ABSTRACT
The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.
Subject(s)
B-Lymphocytes/immunology , Protein Engineering , RNA Phages/immunology , Receptors, Antigen, B-Cell/immunology , Single-Domain Antibodies/immunology , Vaccines, Virus-Like Particle/immunology , Adoptive Transfer , Animals , Antibody Specificity , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , B-Lymphocytes/transplantation , Female , Gene Library , Humans , Ligands , Male , Mice, Transgenic , Proof of Concept Study , RNA Phages/genetics , RNA Phages/metabolism , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/metabolism , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Vaccination , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/metabolismABSTRACT
We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes.
Subject(s)
Drug Delivery Systems/methods , Levivirus/genetics , Levivirus/metabolism , RNA Phages/genetics , RNA Phages/metabolism , Virion/genetics , Virion/metabolism , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line, Tumor , Drug Carriers/metabolism , Endocytosis/genetics , Endocytosis/physiology , Genetic Vectors/genetics , Genetic Vectors/metabolism , HeLa Cells , Humans , RNA, Small Interfering/genetics , Transfection/methods , Transferrin/genetics , Transferrin/metabolismABSTRACT
BACKGROUND: Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. CONCLUSIONS/SIGNIFICANCE: AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
Subject(s)
Epitopes/chemistry , Virion/chemistry , Animals , Cloning, Molecular , Gonadotropin-Releasing Hormone/chemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptides/chemistry , Protein Structure, Tertiary , RNA Phages/metabolism , Recombinant Proteins/chemistry , Vaccination/instrumentation , Vaccination/methodsABSTRACT
AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.
Subject(s)
Hydrostatic Pressure , RNA Phages/physiology , Chelating Agents/pharmacology , Culture Media , Edetic Acid/pharmacology , Genotype , Glucose/metabolism , Laurates/pharmacology , Monoglycerides/pharmacology , RNA Phages/drug effects , RNA Phages/metabolism , Sodium Chloride/metabolism , Sucrose/analogs & derivatives , Sucrose/pharmacology , Surface-Active Agents/pharmacology , Temperature , Virus Inactivation/drug effectsABSTRACT
The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated sludge is presented in this study. Raw and anaerobically digested sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analyzed for total coliforms, E. coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. E. coli concentrations of > or =10(3) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. Likewise, intestinal enterococci concentrations of > or =10(4) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. In the case of SOMCPH, it was not possible to define a threshold, since they were detected with the lowest observed indicator concentrations in all samples.
Subject(s)
Bacteriophages/metabolism , Enterococcus/growth & development , Environmental Monitoring/methods , Escherichia coli/growth & development , Sewage/microbiology , Waste Disposal, Fluid/methods , Coliphages/metabolism , RNA Phages/metabolism , Temperature , Water MicrobiologyABSTRACT
Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell-cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0.1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.
Subject(s)
Escherichia coli K12/metabolism , Escherichia coli Proteins/metabolism , F Factor/genetics , Fimbriae Proteins/metabolism , Microscopy, Fluorescence/methods , RNA Phages/metabolism , Amino Acid Sequence , Conjugation, Genetic , Cysteine , Escherichia coli K12/genetics , Escherichia coli K12/growth & development , Escherichia coli K12/virology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Fimbriae Proteins/chemistry , Fimbriae Proteins/genetics , Fluorescent Dyes/metabolism , Molecular Sequence Data , Mutation , RNA Phages/physiologyABSTRACT
Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.
Subject(s)
DNA Mutational Analysis , Pseudomonas/genetics , RNA Viruses/genetics , Bacteriophage phi 6/metabolism , Biological Evolution , Evolution, Molecular , Gene Frequency , Genes, Viral , Genetic Complementation Test , Genetic Variation , Genome , Genotype , Models, Genetic , Models, Statistical , Mutation , Phenotype , Plants/virology , RNA/chemistry , RNA Phages/metabolism , Selection, GeneticABSTRACT
BACKGROUND: Acute myelogenous leukemia (AML) is the most common type of acute leukemia in adults. Because conventional treatments are associated with substantial side effects, novel therapeutic strategies are required. Antisense oligodeoxynucleotides (ODNs) have shown promise as the basis of emerging therapies for fighting cancer, although in vivo application is hampered by high sensitivity to cellular nuclease degradation. Encapsidation of ODNs in a drug-delivery capsule would reduce such degradation, thereby increasing the potency of therapy. MS2 bacteriophage capsid proteins may be used as novel virus-like particles (VLPs) to deliver ODNs. Here we report an analysis of the uptake mechanism of this VLP system and preliminary examples of its use to deliver a number of ODNs, including some targeting p120 messenger RNAs, a biomarker overexpressed in myelogenous leukemia cells. METHODS: The ODNs were synthesized as covalent extensions to the translational repressor/assembly initiation signal (TR), a 19 nt stem-loop, of the RNA phage MS2. The VLPs were constructed by soaking ODN-TR directly into recombinant RNA-free capsid shells. Targeting of the encapsidated ODNs into cells was achieved by a receptor-mediated endocytosis pathway identified by immunofluorescence microscopy or by transmission electron microscopy with gold-labeled antibodies. RESULTS: After covalent decoration with transferrin on their surface, the VLPs containing ODNs demonstrated increased effectiveness in killing target leukemia cells expressing transferrin receptors, suggesting that this system is worthy of more extensive analysis. CONCLUSIONS: The findings suggest that RNA phage VLPs may be useful as a new nanomaterial for targeted delivery of antisense ODNs, or other macromolecular drug candidates.
