Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.282
Filtrar
1.
Anal Chem ; 92(19): 13396-13404, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32867467

RESUMO

Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent π-π stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent π-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at ∼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ∼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of ∼260 nm and rapid sample-to-answer detection time (∼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.


Assuntos
Bactérias/química , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/síntese química , Nanotecnologia/métodos , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/microbiologia , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Microbiologia de Alimentos/métodos , Humanos , Nanopartículas , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/microbiologia , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Vancomicina/farmacologia
2.
Nucleic Acids Res ; 48(18): 10527-10541, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32845304

RESUMO

YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.


Assuntos
Proteínas de Bactérias/ultraestrutura , Endorribonucleases/ultraestrutura , Proteínas de Escherichia coli/genética , Staphylococcus aureus/ultraestrutura , Sistemas Toxina-Antitoxina/genética , Antitoxinas/genética , Antitoxinas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/química , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Óperon/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Multimerização Proteica/genética , Staphylococcus aureus/química , Staphylococcus aureus/genética
3.
J Dairy Sci ; 103(8): 7407-7410, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32600771

RESUMO

The most clinically relevant staphylococci in veterinary medicine are those that are coagulase-positive, namely Staphylococcus aureus. During microbiological udder health monitoring (2009-2018), a new S. aureus strain (coagulase-positive and maltose-negative) was discovered as an emerging udder pathogen during routine examinations of South African dairy herds. This study challenged the conventional microbiological diagnosis of staphylococci by comparing its results to those of the MALDI-TOF mass spectrometry and 16S rRNA sequencing. Both of these tests confirmed that the maltose-negative staphylococcus (MNS), identified as Staphylococcus pseudintermedius by conventional microbiology, was S. aureus ST2992. Multi locus sequence typing was performed on 3 of the MNS isolates and indicated that these isolates were of single origin. These strains tested positive for both MALA and MALR genes (control: S. aureus ATCC 25923). Although the α-glucosidase gene was present, it was not expressed phenotypically. The latter may be attributed to the abnormal stop codon identified in the MALA gene sequence of S. aureus ST2992 (GenBank accession number, MN531305). The newly identified MNS has a field behavior different to that of maltose-positive S. aureus, and more similar to the low virulence of non-aureus staphylococci.


Assuntos
Mastite Bovina/microbiologia , Staphylococcus aureus/isolamento & purificação , Animais , Bovinos , Coagulase/análise , Feminino , Maltose/análise , Glândulas Mamárias Animais/microbiologia , Espectrometria de Massas , Tipagem de Sequências Multilocus , RNA Bacteriano , RNA Ribossômico 16S , Staphylococcus/classificação , Staphylococcus/isolamento & purificação , Staphylococcus aureus/química , Staphylococcus aureus/classificação
4.
Nature ; 582(7811): 294-297, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32523118

RESUMO

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.


Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/ultraestrutura , Parede Celular/química , Parede Celular/ultraestrutura , Microscopia de Força Atômica , Staphylococcus aureus/citologia , Staphylococcus aureus/ultraestrutura , Bacillus subtilis/química , Viabilidade Microbiana , Peptidoglicano/química , Peptidoglicano/isolamento & purificação , Peptidoglicano/ultraestrutura , Staphylococcus aureus/química
5.
Anal Chim Acta ; 1113: 18-25, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32340665

RESUMO

Magnetic trapping has been employed in the development of analytical methods owing to its ease and simplicity in handling samples. Nevertheless, the generation of functional probes is usually time consuming. A new and simple affinity method that uses gadolinium ion (Gd3+), a magnetic ion, as affinity probe for magnetic tapping of pathogenic bacteria was demonstrated in the present study. Escherichia coli O157:H7, Staphylococcus aureus, and Acinetobacter baumannii were selected as model bacteria. The model bacteria were magnetically isolated after incubation in Tris buffer (pH 8) containing Gd3+ (0.1 M) under microwave heating (power: 180 W, 90 s × 3). The resultant Gd3+-bacterium conjugates possessed sufficient magnetism, resulting in magnetic aggregations by an external magnet (∼4,000 Gauss). For ease of magnetic isolation, the sample containing Gd3+-bacterium complexes was stirred by a small magnet. After 1 h, the magnet attached with precipitates, i.e., Gd3+-bacterium conjugates, was readily removed using a pair of tweezers. The bacteria in the resultant conjugates were characterized by matrix-assisted laser desorption/ionization mass spectrometry. The limits of detection of the current approach toward E. coli O157:H7, S. aureus, and A. baumannii in complex samples were ∼104-105 cells mL-1.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Técnicas Bacteriológicas/métodos , Escherichia coli O157/isolamento & purificação , Gadolínio/química , Staphylococcus aureus/isolamento & purificação , Acinetobacter baumannii/química , Animais , Sangue/microbiologia , Bovinos , Complexos de Coordenação/química , Difosfatos/química , Escherichia coli O157/química , Limite de Detecção , Fenômenos Magnéticos , Microbiologia do Solo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus aureus/química
6.
Chemistry ; 26(34): 7657-7671, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32297355

