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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2614-2622, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472282

RESUMO

Bacteriophages bind to the bacteria receptor through the receptor binding proteins (RBPs), a process that requires the involvement of complex atomic structures and conformational changes. In response to bacteriophage infection, bacteria have developed a variety of resistance mechanisms, while bacteriophages have also evolved multiple antagonistic mechanisms to escape host resistance. The exploration of the "adsorption-anti adsorption-escape process" between bacteriophages and bacteria helps us better understand the co-evolution process of bacteriophages and bacteria, which is important for the development of phage therapeutic technologies and phage-based biotechnologies. This review summarizes the bacteriophage adsorption related proteins, how bacteriophages escape host resistance based on the RBP alternations, and the recent progress of RBP-related biotechnologies.


Assuntos
Receptores de Bacteriófagos , Bacteriófagos , Bactérias , Bacteriófagos/genética , Proteínas de Transporte , Ligação Proteica
2.
Microbiome ; 9(1): 182, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34479645

RESUMO

BACKGROUND: Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown. RESULTS: We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR-Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction-modification systems and dormancy-inducing toxin-antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication. CONCLUSIONS: This study provides new insights into phage-bacteria interplay in intracellular environments of a deep-sea vent snail. Video Abstract.


Assuntos
Bacteriófagos , Animais , Bactérias/genética , Bacteriófagos/genética , Genômica , Proteômica , Caramujos , Transcriptoma/genética
3.
Food Res Int ; 147: 110480, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399476

RESUMO

This study describes the characterization and genomic analysis of six lytic Salmonella phages. To examine the feasibility of using these phages as biocontrol agents, we analyzed their genomes and compared them to those of similar phages. These six phages belong to genus Epseptimavirus, family Demerecviridae. We identified the genes of these six phages by comparing their genomes with those of three type phages in subfamily Markadamsvirinae. All six phages examined in this study were obligately lytic and did not carry undesirable genes. Two phages (vB_SalS_1-23 and vB_SalS_3-29) were selected as the representative phages for general characterization and physiological tests. The biocontrol efficacy of the representative phages was determined by comparing the viable counts of recovered host Salmonella ser. Newlands ZC-S1 from treatment and phage-free control samples. The biocontrol experiment showed that the representative phages were able to reduce the counts of ZC-S1 to below 2 log10 CFU/mL (~4.3 log10 CFU/mL reduction) at 3 h post-infection at 37 °C. Furthermore, we investigated the application of these two phages in the control of ZC-S1 contamination in chicken products and on eggshells. When applied to the surfaces of the samples, the phage cocktail (MOI = 100) reduced the ZC-S1 count to below 2 log10 CFU/mL on chicken skin and to undetectable levels (1 log10 CFU/mL) in chicken breast meat, ground chicken meat and eggshell samples (p < 0.01). Compared to the initial experiment, the phage cocktail reduced the ZC-S1 count by 2-4.08 log10 CFU/mL when applied at an MOI = 1 (except in the ground chicken meat group) and by 4.48-5.67 log10 CFU/mL at an MOI = 100 after 7 h. In conclusion, these two phages with lytic effects show a high potential to inhibit the growth of Salmonella contaminants and can be used as candidate biocontrol agents.


Assuntos
Bacteriófagos , Fagos de Salmonella , Bacteriófagos/genética , Microbiologia de Alimentos , Carne , Salmonella , Fagos de Salmonella/genética
4.
DNA Res ; 28(4)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34390569

RESUMO

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.


Assuntos
Bacteriófagos/genética , Genoma Viral , Klebsiella pneumoniae/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Genômica , Filogenia , Análise de Sequência de DNA , Proteínas Virais/genética
5.
Nat Commun ; 12(1): 4748, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362927

RESUMO

Encapsulins are a class of microbial protein compartments defined by the viral HK97-fold of their capsid protein, self-assembly into icosahedral shells, and dedicated cargo loading mechanism for sequestering specific enzymes. Encapsulins are often misannotated and traditional sequence-based searches yield many false positive hits in the form of phage capsids. Here, we develop an integrated search strategy to carry out a large-scale computational analysis of prokaryotic genomes with the goal of discovering an exhaustive and curated set of all HK97-fold encapsulin-like systems. We find over 6,000 encapsulin-like systems in 31 bacterial and four archaeal phyla, including two novel encapsulin families. We formulate hypotheses about their potential biological functions and biomedical relevance, which range from natural product biosynthesis and stress resistance to carbon metabolism and anaerobic hydrogen production. An evolutionary analysis of encapsulins and related HK97-type virus families shows that they share a common ancestor, and we conclude that encapsulins likely evolved from HK97-type bacteriophages.