Subject(s)
Capsid/chemistry , Drug Carriers/chemistry , Gene Transfer Techniques , Leukemia, Myeloid, Acute/genetics , Oligoribonucleotides, Antisense/administration & dosage , Oligoribonucleotides, Antisense/pharmacokinetics , RNA Phages/metabolism , Cell Line, Tumor , Drug Carriers/administration & dosage , Gene Targeting/methods , Humans , Oligoribonucleotides, Antisense/geneticsABSTRACT
OBJECTIVES: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35-98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). METHODS: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. RESULTS: The HBs*-E7(35-54) protein expressing E7 residues 35-54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae. The HBc Delta-E7(35-98), but not the frCP-E7(35-98), ensured VLP formation in Escherichia coli. In Balb/C mice, the HBs*-E7(35-54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBc Delta-E7(35-98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response. The frCP-E7(35-98) protein elicited equally high antibody responses to both E7 and frCP carrier. Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset. CONCLUSIONS: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes. All chimeric proteins may be regarded as potential vaccine candidates.
Subject(s)
Epitopes/immunology , Hepatitis B Core Antigens/metabolism , Hepatitis B Surface Antigens/metabolism , Oncogene Proteins, Viral/immunology , RNA Phages/metabolism , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Capsid/genetics , Capsid/immunology , Capsid/metabolism , Epitopes/metabolism , Female , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/immunology , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Humans , Immunization , Immunoglobulin Isotypes , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins , RNA Phages/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Virion/genetics , Virion/metabolismABSTRACT
The review deals with some of the transport functions of different systems that have been implicated with several pathological disorders. Membrane transport role in parasitic diseases and metal resistance is discussed as a few selected examples. Among various limitations that are encountered in recombinant technology and in heterologous expression of proteins, transport functions of the host organisms cannot be ignored. Recently, membrane transport has acquired a new emerging role in multidrug resistance. Several membrane transporters, particularly ATP binding cassette (ABC) proteins that are involved in drug resistance, have been identified throughout the evolutionary scale. The review briefly emphasizes that membranes are not only important as structural elements but are also adopted to perform diverse functions.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cell Membrane Permeability/physiology , Cell Membrane/metabolism , Drug Resistance, Multiple , Animals , Bacteria/genetics , Bacteria/metabolism , Biological Transport/physiology , DNA/metabolism , Eukaryotic Cells/metabolism , Genetic Vectors , Leishmania/genetics , Leishmania/metabolism , Monosaccharide Transport Proteins/metabolism , Plasmids/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Prokaryotic Cells/metabolism , RNA Phages/metabolism , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolismABSTRACT
Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region. Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR. The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production. Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them. Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding. In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer. These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint. These mutations alter promoter activity without affecting Mor binding. A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence.
Subject(s)
Bacteriophage mu/genetics , Escherichia coli/genetics , Promoter Regions, Genetic/genetics , RNA Phages/genetics , Transcriptional Activation , Viral Proteins/metabolism , Bacteriophage mu/metabolism , Base Sequence , Escherichia coli/enzymology , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Phages/metabolism , Sequence Analysis , Viral Proteins/geneticsABSTRACT
In order to identify the structural elements important for the activity of the Q beta minus strand RNA as a template for Q beta replicase, a series of minus strand RNAs with internal or external deletions were prepared by in vitro transcription from suitable expression plasmids. The template activities of the deletion mutants were determined by single-round replication assays using purified replicase holoenzyme or core enzyme (lacking subunit alpha) in vitro. Two elements of RNA structure and/or sequence important for template activity were found. The first is a segment in the 5'-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previously recognized to be highly conserved in the small variant MDV-1 RNA and suggested to be involved in its template recognition. The second element is defined by two partially complementary sequence segments in the 3'-terminal region (map positions 557 to 576 and 24 to 35), that appear to be engaged in long-range base-pairing and may form the stem of a large secondary structure domain, whose branches are not necessary for template recognition. The results obtained with replicase holoenzyme and core enzyme were identical within the accuracy of the method. They confirm the absence of any role of S1 protein in the interaction of replicase with minus strand RNA and further emphasize the profound difference in the interactions of replicase with the plus and minus strand.