RESUMO

A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE)) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The cell viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the complexes on mammalian cells. The heteroleptic complexes [BiPh(L)2 ] (in which L=flavonolate) showed good antibacterial activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependent manner. The homoleptic complexes [Bi(L)3 ] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line. Bismuth uptake studies in VRE and COS-7 cells treated with the bismuth flavonolate complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of substitution at the bismuth centre.


Assuntos
Antibacterianos/farmacologia , Bismuto/química , Complexos de Coordenação/química , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Escherichia coli/química , Bactérias Gram-Positivas/química , Humanos , Staphylococcus aureus Resistente à Meticilina/química , Pseudomonas aeruginosa/química , Staphylococcus aureus/química
7.
Immunity ; 52(4): 591-605.e6, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294405

RESUMO

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.


Assuntos
Endorribonucleases/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , RNA Bacteriano/metabolismo , RNA de Protozoário/metabolismo , Receptor 8 Toll-Like/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Endorribonucleases/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Escherichia coli/química , Escherichia coli/imunologia , Edição de Genes/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/imunologia , Monócitos/microbiologia , Monócitos/parasitologia , Neutrófilos/microbiologia , Neutrófilos/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Cultura Primária de Células , Estabilidade de RNA , RNA Bacteriano/imunologia , RNA de Protozoário/imunologia , Serratia marcescens/química , Serratia marcescens/imunologia , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Streptococcus/química , Streptococcus/imunologia , Células THP-1 , Receptor 8 Toll-Like/imunologia
8.
Chemistry ; 26(41): 8958-8968, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32198779

RESUMO

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 µm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Šresolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.


Assuntos
Cetol-Ácido Redutoisomerase/metabolismo , Magnésio/química , Mycobacterium tuberculosis/enzimologia , NADP/química , Staphylococcus aureus/metabolismo , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Humanos , Cetol-Ácido Redutoisomerase/química , Mycobacterium tuberculosis/química , NADP/metabolismo , Staphylococcus aureus/química
9.
Biochim Biophys Acta Biomembr ; 1862(7): 183280, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220553

RESUMO

Short linear antimicrobial peptides are attractive templates for developing new antibiotics. Here, it is described a study of the interaction between two short Trp-rich peptides, horine and verine-L, and model membranes. Isothermal titration calorimetry studies showed that the affinity of these peptides towards large unilamellar vesicles (LUV) having a lipid composition mimicking the lipid composition of S. aureus membranes is ca. 30-fold higher than that towards E. coli mimetics. The former interaction is driven by enthalpy and entropy, while the latter case is driven by entropy, suggesting differences in the forces that play a role in the binding to the two types of model membranes. Upon membrane binding the peptides acquired different conformations according to circular dichroism (CD) studies; however, in both cases CD studies indicated stacked W-residues. Peptide-induced membrane permeabilization, lipid flip-flop, molecular packing at the membrane-water interface, and lateral lipid segregation were observed in all cases. However, the extent of these peptide-induced changes on membrane properties was always higher in S. aureus than E. coli mimetics. Both peptides seem to act via a similar mechanism of membrane permeabilization of S. aureus membrane mimetics, while their mechanisms seem to differ in the case of E. coli. This may be the result of differences in both the peptides´ structure and the membrane lipid composition between both types of bacteria.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Conformação Molecular , Sequência de Aminoácidos/genética , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Biomimética , Calorimetria , Dicroísmo Circular , Escherichia coli/química , Escherichia coli/patogenicidade , Humanos , Staphylococcus aureus/química , Staphylococcus aureus/patogenicidade , Termodinâmica , Triptofano/química , Triptofano/genética , Lipossomas Unilamelares/química
10.
Mikrochim Acta ; 187(2): 119, 2020 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-31927667

RESUMO

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.


