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1.
Harmful Algae ; 132: 102566, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38331538

RESUMO

Pelagic Sargassum in the Gulf of Mexico (GoM) plays an important role in ocean biology and ecology, yet our knowledge of its origins and transport pathways is limited. Here, using satellite observations of Sargassum areal density and ocean surface currents between 2000 and 2023, we show that large amounts of Sargassum in the GoM can either originate from the northwestern GoM or be a result of physical transport from the northwestern Caribbean Sea, both with specific transport pathways. Sargassum of the northwestern GoM can be transported to the eastern GoM by ocean currents and eddies, eventually entering the Sargasso Sea. Sargassum entering the GoM from the northwestern Caribbean Sea can be transported in three different directions, with the northward and eastward transports governed by the Loop Current System (LCS) and westward transport driven by the westward extension of the LCS, the propagation or relaying of ocean eddies, the wind-driven westward currents on the Campeche Bank with or without eddies, and the westward currents with/without currents associated with eddies in the northern/central GoM. Overall, the spatial distribution patterns of pelagic Sargassum in the GoM are strongly influenced by the LCS and relevant eddies.


Assuntos
Sargassum , Golfo do México , Meio Ambiente , Região do Caribe , Ecologia
2.
Analyst ; 149(4): 1289-1296, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38240377

RESUMO

Streptococcus pneumoniae (S. pneumoniae) represents a significant pathogenic threat, often responsible for community-acquired pneumonia with potentially life-threatening consequences if left untreated. This underscores the pressing clinical need for rapid and accurate detection of this harmful bacteria. In this study, we report the screening and discovery of a novel biomarker for S. pneumoniae detection. We used S. pneumoniae nucleases as biomarker and we have identified a specific oligonucleotide that works as substrate. This biomarker relies on a specific nuclease activity found on the bacterial membrane, forming the basis for the development of both fluorescence and electrochemical biosensors. We observed an exceptionally high sensitivity in the performance of the electrochemical biosensor, detecting as low as 102 CFU mL-1, whereas the fluorescence sensor demonstrated comparatively lower efficiency, with a detection limit of 106 CFU mL-1. Moreover, the specificity studies have demonstrated the biosensors' remarkable capacity to identify S. pneumoniae from other pathogenic bacteria. Significantly, both biosensors have demonstrated the ability to identify S. pneumoniae cultured from clinical samples, providing compelling evidence of the potential clinical utility of this innovative detection system.


Assuntos
Bactérias , Streptococcus pneumoniae , Sondas de Oligonucleotídeos , Biomarcadores
3.
J Control Release ; 361: 260-269, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37541593

RESUMO

Nucleoside analogues have been in clinical use since 1960s and they are still used as the first therapeutic option for several cancers and viral infections, due to their high therapeutic efficacy. However, their wide clinical acceptance has been limited due to their high toxicity and severe side effects to patients. Herein, we report on a nanocarrier system that delivers nucleosides analogues in a target-specific manner, making nucleoside-based therapeutics safer and with the possibility to be used in other human conditions. This system, named, Therapeutic OligonUCleotides Activated by Nucleases" (TOUCAN) combines: i) the recognition power of oligonucleotides as substrates, ii) the use of nucleases as enzymatic biomarkers and iii) the clinical efficacy of nucleoside analogues, in a single approach. As a proof-of-concept, we report on a TOUCAN that is activated by a specific nuclease produced by bacteria and releases a therapeutic nucleoside, floxuridine. We demonstrate, for the first time, that, by incorporating a therapeutic nucleoside analogue into oligonucleotide probes, we can specifically inhibit bacterial growth in cultures. In this study, Staphylococcus aureus was selected as the targeted bacteria and the TOUCAN strategy successfully inhibited its growth with minimal inhibitory concentration (MIC) values ranging from 0.62 to 40 mg/L across all tested strains. Moreover, our results indicate that the intravenous administration of TOUCANs at a dose of 20 mg/kg over a 24-h period is a highly effective method for treating bacterial infections in a mouse model of pyomyositis. Importantly, no signs of toxicity were observed in our in vitro and in vivo studies. This work can significantly impact the current management of bacterial infections, laying the grounds for the development of a different class of antibiotics. Furthermore, it can provide a safer delivery platform for clinical nucleoside therapeutics in any human conditions, such as cancer and viral infection, where specific nuclease activity has been reported.


