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1.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
2.
Ecotoxicol Environ Saf ; 203: 111007, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888586

RESUMO

Soil acidification is one of the crucial global environmental problems, affecting sustainable land use, crop yield, and ecosystem stability. Previous research reported the tolerance of crops to acid soil stress. However, the molecular response of woody plant to acid conditions remains largely unclear. Rhododendron L. is a widely distributed woody plant genus and prefers to grow in acidic soils. Herein, weighted gene coexpression network analysis was performed on R. protistum var. giganteum seedlings subjected to five pH treatments (3.5, 4.5, 5.5, 6.0, 7.0), and their ecophysiological characteristics were determined for the identification of their molecular responses to acidic environments. Through pairwise comparison, 855 differentially expressed genes (DEGs) associated with photosynthesis, cell wall, and phenylpropanoid metabolism were identified. Most of the DEGs related to photosynthesis and cell wall were up-regulated after pH 4.5 treatment. Results implied that the species improves its photosynthetic abilities and changes its cell wall characteristics to adapt to acidic conditions. Weighted gene co-expression network analyses showed that most of the hub genes were annotated to the biosynthetic pathways of ribosomal proteins and photosynthesis. Expression pattern analysis showed that genes encoding subunit ribosomal proteins decreased at pH 7.0 treatment, suggesting that pH 7.0 treatment led to cell injury in the seedlings. The species regulates protein synthesis in response to high pH stress (pH 7.0). The present study revealed the molecular response mechanism of woody plant R. protistum var. giganteum to acid environments. These findings can be useful in enriching current knowledge of how woody species adapt to soil acidification under global environmental changes.


Assuntos
Ácidos/farmacologia , Parede Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Madeira/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Fotossíntese/genética , Plântula/efeitos dos fármacos , Plântula/metabolismo , Solo/química , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Madeira/genética , Madeira/metabolismo
3.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908144

RESUMO

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Infecção Hospitalar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Infecção Hospitalar/microbiologia , Análise Mutacional de DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Mutação
4.
PLoS Genet ; 16(7): e1008908, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639995

RESUMO

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1-dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through self-activation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Candida albicans/efeitos dos fármacos , Caspofungina/farmacologia , Proteínas de Domínio MADS/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/patogenicidade , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Fosforilação/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética
5.
Food Chem ; 332: 127416, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619946

RESUMO

Water bamboo shoots quickly deteriorate after harvest as a result of rapid lignification and softening. Nitric oxide (NO) has been used to extend the postharvest life of several other vegetables. Here, we examined the effect of NO on the storage of water bamboo shoots at 4℃ for 28 days. Without NO, fresh weight and firmness decreased quickly, while the cellulose and lignin contents increased sharply during storage. NO treatment delayed softening by maintaining the integrity of the cell wall and inhibiting the degradation of protopectin and the expressions of pectin methylesterase and polygalacturonase. NO treatment also delayed cellulose synthesis by increasing cellulase activity. NO treatment decreased the synthesis of lignin by inhibiting the activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, laccase and peroxidase. These results indicate that NO treatment is effective at suppressing the softening and lignification of water bamboo shoots during postharvest storage.


Assuntos
Armazenamento de Alimentos/métodos , Óxido Nítrico/farmacologia , Poaceae/efeitos dos fármacos , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Celulase/metabolismo , Celulose/metabolismo , Temperatura Baixa , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Poaceae/metabolismo , Poligalacturonase/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(31): 18744-18753, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680963

RESUMO

Morphological profiling is a method to classify target pathways of antibacterials based on how bacteria respond to treatment through changes to cellular shape and spatial organization. Here we utilized the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS classified 94% of tested drugs correctly into broad categories according to modes of action previously identified in the literature. In the other 6%, MorphEUS pointed to key off-target activities. We observed cell wall damage induced by bedaquiline and moxifloxacin through secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly classify three compounds in a blinded study and identified an off-target effect for one compound that was not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify pathways of drug action and the proximal cause of cellular damage in tubercle bacilli will make it applicable to other pathogens and cell types where morphological responses are subtle and heterogeneous.


Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas/métodos , Mycobacterium tuberculosis , Software , Parede Celular/efeitos dos fármacos , Diarilquinolinas , Ensaios de Triagem em Larga Escala , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Transcriptoma/efeitos dos fármacos
7.
Int J Food Microbiol ; 331: 108714, 2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-32544792

RESUMO

Microbiological contamination by spoilage yeasts species are frequent during winemaking, and biological control using antagonistic yeasts is considered a more beneficial alternative to conventional synthetic antimicrobials. Saccharomyces eubayanus killer toxin (SeKT) was produced and purified in a synthetic optimized medium. Purification procedure allowed the identification of SeKT as protein with an apparent molecular mass of 70 kDa and activity at physicochemical conditions suitable for winemaking process. Purified SeKT reduced the levels of volatile phenols produced by the spoilage yeasts Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii and Pichia manshurica in wine-like medium. The putative mode of action of SeKT on sensitive yeast strains comprises cell wall disruption through ß-glucanase and chitinase activities as well as necrotic and apoptotic death in a toxin dose dependent manner. Thus, SeKT appears to be a promising biocontrol agent against spoilage yeasts during wine aging and storing.


Assuntos
Microbiologia de Alimentos , Micotoxinas/química , Micotoxinas/toxicidade , Saccharomyces/química , Vinho/microbiologia , Parede Celular/efeitos dos fármacos , Micotoxinas/isolamento & purificação , Fenóis/metabolismo , Saccharomyces/metabolismo , Leveduras/efeitos dos fármacos
8.
Nat Commun ; 11(1): 2848, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503964

RESUMO

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Estrutura Molecular , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
9.
Int J Nanomedicine ; 15: 3071-3085, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431502

RESUMO

Purpose: Recently, two-dimensional (2D) nanomaterials are gaining tremendous attention as novel antibacterial platforms to combat against continuously evolving antimicrobial resistance levels. Among the family of 2D nanomaterials, black phosphorus (BP) nanosheets have demonstrated promising potential for biomedical applications. However, there is a need to gain nanoscale insights of the antibacterial activity of BP nanosheets which lies at the center of technical challenges. Methods: Ultra-large BP nanosheets were synthesized by liquid-exfoliation method in the eco-friendly deoxygenated water. Synthesized BP nanosheets were characterized by TEM, AFM, and Raman spectroscopy techniques and their chemical stability was evaluated by EDS and EELS elemental analysis. The antibacterial activity of BP nanosheets was evaluated at nanoscale by the ultramicrotome TEM technique. Further, HAADF-STEM image and EDS elemental line map of the damaged bacterium were utilized to analyze the presence of diagnostic ions. Supportive SEM and ATR-FTIR studies were carried out to confirm the bacterial cell wall damage. In vitro colony counting method was utilized to evaluate the antibacterial performance of ultra-large BP nanosheets. Results: Elemental EELS and EDS analysis of BP nanosheets stored in deoxygenated water confirmed the absence of oxygen peak. TEM studies indicate the various events of bacterial cell damage with the lost cellular metabolism and structural integrity. Colony counting test results show that as-synthesized BP nanosheets (100 µg/mL) can kill ~95% bacteria within 12 hours. Conclusion: TEM studies demonstrate the various events of E. coli membrane damage and the loss of structural integrity. These events include the BP nanosheets interaction with the bacterial cell wall, cytoplasmic leakage, detachment of cytoplasm from the cell membrane, reduced density of lipid bilayer and agglomerated DNA structure. The EDS elemental line mapping of the damaged bacterium confirms the disrupted cell membrane permeability and the lost cellular metabolism. SEM micrographs and ATR-FTIR supportive results confirm the bacterial cell wall damage.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Nanoestruturas/química , Fósforo/química , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Escherichia coli/ultraestrutura , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Água/química
10.
Food Chem ; 327: 127057, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32464461

