Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Science ; 384(6691): eadl0635, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38574145

RESUMO

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.


Assuntos
Fímbrias Bacterianas , Fagos de Pseudomonas , Pseudomonas aeruginosa , Vírus de RNA , Internalização do Vírus , Humanos , Microscopia Crioeletrônica , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/virologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/virologia , Vírus de RNA/química , Vírus de RNA/fisiologia , Fagos de Pseudomonas/química , Fagos de Pseudomonas/fisiologia , Proteínas Virais/metabolismo
2.
Mol Microbiol ; 117(5): 1275-1290, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35434837

RESUMO

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSF s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.


Assuntos
Proteínas de Escherichia coli , Fator F , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Plasmídeos/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
3.
Mol Microbiol ; 115(3): 436-452, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33326642

RESUMO

Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.


Assuntos
Conjugação Genética , Fímbrias Bacterianas/fisiologia , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/fisiologia , Sistemas de Translocação de Proteínas/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/fisiologia , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/fisiologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Microscopia Crioeletrônica , Bactérias Gram-Negativas/ultraestrutura , Infecções por Bactérias Gram-Negativas/microbiologia , Helicobacter pylori/química , Helicobacter pylori/fisiologia , Humanos , Legionella pneumophila/química , Legionella pneumophila/fisiologia , Simulação de Acoplamento Molecular , Sistemas de Translocação de Proteínas/química , Sistemas de Translocação de Proteínas/ultraestrutura , Relação Estrutura-Atividade , Sistemas de Secreção Tipo IV/ultraestrutura
4.
Proc Natl Acad Sci U S A ; 117(41): 25751-25758, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989140

RESUMO

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid-encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.


Assuntos
Escherichia coli/virologia , Fímbrias Bacterianas/virologia , Levivirus/fisiologia , Vírus de RNA/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Levivirus/genética , Vírus de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
5.
Microbiol Resour Announc ; 8(41)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601673

RESUMO

Proteus mirabilis is a Gram-negative enteric bacterium associated with complicated human urinary tract infections. Here, we present the complete genome annotation for P. mirabilis siphophage Saba. With a 60,056-bp genome and 75 predicted genes, Saba is most similar at the nucleotide and protein levels to phage Chi and Chi-like viruses.

6.
Microbiol Resour Announc ; 8(29)2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320440

RESUMO

Here, we describe the complete genome sequence of the T4-like Klebsiella pneumoniae myophage Marfa. In its 168,532-bp genome, Marfa has 289 genes, for which 122 gene functions were predicted. Many similar proteins are shared between Marfa and phage T4, as well as its closest phage relatives.

7.
Viruses ; 10(5)2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29883383

RESUMO

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.


Assuntos
Fagos Bacilares/enzimologia , Bacillus/virologia , Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Fagos Bacilares/classificação , Fagos Bacilares/genética , Domínio Catalítico , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/farmacologia , Estabilidade Enzimática , Especificidade de Hospedeiro , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/farmacologia
8.
Proteins ; 85(9): 1645-1655, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28508424

RESUMO

Cholesterol oxidase (ChOx) is a flavoenzyme that oxidizes and isomerizes cholesterol (CHL) to form cholest-4-en-3-one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361-CHL, N485-FAD, and H447-CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin-adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444, and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site-directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non-optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. Proteins 2017; 85:1645-1655. © 2017 Wiley Periodicals, Inc.


Assuntos
Colesterol Oxidase/química , Mutagênese Sítio-Dirigida , Conformação Proteica , Sequência de Aminoácidos/genética , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Colesterol Oxidase/genética , Flavina-Adenina Dinucleotídeo/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Especificidade por Substrato
9.
Genome Announc ; 4(5)2016 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-27738022

RESUMO

Soil from George Town, Grand Cayman Island, yielded the bacteriophage Belinda, isolated on Bacillus thuringiensis DSM 350. We present here the analysis of the complete genome sequence of 162,308 bp, with 298 predicted genes. The genome also contains three tRNA genes. Belinda belongs to the C1 cluster of Bacillus phages.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA