpA with the SpyTag sequence appended to the either the N-terminus, C-terminus, or an internal loop were synthesized by GenScript (Piscataway, NJ) in a pUC57 shuttle vector with flanking NcoI and NotI restriction sites. The truncated OmpA consisted of native OmpA with the unessential C-terminal domain portion deleted.36 The spy tag in each construct was flanked by a spacer amino acid sequence (GGGS). The SpyTag insertion site at the internal loop was chosen based on the published tolerance for insertion at this location.37,38 Synthesized plasmids were digested with G

DOI: 10.1021/acsami.5b08811 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces NcoI-HF and NotI-HF (New England Biolabs, Ipswich, MA) and cloned into identical sites in the pET22b expression vector (Novagen, Billerica, MA). For complete amino acid sequence of constructs, see the Supporting Information. A second expression vector utilizing a compatible origin of replication was constructed for the coexpression of the PTE-SC construct. The pACYC184 vector (New England Biolabs) which contains a p15a origin served as the backbone for this construct. The regulatory elements for arabinose induction were amplified via PCR from the pBAD/Myc-His plasmid (Life Technologies) using primers that also encoded the twin-arginine translocation substrate TMAO reductase (TorA), a hexahistidine sequence, and several unique restriction endonuclease cleavage sites (vector referred to as pACYC184 AraC). The phosphotriesterase and SpyCatcher genes were combined through a series of PCR amplification, restriction digest reactions, and ligations. The SpyCatcher gene was amplified from a bacterial expression vector using primers that generated flanking XhoI sites and a 5′-Acc65I site just upstream of the SpyCatcher sequence. The PCR product was cloned to the pMinit PCR cloning vector (New England Biolabs) which served as the shuttle vector for cloning of the PTE gene.39 PTE was amplified separately using primers to generate flanking Acc65I sites and a short amino acid spacer sequence. The PCR product was digested and cloned to the pMinit SpyCatcher construct whose sequence was confirmed. Both the pMinit PTE-SC and pACYC184 AraC were digested with XhoI and gel purified, and the relevant fragments were ligated using T4 DNA ligase. Protein sequences for each construct can be found in the Supporting Information. Bacterial Growth Conditions. The E. coli strain BL21(DE3) containing the pET22 OmpA-ST (C/I/N) and pACYC184 AraC PTE-SC plasmids was maintained on solid medium and expanded in overnight cultures in the presence of ampicillin (100 μg/mL) and chloramphenicol (25 μg/mL) to ensure plasmid maintenance. For OMV production, 1 mL of overnight culture was used to inoculate 100 mL of TB in baffled culture flasks. The culture was allowed to equilibrate for 10 min and was then split into two separate 50 mL baffled culture flasks at a temperature of 37 °C. The resulting culture was allowed to grow for 3 h until an OD600 of 0.6−0.8 was reached. Where indicated arabinose was added to a 0.2% final concentration. After an additional 3 h incubation IPTG was added to a final concentration of 0.5 mM, and the culture was allowed to grow for an additional 18 h at 37 °C. OMV Purification. All conditioned bacterial culture media was centrifuged two times at 7,000g for 15 min at 4 °C, and then 0.45 μm membrane was filtered to remove intact bacteria and undesired large cellular material. OMVs (36 mL of culture media) were then pelleted at 29,000 rpm (∼150,000g) in a Sorvall WX Ultra 90 centrifuge using an AH-629 rotor for 3 h at 4 °C. The OMV depleted culture media was decanted, and the OMV pellet was resuspended in 1 mL of PBS pH 7.4. SDS-PAGE and Western Blot Analysis. The samples were run on a gradient (4−15%) SDS-PAGE gel with a tris-glycine running buffer under reducing conditions at 90 V for 90 min and were transferred to a nitrocellulose membrane at 90 V for 90 min in a 10% MeOH transfer buffer. The membrane was blocked with 5% dry milk in PBS for 1 h and was then probed for with 1:5,000 dilution of mouse anti6xHis for 1 h and a 1:5,000 dilution of an alkaline phosphatase conjugated antimouse secondary antibody at RT. A chromogenic alkaline phosphatase substrate was used to detect the His-tagged mutant OmpA and PTE. SDS-PAGE gels were GelCode Blue stained for 30 min and destained for 4 h. All gel and blot images were taken on a Bio-Rad imager. SEM. The morphologies of the vesicles were observed by scanning electron microscopy (Zeiss LEO 1550, Jena, Germany). The vesicles were frozen at −80 °C for 12 h followed by lyophilization for 24 h. Samples were cast onto aluminum holders and sputter coated with ca. 5 nm of gold (Cressington 108auto, Hertfordshire, UK). NanoSight. Vesicle size distributions and quantitation were performed on a NanoSight LM10 system (Salisbury, UK) using NTA 2.3 Nanoparticle Tracking and Analysis software. Samples were

