RESUMEN

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay.

Asunto(s)

Genes Reporteros , Interferones , Luciferasas , Regiones Promotoras Genéticas , Elementos de Respuesta , Transducción de Señal , Humanos , Interferones/metabolismo , Interferones/genética , Luciferasas/metabolismo , Luciferasas/genética , Transfección , Animales

RESUMEN

Luciferase reporter systems are commonly used in scientific research to investigate a variety of biological processes, including antiviral innate immunity. These systems employ the use of luciferase enzymes derived from organisms such as fireflies or renilla reniformis, which emit light upon reaction with a substrate. In the context of antiviral innate immunity, the luciferase reporter systems offer a noninvasive and highly sensitive approach for real-time monitoring of immune responses in vitro and in vivo, enabling researchers to delve into the intricate interactions and signaling pathways involved in host-virus dynamic interactions. Here, we describe the methods of the promoter-luciferase reporter and enhancer-luciferase reporter, which provide insights into the transcriptional and post-transcriptional regulation of antiviral innate immunity. Additionally, we outline the split-luciferase complementary reporter method, which was designed to explore protein-protein interactions associated with antiviral immunity. These methodologies offer invaluable knowledge regarding the molecular mechanisms underlying antiviral immune pathways and have the potential to support the development of effective antiviral therapies.

Asunto(s)

Genes Reporteros , Inmunidad Innata , Luciferasas , Humanos , Luciferasas/metabolismo , Luciferasas/genética , Animales , Interferones/metabolismo , Interferones/inmunología , Regiones Promotoras Genéticas , Antivirales/farmacología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética

RESUMEN

Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.

Asunto(s)

Proteasas 3C de Coronavirus , Luciferasas , Humanos , Luciferasas/metabolismo , Luciferasas/genética , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Genes Reporteros , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Antivirales/farmacología , Células HEK293

RESUMEN

A luciferase reporter assay is a cell-based, enzymatic experiment that utilizes an ectopically expressed luciferase in cultured cells or in live animals, with the presence of its substrate luciferin, to generate luminescence in response to gene regulation at the level of transcription. This assay can be easily formatted for high-throughput sample analysis. The reagents are commercially available and routinely used in various applications. We have automated a luciferase reporter assay on the Opentrons OT-2 platform to measure activation of NF-kB in HeLa cells. The Python script-based protocols were developed to perform cell seeding, reporter construct transfection, and enzyme-catalytic reaction to produce detectable signals for quantification with desirable quality of sample handling and minimal hands-on time.

Asunto(s)

Automatización de Laboratorios , Genes Reporteros , Luciferasas , FN-kappa B , Humanos , Luciferasas/metabolismo , Luciferasas/genética , FN-kappa B/metabolismo , Células HeLa , Automatización de Laboratorios/métodos

RESUMEN

Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs.

Asunto(s)

Lípidos , Nanopartículas , Tamaño de la Partícula , Polietilenglicoles , ARN Mensajero , Transfección , Nanopartículas/química , Animales , Polietilenglicoles/química , Inyecciones Intramusculares , Lípidos/química , Transfección/métodos , Ratones , Técnicas de Transferencia de Gen , Humanos , Luciferasas/metabolismo , Luciferasas/genética , Propiedades de Superficie , Liposomas

RESUMEN

Gaussia luciferase (Gluc) is currently known as the smallest naturally secreted luciferase. Due to its small molecular size, high sensitivity, short half-life, and high secretion efficiency, it has become an ideal reporter gene and is widely used in monitoring promoter activity, studying protein-protein interactions, protein localization, high-throughput drug screening, and real-time monitoring of tumor occurrence and development. Although studies have shown that different Gluc mutations exhibit different bioluminescent properties, their mechanisms have not been further investigated. The purpose of this study is to reveal the relationship between the conformational changes of Gluc mutants and their bioluminescent properties through molecular dynamics simulation combined with neural relationship inference (NRI) and Markov models. Our results indicate that, after binding to the luciferin coelenterazine (CTZ), the α-helices of the 109-119 residues of the Gluc Mutant2 (GlucM2, the flash-type mutant) are partially unraveled, while the α-helices of the same part of the Gluc Mutant1 (GlucM1, the glow-type mutant) are clearly formed. The results of Markov flux analysis indicate that the conformational differences between glow-type and flash-type mutants when combined with luciferin substrate CTZ mainly involve the helicity change of α7. The most representative conformation and active pocket distance analysis indicate that compared to the flash-type mutant GlucM2, the glow-type mutant GlucM1 has a higher degree of active site closure and tighter binding. In summary, we provide a theoretical basis for exploring the relationship between the conformational changes of Gluc mutants and their bioluminescent properties, which can serve as a reference for the modification and evolution of luciferases.

