RESUMO
To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.
Assuntos
Antibacterianos/farmacologia , Biguanidas/farmacologia , Cromossomos Bacterianos/genética , Animais , Antibacterianos/metabolismo , Bacillus megaterium/efeitos dos fármacos , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Biguanidas/metabolismo , Células CHO , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Estruturas Cromossômicas/efeitos dos fármacos , Cricetinae , Cricetulus , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Cavalos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to ß-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on ß-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to ß-lactam-mediated inhibition of cell division, rapidly appeared upon ß-lactam mono-treatment and remained stable for up to 18âh. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of ß-lactam treatment. Interestingly, the effect of sugar on ß-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in ß-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.
Assuntos
Antibacterianos/farmacologia , Metabolismo dos Carboidratos , Carboidratos/farmacologia , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , beta-Lactamas/farmacologia , Bacteriólise/efeitos dos fármacos , Carbenicilina/farmacologia , Cefuroxima/farmacologia , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Citometria de Fluxo , MicroscopiaRESUMO
Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance.
RESUMO
For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood. Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated survival, whereas inhibition of chaperonin expression sensitized bacteria. Overexpression of the DnaK/DnaJ/GrpE chaperone system similarly facilitated survival but did not promote growth of aminoglycoside-treated bacteria. Inhibition of chaperonin expression sensitized bacteria to aminoglycosides as measured by reduced minimum inhibitory concentrations, whereas GroEL/GroES overexpression did not increase minimum inhibitory concentrations. Our observations establish misfolding of cytosolic proteins as an effect of aminoglycoside action and reveal that chaperones, chaperonins in particular, help bacteria cope during early exposure to these drugs.
Assuntos
Aminoglicosídeos/farmacologia , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/enzimologia , Dobramento de Proteína/efeitos dos fármacos , Chaperonina 10/genética , Chaperonina 60/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismoRESUMO
Information storage capabilities are key in most aspects of society and the requirement for storage capacity is rapidly expanding. In principle, DNA could be a high-density medium for information storage. Church and coworkers recently demonstrated how binary data can be encoded, stored in, and retrieved from a library of oligonucleotides, increasing by several orders of magnitude the amount and density of manmade information stored in DNA to date. The technology remains in its infancy and important hurdles have yet to be overcome in order to realize its potential. However, DNA may be particularly useful as a storage-medium over long time-scales (centuries), because data-access is compatible with any large-scale DNA-sequencing and -synthesis technology.
Assuntos
Dispositivos de Armazenamento em Computador , DNA , Código Genético , Armazenamento e Recuperação da Informação/métodosRESUMO
A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.
Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , DNA de Cadeia Simples/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanocápsulas/administração & dosagem , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Antígenos HLA/imunologia , Humanos , Fragmentos de Imunoglobulinas/administração & dosagem , Células Jurkat/imunologia , Células Matadoras Naturais/imunologia , Nanocápsulas/química , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/químicaRESUMO
Prominent current ideas on how life emerged on Earth include an RNA world hypothesis in which RNA performed informational as well as catalytic functions in the absence of both DNA and protein. Demonstration of a self-replicative system based on ribonucleic acid polymers as both information carriers and catalysts would lend support to such a scenario. A pivotal component of this system would be an RNA dependent RNA polymerase ribozyme capable of replicating its own RNA gene. Recent work from the Holliger group at the Laboratory for Molecular Biology in Cambridge has provided synthetic ribozymes1 that just might foreshadow the future engineering of such self-replicative systems.
RESUMO
In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output and hence is important to recombinant and, most certainly, endogenous gene expression.
Assuntos
Códon , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Engenharia de Proteínas/métodos , RNA Bacteriano/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/genéticaRESUMO
A recent paper in Science by Li et al. 2011(1) reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized.
Assuntos
DNA/genética , Variação Genética , Genoma Humano , RNA Mensageiro/genética , HumanosRESUMO
BACKGROUND: Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles. METHODOLOGY/PRINCIPAL FINDINGS: The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance. CONCLUSIONS/SIGNIFICANCE: The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.
Assuntos
Sequência de Aminoácidos , Códon , Evolução Molecular Direcionada/métodos , Proteínas/genética , Deleção de Sequência , Sequência de Aminoácidos/genética , Códon/genética , Proteínas de Fluorescência Verde/genética , Mutagênese , Fenilalanina , Engenharia de ProteínasRESUMO
Using LNA in situ hybridization, select mRNAs have been shown to be spatially confined to their chromosomal loci in two distantly related bacterial organisms. Translating ribosomes are diffusion limited by mRNA association.