Assuntos
Bacteriófagos/metabolismo , Capsídeo/metabolismo , Células Procarióticas/metabolismo , Células Procarióticas/virologia , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Evolução Biológica , Vias Biossintéticas/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus de DNA/metabolismo , Filogenia , Virulência
6.
Microb Pathog ; 159: 105135, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34390766

RESUMO

Vibrio alginolyticus is a common opportunistic pathogen that can cause vibriosis of marine aquatic animals. The application of phages or particularly associated protein products for the treatment of vibriosis has shown prominent advantages compared with the treatment with traditional antibiotics. In this study, the function of a holin-endolysin system from V. alginolyticus phage HH109 was characterized by examining the effect of their overexpression on Escherichia coli and V. alginolyticus. Our data revealed that the endolysin of the phage HH109 has stronger bactericidal activity than the holin, as evidenced by observing more cell death and severe structural damage of cells in the endolysin-expressing E. coli. Furthermore, the two proteins displayed the synergistic effect when the holA and lysin were co-expressed in E. coli, although no interaction between them was detected using the bacterial two-hybrid assay. Transmission electron microscopy observation revealed disruptions of cell envelopes accompanied by leakage of intracellular contents. Similarly, the bactericidal activity of the holin and endolysin against V. alginolyticus was also examined whatever the host is sensitive or resistant to phage HH109. Together, our study contributes to a better understanding of the mechanism of phage HH109 destroying the bacterial cell wall to lyse their host and may offer alternative applications potentially for vibriosis treatment.


Assuntos
Bacteriófagos , Animais , Bacteriófagos/genética , Endopeptidases/genética , Escherichia coli , Vibrio alginolyticus
7.
Viruses ; 13(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34372538

RESUMO

Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that "phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system". These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.


Assuntos
Resistência Microbiana a Medicamentos/genética , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/virologia , Antibacterianos/farmacologia , Bacteriófagos/genética , Biofilmes/crescimento & desenvolvimento , Resistência Microbiana a Medicamentos/fisiologia , Humanos , Terapia por Fagos/métodos , Fagocitose/genética , Fenótipo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência
8.
Viruses ; 13(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372537

RESUMO

The increasing prevalence and worldwide distribution of multidrug-resistant bacterial pathogens is an imminent danger to public health and threatens virtually all aspects of modern medicine. Particularly concerning, yet insufficiently addressed, are the members of the Burkholderia cepacia complex (Bcc), a group of at least twenty opportunistic, hospital-transmitted, and notoriously drug-resistant species, which infect and cause morbidity in patients who are immunocompromised and those afflicted with chronic illnesses, including cystic fibrosis (CF) and chronic granulomatous disease (CGD). One potential solution to the antimicrobial resistance crisis is phage therapy-the use of phages for the treatment of bacterial infections. Although phage therapy has a long and somewhat checkered history, an impressive volume of modern research has been amassed in the past decades to show that when applied through specific, scientifically supported treatment strategies, phage therapy is highly efficacious and is a promising avenue against drug-resistant and difficult-to-treat pathogens, such as the Bcc. In this review, we discuss the clinical significance of the Bcc, the advantages of phage therapy, and the theoretical and clinical advancements made in phage therapy in general over the past decades, and apply these concepts specifically to the nascent, but growing and rapidly developing, field of Bcc phage therapy.