Subject(s)
Allolevivirus/genetics , Q beta Replicase/metabolism , RNA Phages/metabolism , Base Sequence , Gene Deletion , Molecular Sequence Data , Plasmids , RNA, Double-Stranded/genetics , Sequence Alignment , Templates, GeneticABSTRACT
The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.
Subject(s)
Capsid Proteins , Capsid/metabolism , RNA Phages/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Binding Sites , Capsid/chemistry , Capsid/genetics , Molecular Sequence Data , Mutation , Protein Conformation , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/geneticsABSTRACT
The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated. Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied. It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent. It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system. In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.
Subject(s)
Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , Base Sequence , DNA , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , RNA Phages/genetics , RNA Phages/metabolism , RNA, Messenger/chemistry , Viral Proteins/geneticsABSTRACT
The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.
Subject(s)
Capsid/metabolism , RNA Phages/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Base Sequence , Binding Sites , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Osmolar ConcentrationABSTRACT
Se comparó la capacidad fagocítica de células ael exudado peritoneal (CEP) de ratones CFW inmunizados con una preparación ribosomal de Salmonella typhi Ty2, con la de ratones protegidos con una vacuna de bacterias inactivadas por calor, ambas en relación con lo obtenido en animales testigo, no inmunizados. Los ribosomas se administraron subcutáneamente en una dosis inicial de 100 µg de ARN y se dio un refuerzo igual a los 14 días, ambos con adyuvantes incompleto de Freund (AIF). Los ratones inmunizados con vacuna de células muertas, recibieron una sola dosis subcutánea con 16***6 bacterias en AIF. Al cabo de 7, 11, 14, 18, 22, 25, y 31 días se indujeron y extrajeron las CEP de los animales de cada grupo e individualmente se cultivaron in vitro junto con S. Typhi Ty2 virulento no opsonizado en relación células-bacterias 1:200. La sobrevida de las bacterias fagocitadas se determinó a las 24 horas de cultivo: las CEP se romperon y por cuenta viable se enumeraron las bacterias no digeridas. Los resultados indican que las CEP de los inmunizados eliminan bacterias con mayor eficiencia que las de testigos. También se demostró que la eficiencia bactericida fue significativamente mayor (P máxima de 0.005) para las CEP de los ratones tratados con la fracción ribosomal que las CEP de los animales vacunados con bacterias intactas no viables. Fiebre tifoidea; vacuna ribosomal; inmunidad a Salmonella; Salmonella typi; fagocitosis por células peritoneales
Subject(s)
Mice , Animals , Peritoneum/cytology , Salmonella typhi/immunology , Immunization, Passive , Mexico , Mice/immunology , RNA Phages/metabolism , Salmonella Phages/immunology , Typhoid-Paratyphoid Vaccines/classificationABSTRACT
RA1 was the only IncC plasmid that was slightly temperature-sensitive for replication and transfer. At 30 degrees C, RA1 determined constitutive synthesis of conjugative pili and yet was transfer-repressed. Attachment of shaft-adsorbing RNA phage C-1 virions prevented the probable retraction of pili under heat stimulus (55 degrees C). Electron microscopy showed single adsorbed virions at pilus bases where they were thought to have stopped retraction.
Subject(s)
Conjugation, Genetic , Fimbriae, Bacterial/metabolism , R Factors/genetics , RNA Phages/genetics , Aeromonas/genetics , Aeromonas/ultrastructure , Coliphages/genetics , Coliphages/ultrastructure , Fimbriae, Bacterial/ultrastructure , Microscopy, Electron , Phenotype , R Factors/physiology , RNA Phages/metabolism , RNA Phages/ultrastructure , TemperatureABSTRACT
The RNA phage MS2 encodes a protein, 75 amino acids long, that is necessary and sufficient for lysis of the host cell. DNA deletion analysis has shown that the lytic activity is confined to the C-terminal half of the protein. We have examined the effects of a synthetic peptide, covering the C-terminal 25 amino acids of the lysis protein, on the electrochemical potential, generated in Escherichia coli membrane vesicles and in liposomes reconstituted with cytochrome c oxidase. In all cases the peptide dissipates the electrochemical potential. The peptide also induces the release of carboxyfluorescein (376 daltons), but not of inuline (5500 daltons), from protein-free liposomes. The phenomena are observed at a lipid to peptide molar ratio of approximately 100:1. The possible connection between the dissipation of the proton-motive force and bacteriolysis is discussed.