Assuntos
Colorimetria/métodos , DNA Bacteriano/análise , Staphylococcus aureus/isolamento & purificação , Animais , Armoracia/enzimologia , Benzidinas/química , Galinhas , Corantes/química , Sondas de DNA/química , Sondas de DNA/genética , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Limite de Detecção , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Aves Domésticas/microbiologia , Staphylococcus aureus/química
11.
Org Biomol Chem ; 18(5): 783-798, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31922180

RESUMO

The S. aureus bacterium is surrounded by capsular polysaccharides. These capsular polysaccharides are important in the pathogenesis of staphylococcal infection. There are 11 serotypes of capsular polysaccharides that have been identified, and a majority of strains express capsular polysaccharides type 5 (CP5) or 8 (CP8). The main focus of this review is to describe recent advances in the area of the chemical synthesis of monosaccharide components of S. aureus CP, oligosaccharide assembly and functionalization. Chemical routes to obtain oligosaccharides derived from CP1, CP5 and CP8 represent a compendium of modern classics of the total synthesis of challenging glycan sequences.


Assuntos
Cápsulas Bacterianas/química , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Staphylococcus aureus/química , Oligossacarídeos/síntese química , Oligossacarídeos/química , Polissacarídeos Bacterianos/síntese química
12.
Carbohydr Polym ; 232: 115801, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952600

RESUMO

The aim of this study was to use of bacterial cellulose/polypyrrole/TiO2-Ag (BC/PPy/TiO2-Ag) nanocomposite film to detect and measure the growth of 5 pathogenic bacteria. For this purpose, at first, 13 BC/PPy/TiO2-Ag films were fabricated, then bacterial suspensions were prepared according to McFarland standard. The results showed that by increasing the bacterial concentration, the electrical resistance of sensors was decreased and there was a relation between bacterial concentration and bacterial type with electrical resistance change of sensors. The obtained data showed that the sensitivity of the sensors was increased with increasing the concentration of polypyrrole and TiO2-Ag. FT-IR and SEM tests were performed to investigate the interaction between nanoparticles and determine the size of nanoparticles. The BC/PPy/TiO2-Ag biosensors are portable and the response time of these sensors is very short for target analysis. Therefore, these sensors have the potential to improve biological safety as diagnostic tools.


Assuntos
Aeromonas hydrophila/química , Celulose/química , Nanocompostos/química , Staphylococcus aureus/química , Staphylococcus epidermidis/química , Aeromonas hydrophila/crescimento & desenvolvimento , Tamanho da Partícula , Polímeros/química , Pirróis/química , Prata/química , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus epidermidis/crescimento & desenvolvimento , Propriedades de Superfície , Titânio/química
13.
Nat Chem Biol ; 16(1): 24-30, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31686030

RESUMO

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.


Assuntos
Lisostafina/química , Peptidoglicano/química , Staphylococcus aureus/química , Bacteriólise/efeitos dos fármacos , Biofilmes , Parede Celular/química , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Glicina/química , Ligantes , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Domínios de Homologia de src
14.
Biosens Bioelectron ; 150: 111945, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31818762

RESUMO

Developing reliable and sensitive detection methods for adenosine triphosphate (ATP) is vital for both clinical diagnosis and food safety. In this work, by coupling aptazyme- and catalytic hairpin assembly (CHA)-based signal amplification and electrochemiluminescence (ECL), an ultrasensitive biosensor for sensing ATP was fabricated using Ru(bpy)32+-doped silica nanoparticles (RuSiO2) as ECL probes and a ferrocene-functionalized hairpin DNA (hairpin-Fc) as quencher. The aptazyme-triggered cleavage of the DNA substrate and the CHA reaction both led to the circular release of trigger DNA, resulting in a significant dual signal amplification, with unprecedented enhancement up to 940-fold. Moreover, the bioconjugation of the DNA substrate with Au@Fe3O4 facilitated the separation and purification of the released trigger DNA, and effectively reduced the background signal. As a result, the as-prepared ECL biosensor exhibited a much lower detection limit of 0.054 pM for ATP, compared to those in previous reports, and showed high reliability for ATP detection in both spiked serum samples and Staphylococcus aureus. This work offers a new perspective for designing nucleic acid-based signal amplification for detecting ATP in bacterial analysis and clinical diagnosis.


Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Complexos de Coordenação/química , DNA Catalítico/química , Nanopartículas/química , Trifosfato de Adenosina/sangue , Técnicas Eletroquímicas/métodos , Compostos Ferrosos/química , Ouro/química , Humanos , Medições Luminescentes/métodos , Metalocenos/química , Dióxido de Silício/química , Staphylococcus aureus/química
15.
J Biol Chem ; 295(7): 1781-1791, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31819010

RESUMO

Iron is an essential nutrient for all living organisms. To acquire iron, many pathogens have developed elaborate systems to steal it from their hosts. The iron acquisition system in the opportunistic pathogen Staphylococcus aureus comprises nine proteins, called iron-regulated surface determinants (Isds). The Isd components enable S. aureus to extract heme from hemoglobin (Hb), transport it into the bacterial cytoplasm, and ultimately release iron from the porphyrin ring. IsdB and IsdH act as hemoglobin receptors and are known to actively extract heme from extracellular Hb. To limit microbial pathogenicity during infection, host organisms attempt to restrict the availability of nutrient metals at the host-pathogen interface. The human acute phase protein haptoglobin (Hp) protects the host from oxidative damage by clearing hemoglobin that has leaked from red blood cells and also restricts the availability of extracellular Hb-bound iron to invading pathogens. To investigate whether Hp serves an additional role in nutritional immunity through a direct inhibition of IsdH-mediated iron acquisition, here we measured heme extraction from the Hp-Hb complex by UV-visible spectroscopy and determined the crystal structure of the Hp-Hb-IsdH complex at 2.9 Å resolution. We found that Hp strongly inhibits IsdH-mediated heme extraction and that Hp binding prevents local unfolding of the Hb heme pocket, leaving IsdH unable to wrest the heme from Hb. Furthermore, we noted that the Hp-Hb binding appears to trap IsdH in an initial state before heme transfer. Our findings provide insights into Hp-mediated IsdH inhibition and the dynamics of IsdH-mediated heme extraction.


Assuntos
Antígenos de Bactérias/química , Haptoglobinas/química , Interações Hospedeiro-Patógeno/genética , Receptores de Superfície Celular/química , Infecções Estafilocócicas/genética , Cristalografia por Raios X , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Haptoglobinas/genética , Haptoglobinas/ultraestrutura , Heme/química , Heme/genética , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Ferro/química , Ferro/metabolismo , Ligação Proteica/genética , Conformação Proteica , Receptores de Superfície Celular/antagonistas & inibidores , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
16.
J Vis Exp ; (153)2019 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-31814606

RESUMO

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.


Assuntos
Bacillus subtilis/citologia , Proteínas de Bactérias/análise , Microscopia de Fluorescência/métodos , Staphylococcus aureus/citologia , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Divisão Celular , Transporte Proteico , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo
17.
Molecules ; 25(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31877955

RESUMO

For the analysis of volatile bacterial compounds, solid phase microextraction (SPME) is currently the most widely used metabolite concentration technique. Recently, the potential of stir bar sorptive extraction (SBSE) for this use has been demonstrated. These two approaches were therefore used in combination with gas-chromatography coupled with mass-spectrometry (GC-MS) for the analysis of volatile and semi-volatile bacterial compounds produced by Staphylococcus aureus. In both cases, SPME and SBSE/headspace sorptive extraction (HSSE) enrichment was carried out in two coating phases. A whole analytical and statistical process was developed to differentiate the metabolites produced from the metabolites consumed. The results obtained with SBSE/HSSE and SPME were compared and showed the recovery of 90% of the compounds by SBSE/HSSE. In addition, we were able to detect the production of 12 volatile/semi-volatile compounds by S. aureus, six of which had never been reported before. The extraction by SBSE/HSSE showed higher concentration capacities and greater sensitivity than SPME concerning bacterial compounds, suggesting that this technique may therefore become the new preferred option for bacterial volatile and semi-volatile compound analysis.