Assuntos
Complexo Burkholderia cepacia/efeitos dos fármacos , Terapia por Fagos/métodos , Terapia por Fagos/tendências , Bacteriófagos/genética , Bacteriófagos/metabolismo , Complexo Burkholderia cepacia/metabolismo , Complexo Burkholderia cepacia/patogenicidade , Humanos
9.
Viruses ; 13(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34372554

RESUMO

Urinary tract infections (UTIs) are among the events that most frequently need medical intervention. Uropathogenic Escherichia coli are frequently their causative agents and the infections are sometimes complicated by the presence of polyresistant nosocomial strains. Phage therapy is a tool that has good prospects for the treatment of these infections. In the present study, we isolated and characterized two bacteriophages with broad host specificity against a panel of local uropathogenic E. coli strains and combined them into a phage cocktail. According to genome sequencing, these phages were closely related and belonged to the Tequatrovirus genus. The newly isolated phages showed very good activity on a panel of local clinical E. coli strains from urinary tract infections. In the form of a two-phage cocktail, they were active on E. coli strains belonging to phylogroups B2 and D, with relatively lower activity in B1 and no response in phylogroup A. Our study is a preliminary step toward the establishment of a national phage bank containing local, well-characterized phages with therapeutic potential for patients in Slovakia.


Assuntos
Myoviridae/genética , Terapia por Fagos/métodos , Escherichia coli Uropatogênica/genética , Antibacterianos/farmacologia , Bacteriófagos/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/terapia , Especificidade de Hospedeiro/genética , Humanos , Eslováquia , Infecções Urinárias/microbiologia , Infecções Urinárias/terapia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/isolamento & purificação , Fatores de Virulência/genética
10.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372577

RESUMO

A novel Enterobacter cloacae phage, EC151, was isolated and characterized. Electron microscopy revealed that EC151 has a siphovirus-like virion morphology. The EC151 nucleotide sequence shows limited similarity to other phage genomes deposited in the NCBI GenBank database. The size of the EC151 genome is 60,753 bp and contains 58 putative genes. Thirty-nine of them encode proteins of predicted function, 18 are defined as hypothetical proteins, and one ORF identifies as the tRNA-Ser-GCT-encoding gene. Six ORFs were predicted to be members of the deazaguanine DNA modification pathway, including the preQ0 transporter. Comparative proteomic phylogenetic analysis revealed that phage EC151 represents a distinct branch within a group of sequences containing clades formed by members of the Seuratvirus, Nonagvirus, and Vidquintavirus genera. In addition, the EC151 genome showed gene synteny typical of the Seuratvirus, Nonagvirus, and Nipunavirus phages. The average genetic distances of EC151/Seuratvirus, EC151/Nonagvirus, and EC151/Vidquintavirus are approximately equal to those between the Seuratvirus, Nonagvirus, and Vidquintavirus genera (~0.7 substitutions per site). Therefore, EC151 may represent a novel genus within the Siphoviridae family. The origin of the deazaguanine DNA modification pathway in the EC151 genome can be traced to Escherichia phages from the Seuratvirus genus.


Assuntos
Bacteriófagos/genética , Enterobacter cloacae/genética , Enterobacter cloacae/virologia , DNA Viral/genética , Enterobacter cloacae/metabolismo , Genoma Viral/genética , Genômica , Especificidade de Hospedeiro , Filogenia , Proteômica , Siphoviridae/genética , Proteínas Virais/genética , Vírion/metabolismo
11.
Front Cell Infect Microbiol ; 11: 698807, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336721

RESUMO

Enterococcus faecalis is a Gram-positive opportunistic pathogen that could cause pneumonia and bacteremia in stroke patients. The development of antibiotic resistance in hospital-associated E. faecalis is a formidable public health threat. Bacteriophage therapy is a renewed solution to treat antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance quite quickly, which is a significant barrier to phage therapy. Here, we characterized a lytic E. faecalis bacteriophage Vb_EfaM_LG1 with lytic activity. Its genome did not contain antibiotic resistance or virulence genes. Vb_EfaM_LG1 effectively inhibits E. faecalis growth for a short period, and phage resistance developed within hours. However, the combination of antibiotics and phage has a tremendous synergistic effect against E. faecalis, prevents the development of phage resistance, and disrupts the biofilm efficiently. Our results show that the phage-antibiotic combination has better killing efficiency against E. faecalis.


