Never underestimate the role of cell culture supernatant!

Go to the profile of Ewa Bielska
Apr 19, 2018
Upvote 2 Comment

The paper in Nature Communications is here:

Cryptococcus is an example of a perfect fungal pathogen, able to colonize, infect and survive in a whole spectrum of organisms including plants, protists, animals and humans. Over millions of years, natural selection has honed its fitness to allow it to overcome host defences and flourish as an intracellular pathogen. That was my first impression about this fungal pathogen three years ago when I joined Prof. Robin May’s lab at the University of Birmingham and when I was introduced to the hypervirulent strain of Cryptococcus gattii. This strain was the case of the Pacific Northwest outbreak of cryptococcosis, which led to many deaths of otherwise healthy humans and animals and caused ripples of fear in North America in 2001. Extensive work since then has shown that this fatal pathogen uses a spectrum of mechanisms to allow it to grow extremely rapidly within mammals. One of these is ‘division of labour’, a strategy in which a subpopulation of dormant ‘guardian’ fungal cells protects other fungi in the population, allowing them to proliferate.  

‘Wow, an altruistic pathogen!’ I thought and immediately started wondering how these cells communicate in order to coordinate this response. Do they need to be in close proximity (e.g. in the same host cell) and how do they send messages to each other? I then read two publications by Rodrigues et al. (2008) and Peres da Silva et al. (2015) where the authors showed that extracellular vesicles (EVs) produced by C. neoformans contain RNA and proteins associated with virulence. The idea that EVs can serve not only as rubbish bags but also as virulence bags for long distance communication between different cells inspired me to look at this in the context of these virulent strains.  

As a complete novice to the field of EVs, I was initially overwhelmed by the number of methods I was not familiar with and how technically challenging they were. However, after 13 hours of painful EV isolations (Figure 1) we had our first promising results, suggesting that EVs from outbreak strains might be able to trigger virulence in less infectious strains.

Figure 1. EV isolation was performed from 5 day old cultures of C. gattii strains. a. India ink staining of cells from 5 days old cultures of the hypervirulent strain R265 and low virulent strain ICB180. Scale bar: 10 μm. b. Ultracentrifuge tube with a tiny EV pellet stained with an orange/red-fluorescent lipophilic dye DiI. 

Long hours spent alongside a motivated Masters student, Marta Arch Sisquella, together with discussions with our local EV expert Dr Paul Harrison and attending some well-timed EV conferences (UKEV and ISEV) gave us some insights how to deal with fungal EVs. Although cryptococcal EVs do not contain typical exosomal tetraspanins like CD9, CD63 and CD81, and therefore they could not be confirmed using standard methods, we managed to confirm the presence of EVs in our samples by electron microscopy and NanoSight, and finally by immunostaining using antibodies which recognize capsular polysacharide GXM. That raised another question – are these really EVs or simply shed capsule (Figure 2)?

Figure 2. Diagram representing a cryptococcal cell shedding capsular material and releasing EVs. GXM – glucuronoxylomannan; GXMGal – glucuronoxylomannogalactan; EV – extracellular vesicle. Picture modified from Bielska & May (2016) What makes Cryptococcus gattii a pathogen? FEMS Yeast Res, 16(1).

By using EVs isolated from an acapsular mutant of the outbreak strain, we found that EVs lacking capsular material were still able to enhance survival of non-outbreak strains. Finally, we tested several combinations of infections with different strains to demonstrate that EV communication is highly specific…in other words, the EV “bullet” can only be used if the recipient strain has the matching “gun”.  

Now, of course, some big questions remain. What is the composition of these EVs? How are they exchanged by cryptococcal cells? What is the mechanism of EV-stimulated proliferation? Ultimately, we hope that finding answers to these questions might help us develop therapies to target EVs and, in that way, neutralise this part of the fungal armament.   

Go to the profile of Ewa Bielska

Ewa Bielska

Postdoctoral Research Fellow, University of Birmingham

No comments yet.