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1.
J Agric Food Chem ; 67(42): 11703-11709, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31578056

RESUMO

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Engenharia Metabólica , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/biossíntese
2.
Cell Mol Life Sci ; 76(21): 4245-4273, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31317204

RESUMO

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.


Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/fisiologia , Transporte Proteico/genética
3.
Biophys Chem ; 252: 106191, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31177024

RESUMO

Bacterial cell characteristics, such as size, morphology, and membrane integrity, are affected by environmental conditions. Thermal treatment results in related structural changes, extent of which is determined by the microorganism's survival skills and inactivation kinetics. The objective of this study was to characterize changes in cell structure of Escherichia coli during heating using the combined analysis of dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM) techniques. The size of E. coli cells increased from 2.3 µm to 3.0 µm with heating up to 50 °C followed by a shrinkage with further heating up to 70 °C. The morphological changes were verified using transmission electron microscopy. Related changes in membrane integrity was quantified via the mobility of 16-doxylstearic acid (16-DSA) spin probe using EPR spectroscopy. Two order parameters S1 and S2 defined on x- and y-axes, respectively, decreased with increasing temperature indicating loss of membrane integrity. The combined techniques as in this study can be used to further understand factors that play role in survival behavior of microorganisms.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/citologia , Calefação , Difusão Dinâmica da Luz , Espectroscopia de Ressonância de Spin Eletrônica , Hidrodinâmica , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Propriedades de Superfície
4.
Int J Nanomedicine ; 14: 3245-3263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190792

RESUMO

Background: Bacterial resistance to antibiotics is one of the biggest challenges facing medicine today. Anti-adhesive therapy, using inhibitors of bacterial adhesion to epithelial cells, one of the first stages of infection, is a promising approximation in this area. The size, shape, number of sugar and their placement are variables that have to be taken into account in order to develop multivalent systems able to inhibit the bacterial adhesion based on sugar-lectin interaction. Materials and methods: In the present work we report a modular approach for the synthesis of water-soluble 1D-carbon nanotube-sugar nanoconstructs, with the necessary flexibility to allow an efficient sugar-lectin interaction. The method is based on the reaction of aryl diazonium salts generated in situ from aniline-substituted mannose and lactose derivatives with single wall carbon nanotubes (SWCNTs) sidewalls. Results: Two hybrid nanosystems, I-II, exposing mannose or lactose and having a tetraethylene glycol spacer between the sugar and the nanotube sidewall were rapidly assembled and adequately characterized. The sweet nano-objects were then tested for their ability to agglutinate and selectively inhibit the growth of uropathogenic Escherichia coli. These studies have shown that nanosystem I, exposing mannose on the nanotube surface is able to agglutinate and to inhibit the bacterial growth unlike nano-objects II exposing lactose. Conclusion: The results reported constitute a proof of principle in using mannose-coated 1D-carbon nanotubes as antiadhesive drugs that compete for FimH binding and prevent the uropathogenic bacteria from adhering to the urothelial surface.


Assuntos
Escherichia coli/citologia , Nanotubos de Carbono/química , Aglutinação , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Manose/química , Nanotubos de Carbono/ultraestrutura , Polissacarídeos/síntese química , Polissacarídeos/química , Propriedades de Superfície
5.
Subcell Biochem ; 92: 127-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214986

RESUMO

The peptidoglycan sacculus is a net-like polymer that surrounds the cytoplasmic membrane in most bacteria. It is essential to maintain the bacterial cell shape and protect from turgor. The peptidoglycan has a basic composition, common to all bacteria, with species-specific variations that can modify its biophysical properties or the pathogenicity of the bacteria. The synthesis of peptidoglycan starts in the cytoplasm and the precursor lipid II is flipped across the cytoplasmic membrane. The new peptidoglycan strands are synthesised and incorporated into the pre-existing sacculus by the coordinated activities of peptidoglycan synthases and hydrolases. In the model organism Escherichia coli there are two complexes required for the elongation and division. Each of them is regulated by different proteins from both the cytoplasmic and periplasmic sides that ensure the well-coordinated synthesis of new peptidoglycan.


Assuntos
Peptidoglicano , Parede Celular/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo
6.
Subcell Biochem ; 92: 337-366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214992

RESUMO

The inner membrane of Gram-negative bacteria is a ~6 nm thick phospholipid bilayer. It forms a semi-permeable barrier between the cytoplasm and periplasm allowing only regulated export and import of ions, sugar polymers, DNA and proteins. Inner membrane proteins, embedded via hydrophobic transmembrane α-helices, play an essential role in this regulated trafficking: they mediate insertion into the membrane (insertases) or complete crossing of the membrane (translocases) or both. The Gram-negative inner membrane is equipped with a variety of different insertases and translocases. Many of them are specialized, taking care of the export of only a few protein substrates, while others have more general roles. Here, we focus on the three general export/insertion pathways, the secretory (Sec) pathway, YidC and the twin-arginine translocation (TAT) pathway, focusing closely on the Escherichia coli (E. coli) paradigm. We only briefly mention dedicated export pathways found in different Gram-negative bacteria. The Sec system deals with the majority of exported proteins and functions both as a translocase for secretory proteins and an insertase for membrane proteins. The insertase YidC assists the Sec system or operates independently on membrane protein clients. Sec and YidC, in common with most export pathways, require their protein clients to be in soluble non-folded states to fit through the translocation channels and grooves. The TAT pathway is an exception, as it translocates folded proteins, some loaded with prosthetic groups.


Assuntos
Membrana Celular/enzimologia , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Canais de Translocação SEC/metabolismo , Sistema de Translocação de Argininas Geminadas/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Transporte Proteico
7.
Talanta ; 202: 384-391, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171199

RESUMO

A novel microfluidic paper-based analytical device (µPAD) was developed with benzoquinone (BQ)-mediated E. coli respiration method to measure the biotoxicities of pollutants. Functional units including sample injection, fluid-cell separation, all-carbon electrode-enabled electrochemical detection, were integrated on a piece of chromatography paper. The three-electrode, working electrode, counter electrode and reference electrode, were simultaneously screen-printed on the µPAD with conductive carbon ink. The satisfying electrochemical performance of the paper-based carbon three-electrode was confirmed by cyclic voltammetry detecting K3 [Fe(CN)6]. The process of cell toxication was considered that toxicants inhibited cell respiration and diminished the electrons on E. coli respiratory chain. It was quantitatively reflected by measuring oxidation current of hydroquinone (HQ) as a reduced state of the redox mediator BQ after the incubation of cells with pollutants. The current detection time, BQ concentration and E. coli incubation time were carefully optimized to establish the systematic optimized operations of BQ-mediated E. coli respiration method. Using the fabricated µPAD the half inhibitory concentration (IC50) were Cu2+ solution 13.5 µg mL-1, Cu2+-soil 21.4 mg kg-1, penicillin sodium-soil 85.1 mg kg-1, and IC30 of Pb2+ solution was 60.0 µg mL-1. Detection of pesticide residues in vegetable juices were accomplished in a similar way. The proposed method is fascinating on three points; 1) The generality in the biotoxicity detection depends on toxicants inducing cellular respiratory inhibition; 2) The portability and affordability make it convenient for practical applications, because of replacing incubators and centrifuges; 3) There is potential applicability in less-developed areas due to its simple operation and low-cost.


Assuntos
Técnicas Eletroquímicas , Poluentes Ambientais/farmacologia , Escherichia coli/efeitos dos fármacos , Técnicas Analíticas Microfluídicas , Papel , Benzoquinonas/química , Eletrodos , Poluentes Ambientais/análise , Escherichia coli/citologia , Escherichia coli/metabolismo
8.
Chem Commun (Camb) ; 55(49): 7001-7004, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31155630

RESUMO

Chiral polyoxometalates (POMs) were deposited onto the inner surface of an ordered cavernous film to construct POM-based chiral interfaces, which exhibit a significantly different chiral influence on the adhesion and proliferation of Escherichia coli (E. coli) cells tuned by protein adsorption. Anti-fouling was also shown for the racemic surface.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Compostos de Tungstênio/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imagem Óptica , Propriedades de Superfície
9.
Can J Microbiol ; 65(9): 691-702, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31075206

RESUMO

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.


Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Sistemas CRISPR-Cas , Escherichia coli/citologia , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Microscopia Eletrônica de Varredura , Deleção de Sequência , Virulência
10.
Phys Rev E ; 99(4-1): 042413, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31108593

RESUMO

Using a population dynamics inspired by an ensemble of growing cells, a set of fluctuation theorems linking observables measured at the lineage and population levels is derived. One of these relations implies specific inequalities comparing the population doubling time with the mean generation time at the lineage or population levels. While these inequalities have been derived before for age-controlled models with negligible mother-daughter correlations, we show that they also hold for a broad class of size-controlled models. We discuss the implications of this result for the interpretation of a recent experiment in which the growth of bacteria strains has been probed at the single-cell level.


Assuntos
Modelos Biológicos , Ciclo Celular , Escherichia coli/citologia , Dinâmica Populacional
11.
Phys Rev E ; 99(4-1): 042409, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31108688

RESUMO

Bacteria communicate with each other to coordinate macroscale behaviors including pathogenesis, biofilm formation, and antibiotic production. Empirical evidence suggests that bacteria are capable of communicating at length scales far exceeding the size of individual cells. Several mechanisms of signal interference have been observed in nature, and how interference influences macroscale activity within microbial populations is unclear. Here we examined the exchange of quorum sensing signals to coordinate microbial activity over long distances in the presence of a variable amount of interference through a neighboring signal-degrading strain. As the level of interference increased, communication over large distances was disrupted and at a critical amount of interference, large-scale communication was suppressed. We explored this transition in experiments and reaction-diffusion models, and confirmed that this transition is a two-dimensional percolation transition. These results demonstrate the utility of applying physical models to emergence in complex biological networks to probe robustness and universal quantitative features.


Assuntos
Escherichia coli/citologia , Modelos Biológicos , Percepção de Quorum , Biofilmes , Difusão , Escherichia coli/genética , Escherichia coli/fisiologia
12.
Methods Mol Biol ; 1968: 123-134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929211

RESUMO

Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should be completed to characterize limits of detection and range of linearity with respect to the plate count method. To demonstrate the calibration process, mixtures of Escherichia coli or Staphylococcus aureus with proportions of live and dead cells ranging from 0% to 100% were prepared. These samples were stained using nucleic acid-binding dyes, and 6 µm reference beads were added (LIVE/DEAD® BacLight kit). The calibration samples were analyzed using bead-based FCM as well as the agar plate count method, and the results from both methods were compared.


Assuntos
Bactérias/citologia , Citometria de Fluxo/métodos , Escherichia coli/citologia , Staphylococcus aureus/citologia
13.
Artif Cells Nanomed Biotechnol ; 47(1): 1603-1609, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31027437

RESUMO

Synthesis of silver and silver based nanoparticles using microorganisms has received profound interest because of obtaining nanoparticles with unique physicochemical and biological properties. In the current study, for the first time, synthesis of silver chloride nanoparticles (AgClNPs) using cell-free supernatant of Escherichia coli culture is reported. Prepared AgClNPs were characterized by EDS, XRD and FESE. Data revealed the synthesized nanoparticles, mostly, have a spherical shape with an average size of 13 nm. Additionally, MTT assay elucidated a dose-dependent cytotoxicity of AgClNPs against MCF-7 cells (IC50 = 44 µg/mL). Quantitative real-time reverse transcription-PCR and colourimetric assays were employed to investigate the mechanism of cell toxicity in several cell death pathways. The results revealed the ability of AgClNPs to upregulate Bax/Bcl-2 ratio and p53 at mRNA level. Moreover, other apoptotic factors such as caspase-3, 8 and 9 were also upregulated at both mRNA and proteome levels. Finally, apoptosis induction was confirmed by Annexin-V/PI detection assay. Based on the obtained data, biosynthesized AgClNPs using E. coli cell-free supernatant exhibit a cytotoxic effect on human breast cancer cells through up-regulation of apoptotic factors, which suggest them as anti-tumour agents for further investigations.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Escherichia coli/metabolismo , Nanopartículas , Nanotecnologia , Compostos de Prata/metabolismo , Compostos de Prata/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/citologia , Células HEK293 , Humanos , Células MCF-7 , Biossíntese de Proteínas/efeitos dos fármacos , Compostos de Prata/química
14.
Int J Biol Macromol ; 132: 759-765, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953720

RESUMO

1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl ß-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/química , Permeabilidade da Membrana Celular , Estabilidade Enzimática , Escherichia coli/citologia , Geobacillus/enzimologia , Rhodothermus/enzimologia
15.
Talanta ; 199: 27-31, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952256

RESUMO

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL-1resin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at -2.7 column volumes compared to wildtype HbF.


