Recombinant Phage Research Articles

Validation of PhageDx™Salmonella Assay in Raw Ground Turkey and Powdered Infant Formula: AOAC Performance Tested MethodSM 121904.

BackgroundThe PhageDx™Salmonella Assay is based on the infection of Salmonella spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 9.5 h for raw ground turkey and 18.5 h for milk-based powdered infant formula (PIF).ObjectiveAn AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Salmonella Assay for the detection of Salmonella in 25 g raw ground turkey and 100 g PIF test portions.MethodThe performance of the PhageDx Salmonella Assay was compared to that of the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 4.10 for raw ground turkey and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 for PIF. Inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted.ResultsThere was no significant difference between the 25 g raw ground turkey and 100 g PIF PhageDx Salmonella Assay and the USDA/FSIS MLG 4.10 and FDA/BAM Chapter 5, respectively. The reporter bacteriophages were specific for Salmonella and infected 108 strains in inclusivity testing. They did not infect 30 non-Salmonella bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least eight months.ConclusionsThe data collected in the validation study demonstrate that the PhageDx Salmonella Assay meets the qualifications for PTM status. HighlightsThe PhageDx Salmonella Assay is a rapid, specific, sensitive assay capable of detecting a wide range of Salmonella spp. with a significantly shorter turn around time than the USDA/FSIS and FDA reference methods.

Validation of PhageDx™ Cronobacter Assay for the Identification of Cronobacter Spp. in Powdered Infant Formula: AOAC Performance Tested MethodSM 051803.

BackgroundThe PhageDx™Cronobacter Assay is based on the infection of Cronobacter spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 18.5 h for powdered infant formula (PIF).ObjectiveAn AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Cronobacter Assay for the detection of Cronobacter in 10, 100, and 300 g milk- and soy-based PIF test portions.MethodThe performance of the PhageDx method was compared to the ISO 22964:2006/2017 Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp. and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 29 Cronobacter: 2012. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted.ResultsThere was no significant difference between the 10, 100, or 300 g test portions for the milk and soy PIF matrixes between the PhageDx Cronobacter Assay, the ISO 22964:2006/2017, and the FDA BAM Chapter 29 Cronobacter: 2012 methods. The reporter bacteriophages were specific for Cronobacter and infected 75 strains in inclusivity testing. They did not infect 35 non-Cronobacter bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 3 months.ConclusionsWork in the submitting and independent laboratories demonstrated that the PhageDx Cronobacter Assay meets the qualifications for PTM status.HighlightsThe PhageDx Cronobacter Assay is a rapid, simple, and specific test that has shown equivalence to both the FDA BAM and ISO reference methods for detecting Cronobacter spp. in PIF.

Validation of the PhageDx™ Listeria Assay for Detection of Listeria Spp. on Stainless Steel and Ceramic Environmental Surfaces AOAC Performance Tested MethodSM 102005.

BackgroundThe PhageDx™ Listeria Assay is a simple, specific, and sensitive assay based on the infection of Listeria spp. by selected bacteriophages and the resultant expression of a luciferase reporter gene. Results are generated in as little as 24.5 h for stainless steel and ceramic environmental surfaces.ObjectiveAn AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Listeria Assay for the detection of Listeria on stainless steel and ceramic surfaces.MethodThe performance of the PhageDx method was compared to that of the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Ch. 10. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted.ResultsInclusivity testing demonstrated that the reporter bacteriophages were specific for Listeria ssp. and detected 58/61 Listeria strains tested, including all 34 L. monocytogenes strains. The reporter bacteriophage also was shown to not detect 46/47 non-Listeria bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 6 months. Matrix studies on stainless steel and ceramic surfaces showed that there was no significant difference between the PhageDx Listeria Assay and the FDA/BAM Chapter 10 reference method.Conclusions and HighlightsThe validation study demonstrates that the PhageDx Listeria Assay is an effective method for the detection of Listeria spp. on stainless steel and ceramic environmental surfaces and meets the qualifications for AOAC PTM status.

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

Whole Genome Sequence Analysis of Phage-Resistant Listeria monocytogenes Serotype 1/2a Strains from Turkey Processing Plants.

