Real-time Infection Research Articles

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load. This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Real-time imaging of bacteria in living mice using a fluorescent dye

A novel technique developed in the laboratories of Bradley D. Smith and David Piwnica-Worms for imaging bacterial infections in intact living nude mice using a novel fluorescent dye, a conjugate of a NIR carbocyanine dye and two zinc(II) dipicolylamine units, allows relatively deep imaging of bacterial infection in real time. The behavior of the mice indicated good tolerance of the probe. The probe's water-octanol partition coefficient calculated by Hansch and Leo's procedure demonstrates that it is slightly lipophilic and therefore could enter mouse cells. Extant values of the physicochemical and spectroscopic parameters relevant to practical use are tabulated.

Invertebrate immunity: Infection in real time

Invertebrate immunity: Infection in real time

Drosophila embryos as model systems for monitoring bacterial infection in real time.

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.

Visualizing bacterial surfaces in real time

The study of the network of proteins, protein complexes, sugars and surfaces organelles has been frustrated by the complexity of bacterial surfaces and the necessity to rely on molecular coordinates. The logical consequence to develop crystallographic methods for large cellular components and the alternative use of cryo‐electron microscopy to solve organelles structure (flagellum, Type IV pili) has been fundamental but inherently slow. Development of new vaccines may also depend on the identification of complexes located at the surface and exposed protective molecules. Prediction of both is part of routine genome analysis using dedicated algorithms. Antibody staining and FACS analysis is a potent tool to visualize exposed molecules while it is heavily dependent from antibody specificity, affinity and outer layer penetration effect (detection of hidden structures by antibody penetration during preparation of the sample). We suggest it is now possible to merge high‐resolution fluorescence and scanning confocal microscopy with sortase tagging (Popp et al., 2007) to generate native molecules bearing a chromophore. This will potentially allow image rendering of the bacterial surface to visualize the dynamics of protein topology during growth and infection in real time. Science and Nature have both expressed their wonder in 2008 about recent advances in light microscopy (Chi, 2008). In addition, white light sources are moving from lasers to inexpensive mass production of LED (http://www.lens.unifi.it). White light is a necessary step to pulse a sample with a wide range of light wavelengths to simultaneously collect signals in the visible spectrum from excited dyes. The resulting live image can integrate all labelled proteins in a single picture and with a different colour tag providing realistic 3D coordinates. The crucial step in closing the loop is in vivo tagging of a target protein. This in theory can provide a repertoire of one strain‐one tagged protein with the final goal to colour‐code all the proteins of a bacterial species. Sortases are bacterial enzymes that predominantly catalyse the attachment of surface proteins to the bacterial cell wall (Telford et al., 2006; Popp et al., 2007). Other sortases polymerize pilin subunits for the construction of the covalently attached pili of the Gram‐positive bacteria (Telford et al., 2006). The sortase recognition sequence of Staphylococcus aureus sortase A, LPXTG, when engrafted near the C‐terminus of proteins without natural sortase specificity, should be part of a sortase‐catalysed transpeptidation reaction using artificial glycine‐based nucleophiles. The chemical modification of such substrates with fluorophores allows modifications of proteins in in vitro and in vivo conditions. This method can be efficiently scaled‐up for high‐throughput data capture. Once the expected wave of new microscopes will be available and tagging with fluorophores will be pervasive, this technology should be ready also for fast and unexpensive real‐time expression analyses.

An experimental in-vivo canine model for adult shunt infection

BackgroundDetailed human studies of the mechanisms and development of shunt infection in real time are not possible, and we have developed a canine hydrocephalus model to overcome this. The intention of this pilot study was to show that the canine hydrocephalus model could be shunted using conventional "human" shunts, and that a shunt infection could be established so that further studies could then be planned.MethodsHydrocephalus was induced in seven dogs (Canis familiaris) by fourth ventricle obstruction. Four weeks later they were shunted using a Hakim Precision valve. Four of the dogs received shunts whose ventricular catheter had been inoculated with Staphylococcus epidermidis, and three were uninoculated controls. Four weeks after shunting the dogs were sacrificed and necropsy was performed. Removed shunts and tissue samples were examined microbiologically and isolates were subjected to detailed identification and genomic comparison.ResultsAll the dogs remained well after shunting. Examination of removed shunt components revealed S. epidermidis in the brain and throughout the shunt system in the four inoculated animals, but in two of these Staphylococcus intermedius was also found. S. intermedius was also isolated from all three "negative" controls. There were slight differences between S. intermedius strains suggesting endogenous infection rather than cross- infection from a point source.ConclusionShunt infection was established in the canine model, and had the experiment been extended beyond four weeks the typical microbiological, pathological and clinical features might have appeared. The occurrence of unplanned shunt infections in control animals due to canine normal skin flora reflects human clinical experience and underlines the usual source of bacteria causing shunt infection.

