Helper Phage Research Articles

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli.

The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 109-1011 independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F+ or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage.

Generation of a Biotin-Tagged Dual-Display Phage.

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems.

ABD-3, the confluence of powerful antibacterial modalities: ABDs delivering and expressing lss, the gene encoding lysostaphin.

In response to the antimicrobial resistance crisis, we have developed a powerful and versatile therapeutic platform, the Antibacterial Drone (ABD) system. The ABD consists of a highly mobile staphylococcal pathogenicity island re-purposed to deliver genes encoding antibacterial proteins. The chromosomally located island is induced by a co-resident helper phage, packaged in phage-like particles, and released in very high numbers upon phage-induced lysis. ABD particles specifically adsorb to bacteria causing an infection and deliver their DNA to these bacteria, where the bactericidal cargo genes are expressed, kill the bacteria, and cure the infection. Here, we report a major advance of the system, incorporation of the gene encoding a secreted, bactericidal, species-specific lytic enzyme, lysostsphin. This ABD not only kills the bacterium that has been attacked by the ABD, but also any surrounding bacteria that are sensitive to the lytic enzyme which is released by secretion and by lysis of the doomed cell. So while the killing field is thus expanded, there are no civilian casualties (bacteria that are insensitive to the ABD and its cargo protein(s) are not inadvertently killed). Without amplifying the number of ABD particles (which are not re-packaged), the expression and release of the cargo gene's product dramatically extend the effective reach of the ABD. A cargo gene that encodes a secreted bactericidal protein also enables the treatment of a mixed bacterial infection in which one of the infecting organisms is insensitive to the ABD delivery system but is sensitive to the ABD's secreted cargo protein.

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Acinetobacter baumannii satellite phage Aci01-2-Phanie depends on a helper myophage for its multiplication.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine

IntroductionCholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. Material and methodsB and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. ResultsThe titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. ConclusionThis study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Problems of creating antibody phage libraries and their solutions.

Phage display has become an efficient, reliable and popular molecular technique for generating libraries encompassing millions or even billions of clones of divergent peptides or proteins. The method is based on the correspondence between phage genotype and phenotype, which ensures the presentation of recombinant proteins of known amino acid composition on the surface of phage particles. The use of affinity selection allows one to choose variants with affinity for different targets from phage libraries. The implementation of the antibody phage display technique has revolutionized the field of clinical immunology, both for developing tools to diagnose infectious diseases and for producing therapeutic agents. It has also become the basis for efficient and relatively inexpensive methods for studying protein-protein interactions, receptor binding sites, as well as epitope and mimotope identification. The antibody phage display technique involves a number of steps, and the final result depends on their successful implementation. The diversity, whether natural or obtained by combinatorial chemistry, is the basis of any library. The choice of molecular techniques is critical to ensure that this diversity is maintained during the phage library preparation step and during the transformation of E. coli cells. After a helper phage is added to the suspension of transformed E. coli cells, a bacteriophage library is formed, which is a working tool for performing the affinity selection procedure and searching for individual molecules. Despite the apparent simplicity of generating phage antibody libraries, a number of subtleties need to be taken into account. First, there are the features of phage vector preparation. Currently, a large number of phagemid vectors have been developed, and their selection is also of great importance. The key step is preparing competent E. coli cells and the technology of their transformation. The choice of a helper phage and the method used to generate it is also important. This article discusses the key challenges faced by researchers in constructing phage antibody libraries.

Antirepressor specificity is shaped by highly efficient dimerization of the staphylococcal pathogenicity island regulating repressors: Stl repressor dimerization perturbed by dUTPases

The excision and replication, thus the life cycle of pathogenicity islands in staphylococci are regulated by Stl master repressors that form strong dimers. It has been recently shown that SaPIbov1-Stl dimers are separated during the activation of the Staphylococcus aureus pathogenicity island (SaPI) transcription via helper phage proteins. To understand the mechanism of this regulation, a quantitative analysis of the dimerization characteristics is required. Due to the highly efficient dimerization process, such an analysis has to involve specific solutions that permit relevant experiments to be performed. In the present work, we focused on two staphylococcal Stls associated with high biomedical interest, namely Stl proteins of Staphylococcus aureus bov1 and Staphylococcus hominis ShoCI794_SEPI pathogenicity islands. Exploiting the interactions of these two Stl proteins with their antirepressor-mimicking interaction partners allowed precise determination of the Stl dimerization constant in the subnanomolar range.

