24 In vivo imaging of fungal infections Flashcards
Definition of in vivo Imaging
non-invasive technique for investigation of cells/pathogens in living animals
how is virulence investigated
in vivo imaging
what are the advantages of in vivo imaging
- Repeated investigation of individual animals
- Monitoring of pathogen and/or immune cells in temporal and spatial resolution
- Insights on disease progression from small animal groups
what are the disadvantages of in vivo imaging
Clinical and environmental isolates require genetic modification (reporter strains)
„High-throughput” not possible due to time-consuming measurements
what are the common techniques in in vivo imaging
Light-based
Fluorescence-Imaging
Bioluminescence-Imaging
what are the advantages of fluorescence imaging
- Large selection of fluorescent reporters available
- Different excitation and emission wavelength allows multiple labellin
what are the disadvantages of fluorescence imaging
- High background fluorescence from animals à low sensitivity
- Low-wavelength excitation reporters difficult to activate within tissues
- Small band-width for detection of emission spectra (overlap with excitation)
Bioluminescence imaging
Advantages
- Hardly background luminescence from animals
- Very high sensitivity
Bioluminescence imaging disadvantages
- Small number of reporters available
- Frequently substrate injection required
- Biochemical reaction required for light production that might affect fitness
what are the Luciferase reporters
Firefly, Renilla and Gaussia commonly used in eukaryotes
what is the lux operon not suitable in
bacterial “lux” operon
Not suitable for eukaryotic systems
what does Gaussia luciferase possess
very high quantum yield
what is the adavantage of secretion - Gaussia luciferases
Secreted luciferase can be detected from fluids
what is the disadvantages of secretion - Gaussia luciferases
- Secretion is not quantitative
- Difficult to detect infection foci
Firefly luciferase for in vivo imaging advantages
Substrate D-luciferin highly water soluble = Transported to all body sites
Light emission in vivo = Reduced absorption by haemoglobin
Codon-optimisation of reporter for optimal sensitivity
what is an example of codon optimisation of luciferase sequences
Example on the filamentous fungus Aspergillus fumigatus
what is Candida albicans like
dimorphic (polymorphic fungus)
where does dissemination occur in candida albicans
host via yeast cells
how does tissue invasion occur in candida albicans
by hyphal filaments
what infection does candida albicans cause
Superficial infections
Invasive Candida albicans infections
Nosocomial acquisition
who gets Candida albicans
Superficial infections
3/4 of all healthy women experience at least one vaginal yeast infection
Oral Candida infections affect > 90% of HIV infected patients
how frequent are Candida albicans infections
Candida sp. are the third most frequent nosocomial bloodstream isolates in the USA
what does Firefly luciferase work as
reporter gene
how does in vivo investigation of C. albicans pathogenicity occur
Generation of bioluminescent C. albicans reporter strains
Selected promoter
how is extra-renal sites of infection detected
Ex vivo
Analysis of mice with signals outside the kidney localisation
Mouse 4 displays an additional signal
how is the suitability of reporter strains for disease monitoring
= Disease progression monitored from individual mice
= Major and minor sites of infection detected
= Light intensity correlates with fungal burden
what is the meaning of luminescence in evalution
Signals decrease to background under therapy
How can C. albicans persist in the gall bladder under therapy
Bile confers resistance against Caspofungin
can other antifungal drugs persist in gall bladder under therapy
Bile confers a decrease in susceptibility against a wide range of different antifungal
what is BLI
powerful tool to study fungal infections
what does the selection of luciferase depend on
study aims
what are firefly luciferase best suited for
deep-seated infections
what does bile form
micelles
what is the effect of micelles
reduced bioavailability of drugs
what may gall bladder form
cryptic reservoir for re-infection