Assuntos
Hidrocarbonetos Aromáticos/química , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Compostos Orgânicos Voláteis/química , Aminas/química , Aminas/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Hidrocarbonetos Aromáticos/isolamento & purificação , Metaboloma/genética , Microextração em Fase Sólida , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/química , Compostos Orgânicos Voláteis/isolamento & purificação
18.
Sci Rep ; 9(1): 19311, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848419

RESUMO

In this study, we develop a real-time PCR strategy to directly detect and quantify DNA aptamers on functionalized graphene surfaces using a Staphylococcus aureus aptamer (SA20) as demonstration case. We show that real-time PCR allowed aptamer quantification in the range of 0.05 fg to 2.5 ng. Using this quantitative technique, it was possible to determine that graphene functionalization with amino modified SA20 (preceded by a graphene surface modification with thionine) was much more efficient than the process using SA20 with a pyrene modification. We also demonstrated that the functionalization methods investigated were selective to graphene as compared to bare silicon dioxide surfaces. The precise quantification of aptamers immobilized on graphene surface was performed for the first time by molecular biology techniques, introducing a novel methodology of wide application.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Grafite/química , Reação em Cadeia da Polimerase em Tempo Real , Ouro/química , Fenotiazinas/farmacologia , Staphylococcus aureus/química , Propriedades de Superfície
19.
ACS Appl Mater Interfaces ; 11(47): 43949-43963, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31684721

RESUMO

A graphene aerogel (GA) with a three-dimensional (3D) structure, ultra-lightweight nature, and high hydrophobicity was simply fabricated by the one-step pyrolysis of glucose and ammonium chloride. The as-synthesized GA exhibited a 3D interconnected microporous architecture with a high surface area of ∼2860 m2 g-1 and pore volume of 2.24 cm3 g-1. The hydrophobic GA (10 mg 100 mL-1) demonstrated rapid and excellent adsorption performance for the removal of food toxins such as various biogenic amines (histamine, cadaverine, and spermine) and the hazardous bacterium Staphylococcus aureus (a food contaminant and a cause of poor wound healing) from a liquid matrix with a maximum simultaneous adsorption capacity for multiple biogenic amines of >85.19% (histamine), 74.1% (cadaverine), and 70.11% (spermidine) and a 100% reduction in the viable cell count of S. aureus within 80 min of interaction. The outstanding adsorption capacity can be attributed to a highly interconnected porous network in the 3D architecture and a high surface-to-volume ratio. A case study using soy sauce spiked with multiple biogenic amines showed successful removal of toxins with excellent recyclability without any loss in absorption performance. Biocompatibility of the GA in terms of cell viability was observed even at high concentrations (83.46% and 75.28% at 25 and 50 mg mL-1, respectively). Confirmatory biocompatibility testing was conducted via live/dead cell evaluation, and the morphology of normal lung epithelial cells was examined via scanning electron microscopy showed no cellular shrinkage. Moreover, GA showed excellent removal of live colonies of S. aureus from the food matrix and immunoblotting analysis showed elevated protein expression levels of ß-catenin and α-SMA (α-smooth muscle actin). The biocompatible sugar-based GA could simultaneously adsorb multiple biogenic amines and live bacteria and was easy to regenerate via simple separation due to its high floatability, hydrophobicity, surface area, and porosity without any structural and functional loss, making it especially relevant for food safety and biomedical applications.


Assuntos
Aminas Biogênicas/química , Grafite/química , Alimentos de Soja/microbiologia , Staphylococcus aureus/química , Actinas/genética , Actinas/metabolismo , Adsorção , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Contaminação de Alimentos/análise , Géis/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação , beta Catenina/genética , beta Catenina/metabolismo
20.
Nat Commun ; 10(1): 4927, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666527

RESUMO

Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/classificação , Infecções Bacterianas/diagnóstico , Aprendizado Profundo , Análise Espectral Raman/métodos , Bactérias/química , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana , Candida/química , Candida/classificação , Enterococcus/química , Enterococcus/classificação , Escherichia coli/química , Escherichia coli/classificação , Humanos , Klebsiella/química , Klebsiella/classificação , Modelos Logísticos , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/classificação , Testes de Sensibilidade Microbiana , Redes Neurais de Computação , Análise de Componente Principal , Proteus mirabilis/química , Proteus mirabilis/classificação , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/classificação , Salmonella enterica/química , Salmonella enterica/classificação , Análise de Célula Única , Staphylococcus aureus/química , Staphylococcus aureus/classificação , Streptococcus/química , Streptococcus/classificação , Máquina de Vetores de Suporte
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA