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10 pages, 1749 KiB

Open AccessArticle

Bacteriophages M13 and T4 Increase the Expression of Anchorage-Dependent Survival Pathway Genes and Down Regulate Androgen Receptor Expression in LNCaP Prostate Cell Line

by Swapnil Ganesh Sanmukh, Nilton José dos Santos, Caroline Nascimento Barquilha, Maira Smaniotto Cucielo, Márcio de Carvalho, Patricia Pintor dos Reis, Flávia Karina Delella, Hernandes F. Carvalho and Sérgio Luis Felisbino

Viruses 2021, 13(9), 1754; https://doi.org/10.3390/v13091754 - 2 Sep 2021

Cited by 11 | Viewed by 3448

Abstract

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and [...] Read more.

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 10⁵ pfu/mL, 1 × 10⁶ pfu/mL, and 1 × 10⁷ pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes AKT, ITGA5, ITGB1, ITGB3, ITGB5, MAPK3, and PI3K were significantly up-regulated, whilst the genes AR, HSPB1, ITGAV, and PGC1A were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with PI3K/AKT pathway inhibitors. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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12 pages, 1169 KiB

Open AccessArticle

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

by Peter Braun, Nadja Rupprich, Diana Neif and Gregor Grass

Viruses 2021, 13(8), 1462; https://doi.org/10.3390/v13081462 - 27 Jul 2021

Cited by 8 | Viewed by 4742

Abstract

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the [...] Read more.

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium’s close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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19 pages, 3652 KiB

Open AccessArticle

Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity

by Sangsang Deng, Qiang Xu, Yajuan Fu, Leiqin Liang, Yan Wu, Fang Peng and Meiying Gao

Viruses 2021, 13(6), 1034; https://doi.org/10.3390/v13061034 - 31 May 2021

Cited by 8 | Viewed by 3798

Abstract

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of [...] Read more.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage’s host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4–85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0–12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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16 pages, 2584 KiB

Open AccessArticle

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

by Jutta Kasurinen, Cindy M. Spruit, Anu Wicklund, Maria I. Pajunen and Mikael Skurnik

Viruses 2021, 13(5), 750; https://doi.org/10.3390/v13050750 - 24 Apr 2021

Cited by 2 | Viewed by 3289

Abstract

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The [...] Read more.

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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21 pages, 2746 KiB

Open AccessArticle

Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans

by Fernando Clavijo-Coppens, Nicolas Ginet, Sophie Cesbron, Martial Briand, Marie-Agnès Jacques and Mireille Ansaldi

Viruses 2021, 13(5), 725; https://doi.org/10.3390/v13050725 - 21 Apr 2021

Cited by 15 | Viewed by 3042

Abstract

Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in [...] Read more.

Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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Review

Jump to: Research

16 pages, 923 KiB

Open AccessReview

Bacteriophages as Fecal Pollution Indicators

by Daniel Toribio-Avedillo, Anicet R. Blanch, Maite Muniesa and Lorena Rodríguez-Rubio

Viruses 2021, 13(6), 1089; https://doi.org/10.3390/v13061089 - 7 Jun 2021

Cited by 22 | Viewed by 3770

Abstract

Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), [...] Read more.

Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), have been proposed as alternatives or complements to fecal indicator bacteria. Here, we provide a general overview of the potential use of enteric bacteriophages as fecal and viral indicators in different environments, as well as the available methods for their detection and enumeration, and the regulations for their application. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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12 pages, 20990 KiB

Open AccessReview

Viruses with U-DNA: New Avenues for Biotechnology

by Kinga K. Nagy, Mikael Skurnik and Beáta G. Vértessy

Viruses 2021, 13(5), 875; https://doi.org/10.3390/v13050875 - 10 May 2021

Cited by 2 | Viewed by 2900

Abstract

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations [...] Read more.

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA–protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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22 pages, 1382 KiB

Open AccessReview

The Advantages and Challenges of Using Endolysins in a Clinical Setting

by Ellen Murray, Lorraine A. Draper, R. Paul Ross and Colin Hill

Viruses 2021, 13(4), 680; https://doi.org/10.3390/v13040680 - 15 Apr 2021

Cited by 106 | Viewed by 12148

Abstract

Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases [...] Read more.

Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that act to lyse bacterial cells by targeting their cell’s wall, particularly in Gram-positive bacteria due to their naturally exposed peptidoglycan layer. These lytic enzymes have received much interest from the scientific community in recent years for their specificity, mode of action, potential for engineering, and lack of resistance mechanisms. Over the past decade, a renewed interest in endolysin therapy has led to a number of successful applications. Recombinant endolysins have been shown to be effective against prominent pathogens such as MRSA, Listeria monocytogenes, Staphylococcus strains in biofilm formation, and Pseudomonas aeruginosa. Endolysins have also been studied in combination with other antimicrobials, giving a synergistic effect. Although endolysin therapy comes with some regulatory and logistical hurdles, the future looks promising, with the emergence of engineered “next-generation” lysins. This review will focus on the likelihood that endolysins will become a viable new antimicrobial therapy and the challenges that may have to be overcome along the way. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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19 pages, 1952 KiB

Open AccessReview

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy

by Annamaria Aloisio, Nancy Nisticò, Selena Mimmi, Domenico Maisano, Eleonora Vecchio, Giuseppe Fiume, Enrico Iaccino and Ileana Quinto

Viruses 2021, 13(4), 649; https://doi.org/10.3390/v13040649 - 9 Apr 2021

Cited by 18 | Viewed by 3570

Abstract

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides [...] Read more.

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins 2021)

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