diluted in PBS pH 7.4 as indicated, and camera shutter (13.8 ms) and gain (324) were manually optimized to enhance data collection. Videos (90 s) were taken, and frame sequences were analyzed under auto particle detection and tracking parameters: detection threshold, pixel blur, min track length, and min expected particle size. All samples were run at RT and allowed to equilibrate prior to analysis. All particle/mL concentrations are 55-fold dilutions from culture media concentrations. PTE Kinetic Assays. All enzymatic assays were conducted in CHES buffer (pH 8) at 25 °C. Paraoxon (1:1,000 dilution) was utilized as a reporter molecule by measuring an absorbance shift from PTE’s cleavage of the chromogenic tag, monitored at 405 nm. KM, Vmax, and kcat were determined at a fixed concentration of PTE (specific to each sample) while varying the paraoxon substrate concentrations (0−1,000 μM). Initial velocities were determined by the slope of the first 3 min of reaction with the paraoxon substrate. A Lineweaver−Burke plot was utilized for determination of kinetic parameters where the y-intercept = 1/Vmax and slope = KM/Vmax. Initial velocities were utilized to compare the relative quantity of PTE in each sample to determine packaging efficiency and total PTE production for each OmpA-ST mutant and initiator combination. Vesicle Rupture. To determine if paraoxon freely entered the OMVs to react with the packaged PTE we assessed the necessary conditions to cause vesicle rupture. A range of Triton X100 concentrations (0−5%) were tested using kinetic assays and NanoSight to determine the impact of the Triton on the vesicle stability and catalytic activity of PTE. Freeze−Thaw Stability Testing. OMVs from the NAI and CAI constrcuts were compared to free PTE-SC for stability against multiple cycles of freeze−thaw. The samples were assayed for initial PTE activity via initial velocity measurements utilizing paraoxon as a substrate. The samples were then snap frozen in liquid nitrogen and placed at −80 °C for 1 h and were then allowed to warm to room temperature for 1 h for a total of four freeze−thaw cycles. PTE activity was assayed at the completion of each thaw to assess the stability of OMV packaged PTE compared to free PTE-SC.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08811. SDS-PAGE and Western Blots of cell pellets and ultracentrifuge supernatants from each OmpA-ST and PTE-SC construct, PTE activity test in increasing concentrations of Triton X-100, and protein sequences for OmpA-N-ST, OmpA-I-ST, OmpA-C-ST, and PTESC (PDF)



AUTHOR INFORMATION

Corresponding Author

*Phone: 202-404-6070. E-mail: [email protected]. Corresponding author address: Center for Bio/Molecular Science & Engineering, Naval Research Laboratory (NRL). 4555 Overlook Avenue, SW, Washington, DC 20375. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was funded by the Office of Naval Research through NRL Base funds, the NRL Nanoscience Institute (NSI), and the Defense Threat Reduction Agency (DTRA) Joint Science and Technology Office MIPR#B112582M. H

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