RESUMO
Molecular biology owes its prominent role in the biological sciences to the tools of recombinant DNA. While the foundations of recombinant DNA were laid in the 1970s with the discovery of type II restriction endonucleases,1,2 development of robust sequencing technology3 and pioneering work on gene synthesis,4,5 it was not until the turn of the new millennium before the first complete synthetic viral genomes saw the light of day including that of hepatitis C,6 poliovirus,7 and bacteriophage PhiX174.8 Recombinant DNA has come of age as entire cellular genomes are sequenced and stored as digitized information. So what's next? One novel branch of recombinant DNA, referred to as synthetic genomics,9 is occupied with (re)construction of entire cellular genomes from virtual sequence information and using chemical components. Here we look at the most recent developments in such de novo construction. For a broader and more extensive review on genome engineering, the reader is referred to the excellent paper by Carr and Church.10.
RESUMO
While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes--mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na(+)). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.
Assuntos
DNA/química , Ácidos Nucleicos Peptídicos/química , Aminacrina/química , Sequência de Bases , Citosina/análogos & derivados , Citosina/química , Concentração de Íons de Hidrogênio , Substâncias Intercalantes/químicaRESUMO
Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine-cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complexes, and also conventional triplexes with the dsDNA target. Triplexes form with much faster kinetics than invasion complexes and prevail at lower PNA concentrations and at shorter incubation times. Furthermore, increasing the ionic strength strongly favour triplex formation over invasion as the latter is severely inhibited by cations. Whereas a single triplex invasion complex is formed with the decameric PNA, two structurally different target-specific invasion complexes were characterized for the dodecameric PNA and more than five for the pentadecameric PNA. Finally, it is shown that isolated triplex complexes can be converted to specific invasion complexes without dissociation of the Hoogsteen base-paired triplex PNA. These result demonstrate a clear example of a 'triplex first' mechanism for PNA helix invasion.
Assuntos
DNA/química , Ácidos Nucleicos Peptídicos/química , Pareamento de Bases , Citosina/química , Ensaio de Desvio de Mobilidade Eletroforética , Cinética , Timina/químicaRESUMO
The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.
Assuntos
RNA/genética , Transcrição Gênica/genética , DNA/genética , Cinética , Conformação de Ácido Nucleico , RNA/química , Ribonuclease H/metabolismo , Moldes GenéticosRESUMO
Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested for in vitro charging of tRNA(Phe) and for protein mutagenesis via the phenylalanyl-tRNA synthetase variant alphaA294G that is able to facilitate in vivo protein synthesis using a range of para-substituted phenylalanine analogues. The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase alphaA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency. The in vivo incorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.
Assuntos
Alanina/análogos & derivados , Aminoácidos/química , Escherichia coli/enzimologia , Escherichia coli/genética , Fenilalanina-tRNA Ligase/genética , Alanina/síntese química , Benzofuranos/síntese química , Cinética , Espectrometria de Massas , Modelos Químicos , Mutagênese , Fenilalanina/química , Ligação Proteica , Engenharia de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Fatores de Tempo , Triazóis/síntese química , Raios UltravioletaRESUMO
"Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as determined by T(m) measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C(50) measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion moiety to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Peptídicos/química , Pareamento Incorreto de Bases , Sequência de Bases , Sítios de Ligação , DNA de Cadeia Simples/química , Ligação de Hidrogênio , Oligodesoxirribonucleotídeos/síntese química , Ácidos Nucleicos Peptídicos/síntese química , Nucleotídeos de Pirimidina/químicaRESUMO
DNA helix invasion by P-loop forming peptide nucleic acids (PNAs) is extremely sensitive to increased ionic strength as this stabilizes the DNA duplex. To address this, the DNA intercalator 9-aminoacridine was conjugated to helix invading PNAs, and the duplex DNA binding efficiency of such constructs was measured at different ionic strength conditions by electrophoretic mobility shift analysis. Remarkably, at physiogically relevant ionic strength (140 mM K+/10 mM Na+, 2 mM Mg2+), acridine conjugated PNAs showed 20-150-fold superior binding to a cognate sequence target as compared to the conventional PNAs. This enhancement occurred without compromising the sequence specificity of binding. Thus, simply conjugating the DNA intercalator 9-aminoacridine to PNA represents a major step toward the development of helix invading constructs for in vivo applications such as gene targeting.