Bacteriophage P22 SieA-mediated superinfection exclusion.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.

Molecular Architecture of Salmonella Typhimurium Virus P22 Genome Ejection Machinery.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted ß-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.

Delayed In Vivo Encapsulation of Enzymes Alters the Catalytic Activity of Virus-Like Particle Nanoreactors.

Encapsulation of enzymes inside protein cage structures, mimicking protein-based organelle structures found in nature, has great potential for the development of new catalytic materials with enhanced properties. In vitro and in vivo methodologies have been developed for the encapsulation of enzymes within protein cage structures of several types, particularly virus-like particles (VLPs), with the ability to retain the activity of the encapsulated enzymes. Here, we examine the in vivo encapsulation of enzymes within the bacteriophage P22 derived VLP and show that some enzymes may require a delay in encapsulation to allow proper folding and maturation before they can be encapsulated inside P22 as fully active enzymes. Using a sequential expression strategy, where enzyme cargoes are first expressed, allowed to fold, and later encapsulated by the expression of the P22 coat protein, altered enzymatic activities are obtained in comparison to enzymes encapsulated in P22 VLPs using a simultaneous coexpression strategy. The strategy and results discussed here highlight important considerations for researchers investigating the encapsulation of enzymes inside confined reaction environments via in vivo routes and provide a potential solution for those that have been unable to produce active enzymes upon encapsulation.

Working with Phage P22.

Transduction experiments in Escherichia coli and Salmonella are usually performed with virulent phage variants. A widely used P1 mutant, called P1 vir, carries one or more uncharacterized mutations that prevent formation of lysogens. In the case of P22, by far the most frequently used variant is named P22 HT105/1 int-201 This phage has a high transducing (HT) frequency due to a mutant nuclease with lower specificity for the pac sequence. As a result, ∼50% of the P22 HT phage heads carry random transducing fragments of chromosomal DNA. The int mutation reduces the formation of stable lysogens. The basic steps in handling the P22 HT105/1 int-201 phage and in performing transduction experiments in Salmonella are described here.

Tryptophan Residues Are Critical for Portal Protein Assembly and Incorporation in Bacteriophage P22.

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly.

Bioinspired Approaches to Self-Assembly of Virus-like Particles: From Molecules to Materials.

When viewed through the lens of materials science, nature provides a vast library of hierarchically organized structures that serve as inspiration and raw materials for new synthetic materials. The structural organization of complex bioarchitectures with advanced functions arises from the association of building blocks and is strongly supported by ubiquitous mechanisms of self-assembly, where interactions among components result in spontaneous assembly into defined structures. Viruses are exemplary, where a capsid structure, often formed from the self-assembly of many individual protein subunits, serves as a vehicle for the transport and protection of the viral genome. Higher-order assemblies of viral particles are also found in nature with unexpected collective behaviors. When the infectious aspect of viruses is removed, the self-assembly of viral particles and their potential for hierarchical assembly become an inspiration for the design and construction of a new class of functional materials at a range of different length scales.Salmonella typhimurium bacteriophage P22 is a well-studied model for understanding viral self-assembly and the construction of virus-like particle (VLP)-based materials. The formation of cage-like P22 VLP structures results from scaffold protein (SP)-directed self-assembly of coat protein (CP) subunits into icosahedral capsids with encapsulation of SP inside the capsid. Employing the CP-SP interaction during self-assembly, the encapsulation of guest protein cargos inside P22 VLPs can be achieved with control over the composition and the number of guest cargos. The morphology of cargo-loaded VLPs can be altered, along with changes in both the physical properties of the capsid and the cargo-capsid interactions, by mimicking aspects of the infectious P22 viral maturation. The structure of the capsid differentiates the inside cavity from the outside environment and serves as a protecting layer for the encapsulated cargos. Pores in the capsid shell regulate molecular exchange between inside and outside, where small molecules can traverse the capsid freely while the diffusion of larger molecules is limited by the pores. The interior cavity of the P22 capsid can be packed with hundreds of copies of cargo proteins (especially enzymes), enforcing intermolecular proximity, making this an ideal model system in which to study enzymatic catalysis in crowded and confined environments. These aspects highlight the development of functional nanomaterials from individual P22 VLPs, through biomimetic design and self-assembly, resulting in fabrication of nanoreactors with controlled catalytic behaviors.Individual P22 VLPs have been used as building blocks for the self-assembly of higher-order structures. This relies on a balance between the intrinsic interparticle repulsion and a tunable interparticle attraction. The ordering of VLPs within three-dimensional assemblies is dependent on the balance between repulsive and attractive interactions: too strong an attraction results in kinetically trapped disordered structures, while decreasing the attraction can lead to more ordered arrays. These higher-order assemblies display collective behavior of high charge density beyond those of the individual VLPs.The development of synthetic nanomaterials based on P22 VLPs demonstrates how the potential for hierarchical self-assembly can be applied to other self-assembling capsid structures across multiple length scales toward future bioinspired functional materials.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.