Listeria monocytogenes is a Gram-positive bacterial pathogen and the causative agent of listeriosis, a severe foodborne infection. L. monocytogenes is notorious for its ability to persist in food processing environments (FPEs) via a variety of adaptive traits. Even though traits such as cold tolerance, biofilm formation and sanitizer resistance have been extensively investigated for their roles in persistence of L. monocytogenes in FPEs, much less is known about resistance to bacteriophages. Previous studies explored phage resistance mechanisms in laboratory-created mutants but it is imperative to investigate phage resistance that is naturally exhibited in FPE-derived strains. Here, we integrated the analysis of whole genome sequence data from a panel of serotype 1/2a strains of sequence types 321 and 391 from turkey processing plants, with the determination of cell surface substituents required for phage adsorption and phage infection assays with the four wide-host-range phages A511, P100, 20422-1 and 805405-1. Using a specific set of recombinant phage protein probes, we discovered that phage-resistant strains lacked one or both of the serogroup 1/2-specific wall teichoic acid carbohydrate decorations, N-acetylglucosamine and rhamnose. Furthermore, these phage-resistant strains harbored substitutions in lmo1080, lmo1081, and lmo2550, which mediate carbohydrate decoration of the wall teichoic acids.

Metisiline Dirençli Staphylococcus aureus (MRSA)’dan Elde Edilen Fajın Karakterizasyonu

Objective: The emergence of Staphylococcus aureus strains resistant to all antimicrobials and failure to discover new antibiotics have led researchers to phage therapy, which lost popularity after the discovery of antibiotics. The development of recombinant technology introduced the idea of creating lysogenic recombinant phages that provide controlled bacterial death and this required small- sized phages that were easy to manipulate. Our aim is to identify small-sized lysogenic bacteriophages that can be used safely in therapy. Method: The gene and protein map of the phage was created by analysis of sequencing after extracting a phage from the MRSA strain that is known to contain a small phage. Results: The phage was classified in Caudovirales spp. as it contains genes encoding tail proteins, and in Podoviridae spp. due to its genomic size and arrangement. Conclusion: To date, there are only sixteen phages from Podoviridae family uploaded on NCBI, and the phage described in this study is the seventeenth one. Only 41.4% of the ORFs (Open Reading Frames) in the genome could be matched with proteins using the NCBI BLAST. Recent studies suggest that 50-75% of bacteriophage ORFs do not correspond to any organism in GenBank. For better understanding of bacteriophages and their utilization in phage therapy, it is essential to sequence greater number of phages, and to discover their genomes and corresponding proteins. Since the genes and proteins of a lysogenic phage that can be used safely in recombinant phage therapies have been identified in our study, it will contribute to the relevant literature.

Phage-Based Assay for the Detection of Salmonella in Brazilian Poultry Products

Salmonella is one of the most common microorganisms responsible for foodborne diseases worldwide, and its rapid and accurate detection is necessary for food safety. Bacteriophages are a promising tool for detecting bacterial foodborne pathogens due to their safety, specificity, rapid propagation, and capacity to differentiate living and dead cells. The PhageDx Salmonella Assay is a new Salmonella detection method composed of recombinant bacteriophages encoding a luciferase reporter gene. While this method has been validated in the United States to detect Salmonella in ground turkey and powdered infant formula, it has not been validated in other countries, and its performance in other matrices is unknown. In this study, the performance of the PhageDx Salmonella Assay was evaluated using Salmonella strains isolated in Brazil. 55 isolates from food and food processing environments in Brazil were examined and successfully detected using the recombinant bacteriophages employed by this method. As Brazil is the number one exporter of chicken globally, this method was also validated in several chicken-based food matrices. Using a pre-enrichment of 7 hours, it was possible to detect one CFU per 25 g on chicken meat, sausage, pâté, and chicken nuggets. The total analysis time was 9 hours, shorter than other Salmonella detection methods currently available. The method proved to be easy to execute, sensitive, and fast, making it a promising tool for the Brazilian poultry industry.

