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At least 19 recordsLinked to original sources
PubMed2026

A fluorescent reporter system for tracking Lactobacillus casei T1 in the murine gastrointestinal tract.

BACKGROUND: Fluorescent reporter systems are useful for studying probiotic colonization and host-microbe interactions. However, their use in lactic acid bacteria is still limited by relatively weak fluorescence signals, insufficient expression stability, and limited resolution during in vivo imaging. In particular, efficient strain-specific tracking systems remain scarce. METHODS: Here, we developed a red fluorescent reporter system for Lactobacillus casei T1 (L.c T1). Lactate dehydrogenase (LDH) promoters identified from the L.c T1 genome were compared with the constitutive P32 promoter to drive expression of the red fluorescent proteins mCherry and mKate. The different promoter-reporter combinations were evaluated in both Escherichia coli DH5α and L.c T1. Fluorescence expression was further examined under different environmental pH conditions. The optimized reporter strains were then evaluated by whole-body fluorescence imaging in living mice and ex vivo imaging of gastrointestinal tissues following oral administration. RESULTS: Among the constructs tested, P32-mKate produced the strongest and most stable fluorescence signal in L.c T1. Fluorescence intensity was influenced by environmental pH, with higher signals observed under mildly alkaline conditions. Whole-body fluorescence imaging showed that the engineered strain could be detected in living mice following oral administration. Ex vivo imaging of gastrointestinal tissues provided clearer localization of fluorescence, with signals mainly detected in the stomach and upper small intestine. CONCLUSION: We established a stable and efficient red fluorescent reporter system for L.c T1. The P32-mKate system enables detection of the engineered strain both in vitro and in vivo and provides a practical approach for tracking probiotic distribution and studying host-microbe interactions in preclinical animal models.

Lactobacillus casei T1↗
PubMed2005

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

Cell Separation↗
PubMed2003

A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis.

We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-ras(m) gene and an N-ras(m) suppressor, respectively. Retroviral vectors were produced in which human N-ras(m) (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-ras(m) was coexpressed with RFP. N-Ras(m) suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-ras(m) suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-ras(m)suppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-ras(m) suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-ras(m)-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.

Animals↗
PubMed1996

Detection of HIV-1 infection with a green fluorescent protein reporter system.

Several systems for the detection of HIV-1 have been described in which HIV-1-susceptible cells contain a reporter gene (chloramphenicol acetyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HIV-1, the expression of the viral tat product increases transcription from the HIV-1 LTR promoter, leading to high-level expression of the reporter gene product. Previously described reporter systems require processing of the cells by lysis, fixation, or other steps following infection to detect the reporter gene product. In the present study, the Aequorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV-1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. The construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.

CD4-Positive T-Lymphocytes↗
PubMed2000

Rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system.

A genetically modified cell line (Vero-ICP10-EGFP) was constructed for detection of herpes simplex virus (HSV) by a simple, rapid and direct method. The cell line was developed by stable transfection of Vero cell with a plasmid encoding the green fluorescent protein (GFP) driven by the promoter of the HSV-2 ICP10 gene. As early as 6 h after infection with HSV, fluorescence-emitting cells can be observed under a fluorescence microscope. A single infected cell emitting fluorescence can be observed with soft agar overlay by inverted fluorescence microscopy. No induction of detectable fluorescence was seen following infections with human cytomegalovirus (HCMV), varicella zoster virus (VZV), coxsackievirus A16 and enterovirus 71. Analysis by flow cytometry also demonstrated that intensity of the triggered fluorescence is proportional to the titer of HSV inoculated. Taken together, this novel GFP reporter system could become a useful means for rapid detection and quantification of HSV in clinical specimens.

Animals↗
PubMed2004

In vivo study of trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems.

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.

Chitin↗
PubMed1998

Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system.

A promoter probe vector, pGFPUV2, with Green Fluorescent Protein (GFP) as the reporter system was constructed to identify potential mycoplasmal promoter-containing regulatory sequences in E. coli. Libraries of M. pneumoniae and M. genitalium DNA constructed in pGFPUV2 and transformed into E. coli resulted in GFP-expressing clones. Primer extension analysis with E. coli RNA from five M. pneumoniae clones and two M. genitalium clones indicated that transcription originated from the insert DNA fragments of these promoter constructs. Primers based on the insert DNA sequences were used in primer extension reactions with total RNA isolated from M. pneumoniae or M. genitalium. Of the seven primers used, three generated products by primer extension with mycoplasmal RNA. However, only one of the DNAs had a 5' end similar to that obtained in a comparable reaction with E. coli RNA, and the start site of this transcript appeared to originate one base prior to the predicted open reading frame. These results indicate that E. coli can identify mycoplasmal promoters which have transcriptional elements resembling E. coli promoters.

Base Sequence↗
PubMed2006

Use of fluorescence resonance energy transfer for rapid detection of enteroviral infection in vivo.

Enteroviruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations. Recent outbreaks associated with enteroviral contamination in aquatic environments have called for the development of a more efficient and accurate virus monitoring system. To develop a simple, rapid, and direct method for identifying enteroviral infections, we generated a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2A protease (2A(pro)), flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein undergoing fluorescence resonance energy transfer. The covalent linkage between two fluorophores is disrupted due to 2A(pro) activity upon viral infection, which results in an increase in CFP intensity. This allows the rapid (within 7.5 h) detection of very low numbers (10 PFU or fewer) of infectious enteroviruses.

