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.

Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora.

Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 (dpoL1-C) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2::luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material.IMPORTANCE Fire blight, caused by Erwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells.

Genome editing for phage design and uses for therapeutic applications.

The over usage of antibiotics leads to antibiotic abuse which in turn eventually raises resistance mechanisms among wide range of pathogens. Due to lack of experimental data of efficacy of phages as potential antimicrobial and therapeutic agent and also more specific and cumbersome isolation process against specific pathogens makes it not so feasible technology to be looked as an alternative therapy. But, recent developments in genome editing techniques enables programmed nuclease enzymes that has effectively improvised our methodology to make accurate changes in the genomes of prokaryote as well as eukaryote cells. It is already strengthening our ability to improvise genetic engineering to disease identification by facilitating the creation of more precise models to identify the root cause. The present chapter discusses on improvisation of phage therapy using recent genome editing tools and also shares data on the methods of usage of phages and their derivatives like proteins and enzymes such as lysins and depolymerases, as a potential therapeutic or prophylaxis agent. Methods involved in recombinant based techniques were also discussed in this chapter. Combination of traditional approach with modern tools has led to a potential development of phage-based therapeutics in near future.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Recombinant phage displaying ToAP2D peptide with antifungal activity against Sporothrix globosa.

We designed and synthesized recombinant phage nanofibers displaying ToAP2D peptide and investigated their antifungal effect on Sporothrix and the corresponding mechanism. Antimicrobial peptide, ToAP2D, was used as the template. The effect of synthesized recombinant phages on the immune function of CD4+ T lymphocytes in mice was tested using an enzyme-linked immunosorbent assay. The therapeutic effect and safety of recombinant phage administration on Sporothrix-infected BALB/c mice were evaluated based on survival analysis, histopathological changes, and renal and liver functions. The successfully prepared recombinant phage displaying ToAP2D peptides significantly inhibited Sporothrix growth. According to the scanning electron microscopy results, the recombinant phage caused shrinkage and rupture of Sporothrix globosa, leading to leakage of the contents. The Hoechst/propidium iodide double staining test indicated that the recombinant phage could induce cell apoptosis of Sporothrix globosa. The apoptotic pathway might be due to the accumulation of reactive oxygen species in large quantities in cells, activating caspase dependence; this reduced inflammation, prolonged the survival time, and enhanced levels of IFN-γ and IL-17 in mice. We believe that recombinant phage inhibits Sporothrix growth by adjusting the immune response of mice, inducing Sporothrix apoptosis and improving animal survival. This study offers a new approach to preparing antimicrobial peptides.

Efficient Treatment of Sporothrix globosa Infection Using the Antibody Elicited by Recombinant Phage Nanofibers.

Antifungal therapy is used to treat sporotrichosis. However, there are several limitations in this therapy, such as development of drug resistance and potential health risks including liver injury. The purpose of our study was to evaluate the antifungal efficacy of antibody against the hybrid phage nanofibers displaying KPVQHALLTPLGLDR (phage-KR) in a fungal-infected mouse model. In this study, we extracte an antibody against hybrid phage nanofibers (phage-KR) from immunized mice and passively inoculate Sporothrix globosa (S. globosa) infected mice. The study shows that the antibody exhibits efficient inhibition efficacy of the S. globosa infection, including reduction of the progressive fungi colonizing, increase of animal survival rate and relief of organ inflammation in the mice. The results indicate that antibody against phage-KR may act as a potential strategy for safe and efficient treatment of S. globosa infections.

The ΦCPG1 chlamydiaphage can infect Chlamydia trachomatis and significantly reduce its infectivity.

Recent years have seen a significant increase in rates of persistent, antibiotic-resistant infection of Chlamydia trachomatis (CT) infections, representing an increasingly serious public health threat. At present there are no effective vaccines or antibodies available to treat CT, prompting the need for novel treatment strategies. One potential solution to this issue is the use of ΦCPG1, a chlamydia-specific lytic phage which has over 90% nucleotide sequence identity with other chlamydiaphages. Previous work has shown the Vp1 capsid protein of ΦCPG1 to exhibit broad inhibitory activity against all CT serotypes, inhibiting CT-mediated host cell toxicity. Patients with CT infections exhibit circulating antibodies against this Vp1 protein, suggesting that this or similar phages may be present in vivo in the context of CT infections, even though no phages have been specifically detected to date. Given these previous findings, we hypothesized that the ΦCPG1 chlamydiaphage may be able to infect CT, thereby inhibiting its growth and proliferation. To test this, we generated a recombinant pGFP-ΦCPG1 phage which we used to explore its effects on CT and chlamydia conjunctivitis of guinea pigs (GPIC). We found that pGFP insertion did not alter the packaging or infectivity of ΦCPG1, and that this recombinant phage was readily able to infect CT and GPIC and inhibit CT and GPIC in a dose-dependent fashion. This inhibition was most pronounced during the mid and late stages of the CT infection, disrupting the reticular body (RB) to EB transition, leading to the formation of enlarged RBs. These results indicate that ΦCPG1 is able to infect CT, highlighting this phage as a novel potential therapeutic agent for treating chlamydia infections. In addition, by engineering pGFP to express ΦCPG1, we have produced an valuable experimental tool useful for future studies of drug resistance, pathogenicity, and vaccine research aimed at improving CT treatment.