Lateral transduction is inherent to the life cycle of the archetypical Salmonella phage P22.

Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with early prophage excision followed by DNA replication and packaging (ERP). This temporal program is considered universal for P22-like temperate phages, though there is no direct evidence to support the timing and sequence of these events. Here we report that the long-standing ERP program is an observation of the experimentally favored Salmonella phage P22 tsc229 heat-inducible mutant, and that wild-type P22 actually follows the replication-packaging-excision (RPE) program. We find that P22 tsc229 excises early after induction, but P22 delays excision to just before it is detrimental to phage production. This allows P22 to engage in lateral transduction. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation.

Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins.

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.

Molecular exclusion limits for diffusion across a porous capsid.

Molecular communication across physical barriers requires pores to connect the environments on either side and discriminate between the diffusants. Here we use porous virus-like particles (VLPs) derived from bacteriophage P22 to investigate the range of molecule sizes able to gain access to its interior. Although there are cryo-EM models of the VLP, they may not accurately depict the parameters of the molecules able to pass across the pores due to the dynamic nature of the P22 particles in the solution. After encapsulating the enzyme AdhD within the P22 VLPs, we use a redox reaction involving PAMAM dendrimer modified NADH/NAD+ to examine the size and charge limitations of molecules entering P22. Utilizing the three different accessible morphologies of the P22 particles, we determine the effective pore sizes of each and demonstrate that negatively charged substrates diffuse across more readily when compared to those that are neutral, despite the negatively charge exterior of the particles.

Development of target-tunable P22 VLP-based delivery nanoplatforms using bacterial superglue.

Protein cage nanoparticles are widely used as targeted delivery nanoplatforms, because they have well-defined symmetric architectures, high biocompatibility, and enough plasticity to be modified to produce a range of different functionalities. Targeting peptides and ligands are often incorporated on the surface of protein cage nanoparticles. In this research, we adopted the SpyTag/SpyCatcher protein ligation system to covalently display target-specific affibody molecules on the exterior surface of bacteriophage P22 virus-like particles (VLP) and evaluated their modularity and efficacy of targeted delivery. We genetically introduced the 13 amino acid SpyTag peptide into the C-terminus of the P22 capsid protein to construct a target-tunable nanoplatform. We constructed two different SpyCatcher-fused affibody molecules as targeting ligands, SC-EGFRAfb and SC-HER2Afb, which selectively bind to EGFR and HER2 surface markers, respectively. We produced target-specific P22 VLP-based delivery nanoplatforms for the target cell lines by selectively combining SpyTagged P22 VLP and SC-fused affibody molecules. We confirmed its target-switchable modularity through cell imaging and verified the target-specific drug delivery efficacy of the affibody molecules displaying P22 VLP using cell viability assays. The P22 VLP-based delivery nanoplatforms can be used as multifunctional delivery vehicles by ligating other functional proteins, as well as affibody molecules. The interior cavity of P22 VLP can be also used to load cargoes like enzymes and therapeutic proteins. We anticipate that the nanoplatforms will provide new opportunities for developing target-specific functional protein delivery systems.

Of capsid structure and stability: The partnership between charged residues of E-loop and P-domain of the bacteriophage P22 coat protein.

Tailed dsDNA bacteriophages and herpesviruses form capsids using coat proteins that have the HK97 fold. In these viruses, the coat proteins first assemble into procapsids, which subsequently mature during DNA packaging. Generally interactions between the coat protein E-loop of one subunit and the P-domain of an adjacent subunit help stabilize both capsomers and capsids. Based on a recent 3.3 Šcryo-EM structure of the bacteriophage P22 virion, E-loop amino acids E52, E59 and E72 were suggested to stabilize the capsid through intra-capsomer salt bridges with the P-domain residues R102, R109 and K118. The glutamic acid residues were each mutated to alanine to test this hypothesis. The substitutions resulted in a WT phenotype and did not destabilize capsids; rather, the alanine substituted coat proteins increased the stability of procapsids and virions. These results indicate that different types of interactions must be used between the E-loop and P-domain to stabilize phage P22 procapsids and virions.