Recombinant bacteriophage T4 Rnl1 impacts Streptococcus mutans biofilm formation

ABSTRACT Bacteriophage T4 RNA ligase 1 (T4 Rnl1) can be stably expressed in many bacteria and has been reported to affect the bioactivity of the host bacteria. Recently, we constructed bacteriophage T4 Rnl1 expressing system in Streptococcus mutans, a crucial biofilm-forming and dental caries-causing oral pathogen. Here, we characterized the function of recombinant bacteriophage T4 Rnl1 in biofilm formation of S. mutans. The T4 Rnl1 mutant exhibited similar growth phenotype but resulted in a significant reduction of biofilm biomass compared to wild type strain and empty plasmid carrying strain. The abnormal biofilm of the T4 Rnl1 mutant harbored loose bacterial clusters with defective production and distribution of exopolysaccharides. Moreover, the expression of several biofilm formation-associated genes was dysregulated at mRNA level in the T4 Rnl1 mutant. These results reveal that the bacteriophage T4 Rnl1 exert antibiofilm activities against the cariogenic bacterium S. mutans, which impacts the spatial organization of the exopolysaccharides and further impairs the three-dimensional biofilm architecture. These findings implicate that manipulation of bacteriophage T4 Rnl1, a biological tool used for RNA ligation, will provide a promising approach to cariogenic biofilm control.

Combining the advantages of prokaryotic expression and T7 phage display systems to obtain antigens for antibody preparation

The gene encoding the phage major capsid protein 10A was cloned into the prokaryotic expression vector pET24a, and a 6XHis-tag was fused to the 3′-end of the 10A gene to verify complete expression. The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and 10A expression was induced by IPTG. SDS-PAGE and Western blot were used to confirm the target protein expression. The T7Select10-3b vector was added to the cultured bacteria expressing 10A at a multiplicity of infection (MOI) ranging from 0.01 to 0.1, and complete lysis of the bacteria was monitored by absorbance changes in the medium. The recombinant phage (reP) was harvested by PEG/NaCl sedimentation and resuspended in PBS. ELISA was performed to verify the presence of the 6XHis-tag on the surface of reP. The 10A-fusion expression vectors (pET10A-flag, pET10A-egfp, and pET10A-pct) were constructed, and fusion proteins were expressed and detected by the same method. The corresponding rePs (reP-Flag, reP-EGFP, and reP-PCT) were prepared by T7Select10-3b infection. After the expression of the peptides/proteins on the reP surfaces was confirmed, reP-Flag and reP-PCT were used to immunize mice to prepare anti-Flag and anti-PCT antibodies. The results showed that rePs prepared using the 10A-fusion vector and T7Select10-3b can be used as antigens to immunize mice and prepare antibodies. This method may be able to meet the rapid antigen preparation requirements for antibody production.Notably, the recombinant phage (reP) described in this study was obtained by the sedimentation method from T7Select10-3b-infected E. coli BL21 (DE3) cells carrying the major capsid protein 10A expression vector or 10A-fusion protein vector.

Mechanisms and Control Strategies of Antibiotic Resistance in Pathological Biofilms.

Bacterial biofilm is a community of bacteria that are embedded and structured in a self-secreted extracellular matrix. An important clinical-related characteristic of bacterial biofilms is that they are much more resistant to antimicrobial agents than the planktonic cells (up to 1,000 times), which is one of the main causes of antibiotic resistance in clinics. Therefore, infections caused by biofilms are notoriously difficult to eradicate, such as lung infection caused by Pseudomonas aeruginosa in cystic fibrosis patients. Understanding the resistance mechanisms of biofilms will provide direct insights into how we overcome such resistance. In this review, we summarize the characteristics of biofilms and chronic infections associated with bacterial biofilms. We examine the current understanding and research progress on the major mechanisms of antibiotic resistance in biofilms, including quorum sensing. We also discuss the potential strategies that may overcome biofilm-related antibiotic resistance, focusing on targeting biofilm EPSs, blocking quorum sensing signaling, and using recombinant phages.

Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes.

Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages.IMPORTANCEListeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.

Identification of β-conglycinin α' subunit antigenic epitopes destroyed by thermal treatments