Phage-inducible chromosomal minimalist islands (PICMIs), a novel family of small marine satellites of virulent phages

Phage satellites are bacterial genetic elements that co-opt phage machinery for their own dissemination. Here we identify a family of satellites, named Phage-Inducible Chromosomal Minimalist Islands (PICMIs), that are broadly distributed in marine bacteria of the family Vibrionaceae. A typical PICMI is characterized by reduced gene content, does not encode genes for capsid remodelling, and packages its DNA as a concatemer. PICMIs integrate in the bacterial host genome next to the fis regulator, and encode three core proteins necessary for excision and replication. PICMIs are dependent on virulent phage particles to spread to other bacteria, and protect their hosts from other competitive phages without interfering with their helper phage. Thus, our work broadens our understanding of phage satellites and narrows down the minimal number of functions necessary to hijack a tailed phage.

Generation of a helper phage for the fluorescent detection of peptide-target interactions by dual-display phages

Phage display is a molecular biology technique that allows the presentation of foreign peptides on the surface of bacteriophages. It is widely utilized for applications such as the discovery of biomarkers, the development of therapeutic antibodies, and the investigation of protein–protein interactions. When employing phages in diagnostic and therapeutic monitoring assays, it is essential to couple them with a detection system capable of revealing and quantifying the interaction between the peptide displayed on the phage capsid and the target of interest. This process is often technically challenging and costly. Here, we generated a fluorescent helper phage vector displaying sfGFP in-frame to the pIII of the capsid proteins. Further, we developed an exchangeable dual-display phage system by combining our newly developed fluorescent helper phage vector with a phagemid vector harboring the engineered pVIII with a peptide-probe. By doing so, the sfGFP and a peptide-probe are displayed on the same phage particle. Notably, our dual-display approach is highly flexible as it allows for easy exchange of the displayed peptide-probe on the pVIII to gain the desired selectivity, while maintaining the sfGFP gene, which allows easy visualization and quantification of the interaction peptide-probe. We anticipate that this system will reduce time and costs compared to the current phage-based detection systems.

Simultaneous entry as an adaptation to virulence in a novel satellite-helper system infecting Streptomyces species

Satellites are mobile genetic elements that are dependent upon the replication machinery of their helper viruses. Bacteriophages have provided many examples of satellite nucleic acids that utilize their helper morphogenic genes for propagation. Here we describe two novel satellite-helper phage systems, Mulch and Flayer, that infect Streptomyces species. The satellites in these systems encode for encapsidation machinery but have an absence of key replication genes, thus providing the first example of bacteriophage satellite viruses. We also show that codon usage of the satellites matches the tRNA gene content of the helpers. The satellite in one of these systems, Flayer, does not appear to integrate into the host genome, which represents the first example of a virulent satellite phage. The Flayer satellite has a unique tail adaptation that allows it to attach to its helper for simultaneous co-infection. These findings demonstrate an ever-increasing array of satellite strategies for genetic dependence on their helpers in the evolutionary arms race between satellite and helper phages.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

Helper-embedded satellites from an integrase clade that repeatedly targets prophage late genes.