A Hydrophobic Network: Intersubunit and Intercapsomer Interactions Stabilizing the Bacteriophage P22 Capsid.

Double-stranded DNA (dsDNA) tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here, we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra- and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra- and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

Encapsulation of Active Enzymes within Bacteriophage P22 Virus-Like Particles.

Virus-like particles (VLPs) are nonpathogenic protein cage structures derived from viral coat proteins that have found utility in the area of biomaterials and nanotechnology. VLPs have been exploited as containers for the sequestration and encapsulation of a wide range of guest molecules in their hollow interiors. The robust nature of VLPs lend them as versatile scaffolds that can be exploited to provide protection to encapsulated guest molecules, such as enzymes which are often susceptible to inactivation and degradation, and for organization and construction of new nanomaterials incorporating the chemical properties of the guest molecules. In this chapter a background and methodology for the encapsulation of enzymes on the interior of the bacteriophage P22 derived VLP is described.

In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620.

Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.

Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release.

Genome ejection proteins are required to facilitate transport of bacteriophage P22 double-stranded DNA safely through membranes of Salmonella. The structures and locations of all proteins in the context of the mature virion are known, with the exception of three ejection proteins. Furthermore, the changes that occur to the proteins residing in the mature virion upon DNA release are not fully understood. We used cryogenic electron microscopy to obtain what is, to our knowledge, the first asymmetric reconstruction of mature bacteriophage P22 after double-stranded DNA has been extruded from the capsid-a state representative of one step during viral infection. Results of icosahedral and asymmetric reconstructions at estimated resolutions of 7.8 and 12.5 Å resolutions, respectively, are presented. The reconstruction shows tube-like protein density extending from the center of the tail assembly. The portal protein does not revert to the more contracted, procapsid state, but instead maintains an extended and splayed barrel structure. These structural details contribute to our understanding of the molecular mechanism of P22 phage infection and also set the foundation for future exploitation serving engineering purposes.

A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain.

P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.

High-resolution structure of podovirus tail adaptor suggests repositioning of an octad motif that mediates the sequential tail assembly.

The sophisticated tail structures of DNA bacteriophages play essential roles in life cycles. Podoviruses P22 and Sf6 have short tails consisting of multiple proteins, among which is a tail adaptor protein that connects the portal protein to the other tail proteins. Assembly of the tail has been shown to occur in a sequential manner to ensure proper molecular interactions, but the underlying mechanism remains to be understood. Here, we report the high-resolution structure of the tail adaptor protein gp7 from phage Sf6. The structure exhibits distinct distribution of opposite charges on two sides of the molecule. A gp7 dodecameric ring model shows an entirely negatively charged surface, suggesting that the assembly of the dodecamer occurs through head-to-tail interactions of the bipolar monomers. The N-terminal helix-loop structure undergoes rearrangement compared with that of the P22 homolog complexed with the portal, which is achieved by repositioning of two consecutive repeats of a conserved octad sequence motif. We propose that the conformation of the N-terminal helix-loop observed in the Sf6-gp7 and P22 portal:gp4 complex represents the pre- and postassembly state, respectively. Such motif repositioning may serve as a conformational switch that creates the docking site for the tail nozzle only after the assembly of adaptor protein to the portal. In addition, the C-terminal portion of gp7 shows conformational flexibility, indicating an induced fit on binding to the portal. These results provide insight into the mechanistic role of the adaptor protein in mediating the sequential assembly of the phage tail.

Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells.

Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli. The VLPs with enzymatic activity were covered with polyethylene glycol that was functionalized in its distal end with folic acid in order to be recognized by folate receptors exhibited on tumor cells. The capacity of biocatalytic VLPs to be recognized and internalized into tumor cells is demonstrated. The VLP-treated cells showed enhanced capacity for the transformation of the pro-drug tamoxifen, which resulted in an increase of the cell sensitivity to this oncological drug. In this work, the potential use of biocatalytic VLPs vehicles as a delivery system of medical relevant enzymes is clearly demonstrated. In addition to cancer treatment, this technology also offers an interesting platform as nano-bioreactors for intracellular delivery of enzymatic activity for other diseases originated by the lack of enzymatic activity.