RESUMO
The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.
Assuntos
Bacteriófago P22 , Viroides , Capsídeo , Proteínas do Capsídeo/genética , Núcleo Celular , NanotecnologiaRESUMO
Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.
Assuntos
Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/fisiologia , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago P22/fisiologia , Glucanos/metabolismo , Guanosina Tetrafosfato/metabolismo , Viabilidade Microbiana , Mutação , Antígenos O/metabolismo , Pressão Osmótica , Salmonella typhimurium/efeitos da radiação , Salmonella typhimurium/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.
Assuntos
Antineoplásicos Hormonais/administração & dosagem , Bacteriófago P22/enzimologia , Neoplasias da Mama/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Tamoxifeno/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Bacteriófago P22/química , Biocatálise , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Terapia Enzimática , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologiaRESUMO
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.
Assuntos
Bacteriófago P22/enzimologia , Capsídeo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Nanopartículas/química , Tamoxifeno/administração & dosagem , Bacteriófago P22/química , Bacteriófago P22/genética , Biocatálise , Capsídeo/química , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/química , Sistemas de Liberação de Medicamentos , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Polietilenoglicóis/químicaRESUMO
BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.
Assuntos
Bacteriófago P22/química , Capsídeo/química , Sistema Enzimático do Citocromo P-450/administração & dosagem , Portadores de Fármacos/química , Proteínas do Capsídeo/química , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/uso terapêutico , Terapia Enzimática , Feminino , Células HeLa , Humanos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/enzimologiaRESUMO
Com a grande expansão da indústria avícola, a salmonelose tornou-se fator limitante na criação de aves, pois as perdas econômicas estão presentes em todas as fases. O aumento mundial da resistência bacteriana tornou necessária a buscapor alternativas para controle desta doença fazendo ressurgir a fagoterapia como opção. O presente artigo revisa o fenômeno da bacteriofagia voltado para sua aplicação no controle da salmonelose aviária, usando como modelo os conhecimentos sobre o bacteriófago P22.(AU)
With the enormous expansion in the poultry industry, salmonellosis became a limiting factor on avian production, because ofthe important economic losses during the production stages. The world wide increase in bacterial resistance stimulated the searchfor alternative strategies to control the disease and so phagoteraphy has been reconsidered as a therapeutic option. This article reviews the phenomenon of bacteriophagia focusing on its usefor the control of avian salmonellosis, using the P22 bacteriophageas a model.(AU)
Assuntos
Animais , Bacteriófago P22 , Salmonelose Animal/diagnóstico , Microbiologia , Aves DomésticasRESUMO
The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.
Assuntos
Bacteriófago P22/fisiologia , Dobramento de Proteína , Proteínas Virais/fisiologia , Cromatografia em Gel , Dicroísmo Circular , Relação Dose-Resposta a Droga , Pressão Hidrostática , Luz , Modelos Químicos , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espalhamento de Radiação , Temperatura , Ureia/farmacologia , Proteínas Virais/química , Proteínas Virais/ultraestruturaRESUMO
To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.
Assuntos
Sequência Conservada , Fagos de Salmonella/genética , Proteínas da Cauda Viral/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Bacteriófago P22/genética , Bacteriófago P22/imunologia , Western Blotting , Impressões Digitais de DNA , DNA Viral/análise , Glicosídeo Hidrolases , Reação em Cadeia da Polimerase , Fagos de Salmonella/classificação , Fagos de Salmonella/imunologia , Salmonella typhi/virologia , Proteínas da Cauda Viral/imunologiaRESUMO
OBJECTIVES: The goals of these studies were to characterize the interaction of the P22 phage particle with the Salmonella cell surface and to determine the phage elements involved in this interaction by mutational analysis. BACKGROUND: The phage P22 has been characterized extensively. The gene and protein for the phage P22 tailspike, which is the phage adsorption organelle, have been intensively studied. The kinetics of the interaction of the tailspike protein with the cell surface has been studied in detail, surprisingly no mutational analysis has ever been reported that has defined these components and their interaction between themselves and the cell surface. The main and perhaps only component needed for this cell surface interaction is the tailspike protein. METHODS: Adsorption to the cell surface has been measured in the wild type phage and in mutant derivatives, isolated in this study. Phage mutants have been isolated after hydroxylamine mutagenesis. RESULTS: The adsorption of P22 to the cell surface is a temperature-independent event. Forty putative phage adsorption mutants have been isolated. A sample of them have been further analyzed. These divide the adsorption process into at least two stages. One stage contains mutants that absorb with essential wild type phage kinetics to the cell surface while the other stage with delayed adsorption kinetics. CONCLUSIONS: The interaction of the phage P22 with the Salmonella cell surface has been shown to be a complicated one which is temperature-independent and multi-stage. Mutants isolated in this study may help dissect this process even further.
Assuntos
Adsorção , Bacteriófago P22/metabolismo , Salmonella typhimurium/virologia , Bacteriófago P22/ultraestrutura , Humanos , Lipopolissacarídeos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Temperatura , Proteínas da Cauda Viral/metabolismoRESUMO
We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products.
Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese Insercional/métodos , Fenótipo , Salmonella typhi/genética , Bacteriófago P22 , Sequência de Bases , Southern Blotting , Primers do DNA , Resolvases de Junção Holliday/genética , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , TetraciclinaRESUMO
This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations.
Assuntos
Bacteriófago P22/genética , Dobramento de Proteína , Proteínas da Cauda Viral/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Bacteriófago P22/fisiologia , DNA Viral/genética , Humanos , Hidrólise , Técnicas In Vitro , Mutação , Conformação Proteica , Salmonella typhimurium/virologiaRESUMO
Bacteriophage P22 belongs to a family of double-stranded DNA viruses that share common morphogenetic features like DNA packaging into a procapsid precursor and maturation. Maturation involves cooperative expansion of the procapsid shell with concomitant lattice stabilization. The expansion is thought to be mediated by movement of two coat protein domains around a hinge. The metastable conformation of subunit within the procapsid lattice is considered to constitute a late folding intermediate. In order to understand the mechanism of expansion it is necessary to characterize the interactions stabilizing procapsid and mature capsid lattices, respectively. We employ pressure dissociation to compare subunit packing within the procapsid and expanded lattice. Procapsid shells contain larger cavities than the expanded shells, presumably due to polypeptide packing defects. These defects contribute to the metastable nature of the procapsid lattice and are cured during expansion. Improved packing contributes to the increased stability of the expanded shell. Comparison of two temperature-sensitive folding (tsf) mutants of coat protein (T294I and W48Q) with wild-type coat revealed that both mutations markedly destabilized the procapsid shell and yet had little effect on relative stability of the monomeric subunit. Thus, the regions affected by these packing defects constitute subunit interfaces of the procapsid shell. The larger activation volume of pressure dissociation observed for both T294I and W48Q indicates that the decreased stability of these particles is due to increase of cavity defects. These defects in the procapsid lattice are cured upon expansion suggesting that the intersubunit contacts affected by tsf mutations are absent or rearranged in the mature shell. The energetics of the in vitro expansion reaction also suggests that entropic stabilization contributes to the large free energy barrier for expansion.
Assuntos
Bacteriófago P22/crescimento & desenvolvimento , Bacteriófago P22/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Bacteriófago P22/genética , Capsídeo/genética , Cinética , Mutação Puntual , Pressão , Conformação Proteica , Dobramento de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Temperatura , TermodinâmicaRESUMO
This brief report describes the isolation and initial characterization of revertants to the most severe temperature sensitive folding mutant known. The revertants or suppressors may describe amino acid interactions that occur during the folding of the P22 tailspike polypeptide chain. Results indicate that several different types of suppressors may have been obtained.
Assuntos
Bacteriófago P22/genética , Genes Supressores/genética , Glicosídeo Hidrolases/genética , Mutação , Dobramento de Proteína , Proteínas da Cauda Viral/genéticaRESUMO
Bacteriophage P22 is a double-stranded DNA containing phage. Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid. The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously [Prevelige, P. E., King, J., & Silva, J. L. (1994) Biophys. J. 66, 1631-1641]. The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar. However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods. While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 degrees C), the G232D mutant shells showed time-dependent dissociation under these conditions. Decreasing the temperature to 1 degree C dramatically accelerated the dissociation of G232D mutant under applied pressure. On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bacteriófago P22/química , Capsídeo/química , Bacteriófago P22/ultraestrutura , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Relação Estrutura-Atividade , Temperatura , Termodinâmica , Ureia/químicaRESUMO
The mechanisms by which regulatory proteins recognize specific DNA sequences are not fully understood. Here we examine the basis for the stability of a protein-DNA complex, using hydrostatic pressure and low temperature. Pressure converts the DNA-binding Arc repressor protein from a native state to a denatured, molten-globule state. Our data show that the folding and dimerization of Arc repressor in the temperature range 0-20 degrees C are favored by a large positive entropy value, so that the reaction proceeds in spite of an unfavorable positive enthalpy. On binding operator DNA, Arc repressor becomes extremely stable against denaturation. However, the Arc repressor-operator DNA complex is cold-denatured at subzero temperatures under pressure, demonstrating that the favorable entropy increases greatly when Arc repressor binds tightly to its operator sequence but not a nonspecific sequence. We show how an increase in entropy may operate to provide the protein with a mechanism to distinguish between a specific and a nonspecific DNA sequence. It is postulated that the formation of the Arc-operator DNA complex is followed by an increase in apolar interactions and release of solvent which would explain its entropy-driven character, whereas this solvent would not be displaced in nonspecific complexes.
Assuntos
DNA/química , DNA/metabolismo , Desnaturação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófago P22/metabolismo , Calorimetria , Clonagem Molecular , Temperatura Baixa , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Modelos Estruturais , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/isolamento & purificação , Espectrofotometria Ultravioleta , Termodinâmica , Proteínas Virais/isolamento & purificação , Proteínas Virais Reguladoras e AcessóriasRESUMO
The effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein-DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG-dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA-dT). However, the maximum stabilization was much larger for operator sequence (delta p = 1,750 bar) as compared for poly(dA-dT) (delta p = 750 bar). The importance of the free-energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc-operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands.