Tool For Protein Delivery Research Articles

A programmable protein delivery tool derived from bacterial syringe

A programmable protein delivery tool derived from bacterial syringe

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Bacterial molecular syringe for drug delivery

Different protein-delivery tools are being actively developed for efficient drug delivery into host cells. A recent study by Kreitz etal. in Nature reported a syringe-like protein delivery vector engineered from natural endosymbiotic bacteria for potential human use.

N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system.

Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, Photorhabdus virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo–electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants.

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants.

Tetraphenylethylene-Featured Fluorescent Supramolecular Nanoparticles for Intracellular Trafficking of Protein Delivery and Neuroprotection.

The delivery of protein into mammalian cells enables the dissection and manipulation of biological processes; however, this potency is challenged by the lack of an efficient protein delivery tool and a means to monitor its intracellular trafficking. Herein, we report that the hierarchical self-assembly of tetraphenylethylene (TPE)-featured metal-organic cages (MOCs) and β-cyclodextrin-conjugated polyethylenimine can generate fluorescent supramolecular nanoparticles (FSNPs) to deliver protein into neural cells, a cell line that is hard to transfect using conventional strategy. Further, the aggregation-induced emission (AIE) of TPE enabled the fluorescent monitoring of cytosolic protein release. It is found that FSNPs can deliver and release protein into cytosol for subcellular targeting as fast as 18 h post-delivery. Moreover, the delivery of molecular chaperone DJ-1 using FSNPs activates MAPK/ERK signaling of neural cells to protect cells from oxidative stress.

Tetraphenylethylene‐Featured Fluorescent Supramolecular Nanoparticles for Intracellular Trafficking of Protein Delivery and Neuroprotection

AbstractThe delivery of protein into mammalian cells enables the dissection and manipulation of biological processes; however, this potency is challenged by the lack of an efficient protein delivery tool and a means to monitor its intracellular trafficking. Herein, we report that the hierarchical self‐assembly of tetraphenylethylene (TPE)‐featured metal–organic cages (MOCs) and β‐cyclodextrin‐conjugated polyethylenimine can generate fluorescent supramolecular nanoparticles (FSNPs) to deliver protein into neural cells, a cell line that is hard to transfect using conventional strategy. Further, the aggregation‐induced emission (AIE) of TPE enabled the fluorescent monitoring of cytosolic protein release. It is found that FSNPs can deliver and release protein into cytosol for subcellular targeting as fast as 18 h post‐delivery. Moreover, the delivery of molecular chaperone DJ‐1 using FSNPs activates MAPK/ERK signaling of neural cells to protect cells from oxidative stress.

Baculovirus-mediated delivery of viral hemorrhagic septicemia virus G protein in forms of envelope-spiked protein and a CMV promoter-driven expression cassette

Viral hemorrhagic septicemia virus (VHSV) has been a main cause of morbidity and mortality in cultured olive flounder (Paralichthys olivaceus) in Korea. The aim of the present study was to construct recombinant baculovirus-based VHSV G protein delivery tools for the immunization of olive flounder. We rescued recombinant A. californica multiple nucleopolyhedrovirus (AcMNPV) i) displaying VHSV G protein on the viral envelop, ii) harboring a CMV promoter-driven VHSV G protein gene cassette, or iii) having a bicistronic construct equipped with both envelope-spiked G protein and a CMV promoter driven G protein cassette. In the immunization experiment, groups of olive flounder immunized with recombinant baculoviruses carrying VHSV G protein on the envelope showed significantly higher survival rates than groups of fish in the control group (medium only) and the control recombinant baculovirus (expressing green florescent protein) immunized group, suggesting the possible use of recombinant baculoviruses carrying foreign antigens on the envelope for the development of fish vaccines. The survival rate of fish immunized with a recombinant baculovirus having a CMV-driven VHSV G protein expressing cassette was higher than that of fish immunized with a baculovirus having a reporter protein-expressing cassette, but was much lower compared to previously reported results of VHSV G protein-based DNA vaccines, suggesting that the delivery efficiency of the present baculovirus-mediated VHSV G protein gene into olive flounder cell's nucleus might not be high compared to plasmid-based DNA vaccines. Although the group of fish immunized with the recombinant baculovirus equipped with both envelope-spiked G protein and a CMV promoter driven G protein cassette showed the highest survival rate against VHSV challenge, further elaborate studies are needed to uncover the synergic effect of the two forms of VHSV G protein delivered by recombinant baculoviruses.

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNFY, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.

Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System.

The Type III Secretion System (T3SS) is a multimeric protein complex composed of over 20 different proteins, utilized by Gram-negative bacteria to infect eukaryotic host cells. The T3SS has been implicated as a virulence factor by which pathogens cause infection and has recently been characterized as a communication tool between bacteria and plant cells in the rhizosphere. The T3SS has been repurposed to be used as a tool for the delivery of non-native or heterologous proteins to eukaryotic cells or the extracellular space for a variety of purposes, including drug discovery and drug delivery. This review covers the methodology of heterologous protein secretion as well as multiple cases of utilizing the T3SS to deliver heterologous proteins or artificial materials. The research covered in this review will serve to outline the scope and limitations of utilizing the T3SS as a tool for protein delivery.

Review of Newly Identified Functions Associated With the Heat-Labile Toxin of Enterotoxigenic Escherichia coli.

Heat-labile toxin (LT) is a well-characterized powerful enterotoxin produced by enterotoxigenic Escherichia coli (ETEC). This toxin is known to contribute to diarrhea in young children in developing countries, international travelers, as well as many different species of young animals. Interestingly, it has also been revealed that LT is involved in other activities in addition to its role in enterotoxicity. Recent studies have indicated that LT toxin enhances enteric pathogen adherence and subsequent intestinal colonization. LT has also been shown to act as a powerful adjuvant capable of upregulating vaccine antigenicity; it also serves as a protein or antigenic peptide display platform for new vaccine development, and can be used as a naturally derived cell targeting and protein delivery tool. This review summarizes the epidemiology, secretion, delivery, and mechanisms of action of LT, while also highlighting new functions revealed by recent studies.

Modular Cloning of the Type III Secretion Gene Cluster from the Plant-Pathogenic Bacterium Xanthomonas euvesicatoria.

Type III secretion (T3S) systems are essential pathogenicity factors of most Gram-negative bacteria and translocate effector proteins into plant or animal cells. T3S systems can, therefore, be used as tools for protein delivery into eukaryotic cells, for instance after transfer of the T3S gene cluster into nonpathogenic recipient strains. Here, we report the modular cloning of the T3S gene cluster from the plant-pathogenic bacterium Xanthomonas euvesicatoria. The resulting multigene construct encoded a functional T3S system and delivered effector proteins into plant cells. The modular design of the T3S gene cluster allowed the efficient replacement and rearrangement of single genes or operons and the insertion of reporter genes for functional studies. In the present study, we used the modular T3S system to analyze the assembly of a fluorescent fusion of the predicted cytoplasmic ring protein HrcQ. Our studies demonstrate the use of the modular T3S gene cluster for functional analyses and mutant approaches in X.euvesicatoria. A potential application of the modular T3S system as protein delivery tool is discussed.

Enhanced differentiation of human pluripotent stem cells into cardiomyocytes by bacteria-mediated transcription factors delivery.

Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.

A highly efficient cell penetrating peptide pVEC-mediated protein delivery system into microalgae

Interest in microalgae has significantly increased due to its potential as a promising bioresource in various fields. However, a technique for intracellular delivery of proteins into microalgae has not established yet, although it is potentially valuable for advanced algal research. Here, we propose a cell penetrating peptide pVEC-mediated protein delivery tool for microalgae. We discovered that the peptide pVEC from vascular endothelial cadherin was more effective in transporting exogenous proteins into algal cells than other peptides including R9, Transportan, TAT and Penetratin. pVEC-mediated tool was shown to deliver proteins of various sizes from 6kDa to 150kDa into wild-type Chlamydomonas reinhardtii and to also work in other algal species such as Nannochloropsis salina and Chlorella vulgaris when delivering a 66kDa protein. In addition, we show that our proposed pVEC-mediated protein delivery system is based on both the endocytic and non-endocytic pathways simultaneously through a mechanism study using inhibitors such as low temperature, N-ethylmaleimide, 5-(N-ethyl-N-isopropyl) amiloride, chlorpromazine, sodium azide, and methyl-beta-cyclodextrin. The results show that the proposed simple and efficient protein delivery tool could contribute to advanced algal research for a wide array of applications for microalgae in various industries.

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.

Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors.

Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.

Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.

Hijacking of microparticle transcellular communication: a means of protein delivery and alteration of cell function (P3257)

Abstract Microparticles are submicron vesicles present in blood and other fluids that influence cells such as lymphocytes, monocytes, endothelial cells, etc. Microparticle composition is derived from the parent cell and determines the “message” communicated to other cells. Platelets and megakaryocytes produce about 80% of circulating microparticles. We aimed to usurp this unique means of transcellular communication by manipulating microparticle composition in order to deliver proteins to target cells to influence their function. We used a lentiviral vector for the transduction of a human megakaryoblastic cell line, thus changing the protein composition of generated platelets and microparticles. We successfully engineered microparticles to overexpress the anti-inflammatory transcription factor peroxisome proliferator activated receptor-γ (PPARγ) along with GFP. These genetically enhanced microparticles were taken up by the monocytic cell line THP-1 and by primary endothelial cells, and this uptake was enhanced by PMA activation. Importantly, the transfer of PPARγ was functional, in that, microparticles that overexpressed PPARγ caused THP-1 cells to increase transcription of a PPARγ target gene, fatty acid binding protein 4. This proof of concept alteration of microparticle composition and subsequent functional changes in target cells provide an important new tool for therapeutic delivery of proteins, which could be developed into new anti-inflammatory or adjuvant therapies.

A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines

Cell-permeable proteins, also called cell-penetrating peptides (CPPs), have the ability to cross cellular membranes, either alone or in association with bioactive cargo. We identified the Yersinia protein YopM as a novel bacterial cell-permeable protein. Here, we describe the ability of isolated recombinant YopM to enter host cells without a requirement for additional factors. This autonomous translocation of YopM was confirmed in several cell types, indicating that it is an intrinsic property of YopM. Using truncated versions of YopM, we show that either of the two N-terminal alpha-helices of YopM mediates translocation into the cells. Furthermore, the two alpha-helices are also able to deliver heterologous cargo, such as GFP or YopE. In addition, we found that, after entering the cells, YopM is functional and efficiently downregulates the transcription of pro-inflammatory cytokines (such as tumor necrosis factor-alpha and interleukins 12, 15 and 18). This finding suggests the potential use of YopM as a tool for protein delivery. Furthermore, it can lead to important advances in understanding and evaluating the intracellular and molecular function of YopM without the need for infection with Yersinia.