Assuntos
Neoplasias , Nucleosídeos , Animais , Camundongos , Humanos , Nucleosídeos/uso terapêutico , Nucleosídeos/farmacologia , Oligonucleotídeos/uso terapêutico , Neoplasias/tratamento farmacológico
4.
Expert Rev Mol Diagn ; 22(3): 265-294, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35240900

RESUMO

INTRODUCTION: In the increasingly challenging field of clinical microbiology, diagnosis is a cornerstone whose accuracy and timing are crucial for the successful management, therapy, and outcome of infectious diseases. Currently employed biomarkers of infectious diseases define the scope and limitations of diagnostic techniques. As such, expanding the biomarker catalog is crucial to address unmet needs and bring about novel diagnostic functionalities and applications. AREAS COVERED: This review describes the extracellular nucleases of 15 relevant bacterial pathogens and discusses the potential use of nuclease activity as a diagnostic biomarker. Articles were searched for in PubMed using the terms: 'nuclease,' 'bacteria,' 'nuclease activity' or 'biomarker.' For overview sections, original and review articles between 2000 and 2019 were searched for using the terms: 'infections,' 'diagnosis,' 'bacterial,' 'burden,' 'challenges.' Informative articles were selected. EXPERT OPINION: Using the catalytic activity of nucleases offers new possibilities compared to established biomarkers. Nucleic acid activatable reporters in combination with different transduction platforms and delivery methods can be used to detect disease-associated nuclease activity patterns in vitro and in vivo for prognostic and diagnostic applications. Even when these patterns are not obvious or of unknown etiology, screening platforms could be used to identify new disease reporters.


Assuntos
Infecções Bacterianas , Doenças Transmissíveis , Bactérias/genética , Infecções Bacterianas/diagnóstico , Biomarcadores , Endonucleases , Humanos
5.
Biomark Res ; 9(1): 86, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34809722

RESUMO

Early cancer diagnosis is a crucial element to improved treatment options and survival. Great research efforts have been made in the search for better performing cancer diagnostic biomarkers. However, the quest continues as novel biomarkers with high accuracy for an early diagnosis remain an unmet clinical need. Nucleases, which are enzymes capable of cleaving nucleic acids, have been long considered as potential cancer biomarkers. The implications of nucleases are key for biological functions, their presence in different cellular counterparts and catalytic activity led the enthusiasm towards investigating the role of nucleases as promising cancer biomarkers. However, the most essential feature of these proteins, which is their enzymatic activity, has not been fully exploited. This review discusses nucleases interrogated as cancer biomarkers, providing a glimpse of their physiological roles. Moreover, it highlights the potential of harnessing the enzymatic activity of cancer-associated nucleases as a novel diagnostic biomarker using nucleic acid probes as substrates.

6.
Diagnostics (Basel) ; 11(11)2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34829369

RESUMO

The identification of pathogens causing infectious diseases is still based on laborious and time-consuming techniques. Therefore, there is an urgent need for the development of novel methods and devices that can considerably reduce detection times, allowing the health professionals to administer the right treatment at the right time. Lateral flow-based systems provide fast, cheap and easy to use alternatives for diagnosis. Herein, we report on a lateral flow approach for specifically detecting S. aureus bacteria within 6 h.

7.
Anal Bioanal Chem ; 413(30): 7353-7362, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34704109

RESUMO

Activatable fluorescent probes have been successfully used as molecular tools for biomedical research in the last decades. Fluorescent probes allow the detection of molecular events, providing an extraordinary platform for protein and cellular research. Nevertheless, most of the fluorescent probes reported are susceptible to interferences from endogenous fluorescence (background signal) and limited tissue penetration is expected. These drawbacks prevent the use of fluorescent tracers in the clinical setting. To overcome the limitation of fluorescent probes, we and others have developed activatable magnetic resonance probes. Herein, we report for the first time, an oligonucleotide-based probe with the capability to detect bacteria using magnetic resonance imaging (MRI). The activatable MRI probe consists of a specific oligonucleotide that targets micrococcal nuclease (MN), a nuclease derived from Staphylococcus aureus. The oligonucleotide is flanked by a superparamagnetic iron oxide nanoparticle (SPION) at one end, and by a dendron functionalized with several gadolinium complexes as enhancers, at the other end. Therefore, only upon recognition of the MRI probe by the specific bacteria is the probe activated and the MRI signal can be detected. This approach may be widely applied to detect bacterial infections or other human conditions with the potential to be translated into the clinic as an activatable contrast agent.


Assuntos
Corantes Fluorescentes/química , Imageamento por Ressonância Magnética/métodos , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Limite de Detecção , Microscopia Eletrônica de Transmissão , Espectrofotometria Ultravioleta
8.
J Med Chem ; 64(17): 12855-12864, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34460263

RESUMO

An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure-activity relationship analysis, propose a catalytically active substrate-enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme-biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process.