RESUMO

Chilling injury (CI) restricts the quality and shelf life of bell pepper fruits; reducing these CI-induced detrimental effects is therefore of high economic and agricultural relevance. Here, we investigated the effects of trisodium phosphate (TSP), salicylic acid (SA), and TSP + SA treatments on pepper fruits under cold stress at 4 °C for 25 d. Combined TSP + SA treatment performed an optimal effect. Specifically, TSP + SA treatment enhanced fatty-acid desaturation efficiency, as indicated by the increased expression of key fatty acid desaturase genes, and higher content of unsaturated fatty acids. Meanwhile, TSP + SA treatment inhibited the CI-induced membrane damage, manifested as lower electrolyte leakage and malondialdehyde content. Furthermore, low field-nuclear magnetic resonance and proline content also revealed that TSP + SA treatment mitigated CI through enhancing water retention in pepper fruits. Collectively, our results may shed new light on optimizing the low-temperature storage conditions of post-harvest peppers.


Assuntos
Capsicum/química , Ácidos Graxos/química , Fosfatos/química , Ácido Salicílico/química , Água/química , Capsicum/efeitos dos fármacos , Capsicum/metabolismo , Parede Celular/efeitos dos fármacos , Temperatura Baixa , Ácidos Graxos/metabolismo , Frutas/química , Frutas/efeitos dos fármacos , Frutas/metabolismo , Malondialdeído/química , Malondialdeído/metabolismo , Permeabilidade/efeitos dos fármacos , Fosfatos/farmacologia , Prolina/química , Ácido Salicílico/farmacologia
11.
Ecotoxicol Environ Saf ; 199: 110750, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32446103

RESUMO

Both antibiotics and surfactants commonly exist in natural environment and have generated great concerns due to their biological influence on the ecosystem. A major concern lies in the capacity of antibiotics to induce bacterial filaments formation, which has potential health risks. However, their joint effect is not clear so far. Here, we studied the joint effect of cephalexin (Cex), a typical antibiotic, and differently charged surfactants on the formation of E. coli filaments. Three kinds of surfactants characterized by different charges were used: cationic surfactant (CTAB), anionic surfactant (SDS) and nonionic surfactant (Tween). Data showed that Cex alone caused the formation of E. coli filaments, elongating their maximum profile from ca. 2 µm (a single E. coli cell) to tens of micrometers (an E. coli filament). A joint use of surfactants with Cex could produce even longer E. coli filaments, elongating the maximum length of the bacteria to larger than 100 µm. The capacity order of different surfactants under their optimum concentrations to produce elongated E. coli filaments was Tween > SDS > CTAB. The E. coli filaments were characterized with a normal DNA distribution and a good cell membrane integrity. We measured the stiffness of bacterial cell wall by atomic force microscopy and correlated the elongation capacity of the E. coli filaments to the stiffness of cell wall. Zeta potential measurement indicated that inserting into or being bound to the cell surface in a large quantity was tested not to be the major way that surfactants interacted with bacteria.


Assuntos
Antibacterianos/toxicidade , Cefalexina/toxicidade , Poluentes Ambientais/toxicidade , Escherichia coli/efeitos dos fármacos , Polissorbatos/toxicidade , Tensoativos/toxicidade , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Sinergismo Farmacológico , Ecossistema , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura
12.
Biofouling ; 36(3): 319-331, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32410461

RESUMO

Traditional herbal monomers (THMs) are widely distributed in many traditional Chinese formulas (TCFs) and decoctions (TCDs) and are frequently used for the prevention and treatment of fungal infections. The antifungal activities of five common THMs, including sodium houttuyfonate (SH), berberine (BER), palmatine (PAL), jatrorrhizine (JAT) and cinnamaldehyde (CIN), and their potential for inducing cell wall remodeling (CWR), were evaluated against Candida albicans SC5314 and Candida auris 12372. SH/CIN plus BER/PAL/JAT showed synergistic antifungal activity against both Candida isolates. Furthermore, SH-associated combinations (SH plus BER/PAL/JAT) induced stronger exposure of ß-glucan and chitin than their counterparts, while CIN triggered more marked exposure compared with CIN-associated combinations (CIN plus BER/PAL/JAT). Collectively, this study demonstrated the anti-Candida effect and the CWR induction potential of the five THMs and their associated combinations, providing a possibility of their in vivo application against fungal-associated infections.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Alcanos/farmacologia , Berberina/análogos & derivados , Berberina/farmacologia , Alcaloides de Berberina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Sulfitos/farmacologia
13.
Eur J Med Chem ; 200: 112390, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32447196

RESUMO

The mycobacterial membrane protein Large 3 (MmpL3) is an inner membrane protein that transports trehalose-monomycolates, precursors for trehalose-dimycolates and mycolic acids that make up essential components of the mycobacterial outer membrane. Inhibition of MmpL3 weakens the mycobacterial cell wall and ultimately results in cell death in both in vitro and in vivo infection models. This highlights the therapeutic potential of MmpL3 as a drug target. High-throughput whole-cell screening along with whole genome sequencing of resistant mutants has identified numerous classes of compounds that can be classified as MmpL3 inhibitors. In this review, we provide insights into the current development of various MmpL3 inhibitors and discuss the potential challenges in this area.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Parede Celular/efeitos dos fármacos , Antituberculosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Fatores Corda/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo
14.
Ecotoxicol Environ Saf ; 200: 110748, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32470678

RESUMO

Cadmium (Cd) is an inauspicious abiotic traction that not only influences crop productivity and its growth parameters, but also has adverse effects on human health if these crops are consumed. Among crops, leafy vegetables which are the good source of mineral and vitamins accumulate more Cd than other vegetables. It is thus important to study photosynthetic variables, amino acid composition, and ultrastructural localization of Cd differences in response to Cd accumulation between two low and high Cd accumulating Brassica rapa ssp. chinensis L. (pak choi) cultivars, differing in Cd accumulation ability. Elevated Cd concentrations significantly lowered plant growth rate, biomass, leaf gas exchange and concentrations of amino acids collated to respective controls of both cultivars. Electron microscopy indicated that the impact of high Cd level on ultrastructure of leaf cells was associated to affecting cell functionalities, i.e. irregular cell wall, withdrawal of cell membrane, and chloroplast structure which has negative impact on photosynthetic activities, thus causing considerable plant growth suppression. Damage in root cells were observed in the form of enlargement of vacuole. The energy dispersive micro X-ray spectroscopy of both cultivars leaves indicated that cellular structure exhibited exudates of Cd-dense material. Ultrastructural damages and phytotoxicity were more pronounced in high accumulator cultivar as compared to the low accumulator cultivar. These findings are useful in determining the mechanisms of differential Cd-tolerance among cultivars with different Cd tolerance abilities at cellular level.


Assuntos
Brassica rapa/efeitos dos fármacos , Brassica rapa/metabolismo , Cádmio/toxicidade , Aminoácidos/análise , Biomassa , Brassica rapa/crescimento & desenvolvimento , Brassica rapa/ultraestrutura , Cádmio/farmacocinética , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Humanos , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura
15.
Biochim Biophys Acta Biomembr ; 1862(8): 183291, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234322

RESUMO

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Parede Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Sequência de Aminoácidos/genética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/patogenicidade , Humanos , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana
16.
Artigo em Inglês | MEDLINE | ID: mdl-32289635

RESUMO

Caffeate 3-O-methyltransferase (COMT) catalyzes the methylation of the 3-hydroxyl group of caffeate to produce ferulate, an important precursor of the lignin biosynthesis. As a crucial drawback for biofuel production, lignin limits the enzymatic hydrolysis of polysaccharides to result in fermentable sugars. We hypothesized that a controlled inhibition of maize COMT can be an efficient approach to reduce ferulate and lignin, thus improving the saccharification process. First, we applied in silico techniques to prospect potential inhibitors of ZmaysCOMT, and the nitrocatechol entacapone was selected. Second, in vitro assays confirmed the inhibitory effect of entacapone on maize COMT. Finally, in vivo experiments revealed that entacapone reduced the contents of cell-wall-esterified hydroxycinnamates and increased saccharification of stems (18%) and leaves (70%), without negatively affecting maize growth and lignin biosynthesis. This non-genetically modified approach can be an alternative strategy to facilitate the enzymatic hydrolysis of biomass polysaccharides and increase saccharification for bioethanol production.


