A hybrid vector for ligand-directed tumor targeting and molecular imaging

Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.     

Experimental phage therapy against lethal lung-derived septicemia caused by Staphylococcus aureus in mice

Nosocomial respiratory infections caused by methicillin-resistant Staphylococcus aureus (MRSA) can progress to lethal systemic infections. Bacteriophage (phage) therapy is expected to be effective against these critical infections. Previously, phage S13′ was proposed as a potential therapeutic phage. We here examined phage treatment in a mouse model of lung-derived septicemia using phage S13′. Intraperitoneal phage administration at 6 h postinfection reduced the severity of infection and rescued the infected mice. Phage S13′ can efficiently lyse hospital-acquired MRSA strains causing pneumonia-associated bacteremia in vitro. Thus, phage therapy may be a possible therapeutic intervention in staphylococcal lung-derived septicemia.     

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvsY) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication. Received: 3 February 1999 / Accepted: 20 July 1999     

Design and construction of targeted AAVP vectors for mammalian cell transduction

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.     

phiGENOME: an integrative navigation throughout bacteriophage genomes

phiGENOME is a web-based genome browser generating dynamic and interactive graphical representation of phage genomes stored in the phiSITE, database of gene regulation in bacteriophages. phiGENOME is an integral part of the phiSITE web portal (http://www.phisite.org/phigenome) and it was optimised for visualisation of phage genomes with the emphasis on the gene regulatory elements. phiGENOME consists of three components: (i) genome map viewer built using Adobe Flash technology, providing dynamic and interactive graphical display of phage genomes; (ii) sequence browser based on precisely formatted HTML tags, providing detailed exploration of genome features on the sequence level and (iii) regulation illustrator, based on Scalable Vector Graphics (SVG) and designed for graphical representation of gene regulations. Bringing 542 complete genome sequences accompanied with their rich annotations and references, makes phiGENOME a unique information resource in the field of phage genomics.     

Removal of microbial indicators from municipal wastewater by a membrane bioreactor (MBR)

The impact of removable and irremovable fouling on the retention of viral and bacterial indicators by the submerged microfiltration membrane in an MBR pilot plant was evaluated. Escherichia coli, sulphite-reducing Clostridium spores, somatic coliphages and F-specific RNA bacteriophages were used as indicators. The membrane demonstrated almost complete removal of E. coli and sulphite-reducing Clostridium spores. However, there was no correlation with membrane fouling. The phage removal varied in accordance with the irremovable fouling, rising from 2.6 to 5.6 log₁₀ units as the irremovable fouling increased (measured by the change in the transmembrane pressure). In contrast, removable fouling did not have any effect on the retention of viruses by the membrane. These results indicate that irremovable membrane fouling may affect the removal efficiency of MBRs and, therefore, their capacity to ensure the required microbiological standards for the permeate achieved.     

Automated classification of tailed bacteriophages according to their neck organization

Background

The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database.

Results

We show that using a combination of iterative profile-profile comparison and gene context analyses, we can identify a set of head, neck and tail proteins in most tailed bacteriophages of our database. Classification of phages based on neck protein sequences delineates 4 Types corresponding to known morphological subfamilies. Further analysis of the most abundant Type 1 yields 10 Clusters characterized by consistent sets of head, neck and tail proteins. We developed Virfam, a webserver that automatically identifies proteins of the phage head-neck-tail module and assign phages to the most closely related cluster of phages. This server was tested against 624 new phages from the NCBI database. 93% of the tailed and unclassified phages could be assigned to our head-neck-tail based categories, thus highlighting the large representativeness of the identified virion architectures. Types and Clusters delineate consistent subgroups of Caudovirales, which correlate with several virion properties.

Conclusions

Our method and webserver have the capacity to automatically classify most tailed phages, detect their structural module, assign a function to a set of their head, neck and tail genes, provide their morphologic subtype and localize these phages within a “head-neck-tail” based classification. It should enable analysis of large sets of phage genomes. In particular, it should contribute to the classification of the abundant unknown viruses found on assembled contigs of metagenomic samples.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1027) contains supplementary material, which is available to authorized users.     

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i × j) and three-factor (i k × j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1 × i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1–i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1 × i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1 × i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII⁺ recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3–8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.     

Design,preparation and application of nucleic acid delivery carriers

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy.     

Planctopirus ephydatiae,a novel Planctomycete isolated from a freshwater sponge

The microbiome of freshwater sponges is rarely studied, and not a single novel bacterial species has been isolated and subsequently characterized from a freshwater sponge to date. A previous study showed that 14.4% of the microbiome from Ephydatia fluviatilis belong to the phylum Planctomycetes. Therefore, we sampled an Ephydatia sponge from a freshwater lake and employed enrichment techniques targeting bacteria from the phylum Planctomycetes. The obtained strain spb1^T was subject to genomic and phenomic characterization and found to represent a novel planctomycetal species proposed as Planctopirus ephydatiae sp. nov. (DSM 106606 = CECT 9866). In the process of differentiating spb1^T from its next relative Planctopirus limnophila DSM 3776^T, we identified and characterized the first phage – Planctopirus phage vB_PlimS_J1 – infecting planctomycetes that was only mentioned anecdotally before. Interestingly, classical chemotaxonomic methods would have failed to distinguish Planctopirus ephydatiae strain spb1^T from Planctopirus limnophila DSM 3776^T. Our findings demonstrate and underpin the need for whole genome-based taxonomy to detect and differentiate planctomycetal species.     

A filamentous phage display system for N-linked glycoproteins

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X₁-N-X₂-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10⁵ per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the −2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype–phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.     

Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides

We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.     

Involvement of cellular proteins in Junin arenavirus entry

Junin arenavirus (JUNV) entry is dependent on clathrin-mediated pathways and it relies on the integrity of the actin cytoskeleton as well as the dynamics of microtubules. To determine the method of entry used by this human pathogen, we have demonstrated that in Vero cells JUNV is trafficked via the cellular dynamin 2 (dyn2) endocytic pathway and it is dependent on the Eps15 GTPase. In addition, we have shown that the virus travels through Rab5-mediated early and Rab7-mediated late endosomes in its pH-dependent entry. Altogether, this study gives further inside into the endocytic pathway utilized by the arenavirus JUNV     

Bacteriophage MB78 DNA synthesis is specifically inhibited by the chelating agent ethylene diamine tetraacetic acid

The growth of bacteriophage MB78, a virulent phage of Salmonella typhimurium is extremely sensitive to the chelating agent EDTA. Other chelating agents like EGTA, a specific chelator for Ca²⁺ and orthophenanthroline which chelates Zn²⁺ and Fe²⁺ have no effect. EDTA stops phage MB78 DNA synthesis while synthesis of host DNA and other Salmonella phage DNA are not affected in presence of such low concentrations of EDTA. The present report indicates that some early phage function(s) and most probably the phage DNA synthesis are sensitive to EDTA which is probably due to chelation of Mg²⁺.     

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.