Assuntos
Proteínas de Bactérias/química , Desoxirribonucleases/metabolismo , Desenho de Fármacos , Oligonucleotídeos/química , Staphylococcus aureus/metabolismo , Técnicas Biossensoriais , Cromatografia Líquida , Desoxirribonucleases/genética , Espectrometria de Massas , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Especificidade por Substrato
9.
Cancers (Basel) ; 13(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33451046

RESUMO

Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic.

10.
J Vis Exp ; (153)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31736483

RESUMO

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable. Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes' design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.


Assuntos
Endonucleases/metabolismo , Escherichia coli/enzimologia , Sondas de Ácido Nucleico/metabolismo , Ácidos Nucleicos/análise , Salmonella/enzimologia , Endonucleases/isolamento & purificação , Humanos , Cinética , Sondas de Ácido Nucleico/química
11.
Anal Chim Acta ; 1054: 157-166, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30712586

RESUMO

Salmonella is a leading source of bacterial foodborne illness in humans, causing gastroenteritis outbreaks with bacteraemia occurrences that can lead to clinical complications and death. Eggs, poultry and pig products are considered as the main carriers of the pathogenic Salmonella for humans. To prevent this relevant zoonosis, key changes in food safety regulations were undertaken to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis were reported worldwide in the last decade. Thus, new strategies for Salmonella detection are a priority for both, food safety and public health authorities. Such detection systems should provide significant reduction in diagnostic time (hours) compared to the currently available methods (days). Herein, we report on the discovery and characterization of nucleic acid probes for the sensitive and specific detection of live Salmonella within less than 8 h of incubation. We are the first to postulate the nuclease activity derived from Salmonella as biomarker of infection and its utility to develop innovative detection strategies. Our results have shown the screening and identification of two oligonucleotide sequences (substrates) as the most promising probes for detecting Salmonella - Sal-3 and Sal-5. The detection limits for both probes were determined with the reference Salmonella Typhimurium (STM 1) and Salmonella Enteritidis (SE 1) cultures. Sal-3 has reported LOD values around 105 CFU mL-1 for STM 1 and 104 CFU mL-1 for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 104 CFU mL-1 for STM 1 and 104 CFU mL-1 for SE 1. Both selected probes have shown the capability to recognize 49 out of 51 different Salmonella serotypes tested in vitro and the most frequent serotypes in porcine mesenteric lymph nodes as a standard sample used in fattening-pig salmonellosis baseline studies. Notably, our results showed 100% correlation between nuclease detection and the PCR-InvA or ISO-6579 standard method, underlining the great potential of this innovative nucleic acids technology to be implemented as a rapid method for food safety testing.


Assuntos
Microbiologia de Alimentos/métodos , Sondas de Oligonucleotídeos/metabolismo , Salmonella/isolamento & purificação , Salmonella/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Inocuidade dos Alimentos , Limite de Detecção , Modelos Moleculares , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Conformação Proteica , Salmonella/enzimologia , Fatores de Tempo
12.
Sci Rep ; 9(1): 178, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655587

RESUMO

The northern Gulf of Mexico (GoM) is a region strongly influenced by river discharges of freshwater and nutrients, which promote a highly productive coastal ecosystem that host commercially valuable marine species. A variety of climate and weather processes could potentially influence the river discharges into the northern GoM. However, their impacts on the coastal ecosystem remain poorly described. By using a regional ocean-biogeochemical model, complemented with satellite and in situ observations, here we show that El Niño - Southern Oscillation (ENSO) is a main driver of the interannual variability in salinity and plankton biomass during winter and spring. Composite analysis of salinity and plankton biomass anomalies shows a strong asymmetry between El Niño and La Niña impacts, with much larger amplitude and broader areas affected during El Niño conditions. Further analysis of the model simulation reveals significant coastal circulation anomalies driven by changes in salinity and winds. The coastal circulation anomalies in turn largely determine the spatial extent and distribution of the ENSO-induced plankton biomass variability. These findings highlight that ENSO-induced changes in salinity, plankton biomass, and coastal circulation across the northern GoM are closely interlinked and may significantly impact the abundance and distribution of fish and invertebrates.

