When studying how infections grow and spread it is always helpful to be able to see the organism causing the disease. There are currently a range of microbial and labelling techniques available to view micro-organisms within the cells they infect, and one of the most useful is bioluminescence imaging. This takes advantage of a natural reaction that creates visible light in the presence of oxygen found in various bioluminescent organisms including certain bacteria, fish, insects, algae and squid.
In order to develop a system of bioluminescence imaging for fungi you need to develop genes for proteins that produce the glowing substrate at high enough levels to view. There are currently three major ones in use; one from fireflies, one from a sea pansy, and one from a small sea crustacean. While all three of these function well for reporting gene regulation of proteins, in order to view the entire fungi during the infection of an organism the glowing light needs to be very bright and sustained. The firefly gene also contained a section of extra sequence localising the protein to a specific area of the cell away from the substrate needed to create glowing. Removing this, and creating synthetic genes more suited to the internal environment of a fungi, created genes that produced enough signal to be used for imaging.
The first glowing fungi used for studies of infection was a yeast (Candida albicans – which causes opportunistic yeast infections in the throat and genitals) containing the firefly gene. While the bioluminescence is bright enough to view, it runs into problems with infections located deep within the body. The glow from the firefly gene is absorbed by haemoglobin making it invisible when the infection gets too deep. This particular firefly gene has also been introduced to filamentous fungi, which tend to spread across the surface. Once again, the infection can be viewed spreading and developing, but the signal is lost if the fungi burrow deep into the body.
The latest advances in fungi imaging have focused on increasing the amount of light produced, so that the fungi can be viewed even when deep inside the body. Much of this work has focused on the process of ‘codon optimisation’. Different organisms sometimes use slight variations in the code for creating proteins from the genetic blueprint. By altering the synthetically produced genetic sequence it can be changed from a sequence that works best in fireflies to a sequence that works best in fungi.
Currently these models are best used for studying surface infections and the development of fungal biofilms. As well as the challenges of loosing the signal in deep infections there is also the complication that the firefly luciferase (the protein that creates the glow) requires at least a small amount of oxygen which may be difficult to find deep in an infected niche surrounded by the workings of the immune system. The firefly luciferase also uses up a lot of cellular energy. Work to adapt to these challenges, and to create bioluminescence imaging models in other fungi species, would be a useful and valuable tool for studying the spread of internal fungal infections.
Reference: Papon N, Courdavault V, Lanoue A, Clastre M, Brock M (2014) Illuminating Fungal Infections with Bioluminescence. PLoS Pathog 10(7): e1004179. doi:10.1371/journal.ppat.1004179
Credit link for image 1
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