Zebrafish and Frog Models of Mycobacterium marinum Infection

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium-host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

Infection in Real Time

Infection in Real Time

Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging

Toxoplasma gondii infection can lead to life-threatening systemic disease in the immunocompromised individual and in the developing fetus. Despite intensive investigation in animal models of toxoplasmosis, the processes leading to systemic dissemination remain poorly characterized. In the present study, in vivo bioluminescence imaging (BLI) was applied to the Toxoplasma mouse model to study the dynamics of infection in real time. Photon emission analyses revealed rapid dissemination of parasites in the organism and dissemination to immunoprivileged organs (brain, eyes and testes). Spatio-temporal analysis by BLI in individual mice showed that the virulent RH strain (type I) and the non-virulent ME49/PTG strain (type II) disseminate widely, but the virulent RH strain (type I) exhibits a more dramatic expansion of parasite biomass. Assessment by BLI of the Toll/interleukin-1 receptor (TIR) signalling pathway in host resistance to T. gondii revealed that signal transduction to the adaptor protein MyD88 is probably mediated by Toll-like receptor(s) rather than by IL-1R or IL-18R signalling. However, TLR1(-/-), TLR2(-/-), TLR4(-/-), TLR6(-/-) and TLR9(-/-) animals did not exhibit increased susceptibility to infection. These results suggest that intricate mechanisms regulate TIR-mediated responses during Toxoplasma infection.

Infection of human oligodendroglioma cells by a recombinant measles virus expressing enhanced green fluorescent protein.

One of the hallmarks of the human CNS disease subacute sclerosing panencephalitis (SSPE) is a high level of measles virus (MV) infection of oligodendrocytes. It is therefore surprising that there is only one previous report of MV infection of rat oligodendrocytes in culture and no reports of human oligodendrocyte infection in culture. In an attempt to develop a model system to study MV infection of oligodendrocytes, time-lapse confocal microscopy, immunocytochemistry, and electron microscopy (EM) were used to study infection of the human oligodendroglioma cell line, MO3.13. A rat oligodendrocyte cell line, OLN-93, was also studied as a control. MO3.13 cells were shown to be highly susceptible to MV infection and virus budding was observed from the surface of infected MO3.13 cells by EM. Analysis of the infection in real time and by immunocytochemistry revealed that virus spread occurred by cell-to-cell fusion and was also facilitated by virus transport in cell processes. MO3.13 cells were shown to express CD46, a MV receptor, but were negative for the recently discovered MV receptor, signaling leucocyte activation molecule (SLAM). Immunohistochemical studies on SSPE tissue sections demonstrated that CD46 was also expressed on populations of human oligodendrocytes. SLAM expression was not detected on oligodendrocytes. These studies, which are the first to show MV infection of human oligodendrocytes in culture, show that the cells are highly susceptible to MV infection and this model cell line has been used to further our understanding of MV spread in the CNS.

Video imaging of firefly luciferase activity to identify and monitor herpesvirus infection in cell culture

Expression of reporter genes incorporated into complex viral genomes is being used increasingly to monitor virus infection in cell culture and in the organism. One of the most sensitive markers is luciferase from Photinus pyralis which catalyzes a luminescent reaction that can be traced in cell and organ extracts in luminometers. A novel method is described for monitoring Iuciferase activity after infection of cells with recombinant herpesvirus (pseudorabies virus) which carries stably and expresses luciferase using ultra high sensitive photon-counting enhanced video image acquisition. The data show that firefly luciferase activity in virus-infected cells can be monitored without destruction of the cells using video equipment for either macroscopic image acquisition or microscopy. Resolution down to a single-cell level can thereby be achieved. This method increases greatly the potential of monitoring virus infection in real time with a non-destructive highly sensitive method in cell culture and should also help to assay viral spread in the animal.