Asunto(s)

Luciferasas , Cadenas de Markov , Simulación de Dinámica Molecular , Luciferasas/metabolismo , Luciferasas/genética , Luciferasas/química , Conformación Proteica , Mutación , Animales , Copépodos/enzimología , Copépodos/genética , Imidazoles/química , Imidazoles/metabolismo , Unión Proteica , Mediciones Luminiscentes , Pirazinas

RESUMEN

Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours.

Asunto(s)

Represión Catabólica , Genes Reporteros , Luciferasas , Neurospora crassa , Luciferasas/genética , Luciferasas/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Regulación Fúngica de la Expresión Génica , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/genética , Hongos/metabolismo , Carbono/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Represoras

RESUMEN

Bioluminescence imaging (BLI) is an important noninvasive optical imaging technique that has been widely used to monitor many biological processes due to its high sensitivity, resolution, and signal-to-noise ratio. However, the BLI technique based on the firefly luciferin-luciferase system is limited by the expression of exogenous luciferase and the short half-life of firefly luciferin, which pose challenges for long-term tracking in vivo. To solve the problems, here we rationally designed an intelligent strategy for persistent BLI in tumors by combining luciferase-loaded calcium phosphate nanoparticles (Luc@CaP NPs) to provide luciferase and the probe Cys(SEt)-Lys-CBT (CKCBT) to slowly produce the luciferase substrate amino luciferin (Am-luciferin). Luc@CaP NPs constructed with CaP as a carrier could enable luciferase activity to be maintained in vivo for at least 12 h. And compared to the conventional substrate luciferin, CKCBT apparently prolonged the BL time by up to 2 h through GSH-induced intracellular self-assembly and subsequent protease degradation-induced release of Am-luciferin. We anticipate that this strategy could be applied for clinical translation in more disease diagnosis and treatment in the near future.

Asunto(s)

Neoplasias de la Mama , Fosfatos de Calcio , Luciferasas , Mediciones Luminiscentes , Nanopartículas , Fosfatos de Calcio/química , Nanopartículas/química , Animales , Mediciones Luminiscentes/métodos , Humanos , Luciferasas/metabolismo , Luciferasas/química , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Femenino , Ratones , Imagen Óptica , Ratones Endogámicos BALB C , Línea Celular Tumoral , Ratones Desnudos

RESUMEN

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

Asunto(s)

Genes Reporteros , Luciferasas , Animales , Ratones , Luciferasas/metabolismo , Luciferasas/genética , Humanos , Distribución Tisular , Imagen Óptica/métodos , Mediciones Luminiscentes/métodos , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Cintigrafía/métodos , Línea Celular Tumoral

RESUMEN

In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Oryza , Proteínas de Plantas , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Regiones Promotoras Genéticas

RESUMEN

The outbreak of highly pathogenic influenza virus subtypes, such as H7 and H5, presents a significant global health challenge, necessitating the development of rapid and sensitive diagnostic methods. In this study, we have developed a novel dual-component biosensor assembly, each component of which incorporates an antibody fused with a nano-luciferase subunit. Our results demonstrate the effectiveness of this biosensor in enabling the rapid and sensitive detection of influenza H7 and other subtypes. Additionally, we successfully applied the biosensor in paper-based assay and lateral flow assay formats, expanding its versatility and potential for field-deployable applications. Notably, we achieved effective detection of the H7N9 virus using this biosensor. Furthermore, we designed and optimized a dedicated biosensor to the sensitive detection of the influenza H5 subtype. Collectively, our findings underscore the significant potential of this dual-component biosensor assembly as a valuable and versatile tool for accurate and timely diagnosis of influenza virus infections, promising to advance the field of influenza diagnostics and contribute to outbreak management and surveillance efforts.

Asunto(s)

Técnicas Biosensibles , Técnicas Biosensibles/métodos , Humanos , Gripe Humana/diagnóstico , Glicoproteínas Hemaglutininas del Virus de la Influenza/análisis , Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Luciferasas/química , Luciferasas/metabolismo , Luciferasas/genética

RESUMEN

Nipah virus (NiV) is an emerging zoonotic RNA virus that can cause fatal respiratory and neurological diseases in animals and humans. Accurate NiV diagnostics and surveillance tools are crucial for the identification of acute and resolved infections and to improve our understanding of NiV transmission and circulation. Here, we have developed and validated a split NanoLuc luciferase NiV glycoprotein (G) biosensor for detecting antibodies in clinical and animal samples. This assay is performed by simply mixing reagents and measuring luminescence, which depends on the complementation of the split NanoLuc luciferase G biosensor following its binding to antibodies. This anti-NiV-G "mix-and-read" assay was validated using the WHO's first international standard for anti-NiV antibodies and more than 700 serum samples from the NiV-endemic country of Bangladesh. Anti-NiV antibodies from survivors persisted for at least 8 years according to both ⍺NiV-G mix-and-read and NiV neutralization assays. The ⍺NiV-G mix-and-read assay sensitivity (98.6%) and specificity (100%) were comparable to anti-NiV IgG ELISA performance but failed to detect anti-NiV antibodies in samples collected less than a week following the appearance of symptoms. Overall, the anti-NiV-G biosensor represents a simple, fast, and reliable tool that could support the expansion of NiV surveillance and retrospective outbreak investigations.