Amino Acid Sequence↗
PubMed2005

Release and report: a new photolabile caging system with a two-photon fluorescence reporting function.

A new efficient photocaging system with a fluorescence reporting function has been developed. The photolabile latch is based on adducts of C-nucleophiles with aromatic ketones, such as thioxanthones and xanthones. The system is designed to quantify the release of biological effectors and to monitor their spatial distribution and localization by single- and two-photon fluorescence microscopy. In the armed state the ketone's conjugation is disrupted by nucleophilic addition, resulting in a blue shift of the absorption maxima and a dramatic decrease in fluorescence intensity. The mechanism of the photoinduced uncaging involves homolytic C-C bond fragmentation followed by radical disproportionation, regenerating the carbonyl moiety and restoring fluorescence. The uncaging can be initiated via either a one- or two-photon process, offering a new powerful tool for molecular life sciences. The synthesis and uncaging of dendrimer- and polymeric bead-based model systems are described.

Ketones↗
PubMed2026

A Fibroblast-Based Adenoviral Reporter System Driven by the Mouse Collagen Type I Alpha 1 Promoter for Antifibrotic Drug Screening.

Cardiac fibrosis, characterized by aberrant fibroblast activation and excessive extracellular matrix deposition, lacks target-specific therapies, largely due to the absence of longitudinal, scalable, and non-destructive in vitro screening platforms. Traditional end-point assays and resource-intensive stem cell models inherently preclude real-time monitoring of fibrotic progression. To overcome these limitations, this protocol describes the generation, optimization, and validation of a mouse collagen type I alpha 1 (Col1a1) promoter-driven adenoviral mCherry fluorescent reporter system (Ad-mCol1a1p-mCherry) in NIH/3T3 fibroblasts. The critical steps for recombinant adenovirus packaging, transduction optimization (multiplicity of infection) to minimize cytotoxicity, and the establishment of a robust transforming growth factor beta (TGF-β)-induced fibrosis model are detailed. By circumventing the need for cell fixation, this system enables direct and longitudinal monitoring of collagen transcription in live cells. The model's specificity and reliability are pharmacologically validated using the TGF‑β type I receptor (ALK5) inhibitor SB431542, with fluorescent readouts correlating with endogenous fibrotic markers quantified via reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Ultimately, this cost-effective platform provides an accessible tool for the high-throughput screening of novel antifibrotic agents, thereby accelerating translational cardiovascular research.

Animals↗
PubMed1999

cobA, a red fluorescent transcriptional reporter for Escherichia coli, yeast, and mammalian cells.

We demonstrate the use of Propionibacterium freudenreichii uroporphyrinogen III methyltransferase (cobA) as a reporter of gene expression in Escherichia coli, fission yeast, and mammalian cells. Overexpression of cobA in cells resulted in bright red fluorescence that was visualized with standard fluorescence microscopy and fluorescence-activated cell sorting analysis at the single-cell level. As with green fluorescent protein (GFP), no addition of exogenous substrate was required. When expressed in Chinese hamster ovary cells from a bicistronic transcript, cobA and GFP gave rise to fluorescence signals of similar intensity. The bright red fluorescence generated by the cobA reporter promises a better signal-to-noise ratio than blue and green fluorescent reporter systems, as autofluorescence and light scattering of cells, media, and materials are reduced in the red wavelengths.

Animals↗
PubMed1998

Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.

The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.

Animals↗
PubMed2024

A Proteogenomic Pipeline for the Analysis of Protein Biosynthesis Errors in the Human Pathogen Candida albicans.

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37 °C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C. albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an in vivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C. albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C. albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity.

Candida albicans↗
PubMed2002

Molecular and cellular interactions between Brucella abortus antigens and host immune responses.

Host protection against Brucella abortus, is thought to be mediated primarily by a Th1 type immune response. Unfortunately, only few specific bacterial antigens involved in stimulating protective cellular immunity against Brucella are known. Therefore, identifying bacterial proteins that induce a T-lymphocyte mediated response is critical to determine Brucella immunity. Several library screening methods are discussed that have been used to identify Brucella proteins that stimulate T lymphocytes including cellular immunoblotting, Escherichia coli expressed Brucella proteins, green fluorescence reporter systems, and signature tagged mutagenesis. Future studies would likely examine how bacterial proteins expressed within host cells aid pathogen survival and/or induce host responses. Some of these newly identified bacterial gene products may serve as antigens to activate a protective host immune response. Also, identifying Brucella proteins expressed at particular times during infection will also yield insights into Brucella pathogenesis.

Animals↗
PubMed1998

Flow cytometry and bacterial pathogenesis.

Our understanding of microbial adaptations to diverse and threatening environments is limited by the assumption that the behavior of individual bacteria can be accurately determined by measuring the behavior of populations. Recent advances in gene expression reporter systems, fluorescence microscopy and flow cytometry allow microbiologists to explore the complex interactions between bacteria and their environment with single cell resolution. The application of these technologies has been particularly useful in systems, such as host-pathogen interactions, where genetic analysis is often cumbersome. Recently, flow cytometry is increasingly being applied to study host-pathogen interactions.

Bacterial Infections↗
PubMed2000

Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila.

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

Acanthamoeba↗
PubMed1997

Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications.

The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria. This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions. To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP. This modified gfp gene was also equipped with the strong translation signals of the atpE gene. Transfer of the mini-transposon into two different Pseudomonas spp. and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye. Individual GFP+ cells were readily observed by fluorescence microscopy.

Bacteria↗