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1.
Proc Natl Acad Sci U S A ; 117(9): 4724-4731, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071243

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of antibiotic-tolerant infections in humans. P. aeruginosa evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens including Neisseria meningitidis, Vibrio cholerae, and Salmonella enterica harbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.


Assuntos
Bactérias/virologia , Bacteriófagos/genética , Bacteriófagos/fisiologia , DNA Viral/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Capsídeo , Doenças Transmissíveis , Microscopia Crioeletrônica , Farmacorresistência Bacteriana/genética , Genoma Viral , Inovirus/genética , Inovirus/fisiologia , Modelos Moleculares , Neisseria meningitidis , Prófagos/genética , Prófagos/fisiologia , Salmonella enterica , Vibrio cholerae
3.
Sci Transl Med ; 11(488)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996083

RESUMO

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.


Assuntos
Fibrose Cística/microbiologia , Pulmão/microbiologia , Pseudomonas/patogenicidade , Animais , Antibacterianos/farmacologia , Estudos Transversais , Resistência Microbiana a Medicamentos/genética , Resistência Microbiana a Medicamentos/fisiologia , Humanos , Inovirus , Testes de Sensibilidade Microbiana , Estudos Prospectivos
4.
J Cancer Res Ther ; 15(Supplement): S1-S10, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30900613

RESUMO

A new modality of targeting therapeutic drugs based on the use of bacteriophage (virus), as an emerging tool for specific targeting and for vaccine development, has been an area of interest for genetic and cancer research. The approach is based on genetic manipulation and modification in the chemical structure of a filamentous bacteriophage that facilitates its application not only for in vivo imaging but also for therapeutic purpose, as a gene delivery vehicle, as drug carriers, and also as an immunomodulatory agent. Filamentous bacteriophage on account of its high surface holding ability with adaptable genetic engineering properties can effectively be used in loading of chemical and genetic drugs specifically on to the targeted lesion location. Moreover, the specific peptides/proteins exhibited on the phage surface can be applied directly as self-navigating drug delivery nanovehicles. The present review article has been framed with an objective to summarize the importance of bacteriophage in phage cancer therapy and to understand the possible future prospective of this approach in developing new tools for biotechnological and genetic research, especially in phage -mediated cancer therapy. Importantly, the peptides or proteins emerging from the surface of a nano carrier will make the expense of such peptides economically more effective as compared to other immunological tools, and this seems to be a potential approach for developing a new nanodrug carrier platform.


Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Vetores Genéticos/genética , Inovirus/genética , Neoplasias/terapia , Animais , Engenharia Genética , Terapia Genética/métodos , Vetores Genéticos/química , Vetores Genéticos/imunologia , Humanos , Inovirus/química , Inovirus/imunologia , Nanopartículas , Neoplasias/genética , Neoplasias/imunologia
5.
J Mol Biol ; 431(6): 1113-1126, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738893

RESUMO

SW1 is the first filamentous phage isolated from a deep-sea environment. Nevertheless, the mechanism by which the SW1 genetic switch is controlled is largely unknown. In this study, the function of the phage-encoded FpsR protein was characterized by molecular biological and biochemical analyses. The deletion of fpsR increased the copy number of SW1 ssDNA and mRNA, indicating that FpsR functions as a repressor. In addition, transcription from the fpsR promoter was shown to be increased in an fpsR deletion mutant, suggesting self-repression by FpsR. Purified FpsR bound to four adjacent operator sites (O1-O4) embedded within the fpsA promoter and the fpsA-fpsR intergenic region. A surface plasmon resonance experiment showed that FpsR can bind to the O1-O4 operators separately and with different binding affinity, and the dissociation constants of FpsR with O2 and O3 were found to be lower at 4 °C than at 20 °C. A gel permeation chromatography assay revealed that FpsR oligomerized to form tetramers. Point mutation analysis indicated that the C-terminal domain influenced the binding affinity and regulatory function of FpsR. Collectively, these data support a model in which FpsR actively regulates phage production by interacting with the corresponding operators, thus playing a crucial role in the SW1 genetic switch.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Inovirus/genética , RNA Viral/genética , Proteínas Virais/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Inovirus/metabolismo , Ativação Transcricional , Proteínas Virais/genética
6.
Mol Microbiol ; 111(5): 1132-1138, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30556628