Assuntos
Catecóis , Lignina , Nitrilos , Polissacarídeos , Zea mays , Biocombustíveis , Biomassa , Inibidores de Catecol O-Metiltransferase/farmacologia , Catecóis/farmacologia , Parede Celular/efeitos dos fármacos , Nitrilos/farmacologia , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Zea mays/efeitos dos fármacos
17.
Transl Res ; 220: 68-97, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32275897

RESUMO

The current tuberculosis (TB) predicament poses numerous challenges and therefore every incremental scientific work and all positive socio-political engagements, are steps taken in the right direction to eradicate TB. Progression of the late stage TB-drug pipeline into the clinics is an immediate deliverable of this global effort. At the same time, fueling basic research and pursuing early discovery work must be sustained to maintain a healthy TB-drug pipeline. This review encompasses a broad analysis of chemotherapeutic strategies that target the DNA replication, protein synthesis, cell wall biosynthesis, energy metabolism and proteolysis of Mycobacterium tuberculosis (Mtb). It includes a status check of the current TB-drug pipeline with a focus on the associated biology, emerging targets, and their promising chemical inhibitors. Potential synergies and/or gaps within or across different chemotherapeutic strategies are systematically reviewed as well.


Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Micólicos/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Proteólise/efeitos dos fármacos
18.
J Food Sci ; 85(4): 1223-1230, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32147836

RESUMO

The present study aims to study the antibacterial activity of food-grade lipidic nanoemulsion (noncationized/cationized) against Bacillus subtilis (BS). Bactericidal activity was ascertained by studying the morphological transitions on BS using transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM). Morphological changes were witnessed by cell wall breakage, oozing out of cellular contents, loss of cell turgidity and contour. Furthermore, aggregation of cationic nanoemulsion (CaNM) was preferentially observed at apical side of BS construing comparatively more electrostatic attraction between electronegative apical side and CaNM. Resistance response of BS exhibited by apical cell-wall thickening was not able to protect the bacteria due to leakage of cellular content. AFM corroborated its importance in bacteriology, wherein the fragmented cell wall can be "piece-by-piece" identified and sutured back to its appropriate vacant places, thereby, completing the cell wall contour of the ghost cell. Such postmortem analysis of bacterial cell using AFM studies can throw light toward mechanism of cell fragmentation of bacterial cells. SEM study also demonstrated the deformed, fragmented, and amorphous nature of BS construing the bactericidal effect of prepared nanoemulsion.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Lipídeos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Emulsões/química , Emulsões/farmacologia , Lipídeos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão
19.
Nat Commun ; 11(1): 1455, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193379

RESUMO

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Daptomicina/farmacologia , Lipídeos de Membrana/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Humanos , Membranas Artificiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-32114399

RESUMO

Having a central role in cell wall pectin cross-linking, calcium has been increasingly used as supplement to promote fruit firmness and extended shelf-life. However, the molecular rearrangements associated to increased fruit robustness are still a matter of debate. In this study, mechanical, histochemical and molecular assays were conducted to understand the mechanisms underlying the effects of Ca in fruit physical properties. In a two-year field trial, grapevines were sprayed with exogenous CaCl2 throughout the fruiting season. Results showed an increase in berry Ca concentration at harvest, associated to increased fruit consistency and skin resistance. Scanning electron microscopy showed that fruits from Ca-treated plants had smoother skin surfaces than control fruits, and that microcracks encircling the lenticels were less prominent. Histochemistry assays suggested higher deposition of pectin-like material in skin cell walls in grapes from Ca-treated vines, but no evident modifications in cellulose content were observed. Accordingly, the expression of cellulose synthase family gene CesA3 was not affected by exogenous Ca, while polygalacturonase-encoding genes PG1 and PG2 were downregulated, together with EXP6 belonging to expansin family, and CER9 and CYP15 involved in cuticle biosynthesis. These results suggested that Ca acts by inhibiting pectin degradation and cell wall loosening, while remodeling cuticle structure.


Assuntos
Cloreto de Cálcio , Frutas , Vitis , Cálcio/metabolismo , Cloreto de Cálcio/farmacologia , Parede Celular/efeitos dos fármacos , Frutas/efeitos dos fármacos , Frutas/ultraestrutura , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Poligalacturonase/genética , Vitis/efeitos dos fármacos
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