In the present study, phage display technology (PDT) was used to examine β-conglycinin α' subunit epitopes destroyed by heat treatments. The overlapping gene fragments of the α' subunits were amplified using a polymerase chain reaction (PCR) method, and then the recombinant T7 phages containing the target genes were expressed on the phage surface. In addition, an indirect enzyme-linked immunosorbent assay (iELISA) method was adopted to screen the α' subunit epitopes which had been damaged by the heat treatments. Then, after three rounds of the expression and screening processes, the C1-Y fragments (488PHFNSKAIVVLVINEGEANIELVG511) were identified as α' subunit damaged epitopes. In order to confirm the types of epitopes, three peptides of the C1-Y fragments were synthesized using a solid-phase peptide synthesis method. The results of the iELISA showed that there was a linear epitope of 488PHFNSKAIVVLV499 in the C1-Y fragments. The results obtained in this study confirmed that the C1-Y fragments contained both linear and conformational epitopes, and that the conformational epitopes could be destroyed by thermal treatments. However, it was found that the linear epitopes could not be destroyed. The phage display protein Pt-C1-Y, coupling peptide BSA-Pep-Y, BSA-Pep-1, BSA-Pep-2, and soybean allergy patients' serum reaction results showed that only the Pt-C1-Y had reacted positively with the soybean allergy patients' serum. These findings further indicated that heat treatments can reduce not only the antigenicity, but also the allergenicity, of α' subunits in β-conglycinin.

Engineered Bacteriophage T7 as a Potent Anticancer Agent in vivo.

Oncolytic viruses (OVs) induce antitumor effect by both direct lysis of target cells and eliciting immunogenic response to the virus and ultimately to the target cells. These viruses are usually natural human pathogens. Bacteriophages are natural pathogens of bacteria that do not infect human and have greater advantages in safety, manipulation, and production over human viruses. We constructed an engineered bacteriophage T7 displaying a peptide, which targets murine melanoma cells and harbors a mammalian expression cassette of the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) in viral genomic DNA. The engineered phage was successfully transduced to B16F10 melanoma cells both in vitro and in vivo. GM-CSF was expressed from the transduced phage DNA. All mice treated with the phage intravenously survived for 25 days until the end of experiment, while only 40% of those not treated survived. During the 16 days of phage treatment, phage T7 displaying homing peptide and expressing GM-CSF inhibited tumor growth by 72% compared to the untreated control. Serum cytokine levels of IL-1α, TNF-α, and GM-CSF were seen to increase during the treatment. Immunohistochemical analysis of tumor tissue revealed infiltration by macrophages, dendritic cells (DCs), and CD8+ T cells. Migration of murine macrophages to bacteriophages was also observed in in vitro transwell assays in both time- and dose-dependent manners. Taken together, the recombinant bacteriophage T7 efficiently inhibited tumor growth by changing the tumor microenvironment and recruiting anti-tumor immune cells.

Amino acid sequence associated with bacteriophage recombination site helps to reveal genes potentially acquired through horizontal gene transfer

BackgroundHorizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer.ResultsHMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events.ConclusionsCysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.

Articles of Significant Interest in This Issue

The alarming increase in antibiotic resistance has motivated the search for alternative antimicrobial strategies. A study developed by Oliveira et al. (e00073-20) proposes an antivirulence approach to tackle infectious diseases. They show that a recombinant bacteriophage tailspike protein can be used to remove bacterial surface polysaccharides of Providencia stuartii, thereby making the bacterium susceptible to serum killing. Being antivirulence agents, tailspikes do not select for resistance, and thus they represent a promising antibacterial strategy.

Recombination of T4-like Phages and Its Activity against Pathogenic Escherichia coli in Planktonic and Biofilm Forms.

The increasing emergence of multi-drug resistant Escherichia coli (E. coli) has become a global concern, primarily due to the limitation of antimicrobial treatment options. Phage therapy has been considered as a promising alternative for treating infections caused by multi-drug resistant E. coli. However, the application of phages as a promising antimicrobial agent is limited by their narrow host range and specificity. In this research, a recombinant T4-like phage, named WGqlae, has been obtained by changingthe receptor specificity determinant region of gene 37, using a homologous recombination platform of T4-like phages established by our laboratory previously. The engineered phage WGqlae can lyse four additional hosts, comparing to its parental phages WG01 and QL01. WGqlae showed similar characteristics, including thermo and pH stability, optimal multiplicity of infection and one-step growth curve, to the donor phage QL01. In addition, sequencing results showed that gene 37 of recombinant phage WGqlae had genetically stable even after 20 generations. In planktonic test, phage WGqlae had significant antimicrobial effects on E. coli DE192 and DE205B. The optical density at 600nm (OD600) of E. coli in phage WGqlae treating group was significantly lower than that of the control group (P < 0.01). Besides, phage WGqlae demonstrated an obvious inhibitory effect on the biofilm formation and the clearance of mature biofilms. Our study suggested that engineered phages may be promising candidates for future phage therapy applications against pathogenic E. coli in planktonic and biofilm forms.

Deciphering the Role of Holin in Mycobacteriophage D29 Physiology.

In the era of antibiotic resistance, phage therapy is gaining attention for the treatment of pathogenic organisms such as Mycobacterium tuberculosis. The selection of phages for therapeutic purposes depends upon several factors such as the host range that a phage can infect, which can be narrow or broad, time required for the host cell lysis, and the burst size. Mycobacteriophage D29 is a virulent phage that has the ability to infect and kill several slow- and fast-growing mycobacterial species including the pathogenic M. tuberculosis. It, therefore, has the potential to be used in phage therapy against M. tuberculosis. D29 lytic cassette encodes three proteins viz. peptidoglycan hydrolase (LysA), mycolylarabinogalactan esterase (LysB), and holin, which together ensure host cell lysis in a timely manner. In this work, we have scrutinized the importance of holin in mycobacteriophage D29 physiology. Bacteriophage Recombineering of Electroporated DNA (BRED) approach was used to generate D29 holin knockout (D29Δgp11), which was further confirmed by the Deletion amplification detection assay (DADA)-PCR. Our results show that D29Δgp11 is viable and retains plaque-forming ability, although with reduced plaque size. Additionally, the host cell lysis governed by the mutant phage is significantly delayed as compared to the wild-type D29. In the absence of holin, D29 shows increased latent period and reduced burst size. Thus, our experiments show that while holin is dispensable for phage viability, it is essential for the optimal phage-mediated host cell lysis and phage propagation, which further points to the significance of the “clock” function of holin. Taken together, we show the importance of holin in governing timely and efficient host cell lysis for efficient progeny phage release, which further dictates its critical role in phage biology.

Recombinant Filamentous Bacteriophages Encapsulated in Biodegradable Polymeric Microparticles for Stimulation of Innate and Adaptive Immune Responses.

Escherichia coli filamentous bacteriophages (M13, f1, or fd) have attracted tremendous attention from vaccinologists as a promising immunogenic carrier and vaccine delivery vehicle with vast possible applications in the development of vaccines. The use of fd bacteriophage as an antigen delivery system is based on a modification of bacteriophage display technology. In particular, it is designed to express multiple copies of exogenous peptides (or polypeptides) covalently linked to viral capsid proteins. This study for the first time proposes the use of microparticles (MPs) made of poly (lactic-co-glycolic acid) (PLGA) to encapsulate fd bacteriophage. Bacteriophage–PLGA MPs were synthesized by a water in oil in water (w1/o/w2) emulsion technique, and their morphological properties were analyzed by confocal and scanning electron microscopy (SEM). Moreover, phage integrity, encapsulation efficiency, and release were investigated. Using recombinant bacteriophages expressing the ovalbumin (OVA) antigenic determinant, we demonstrated the immunogenicity of the encapsulated bacteriophage after being released by MPs. Our results reveal that encapsulated bacteriophages are stable and retain their immunogenic properties. Bacteriophage-encapsulated PLGA microparticles may thus represent an important tool for the development of different bacteriophage-based vaccine platforms.

Location of destroyed antigenic sites of Gly m Bd 60 K after three processing technologies

Gly m Bd 60 K, which is the α subunit of β-conglycinin, is a major soybean (Glycine max) allergen. We used high hydrostatic pressure (HHP), thermal techniques, and glycation to treat β-conglycinin, which can effectively reduce the antigenicity of β-conglycinin. β-conglycinin was used to immunize New Zealand rabbits, and the antiserum had a titer > 1: 1 × 105 and an IC50 of 2.254 μg/mL. β-conglycinin was subjected to HHP, thermal techniques, and glycation and mixed with rabbit antiserum against β-conglycinin to obtain the site-specific antiserum. The overlapping gene fragments of Gly m Bd 60 K were amplified by polymerase chain reaction (PCR), then cloned into a T7 phage vector and packaged in vitro, the recombinant T7 phages were constructed. Indirect ELISA (iELISA) was used to locate the destroyed antigenic sites and, after three rounds of segment expression and identification, the C2-1 and C2-2 fragments were identified as destroyed antigenic sites of Gly m Bd 60 K. Allergenicity analysis showed that the C2-1 and C2-2 fragments reacted with allergic patients' serum, which indicated that the destroyed sites were allergic sites.

In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.