RESUMO

With the recently awarded Nobel Prize to the inventor of Phage Display, George Smith, the technique has once more gained attention. However, one should not forget about the biology behind the method. Almost always ignored is how the structure of this bacterial virus is assembled. In contrast to lytic phages, filamentous phages are constantly being extruded through the bacterial membranes without lysis. Such filamentous phages are found in all aquatic environments, such as rivers and lakes, in the deep sea, in arctic ice, in hot springs and, associated with their hosts, in plants and animals including humans. While most filamentous phages infect Gram-negative hosts, inoviruses of Gram-positive hosts have also been described. Despite being among the minority within the phage family with an estimate of less than 5%, filamentous phages are real parasites as they exist at the expense of the host, but do not kill it. In contrast to lytic bacteriophages, filamentous phages are assembled in the host's membrane and extruded across the cellular envelope while the bacterium continues to grow. In this review, we focus on this complex and yet poorly understood process of assembly and secretion of filamentous phages.


Assuntos
Bactérias/virologia , Técnicas de Visualização da Superfície Celular , Inovirus/fisiologia , Montagem de Vírus
7.
Subcell Biochem ; 88: 261-279, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29900501

RESUMO

Filamentous bacteriophages, also known as filamentous bacterial viruses or Inoviruses, have been studied extensively over the years. They are interesting paradigms in structural molecular biology and offer insight into molecular assembly, a process that remains to be fully understood. In this chapter, an overview on filamentous bacteriophages will be provided. In particular, we review the constituent proteins of filamentous bacteriophage and discuss assembly by examining the structure of the major coat protein at various stages of the process. The minor coat proteins will also be briefly reviewed. Structural information provides key snapshots into the dynamic process of assembly.


Assuntos
Proteínas do Capsídeo , Inovirus , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Inovirus/química , Inovirus/fisiologia
8.
Methods Mol Biol ; 1688: 67-97, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29151205

RESUMO

Filamentous bacteriophages are elongated semi-flexible viruses that infect bacteria. They consist of a circular single-stranded DNA (ssDNA) wrapped by a capsid consisting of thousands of copies of a major coat protein subunit. Given the increasing number of discovered phages and the existence of only a handful of structures, the development of methods for phage structure determination is valuable for biophysics and structural virology. In recent years, we developed and applied techniques to elucidate the 3D atomic-resolution structures of intact bacteriophages using experimental magic-angle spinning (MAS) solid-state NMR data. The flexibility in sample preparation - precipitated homogeneous solids - and the fact that ssNMR presents no limitation on the size, weight or morphology of the system under study makes it an ideal approach to study phage systems in detail.In this contribution, we describe approaches to prepare isotopically carbon-13 and nitrogen-15 enriched intact phage samples in high yield and purity, and we present experimental MAS NMR methods to study the capsid secondary and tertiary structure, and the DNA-capsid interface. Protocols for the capsid structure determination using the Rosetta modeling software are provided. Specific examples are given from studies of the M13 and fd filamentous bacteriophage viruses.


Assuntos
Bacteriófago M13/ultraestrutura , Capsídeo/ultraestrutura , DNA de Cadeia Simples/metabolismo , Inovirus/ultraestrutura , Espectroscopia de Ressonância Magnética/métodos , Bacteriófago M13/metabolismo , Capsídeo/metabolismo , Isótopos de Carbono , DNA Viral/metabolismo , Inovirus/metabolismo , Isótopos de Nitrogênio
9.
Protein Pept Lett ; 25(1): 64-67, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29237366