Construção do fago recombinante D29:: gfp com potencial de aplicação nos testes de sensibilidade pela concentração inibitória mínima para o Mycobacterium spp / Construction of the recombinant phage D29:: gfp with application potential in sensitivity tests by the minimum inhibitory concentration for Mycobacterium spp

O objetivo desse trabalho foi construir o fago recombinante D29::gfp e testar a sua utilização como um agente revelador da viabilidade bacilar na determinação da concentração inibitória mínima (CIM) aos principais fármacos administrados no tratamento da tuberculose. O fago recombinante contém o promotor hsp70 e o gene da proteína verde fluorescente (gfp) e foi construído através da restrição pela Spe I em uma região intergênica a extremidade coesiva direita no genoma do fago D29. O promotor hsp70 e gfp clonados no pYL GFP foram amplificados pela PCR utilizando iniciadores com sítios para Spe I. O DNA do fago D29 digerido pela Spe I foi ligado com o fragmento hsp70-gfp empregando a T4 DNA ligase e os produtos da reação de ligação foram transformados de acordo com o protocolo de encapsulamento. A infecção do M. smegmatis com esse fago recombinante induziu a expressão da proteína verde fluorescente (GFP). Para avaliar o uso do fago recombinante em teste de sensibilidade aos fármacos anti-tuberculose, 100 isolados clínicos foram testados quanto ao perfil de sensibilidade a isoniazida (H), rifampicina (R), estreptomicina (S) e etambutol (E), utilizando o método das proporções em Lowenstein-Jensen (L-J), técnica em microplaca com a resazurina (REMA) e técnica em microplaca com D29::gfp. Os resultados do REMA demonstraram que 30 isolados clínicos foram sensíveis à H e 58 (66%) isolados clínicos foram resistentes, dentre os quais a CIM foi 1μg/mL ou maior para 41 (71%). A CIM da R para 49 (56%) dos isolados clínicos resistentes foi de 0,5μg/mL para 17 (35%).

The objective of this work was to construct the recombinant phage D29::gfp and to use this phage as an indicator agent of cell viability in a minimal inhibitory concentration (MIC) assay for the mains drugs used for tuberculosis treatment. The recombinant phage contains the mycobacteria-specific hsp70 promoter controlling the green fluorescent protein gene (gfp) and was constructed by Spe I restriction in the intergenic region next to the right cohesive termini of the D29 phage genome. An hsp 70 promoter and gfp previously cloned in p YL GFP was amplified by PCR using primers with Spe I sites. The Spe I-restricted D29 phage DNA was ligated with the hsp 70-gfp fragment using T4 DNA ligase and ligated product was transformed using the packing protocol. Infection of M.smegmatis with this recombinant phage indicated the expression of green f1uorescent protein (GFP). To use the recombinant phage for assaying the activity of anti-TB drugs, 100 clinical isolates was tested for susceptibility to isoniazid (H), rifampicin (R), streptomycin (S), and ethambutol (E) using both the proportion method on Lowenstein-Jensen (L-J) medium, resazurin microtiter assay plate (REMA), as well as a microplate assay using D29::gfp. The REMA plate method showed that 30 clinical isolates were susceptible to H and 58 (66%) clinical isolates were resistant, where the MICs were 1 µg/mL or higher for 41 (71%). The R MICs for 49 (56%) resistant clinical isolates were 0,5 µg/mL for 17 (35%). The S MICs for 33 (37%) resistant clinical isolates were 2 µg/mL for 13 (40%) and E MICs for 34 (39%) resistant clinical isolates were 16 µg/mL or higher for 19 (56%). Molecular characterization by PCR IS6110 showed that 88 clinical isolates were M.tuberculosis and by PRA hsp65 were seven clinical isolates were M.kansasii and four was M.abscessus, and one M.zulgai. After using the recombinant phage as an indicator agent of cell viability for assaying the activitity of anti-TB drugs we can conclude that the expression of green fluorescent protein was non-specific and not reproducible, rendering it not useful for the determination of the MIC of the principal drugs used for the treatment of ...