Assuntos
Bacteriófagos , Enterococcus faecalis , Antibacterianos/farmacologia , Bacteriófagos/genética , Biofilmes , Humanos
12.
Front Cell Infect Microbiol ; 11: 686090, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222050

RESUMO

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.


Assuntos
Bacteriófagos , Infecções por Klebsiella , Cápsulas Bacterianas , Bacteriófagos/genética , Genoma Viral , Especificidade de Hospedeiro , Humanos , Klebsiella pneumoniae/genética , Tailândia
13.
Curr Microbiol ; 78(8): 3192-3200, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34213617

RESUMO

Xanthomonas oryzae pv. oryzae (X. oryzae) is a bacterial pathovar of rice diseases all over the world. Owing to emerging antibacterial resistance, phage therapies have gained significant attention to treat various bacterial infections. Nevertheless, comprehensive research is needed for their use as a safe biocontrol agent. In this study, isolation and characterization of a novel phage Xoo-sp15, that infects X. oryzae was ascertained through experimental and bioinformatics analyses to determine its virulent potency and reliability. High throughput sequencing demonstrated that Xoo-sp15 has a dsDNA genome with a total size of 157,091 bp and 39.9% GC content lower than its host (63.6%). Morphological and phylogenetic analyses characterized it as a new member of the Bastille-like group within the family Herelleviridae. In silico analysis revealed that it contains 229 open reading frames and 16 tRNAs. Additionally, this novel phage does not contain any resistant determinants and can infect nine X. oryzae strains. Therefore, Xoo-sp15 has the potential to serve as a novel candidate for phage therapy.


Assuntos
Bacteriófagos , Oryza , Xanthomonas , Bacteriófagos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Oryza/genética , Filogenia , Doenças das Plantas , Reprodutibilidade dos Testes , Xanthomonas/genética
14.
Nat Commun ; 12(1): 4531, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312383

RESUMO

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.


Assuntos
Células Artificiais , Mimetismo Biológico/genética , Ciclo Celular/genética , Replicação do DNA/genética , Modelos Genéticos , Biologia Sintética/métodos , Bacteriófagos/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , Biologia Sintética/tendências , Transcrição Genética/genética
15.
Bioinformatics ; 37(Suppl_1): i25-i33, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252923

RESUMO

MOTIVATION: Bacteriophages (aka phages), which mainly infect bacteria, play key roles in the biology of microbes. As the most abundant biological entities on the planet, the number of discovered phages is only the tip of the iceberg. Recently, many new phages have been revealed using high-throughput sequencing, particularly metagenomic sequencing. Compared to the fast accumulation of phage-like sequences, there is a serious lag in taxonomic classification of phages. High diversity, abundance and limited known phages pose great challenges for taxonomic analysis. In particular, alignment-based tools have difficulty in classifying fast accumulating contigs assembled from metagenomic data. RESULTS: In this work, we present a novel semi-supervised learning model, named PhaGCN, to conduct taxonomic classification for phage contigs. In this learning model, we construct a knowledge graph by combining the DNA sequence features learned by convolutional neural network and protein sequence similarity gained from gene-sharing network. Then we apply graph convolutional network to utilize both the labeled and unlabeled samples in training to enhance the learning ability. We tested PhaGCN on both simulated and real sequencing data. The results clearly show that our method competes favorably against available phage classification tools. AVAILABILITY AND IMPLEMENTATION: The source code of PhaGCN is available via: https://github.com/KennthShang/PhaGCN.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Metagenoma , Metagenômica , Software
16.
Biophys J ; 120(16): 3292-3302, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34265262

RESUMO

Bacteriophages densely pack their long double-stranded DNA genome inside a protein capsid. The conformation of the viral genome inside the capsid is consistent with a hexagonal liquid crystalline structure. Experiments have confirmed that the details of the hexagonal packing depend on the electrochemistry of the capsid and its environment. In this work, we propose a biophysical model that quantifies the relationship between DNA configurations inside bacteriophage capsids and the types and concentrations of ions present in a biological system. We introduce an expression for the free energy that combines the electrostatic energy with contributions from bending of individual segments of DNA and Lennard-Jones-type interactions between these segments. The equilibrium points of this energy solve a partial differential equation that defines the distributions of DNA and the ions inside the capsid. We develop a computational approach that allows us to simulate much larger systems than what is possible using the existing molecular-level methods. In particular, we are able to estimate bending and repulsion between the DNA segments as well as the full electrochemistry of the solution, both inside and outside of the capsid. The numerical results show good agreement with existing experiments and with molecular dynamics simulations for small capsids.


Assuntos
Bacteriófagos , Capsídeo , Bacteriófagos/genética , DNA Viral/genética , Íons , Conformação de Ácido Nucleico
17.
Curr Protoc ; 1(7): e182, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34232564

RESUMO

Phage display is a powerful platform for the discovery of novel biologics with high binding affinities to a specific target protein. Here, we describe methods to construct a phage display library containing diverse single-chain variable antibody fragments (scFvs). Specifically, updated methods for polymerase chain reaction (PCR) amplification and fusion of human antibody genes, their ligation into the pComb3X vector for transformation into 5αF'Iq competent bacterial cells, and their expression in M13KO7 helper phage are presented. Additionally, we describe how to amplify and quantify the phage library and to prepare it in various formats for short- and long-term storage. © 2021 Wiley Periodicals LLC. Basic Protocol 1: First-round polymerase chain reaction (PCR) for isolation of antibody fragments Basic Protocol 2: Ethanol precipitation and pooling of fragment DNA Basic Protocol 3: Second-round polymerase chain reaction with splicing by overlap extension (SOE) for antibody fragment fusion Basic Protocol 4: Restriction digestion of individual scFv constructs and pComb3XSS vector Basic Protocol 5: Directional ligation of the scFv constructs and pComb3X backbone Basic Protocol 6: Transformation of pComb-scFv plasmids into 5αF'Iq competent cells Basic Protocol 7: Collection of bacteria containing the scFv library Basic Protocol 8: Preparation of bacterial glycerol stock Basic Protocol 9: Preparation of phage library glycerol stock Basic Protocol 10: Preparation of plasmid DNA stock Basic Protocol 11: Amplification of M13KO7 helper phage Basic Protocol 12: Phage titer by plate assay Alternate Protocol: One-plate phage plaque assay.


Assuntos
Bacteriófagos , Anticorpos de Cadeia Única , Bacteriófagos/genética , Técnicas de Visualização da Superfície Celular , Humanos , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Anticorpos de Cadeia Única/genética
18.
Ying Yong Sheng Tai Xue Bao ; 32(6): 2267-2274, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34212633

RESUMO

The spread of antibiotic resistance in soil is a global threat to public health and food safety, challenging the prevention and treatment of human infectious disease. The horizontal transfer of ARGs mediated by bacteriophages (phages) is an important pathway for the spread of antibiotic resistance genes (ARGs). However, the knowledge on the contribution of phages to ARGs transmission in soil is elusive. Here, we reviewed the distribution characteristics of phages in soil and its driving factors. We summarized the main methods for purification and enrichment of soil phage, reviewed recent achievements in the mechanism of phage-mediated horizontal transfer of ARGs in soil and proposed some outstanding questions. This review would contribute to understanding the important ecological role of phages in driving the horizontal transfer of ARGs, and provide a basis for developing management strategies to mitigate ARGs pollution.


Assuntos
Bacteriófagos , Antibacterianos/farmacologia , Bacteriófagos/genética , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Humanos , Solo , Microbiologia do Solo
19.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34234013

RESUMO

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.


Assuntos
Bacteriófagos/genética , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Programas de Imunização , Administração por Inalação , Animais , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Dependovirus/genética , Armazenamento de Medicamentos , Feminino , Programas de Imunização/métodos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Estudo de Prova de Conceito , Temperatura
20.
Viruses ; 13(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200458

RESUMO

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.


Assuntos
Bacteriófagos , Indicadores Ambientais , Monitoramento Ambiental , Poluição Ambiental , Fezes/virologia , Microbiologia , Animais , Bacteriófagos/classificação , Bacteriófagos/genética , Monitoramento Ambiental/métodos , Fezes/microbiologia , Humanos , Técnicas Microbiológicas
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