Asunto(s)

Anticuerpos Antivirales , Técnicas Biosensibles , Infecciones por Henipavirus , Virus Nipah , Virus Nipah/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Henipavirus/diagnóstico , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Técnicas Biosensibles/métodos , Animales , Bangladesh/epidemiología , Luciferasas/genética , Sensibilidad y Especificidad , Femenino , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Masculino , Adolescente , Adulto Joven , Persona de Mediana Edad

RESUMEN

BACKGROUND/AIM: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. RESULTS: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. CONCLUSION: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal.

Asunto(s)

Luciferasas , Mediciones Luminiscentes , Neoplasias Pulmonares , Ratones Desnudos , Animales , Ratones , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/diagnóstico por imagen , Luciferasas/metabolismo , Luciferasas/genética , Línea Celular Tumoral , Mediciones Luminiscentes/métodos , Modelos Animales de Enfermedad , Humanos , Xenoinjertos , Benzotiazoles , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Asunto(s)

Fosfatasa Alcalina , COVID-19 , Luciferasas , SARS-CoV-2 , Anticuerpos de Cadena Única , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Fosfatasa Alcalina/química , Humanos , Luciferasas/química , Luciferasas/genética , COVID-19/diagnóstico , COVID-19/virología , Límite de Detección , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Biblioteca de Péptidos , Técnicas Biosensibles/métodos , Inmunoensayo/métodos

RESUMEN

Luminescent technology based on the luciferin-luciferase reaction has been extensively employed across various disciplines as a quantitative imaging modality. Owing to its non-invasive imaging capacity, it has evolved as a valuable in vivo bioimaging tool, particularly in small animal models in fields such as gene and cell therapies. We have previously successfully generated rats with a systemic expression of the luciferase gene at the Rosa26 locus. In this study, we transplanted bone marrow from these rats into micro-mini pigs and used in vivo imaging to non-invasively analyze the dynamics of the transplanted cells. In addition, we established that the rat-to-pig transplantation system is a discordant system, similar to the pig-to-human transplantation system. Thus, rat-to-pig transplantation may provide a clinically appropriate large animal model for pig-to-human xenotransplantation.

Asunto(s)

Trasplante de Médula Ósea , Luciferasas , Porcinos Enanos , Trasplante Heterólogo , Animales , Porcinos , Ratas , Trasplante de Médula Ósea/métodos , Trasplante Heterólogo/métodos , Luciferasas/metabolismo , Luciferasas/genética , Humanos , Mediciones Luminiscentes/métodos , Xenoinjertos , Luciferina de Luciérnaga/metabolismo , Luciferina de Luciérnaga/química

RESUMEN

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein-protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.

Asunto(s)

Virus de la Fiebre Aftosa , Fiebre Aftosa , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Línea Celular , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Luciferasas/genética , Luciferasas/metabolismo

RESUMEN

Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene Nluc encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, Bacillus subtilis, to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of Nluc mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The B. subtilis reporter strain BS2019 successfully revealed a decline in Nluc expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant Staphylococcus aureus. Production of Nluc mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work. IMPORTANCE: Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress.

Asunto(s)

Antibacterianos , Bacillus subtilis , Genes Reporteros , Luciferasas , Bacillus subtilis/genética , Bacillus subtilis/efectos de los fármacos , Antibacterianos/farmacología , Luciferasas/genética , Pruebas de Sensibilidad Microbiana

RESUMEN

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN , Reparación del ADN/fisiología , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Mediciones Luminiscentes/métodos , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo

RESUMEN

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.

Asunto(s)

Epilepsia , Neuronas , Optogenética , Animales , Concentración de Iones de Hidrógeno , Ratones , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Epilepsia/fisiopatología , Epilepsia/metabolismo , Epilepsia/tratamiento farmacológico , Humanos , Convulsiones/tratamiento farmacológico , Convulsiones/fisiopatología , Convulsiones/metabolismo , Halorrodopsinas/metabolismo , Halorrodopsinas/genética , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Masculino , Luciferasas/metabolismo , Luciferasas/genética , Células Piramidales/metabolismo , Células Piramidales/efectos de los fármacos , Imidazoles/farmacología , Pilocarpina/farmacología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Células HEK293 , Pirazinas

RESUMEN

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal ß-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

Asunto(s)

Técnicas Biosensibles , Luciferasas , Mediciones Luminiscentes , Humanos , Luciferasas/química , Luciferasas/metabolismo , Luciferasas/genética , Técnicas Biosensibles/métodos , Mediciones Luminiscentes/métodos , Conectina/química , Conectina/metabolismo , Vinculina/metabolismo , Vinculina/química