Assuntos
Líquidos Corporais/química , Hemoglobinas/química , Hemoglobinas/isolamento & purificação , Impressão Molecular , Polímeros/química , Cromatografia , Escherichia coli/química , Escherichia coli/citologia , Hemoglobinas/genética , Humanos , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
16.
Nat Commun ; 10(1): 1901, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015409

RESUMO

Asymmetric cell division is a major mechanism generating cell diversity. As cell cycle duration varies among cells in mammalian tissue culture cells, we asked whether their division asymmetry contributes to this variability. We identify among sibling cells an outlier using hierarchical clustering on cell cycle durations of granddaughter cells obtained by lineage tracking of single histone2B-labelled MDCKs. Remarkably, divisions involving outlier cells are not uniformly distributed in lineages, as shown by permutation tests, but appear to emerge from asymmetric divisions taking place at non-stochastic levels: a parent cell influences with 95% confidence and 0.5% error the unequal partitioning of the cell cycle duration in its two progenies. Upon ninein downregulation, this variability propagation is lost, and outlier frequency and variability in cell cycle durations in lineages is reduced. As external influences are not detectable, we propose that a cell-autonomous process, possibly involved in cell specialisation, determines cell cycle duration variability.


Assuntos
Divisão Celular Assimétrica , Linhagem da Célula/genética , Proteínas do Citoesqueleto/genética , Escherichia coli/citologia , Histonas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Rastreamento de Células/métodos , Proteínas do Citoesqueleto/metabolismo , Cães , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Histonas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células Madin Darby de Rim Canino , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo
17.
Eur J Med Chem ; 173: 154-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995568

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleosídeos/farmacologia , Pirimidinas/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/síntese química , Escherichia coli/citologia , Escherichia coli/enzimologia , Estrutura Molecular , Nucleosídeos/análise , Nucleosídeos/síntese química , Pirimidinas/análise , Pirimidinas/síntese química , Relação Estrutura-Atividade
18.
Opt Lett ; 44(7): 1868-1871, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30933168

RESUMO

In advanced biomedicine and microfluidics, there is a strong desire to sort and manipulate various cells and bacteria based on miniaturized microfluidic chips. Here, by integrating fiber tweezers into a T-type microfluidic channel, we report an optofluidic chip to selectively trap Escherichia coli in human blood solution based on different sizes and shapes. Furthermore, we simulate the trapping and pushing regions of other cells and bacteria, including rod-shaped bacteria, sphere-shaped bacteria, and cancer cells based on finite-difference analysis. With the advantages of controllability, low optical power, and compact construction, the strategy may be possibly applied in the fields of optical separation, cell transportation, and water quality analysis.


Assuntos
Separação Celular/instrumentação , Miniaturização/instrumentação , Fibras Ópticas , Pinças Ópticas , Animais , Desenho de Equipamento , Eritrócitos/microbiologia , Escherichia coli/citologia , Humanos
19.
Lab Chip ; 19(8): 1417-1426, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30869093

RESUMO

There is an urgent need to develop novel methods for assessing the response of bacteria to antibiotics in a timely manner. Antibiotics are traditionally assessed via their effect on bacteria in a culture medium, which takes 24-48 h and exploits only a single parameter, i.e. growth. Here, we present a multiparameter approach at the single-cell level that takes approximately an hour from spiking the culture to correctly classify susceptible and resistant strains. By hydrodynamically trapping hundreds of bacteria, we simultaneously monitor the evolution of motility and morphology of individual bacteria upon drug administration. We show how this combined detection method provides insights into the activity of antimicrobials at the onset of their action which single parameter and traditional tests cannot offer. Our observations complement the current growth-based methods and highlight the need for future antimicrobial susceptibility tests to take multiple parameters into account.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Células Imobilizadas/efeitos dos fármacos , Hidrodinâmica , Movimento , Fatores de Tempo
20.
Lab Chip ; 19(8): 1352-1358, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30907393

RESUMO

We report a proof-of-principle demonstration of particle concentration to achieve high-throughput resistive pulse detections of bacteria using a microfluidic-channel-integrated micropore. We fabricated polymeric nanochannels to trap micrometer-sized bioparticles via a simple water pumping mechanism that allowed aggregation-free size-selective particle concentration with negligible loss. Single-bioparticle detections by ionic current measurements were then implemented through releasing and transporting the thus-collected analytes to the micropore. As a result, we attained two orders of magnitude enhancement in the detection throughput by virtue of an accumulation effect via hydrodynamic control. The device concept presented may be useful in developing nanopores and nanochannels for high-throughput single-particle and -molecule analyses.


Assuntos
Dispositivos Lab-On-A-Chip , Escherichia coli/citologia , Hidrodinâmica , Porosidade
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