Undergraduate Student Report 2021

Dymtro Prasolov worked with Dr Carolina Coelho, University of Exeter, to investigate the impact of gene deletion on the growth of Cryptococcus neoformans, a fungal pathogen of mammals.

Cryptococcus neoformans (CN) is basidiomycete fungus that can cause fungal infection (cryptococcosis) of lungs and central nervous system in animals as well as humans (Rajasingham et al., 2017; Chaoyang et al., 2012). This pathogen particularly affects immunocompromised individuals, with estimates suggesting there are 181,100 annual deaths around the world (Rajasingham et al., 2017). Notably, it is particularly prevalent in Africa, with about 73% of all worldwide cases of cryptococcal meningitis occurring in this region (Rajasingham et al., 2017). One of the groups most affected by cryptococcosis are AIDS patients, with 15% of AIDS related deaths being as a result of cryptococcal infection (Rajasingham et al., 2017). Importantly, other immunocompromised groups such as organ transplant recipients also suffer from cryptococcosis, with estimations of up to 20% of these individuals being affected in the USA (Singh et al., 2008).

With strains resistant to antifungal drugs being identified as early as 1999, it is vitally important to identify new targets and pathways that may be used as targets for new therapeutic drugs. (Perfect et al., 1999) One way this could be done is through identification of novel pathways that may be essential for CN virulence. Before start of my project, my supervisor identified several proteins which may have relevance for virulence and has prepared such knockout strains for these proteins. Ultimately, my project aimed to determine a deficiency in virulence through two ways: first by determining whether the knockout strains show reduced virulence on wax moth larvae (Galleria mellonella) model and secondly assessing whether the knockout strains have a reduced capability to form a capsule.

Galleria mellonella model experiments

For this experiment, the strains were chosen based on the data from previous experiment, where a large number of strains were screened for deficiency in virulence. 15 galleria per group were used and two concentrations of were tested, 10⁴ Cn per galleria, and 10⁵ Cn per galleria. This was done in order to investigate whether these deletion strains and wild type have higher virulence at a greater concentration. At 10⁴ Cn per galleria, a difference was found between wild type and strain 2. A difference was also found between wild type and strain 3, although such difference only occurred in the early stage of the infection. At 10⁵ Cn per galleria, a difference was found between all 5 tested strains and wild type. Interestingly, there was no difference in virulence between each individual strain at two concentrations, when pairwise comparisons were performed. This therefore suggests that a further 10 fold increase in concentration of Cn per galleria does not have an effect on virulence of each strain. However, at higher concentration of Cn per galleria, the hypovirulence of knockout strains compared to wild type remains present.

Capsule measurements

During a project that we worked on previously, it became apparent that some strains show deficient growth in host mimicking conditions (DMEM). We hypothesised that such deficiency may be related to lack of capsule production. Capsule is an important virulence factor in CN, as it protects it from oxidative stress. (Zaragoza et al., 2008). In this experiment, we visualised it using India ink, which resulted in capsule having a halo-like appearance (Figure 1).

Strains 4 and 5 showed reduced capsule size. However, it is worth noting that strain 4 has also shown deficient growth in DMEM, so it is reasonable to suggest that the reduced capsule size in this strain is due to lack of cellular growth in this environment. During take 2, all knockout strains that were tested showed a significantly reduced size of capsule in minimal media. Take 2 showed a capsule defect in strains 3, 5, 6 and 7. Interestingly, strain 3 has also shown a reduced virulence during galleria experiments, hence making it plausible that, at least in part, deficient capsule development has contribution to reduced virulence. It is also worth noting that some strains, such as 3 and 5 have formed noticeable clumps. We hypothesise that cells not dividing properly may explain reduced virulence in these two strains. Future experiments should test the fungal growth at 30 degrees Celsius instead of 37 to see if temperature change influences ability to develop capsule.

Figure 1a. Capsule around cryptococcal cells appearing as halo.
Figure 1b. Capsule around cryptococcal cells appearing as halo.
Figure 2. Example of cryptococcal cells with a deficient growth of capsule (strain 3). 
Figure 3. Clumps of cells formed by strains 3 and 5. 

Acknowledgments

I thank my supervisor, Dr.Carolina Coelho, for the idea for this project, as well as teaching me the techniques and skills necessary for this project. I also sincerely thank British Mycological Society for their generous funding which enabled me to undertake this project.

References

1.       Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, Chiller TM et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis [Internet]. 2017 Aug 17 [cited 2021 Oct 11]; (8):873-881. Available from: https://doi.org/10.1016/S1473-3099(17)30243-8

2.       Xue C. Cryptococcus and beyond--inositol utilization and its implications for the emergence of fungal virulence. PLoS Pathog [Internet]. 2012 Sep [cited 2021 Oct 11]; 8(9): e1002869. Available from: https://doi.org/10.1371/journal.ppat.1002869 

3.       Singh N, Alexander BD, Lortholary O, Dromer F, Gupta KL, John GT et al. Pulmonary cryptococcosis in solid organ transplant recipients: clinical relevance of serum cryptococcal antigen. Clin Infect Dis. [Internet] 2008 Jan 15 [cited 2021 Oct 11]; 46(2):12-18. Available from: https://doi.org/10.1086/524738  

4.       Perfect JR, Cox GM. Drug resistance in Cryptococcus neoformans. Drug Resist Updat [Internet]. 1999 Aug [cited 2021 Oct 11]; 2(4):259-269. . Available from: https://doi.org/10.1054/drup.1999.0090  

5.       Kevin Kavanagh, John P. Fallon. Galleria mellonella larvae as models for studying fungal virulence. Fungal Biology Reviews, Volume 24, Issues 1–2, 2010, Pages 79-83. Available from: https://doi.org/10.1016/j.fbr.2010.04.001.

6.       Zaragoza O, Chrisman CJ, Castelli MV, et al. Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival. Cell Microbiol. 2008;10(10):2043-2057.doi:10.1111/j.1462-5822.2008.01186.x

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