13.
Chem Sci ; 8(10): 7038-7046, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29147531

RESUMO

A novel catalytic system based on covalently modified DNA is described. This catalyst promotes 1,3-dipolar reactions between azomethine ylides and maleimides. The catalytic system is based on the distortion of the double helix of DNA by means of the formation of Pt(ii) adducts with guanine units. This distortion, similar to that generated in the interaction of DNA with platinum chemotherapeutic drugs, generates active sites that can accommodate N-metallated azomethine ylides. The proposed reaction mechanism, based on QM(DFT)/MM calculations, is compatible with thermally allowed concerted (but asynchronous) [π4s + π2s] mechanisms leading to the exclusive formation of racemic endo-cycloadducts.

14.
Chem Commun (Camb) ; 52(83): 12346-12349, 2016 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-27722495

RESUMO

We report on the activity of nucleases derived from cancer cells as a means for specific targeting using nucleic acid probes (substrates). We hypothesize that cancer cells can be differentiated from healthy cells based on their nuclease activity profile, and thus, any method based on this property represents a novel alternative for diagnostic and therapeutic intervention.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Desoxirribonucleases/análise , Sondas de Ácido Nucleico/química , Biomarcadores Tumorais/metabolismo , Desoxirribonucleases/metabolismo , Feminino , Humanos , Sondas de Ácido Nucleico/metabolismo
15.
Biosens Bioelectron ; 86: 27-32, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27318566

RESUMO

A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood.


Assuntos
Bacteriemia/sangue , Bacteriemia/diagnóstico , Técnicas de Tipagem Bacteriana/instrumentação , Sondas de DNA/genética , DNA Bacteriano/sangue , Staphylococcus aureus/isolamento & purificação , Análise Química do Sangue/instrumentação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Nanopartículas de Magnetita/química , Nanoconjugados/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício/química , Staphylococcus aureus/genética
16.
Analyst ; 141(8): 2595-9, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27041474

RESUMO

A fast, sensitive and ratiometric biosensor strategy for small molecule detection was developed through nanopore actuation. The new platform engineers together, a highly selective molecular recognition element, aptamers, and a novel signal amplification mechanism, gated nanopores. As a proof of concept, aptamer gated silica nanoparticles have been successfully used as a sensing platform for the detection of ATP concentrations at a wide linear range from 100 µM up to 2 mM.

17.
Curr Top Med Chem ; 15(12): 1066-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25866270

RESUMO

Apatamer technology has been around for a quarter of a century and the field had matured enough to start seeing real applications, especially in the medical field. Since their discovery, aptamers rapidly emerged as key players in many fields, such as diagnostics, drug discovery, food science, drug delivery and therapeutics. Because of their synthetic nature, aptamers are evolving at an exponential rate gaining from the newest advances in chemistry, nanotechnology, biology and medicine. This review is meant to give an overview of the aptamer field, by including general aspects of aptamer identification and applications as well as highlighting certain features that contribute to their quick deployment in the biomedical field.


Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos/métodos , Terapia de Alvo Molecular/métodos , Aptâmeros de Nucleotídeos/uso terapêutico , Bioensaio , Descoberta de Drogas , Tecnologia de Alimentos/métodos , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/patologia , Kit de Reagentes para Diagnóstico , Técnica de Seleção de Aptâmeros , Bibliotecas de Moléculas Pequenas/uso terapêutico , Coloração e Rotulagem/métodos
18.
Chem Commun (Camb) ; 50(67): 9489-92, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25008577

RESUMO

Bacterial resistance is a high priority clinical issue worldwide. Thus, an effective system that rapidly provides specific treatment for bacterial infections using controlled dose release remains an unmet clinical need. Herein, we report on the NanoKeepers approach for the specific targeting of S. aureus with controlled release of antibiotics based on nuclease activity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Portadores de Fármacos/química , Nanocápsulas/química , Antibacterianos/metabolismo , Preparações de Ação Retardada , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Conformação Molecular , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Vancomicina/química , Vancomicina/metabolismo , Vancomicina/farmacologia
19.
Mol Ther ; 22(11): 1910-22, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24954476

RESUMO

Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.


Assuntos
Antígenos de Superfície/metabolismo , Aptâmeros de Nucleotídeos/administração & dosagem , Glutamato Carboxipeptidase II/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Animais , Aptâmeros de Nucleotídeos/farmacocinética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Metástase Neoplásica , Ensaios Antitumorais Modelo de Xenoenxerto
20.
PLoS One ; 9(4): e95574, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24752186

RESUMO

Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Endonucleases/metabolismo , Espaço Extracelular/enzimologia , Staphylococcus aureus/enzimologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biofilmes , Endonucleases/química , Endonucleases/isolamento & purificação , Camundongos , Transporte Proteico , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Homologia Estrutural de Proteína
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