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1.
Front Microbiol ; 13: 864720, 2022.
Article in English | MEDLINE | ID: mdl-35432276

ABSTRACT

Probiotics have been shown to be effective against infectious diseases in clinical trials, with either intestinal or extraintestinal health benefits. Even though probiotic effects are strain-specific, some "widespread effects" include: pathogen inhibition, enhancement of barrier integrity and regulation of immune responses. The mechanisms involved in the health benefits of probiotics are not completely understood, but these effects can be mediated, at least in part, by probiotic-derived extracellular vesicles (EVs). However, to date, there are no clinical trials examining probiotic-derived EVs health benefits against infectious diseases. There is still a long way to go to bridge the gap between basic research and clinical practice. This review attempts to summarize the current knowledge about EVs released by probiotic bacteria to understand their possible role in the prevention and/or treatment of infectious diseases. A better understanding of the mechanisms whereby EVs package their cargo and the process involved in communication with host cells (inter-kingdom communication), would allow further advances in this field. In addition, we comment on the potential use and missing knowledge of EVs as therapeutic agents (postbiotics) against infectious diseases. Future research on probiotic-derived EVs is needed to open new avenues for the encapsulation of bioactives inside EVs from GRAS (Generally Regarded as Safe) bacteria. This could be a scientific novelty with applications in functional foods and pharmaceutical industries.

2.
Biochim Biophys Acta Proteins Proteom ; 1870(2): 140745, 2022 02 01.
Article in English | MEDLINE | ID: mdl-34906734

ABSTRACT

Bacteriophage endolysins are crucial for progeny release at the end of the lytic cycle. Mycobacteriophage's genomes carry a lysin A essential gene, whose product cleaves the peptidoglycan (PG) layer and a lysin B, coding for an esterase, that cleaves the linkage between the mycolic acids and the arabinogalactan-PG complex. Lysin A mycobacteriophage proteins are highly modular and in gp29 (LysA) of phage TM4 three distinctive domains were identified. By bioinformatics analysis the central module was previously found to be similar to an amidase-2 domain family with an N-acetylmuramoyl -L-alanine amidase activity. We demonstrated experimentally that purified LysA is able to lyse a suspension of Micrococcus lysodeikticus and can promote cell lysis when expressed in E. coli and Mycobacterium smegmatis. After incubation of LysA with MDP (Muramyl dipeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine) we detected the presence of N-acetylmuramic acid (NAcMur) and L-Ala- D- isoGlutamine (L-Ala-D-isoGln) corroborating the proposed muramidase activity of this enzyme. This protein was stabilized at acidic pH in the presence of Zn consistent with the increase of the enzymatic activity under these conditions. By homology modeling, we predicted that the Zn ion is coordinated by His 226, His 335, and Asp 347 and we also identified the amino acid Glu 290 as the catalytic residue. LysA activity was completely abolished in derived mutants on these key residues, suggesting that the PG hydrolysis solely relies on the central domain of the protein.


Subject(s)
Endopeptidases/metabolism , Mycobacteriophages/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Peptidoglycan/metabolism , Viral Proteins/metabolism , Computational Biology/methods , Endopeptidases/chemistry , Escherichia coli/metabolism , Galactans , Hydrolysis , Mass Spectrometry/methods , Micrococcus/metabolism , Muramic Acids/metabolism , Mycobacterium smegmatis/metabolism , Viral Proteins/chemistry
3.
Phage (New Rochelle) ; 2(1): 7-10, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-36148437

ABSTRACT

Between 2015 and 2019, we hosted an International Phage Course at Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. The 2-week full-time course was hands-on and included lectures from renowned phage biologists. Participating students were able to meet and discuss with recognized experts from around the world in a familiar setting, facilitating the establishment of scientific collaborations and the expansion of their networks. Eighty-four students from 14 Latin American countries have participated in the course, which included isolation, characterization, genome sequencing, and annotation of novel phages. We have successfully created a coursework that enabled the acquisition of new knowledge and expertise in bacteriophage biology and strengthened ties among Latin American colleagues.

4.
Phage (New Rochelle) ; 2(1): 43-49, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-36148439

ABSTRACT

Introduction: Because of the clinical relevance of Mycobacteria, and from a therapeutic perspective, there is an increasing interest to study phages that infect bacteria belonging to this genus. Materials and Methods: A phage was isolated from a soil sample, using Mycobacterium smegmatis as host. Its characterization included sequencing, annotation, and analysis of the genome, host range determination, and electron microscopy imaging. Results: Mycobacterium phage vB_MsmS_Celfi is a temperate phage able to infect Mycobacterium tuberculosis with high efficiency. From electron microscopy images, Celfi belongs to the Siphoviridae family. Genome analysis classified phage Celfi into cluster L, subcluster L2 of Actinobacteriophage clusters. Mycobacterium phage Celfi exhibits a Lysin B distant to those present in other members of the subcluster and other mycobacteriophages. Conclusions: The discovery of new phages that infect M. tuberculosis could contribute to the development of novel tools for detection systems and future treatment of the disease.

5.
Phage (New Rochelle) ; 2(1): 57-63, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-36148441

ABSTRACT

Introduction: Only a few Lactobacillus casei phages have so far been characterized. As several L. casei strains are part of probiotic formulations, bacteriophage outbreaks targeting these strains can lead to critical losses within the dairy industry. Materials and Methods: A new L. casei phage was isolated from raw milk obtained from a milking yard from the province of Buenos Aires. The phage genome was sequenced, annotated, and analyzed. Morphology was determined by electron microscopy and the host range was established. Results: Lactobacillus phage vB_LcaM_Lbab1 is a member of the Herelleviridae family and features a host range including L. casei/Lactobacillus paracasei and Lactobacillus kefiri strains. We further analyzed the baseplate proteins in silico and found putative carbohydrate binding modules that are responsible for host recognition in other Lactobacillus phages. Conclusions: A new Lactobacillus phage was isolated and characterized. The focus was made on its host recognition mechanism, pointing toward the development of future strategies to avoid deleterious infections in the dairy industry.

6.
ISME J ; 15(3): 636-648, 2021 03.
Article in English | MEDLINE | ID: mdl-33067586

ABSTRACT

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage-host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage-host interactions have been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction between Gordonia and their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Coevolution was followed by monitoring changes over time in the CRISPR loci of Gordonia metagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability of Gordonia, and almost clonal conservation of the trailer end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. The host population carrying a shorter CRISPR locus that contain only ancestral spacers, which has not acquired newer spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and viral genomes revealed how the complexity at the population level has important consequences for bacteria-phage coexistence.


Subject(s)
Bacteriophages , Bacteria , Bacteriophages/genetics , Biotechnology , Clustered Regularly Interspaced Short Palindromic Repeats , Ecosystem
7.
Curr Opin Biotechnol ; 68: 8-14, 2021 04.
Article in English | MEDLINE | ID: mdl-33039679

ABSTRACT

The alarming increase in antibiotic resistance has placed the focus on phages as an alternative antimicrobial therapy. Recently, the first patient treatment using engineered phages to combat a mycobacterial infection was successfully performed; genetic modifications were made using Bacteriophage Recombineering of Electroporated DNA (BRED). BRED is a simple technique that allows genetic manipulation of phages. The phage DNA and a recombination substrate, with short homology to the target, are co-electroporated into recombineering proficient bacteria promoting high levels of recombination. After electroporation, cells are recovered and plated in an infectious centre assay. Individual plaques are then screened by PCR to identify the mutant phage. The main characteristics of this technique, the advantages of engineered versus wild type phages for therapeutic purposes and the future perspective of BRED for doing such modifications, are reviewed here.


Subject(s)
Bacteriophages , Anti-Bacterial Agents/pharmacology , Bacteria , Bacteriophages/genetics , DNA , Drug Resistance, Microbial , Humans
8.
Sci Rep ; 10(1): 3120, 2020 02 20.
Article in English | MEDLINE | ID: mdl-32080346

ABSTRACT

Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.