RESUMO

BACKGROUND: Filamentous M13 phages have recently been utilized as components for developing novel functional soft materials in various fields such as sensor, device, and biomedical applications. Recently, we have developed liquid crystalline hydrogels composed of M13 phages and gold nanoparticles (GNPs) based on specific interactions between the components. OBJECTIVES: The main objective of this study was to clarify the self-healing capability of the hydrogels composed of M13 phages and GNPs. METHODS: M13 phages displaying tag peptides with a sequence of YPYDVPDYA (HA phages) were genetically constructed through general molecular biology. The mechanical strength of hydrogels composed of the HA phages and anti-HA peptide antibodies-immobilized GNPs (HA-GNPs) was measured by indentation tests. The rupture point of the hydrogels was visually observed. An aliquot of buffer solution was added into the rupture point of the hydrogels after the indentation test. After incubation for 2 days, self-healing of the rupture point was checked visually. The indentation test was also performed after self-healing. To clarify the assembled structures of the components in the hydrogels, transmission electron microscopy (TEM) observation was performed by transferring the hydrogel onto a TEM grid before and after healing. RESULTS: The strength of the original hydrogel (before self-healing) required for rupture was approximately 55 mN. Self-healing of the rupture point was confirmed visually, and the hydrogels behaved as uniform hydrogels again during the vial inversion tests. As a result of the indentation test for the self-healed points of the hydrogels, the rupture force of approximately 45 mN was detected, indicating the self-healing capability of the hydrogels. TEM observation of the before and after self-healing exhibited the regularly assembled structures composed of the HA-GNPs, suggesting that the ruptured networks were recovered into regularly assembled network structures. Importantly, control of the concentration of the HA-GNPs resulted in suppression of decreasing the rupture forces during the repetitive self-healing processes. CONCLUSION: Our results demonstrated the self-healing capability of structurally regular hybrid hydrogels composed of genetically engineered filamentous viruses displaying antigen peptides and antibody-immobilized GNPs. The results indicated that supramolecular hydrogels containing filamentous viruses would expand the applicability of virus-based soft materials.


Assuntos
Bacteriófago M13/química , Ouro/química , Hidrogéis/química , Doenças do Complexo Imune/tratamento farmacológico , Inovirus/química , Nanopartículas Metálicas/química , Oligopeptídeos/química , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo , Bacteriófago M13/genética , Escherichia coli , Inovirus/genética , Microscopia Eletrônica de Transmissão/métodos , Oligopeptídeos/genética , Biblioteca de Peptídeos
10.
Cancer Immunol Immunother ; 67(2): 183-193, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29026949

RESUMO

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.


Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Inovirus/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL
11.
Drug Deliv ; 24(1): 1898-1908, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29191048

RESUMO

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.


Assuntos
Bacteriófagos/genética , Vetores Genéticos/administração & dosagem , Inovirus/genética , Nanopartículas/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Nanomedicina/métodos
12.
Sci Rep ; 7(1): 16212, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29176625

RESUMO

Biological systems often generate unique and useful structures, which can have industrial relevance either as direct components or as an inspiration for biomimetic materials. For fabrication of nanoscale silica structures, we explored the use of the silaffin R5 peptide from Cylindrotheca fusiformis expressed on the surface of the fd bacteriophage. By utilizing the biomineralizing peptide component displayed on the bacteriophage surface, we found that low concentrations (0.09 mg/mL of the R5 bacteriophage, below the concentration range used in other studies) could be used to create silica nanofibers. An additional benefit of this approach is the ability of our R5-displaying phage to form silica materials without the need for supplementary components, such as aminopropyl triethoxysilane, that are typically used in such processes. Because this method for silica formation can occur under mild conditions when implementing our R5 displaying phage system, we may provide a relatively simple, economical, and environmentally friendly process for creating silica nanomaterials.


Assuntos
Inovirus/química , Nanofibras/química , Fragmentos de Peptídeos/química , Precursores de Proteínas/química , Dióxido de Silício/química , Inovirus/metabolismo
13.
PLoS One ; 12(9): e0185034, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28934297

RESUMO

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.


Assuntos
Genoma Viral , Inovirus/genética , Inovirus/isolamento & purificação , Doenças das Plantas/virologia , Ralstonia solanacearum/virologia , Virulência/genética , DNA Viral/genética , Índia , Inovirus/metabolismo , Filogenia , Prófagos/genética , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/patogenicidade
14.
Curr Med Chem ; 24(35): 3907-3920, 2017 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-28901276

RESUMO

BACKGROUND: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. OBJECTIVE: In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. RESULTS: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. CONCLUSION: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents.


Assuntos
Anticoncepção Imunológica , Inovirus/metabolismo , Vacinas Anticoncepcionais/imunologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Inovirus/química , Inovirus/imunologia , Biblioteca de Peptídeos , Vacinas de Subunidades/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia
16.
PLoS Pathog ; 13(7): e1006495, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28704569

RESUMO

Neisseria meningitidis is a commensal of human nasopharynx. In some circumstances, this bacteria can invade the bloodstream and, after crossing the blood brain barrier, the meninges. A filamentous phage, designated MDAΦ for Meningococcal Disease Associated, has been associated with invasive disease. In this work we show that the prophage is not associated with a higher virulence during the bloodstream phase of the disease. However, looking at the interaction of N. meningitidis with epithelial cells, a step essential for colonization of the nasopharynx, we demonstrate that the presence of the prophage, via the production of viruses, increases colonization of encapsulated meningococci onto monolayers of epithelial cells. The analysis of the biomass covering the epithelial cells revealed that meningococci are bound to the apical surface of host cells by few layers of heavily piliated bacteria, whereas, in the upper layers, bacteria are non-piliated but surrounded by phage particles which (i) form bundles of filaments, and/or (ii) are in some places associated with bacteria. The latter are likely to correspond to growing bacteriophages during their extrusion through the outer membrane. These data suggest that, as the biomass increases, the loss of piliation in the upper layers of the biomass does not allow type IV pilus bacterial aggregation, but is compensated by a large production of phage particles that promote bacterial aggregation via the formation of bundles of phage filaments linked to the bacterial cell walls. We propose that MDAΦ by increasing bacterial colonization in the mucosa at the site-of-entry, increase the occurrence of diseases.


Assuntos
Inovirus/fisiologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/patogenicidade , Neisseria meningitidis/virologia , Animais , Aderência Bacteriana , Células Epiteliais/microbiologia , Feminino , Fímbrias Bacterianas/fisiologia , Humanos , Camundongos , Camundongos SCID , Nasofaringe/microbiologia , Neisseria meningitidis/crescimento & desenvolvimento , Neisseria meningitidis/fisiologia , Prófagos/fisiologia , Virulência
17.
Viruses ; 9(4)2017 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-28397779

RESUMO

In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of Escherichia coli, the protein gp4 forms a pore-like structure, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggests that gp1 might be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.


Assuntos
Bacteriófago M13/genética , Bacteriófago M13/fisiologia , Inovirus/genética , Inovirus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus , Motivos de Aminoácidos , Bacteriófago M13/química , Sequência Conservada , Análise Mutacional de DNA , Escherichia coli/metabolismo , Escherichia coli/virologia , Inovirus/química
18.
Infect Immun ; 85(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27795361

RESUMO

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.


Assuntos
Inflamação/microbiologia , Inflamação/virologia , Inovirus/crescimento & desenvolvimento , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/virologia , Pseudomonas aeruginosa/virologia , Animais , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/microbiologia , Fibrose Cística/virologia , Pulmão/microbiologia , Pulmão/virologia , Macrófagos/microbiologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia
19.
Arch Virol ; 162(2): 567-572, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27743252

RESUMO

Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.


Assuntos
Cromossomos Bacterianos/química , DNA Viral/genética , Genoma Viral , Inovirus/genética , Mutagênese Insercional , Xanthomonas/virologia , Sequência de Bases , Proteínas do Capsídeo/genética , Mapeamento Cromossômico , Expressão Gênica , Inovirus/isolamento & purificação , Lisogenia , Fases de Leitura Aberta , Oryza/microbiologia , Alinhamento de Sequência , Vírion/genética
20.
J Bacteriol ; 198(23): 3209-3219, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27645387

RESUMO

Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTXϕ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains of Acinetobacter baumannii These CRAϕ-like A. baumannii prophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. IMPORTANCE: We observed the emergence of a novel filamentous phage (CRAϕ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAϕ reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRAϕ is the Vibrio cholerae CTXϕ phage. Variants of CRAϕ are found in the genomes of Acinetobacter baumannii strains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.


Assuntos
Acinetobacter baumannii/virologia , Inovirus/isolamento & purificação , Prófagos/isolamento & purificação , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Genoma Viral , Inovirus/classificação , Inovirus/genética , Inovirus/fisiologia , Prófagos/classificação , Prófagos/genética , Prófagos/fisiologia
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