Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence

Man Shun Fu, Livia C. Liporagi-Lopes, Samuel R. dos Santos Júnior, Jennifer L. Tenor, John R. Perfect, Christina A. Cuomo, Arturo Casadevall

Preprint posted on August 07, 2020

Survival in soil promotes human infection: The evolution of cryptococcal virulence

Selected by Josie Gibson

Categories: microbiology


Cryptococcus neoformans is an environmental fungus, which is also an opportunistic human fungal pathogen. As such, C. neoformans infection can cause life-threatening disease, usually in immunocompromised individuals. To cause infection, C. neoformans has evolved multiple virulence factors, allowing survival within the host. Of particular importance is the ability for cryptococci to survive within, and then escape from, macrophages. However, C. neoformans is an environmental yeast, which does not require human infection as part of its life-cycle, raising the question how C. neoformans evolved virulence factors to survive within macrophages. It has previously been demonstrated that exposure to amoebae, which are soil resident predators sharing similar properties to macrophages, increases cryptococcal virulence [1]. In this study, the selective pressure of exposure to amoebae is studied to examine the mechanisms of how amoebae-cryptoccocal interactions alter cryptoccocal virulence factor selection.

Key findings:

This study shows that exposure of amoebae to C. neoformans leads to increased diversity in cryptococcal traits and virulence factors. This may enable continued environmental survival, since increased diversity may provide a higher chance of some cryptococcal survival. Virulence factor selection driven by amoebae predation may also be a cause of improved human infection, specifically in macrophage resistance.

Using lab and environmental cryptococcal strains, cryptococcal cells were left in the presence of amoebae for a month, after which, cryptococcal colonies had formed that were composed of multiple morphologies. In general, the surviving cryptococcal cells were more resistant to amoebae predation, however, environmental strains were less likely than the lab strain to gain resistance. Pseudohyphae or large cell volume phenotypes formed in resistant strains, which the authors suggest to be important for amoeba resistance. Moreover, known human virulence factors were commonly present in the resistant cryptococcal strains.

Image of cryptococcal cells with altered morphologies (red arrow) after being left in the presence of amoebae (highlighted in the dashed line area). Taken from Figure 1A of the preprint, made available under a CC-BY 4.0 license.



Next, the authors show DNA mutations can occur in cryptococcal isolates following predation, altering roles in quorum sensing, hyphae formation and stress responses. Interestingly, some resistant cryptococci did not have mutations, suggesting an alternative cause of resistance phenotypes than mutations alone. A small number of resistant strains had a chromosome duplication, with roles in increased amoebae resistance. Interestingly, epigenetic modifications were not associated with amoeba resistance.

Finally, the impact of the virulence factors obtained by amoebae resistant cryptococcal strains was examined using macrophages and in vivo infection. Unexpectedly, none of the resistant strains had improved intracellular survival in macrophages, although some isolates caused physical damage to macrophages. Furthermore, the isolates which did cause macrophage damage, did not have increased virulence in murine infection. The authors determined the lack of murine virulence was due to induction of a more effective immune response.

Why I chose this Preprint:

The authors examine mechanisms of cryptoccocal environmental evolution which may promote macrophage evasion in human infection. The study demonstrates that amoebae pressure drives rapid selection of diverse cryptococcal virulence factors (many which are associated with human infection), and determines some are caused by DNA mutations and aneuploidy. This study furthers our understanding of how environmental evolution of C. neoformans may lead to the ability of an environmental fungus to survive within macrophages and cause human disease. Furthermore, the authors suggest that mechanisms driving diversity of virulence factors studied here, may represent an alternative antimicrobial target.

Questions to the authors:

  1. Could differences in soil predators in different locations worldwide be important in outbreaks of cryptoccocal infections?
  2. Considering that epigenetic modifications could lead to rapid cryptococcal adaptation, do you have any suggestions why it is not involved in amoeba resistance?
  3. There was no increase in macrophage persistence or in vivo infection virulence in resistant strains. Do you think that using longer predation times with increased variety of amoebae predators could increase the likelihood of finding a virulent strain?


  1. Chrisman CJ, Albuquerque P, Guimaraes AJ, Nieves E, Casadevall A (2011) Phospholipids Trigger Cryptococcus neoformansCapsular Enlargement during Interactions with Amoebae and Macrophages. PLoS Pathog 7(5): e1002047.

Tags: c. neoformans

Posted on: 2nd September 2020 , updated on: 25th March 2021


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Author's response

Man Shun Fu shared


  1. Quite possible. Many bacterial and viral outbreak are linked to the interaction with amoebae. For examples, the investigation of an outbreak of Pontiac fever which is caused by Legionella anisa, in a conference at a hotel in California showed that anisaand Hartmanella vermiformis were isolated from the decorative fountain in the hotel lobby. Also, identification of Parachlamydia sp. strain Hall coccus within an amoeba isolated from the source of an outbreak of humidifier fever in the United States. In 1992, during an investigation of an outbreak of pneumonia, a giant double-stranded DNA virus within a free-living amoeba have been recovered from the water of a cooling water in Bradford (England). It is quite likely that the outbreaks of fungal infections are associated with amoebae interaction. In addition, there are many different strains of amoebae or different kinds of invertebrate predators in soil. Different kinds of the interaction will increase the diversity of fungi and thus may increase the chance of the emerging. Of course, other factors e.g. temperature could also play an important role in the emerging of fungal pathogens.
  2. In our paper, we only investigate the global histone H3 acetylation, and there are many other epigenetic modifications or gene silencing mechanisms such as polycomb system and RNAi-dependent mechanisms. However, those mechanisms in yeast, especially in Cryptococcus are still poorly understanding. We do not completely rule out the possibility that epigenetic modifications, along with genetic modifications, are also involved in cryptococcal adaptation.
  3. There is an investigation of the genome evolution of amoebal intracellular microorganisms, Lausannevirus and bacteria Estrella lausannesis with the co-incubation of amoebe castellanii for one year. Genome sequencing was performed every three months and revealed that both, Lausannevirus and E. lausannensis, show stable genome with slightly increased mutations. Therefore, I predict longer predation times may slightly increase the mutations of Cryptococcus, but may not be significantly increase the chance of selecting a virulent strain. However, there is another study on investigating role of host genetic diversity in the evolution of virulence of a plant virus [1]. They found faster TuMV adaptation to homogeneous than to heterogeneous host A. thaliana populations. However, viruses evolved in heterogeneous host populations were more pathogenic and infectious than viruses evolved in the homogeneous population. These results illustrate how the genetic diversity of hosts in an experimental ecosystem favour the evolution of virulence of a pathogen. In overall, I believe longer predation times with increase variety of amoebae predators could increase the likelihood of finding a virulent strain.


  1. González R, Butković A, Elena SF. Role of host genetic diversity for susceptibility-to-infection in the evolution of virulence of a plant virus†. Virus Evol. 2019 Jul 1;5(2).

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