Subject(s)
Bacillus subtilis/metabolism , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , Transcytosis , Caco-2 Cells , Cell Polarity , Cell Proliferation , Cell Survival , Epithelium/metabolism , Functional Food , Humans , Intestines , Microscopy, Confocal , Models, Biological , Probiotics
9.
J Proteomics ; 192: 321-333, 2019 02 10.
Article in English | MEDLINE | ID: mdl-30267874

ABSTRACT

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.


Subject(s)
Bacterial Proteins/metabolism , Mycobacterium tuberculosis/enzymology , Protein Serine-Threonine Kinases/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Mutation , Mycobacterium tuberculosis/genetics , Phosphorylation , Protein Domains , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Substrate Specificity
10.
Methods Mol Biol ; 1898: 27-36, 2019.
Article in English | MEDLINE | ID: mdl-30570720

ABSTRACT

Fluoromycobacteriophages are a new class of reporter phages that contain Laboratorio fluorescent reporter genes (gfp, ZsYellow, and mCherry) and provide a simple means of revealing the metabolic state of mycobacterial cells and therefore their response to antibiotics. Here we described a simple and rapid method for drug susceptibility testing (DST) of Mycobacterium spp using a fluorescence microscope, a flow cytometer, or a fluorimeter in a convenient multiwell format.


Subject(s)
Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests/methods , Mycobacteriophages/drug effects , Mycobacterium tuberculosis/drug effects , Green Fluorescent Proteins/genetics , Humans , Microscopy, Fluorescence , Mycobacteriophages/genetics , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/virology
11.
Methods Mol Biol ; 1898: 69-80, 2019.
Article in English | MEDLINE | ID: mdl-30570724

ABSTRACT

We describe a recombineering-based method for the genetic manipulation of lytically replicating bacteriophages, focusing on mycobacteriophages. The approach utilizes recombineering-proficient strains of Mycobacterium smegmatis and employs a cotransformation strategy with purified phage genomic DNA and a mutagenic substrate, which selects for only those cells that are competent to take up DNA. The cotransformation method, combined with the high rates of recombination obtained in M. smegmatis recombineering strains, allows for the efficient and rapid generation of bacteriophage mutants.


Subject(s)
Bacteriophages/genetics , DNA/genetics , Mycobacterium smegmatis/genetics , Recombination, Genetic , Bacteriophages/chemistry , DNA/chemistry , Electrochemotherapy , Electroporation , Genetic Engineering , Mutagenesis/genetics , Mycobacterium smegmatis/virology
12.
Front Microbiol ; 9: 1471, 2018.
Article in English | MEDLINE | ID: mdl-30026735

ABSTRACT

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybomb ϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybomb Φ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybomb Φ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

13.
Tuberculosis (Edinb) ; 109: 35-40, 2018 03.
Article in English | MEDLINE | ID: mdl-29559119

ABSTRACT

Nitroxyl (HNO) is a highly elusive and reactive molecule. Nitroxyl biological effects and pharmacological potential are becoming increasingly relevant. Mycobacterium tuberculosis infection needs new and more efficient drugs. Reactive Nitrogen and Oxygen Species (RNOS) are key compounds used by the immune system to fight intracellular infections, particularly Mycobacterium tuberculosis. In this context, we analyzed HNO potential to kill mycobacteria. We evaluated the viability and biological response of mycobacteria towards HNO releasing compounds. Our results show that HNO donors can affect mycobacterial growth, for both Mycobacterium smegmatis and Mycobacterium tuberculosis. The effect can be observed using a single dose or with successive additions of lower concentrations of the donor, mimicking continuous HNO exposure. When analyzing the effect of the simultaneous addition of sub-inhibitory concentrations of HNO with antibiotics commonly used for Mycobacterium tuberculosis infection treatment we observed: a positive effect on Rifampicin, Kanamycin and Delamanid activity; and a negative effect on Isoniazid and Ethambutol activity. Regarding a possible mechanism of action, based on the recently developed fluoromycobacteriophage assay, we propose that HNO acts by interfering with general mycobacterial physiological state. The results of this study positions HNO donors as potential candidates as new drugs for a new tuberculosis treatment.


Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Nitrogen Oxides/pharmacology , Antibiotics, Antitubercular/pharmacology , Antitubercular Agents/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Microbial Viability/drug effects , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Nitrogen Oxides/metabolism
14.
Front Microbiol ; 8: 1783, 2017.
Article in English | MEDLINE | ID: mdl-28979244

ABSTRACT

Archaea, bacteria, and eukarya secrete membrane microvesicles (MVs) as a mechanism for intercellular communication. We report the isolation and characterization of MVs from the probiotic strain Lactobacillus casei BL23. MVs were characterized using analytical high performance techniques, DLS, AFM and TEM. Similar to what has been described for other Gram-positive bacteria, MVs were on the nanometric size range (30-50 nm). MVs carried cytoplasmic components such as DNA, RNA and proteins. Using a proteomic approach (LC-MS), we identified a total of 103 proteins; 13 exclusively present in the MVs. The MVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. In particular, we identified proteins described as mediators of Lactobacillus' probiotic effects such as p40, p75 and the product of LCABL_31160, annotated as an adhesion protein. The presence of these proteins suggests a role for the MVs in the bacteria-gastrointestinal cells interface. The expression and further encapsulation of proteins into MVs of GRAS (Generally Recognized as Safe) bacteria could represent a scientific novelty, with applications in food, nutraceuticals and clinical therapies.

15.
Mol Microbiol ; 104(4): 608-620, 2017 05.
Article in English | MEDLINE | ID: mdl-28196397

ABSTRACT

Bacteriophage replication requires specific host-recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non-contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell-wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J-1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J-1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with 'classical' Dits. The first insertion is similar to carbohydrate-binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J-1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J-1 evolved Dit as the phage RBP.


Subject(s)
Viral Tail Proteins/metabolism , Viral Tail Proteins/ultrastructure , Bacteriophages/metabolism , Carbohydrates , Host Specificity , Lactic Acid , Lactobacillus , Lacticaseibacillus casei/metabolism , Lactococcus lactis/metabolism , Microscopy, Electron , Protein Binding , Protein Conformation , Structure-Activity Relationship , Viral Tail Proteins/genetics , Virion
16.
Appl Microbiol Biotechnol ; 100(21): 9201-9215, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27448399

ABSTRACT

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.


Subject(s)
Lacticaseibacillus casei/genetics , Lacticaseibacillus casei/virology , Prophages/genetics , Prophages/physiology , Virus Activation , Food Microbiology , Mitomycin/metabolism , Nucleic Acid Synthesis Inhibitors/metabolism , Viral Tail Proteins/genetics
18.
Appl Environ Microbiol ; 80(22): 7107-21, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25217012

ABSTRACT

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using Lactobacillus casei strain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other Siphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically to Lactobacillus casei/paracasei cells, and the addition of l-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls of L. casei ATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into how Lactobacillus phages interact with their hosts at the first steps of infection.


Subject(s)
Bacteriophages/chemistry , Bacteriophages/genetics , Lacticaseibacillus casei/virology , Siphoviridae/chemistry , Siphoviridae/genetics , Amino Acid Sequence , Bacteriophages/physiology , Base Sequence , Genome, Viral , Genomics , Molecular Sequence Data , Sequence Alignment , Siphoviridae/physiology , Viral Proteins/genetics , Viral Proteins/metabolism
19.
Genome Announc ; 2(1)2014 Jan 02.
Article in English | MEDLINE | ID: mdl-24385573

ABSTRACT

Lactobacillus phages J-1 and PL-1 were isolated during the 1960s from abnormal fermentations of Yakult. The genomes are almost identical, but PL-1 has a deletion in the genetic switch region and also differs in a gene coding for a putative tail protein.

20.
Appl Environ Microbiol ; 79(18): 5608-15, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23851082

ABSTRACT

Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence.


Subject(s)
Bacteriological Techniques/methods , Capsid Proteins/metabolism , Capsid/metabolism , Mycobacteriophages/physiology , Mycobacterium/isolation & purification , Staining and Labeling/methods , Virus Assembly , Capsid Proteins/genetics , Fluorescence , Mycobacterium/virology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
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