Satellites such as phage-induced chromosomal islands (PICIs) are mobile genetic elements relying on helper phages for their mobilization, through trans-regulatory interactions. We discovered a PICI with a more intimate cis-regulatory configuration, integrated within a late gene of its helper prophage. This helper-embedded PICI (HE-PICI) configuration delays expression of the interrupted helper late gene until the satellite excises and provides passive helper-driven components to both HE-PICI replication and late transcription. Upon induction of a helper-satellite composite, precise excision of the entire composite was observed, followed by composite replication, then satellite excision. We mapped 491 additional HE-PICIs to one of 14 sites in cognates of phage lambda late genes. Associated integrases form a single phylogenetic clade with subclades respecting the 14 site groups, exhibiting repeated tropism for prophage late genes as new integration sites evolve. Four ordered zones in a general gram-negative PICI genome organization are: an integration zone encoding integrase and AlpA, a dynamic zone encoding members of the Bro-N network of domain-swapping DNA-interactive proteins and immunity repressor RNAs, a replication zone, and a dynamic late zone in which clusters as large as 17 consecutive helper prophage late genes have been captured. Helper-embedded satellites present new dimensions in satellite/helper relationships.

Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens.

Bacteriophages can be reprogrammed to deliver antimicrobials for therapeutic and biocontrol purposes and are a promising alternative treatment to antimicrobial-resistant bacteria. Here, we developed a bacteriophage P4 cosmid system for the delivery of a Cas9 antimicrobial into clinically relevant human gut pathogens Shigella flexneri and Escherichia coli O157:H7. Our P4 cosmid design produces a high titer of cosmid-transducing units without contamination by a helper phage. Further, we demonstrate that genetic engineering of the phage tail fiber improves the transduction efficiency of cosmid DNA in S. flexneri M90T as well as allows recognition of a nonnative host, E. coli O157:H7. We show that the transducing units with the chimeric tails enhanced the overall Cas9-mediated killing of both pathogens. This study demonstrates the potential of our P4 cas9 cosmid system as a DNA sequence-specific antimicrobial against clinically relevant gut pathogenic bacteria.

The Bacteriophage-Phage-Inducible Chromosomal Island Arms Race Designs an Interkingdom Inhibitor of dUTPases.

Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI's repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. In silico and in vitro analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life. IMPORTANCE Stl, the Staphylococcus aureus pathogenicity island (SaPI) master repressor, targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. This fascinating phage-SaPI arms race is exemplified by the Stl from SaPIbov1 which targets phage dimeric and trimeric dUTPases (Duts), structurally unrelated proteins with identical functions in the phages. By solving the structure of the Stl in complex with a prokaryotic (M. tuberculosis) and a eukaryotic (human) trimeric Dut, we showed that Stl has developed a sophisticated substrate mimicry strategy to target trimeric Duts. Since all these Duts present identical catalytic mechanisms, Stl is able to interact with Duts from different kingdoms. In addition, in silico modeling with monomeric Dut supports the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor.

PICI thieves: Molecular piracy and cooperation

Phage-inducible chromosomal islands (PICIs) steal structural proteins from helper phages. In two related studies, Penadés and coworkers reveal that PICIs are not parasites but mutualists. Some PICIs mobilize defense systems that restrict niche competitors, while other PICIs encode their own capsids and steal helper phage tails without affecting their fitness.

Construction of Chicken and Ostrich Antibody Libraries.

Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage-displayed scFvs for use in veterinary diagnostics and research. To add diversity to the scFv repertoire, we expanded the library to include genes from the ostrich, indigenous to southern Africa. The libraries described in this chapter are based on the chicken and ostrich variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.

A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature

Phage satellites are genetic elements that couple their life cycle to that of helper phages they parasitize, interfering with phage packaging through the production of small capsids, where only satellites are packaged. So far, in all analyzed systems, the satellite-sized capsids are composed of phage proteins. Here, we report that a family of phage-inducible chromosomal islands (PICIs), a type of satellites, encodes all the proteins required for both the production of small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, named capsid-forming PICIs (cf-PICIs), only requires phage tails to generate PICI particles. Remarkably, the representative cf-PICIs are produced with no cost from their helper phages, suggesting that the relationship between these elements is not parasitic. Finally, our phylogenomic studies indicate that cf-PICIs are present both in gram-positive and gram-negative bacteria and have evolved at least three times independently to spread in nature.

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.